INCORPORATION BY REFERENCE OF SEQUENCE LISTING
This application contains a Sequence Listing which has been submitted electronically in ST26 format and is hereby incorporated by reference in its entirety. Said ST26 file, created on Apr. 1, 2024, is named “1676192US1.xml” and is 62,496 bytes in size.
BACKGROUND
Breast cancer is one of the most prevalent malignancies with an estimated 2.3 million new cases and 685,000 deaths per year globally. Approximately 60 to 70% of breast cancers express the estrogen receptor (ER+), 10 to 15% are ERBB2-amplified (HER2+) and 10 to 15% are triple-negative breast cancers (TNBC) that are ER−, PR−, and Her2−. While there are targeted therapies available for ER+ and HER2+ breast cancer, no targeted therapy is available for TNBC or IBC, which have a worse outcome than other subtypes.
The major cause of death in all subtypes of breast cancer is metastatic spread of the cancer cells through lymphatics and blood vessels to other organs. A very aggressive and lethal clinical type of cancer, named inflammatory breast cancer (IBC), is more common in women of African-American descent, younger than 40, and overweight patients and is associated with extensive dermal lympho-vascular invasion (LVI) and high metastatic risk. It is worth mentioning that the term pseudo-inflammatory may have been more accurate for this entity; since the edema, erythema and tenderness that mimics breast inflammation (mastitis) is due to dermal tumor emboli plugging the lympho-vascular circulation, rather than true inflammation. Nevertheless, IBC is a very difficult challenge in the clinic; despite the distinct biology of IBC there is no specific therapy for this disease. The patients with IBC are treated with systemic treatments similar to other non-inflammatory breast cancers[1].
The example of breast cancer suggests that targeted therapies can leverage both acquired targets (HER2 amplification) and intrinsic targets (ER expression). The breast cancer acquired amplification of ERBB2 has been targeted with anti-HER2 antibody, biologics and small molecule ERBB2 kinase inhibitors. In the case of hormonally driven breast cancer, the over-expression of ER seems to be a reflection of the normal cell-origin or differentiation lineage since ER mutations or amplifications are exceedingly rare in untreated patients. In some cases, ER mutations emerge after anti-estrogen treatment as a resistance mechanism. Nevertheless, the anti-estrogens that block ER transcriptional activity or suppress estrogen production have been highly-effective in ER+ breast cancer.
SUMMARY
The human body is composed of hundreds of different normal cell types. Some of the important and unique features of these normal cell types are inherited by the cancer cells that arise from them. Indeed, the activities of the tumor gene mutations are constrained by this normal cell inheritance. For example, it was found that the same oncogenes produce highly malignant tumors in some cell types, but not in others. In this study it is demonstrated that this cell-of-origin difference can be used to develop cell-targeted therapies, which is a novel concept that differs from the gene-targeted therapies. This cell-based approach led to a surprising discovery that a drug that was approved for treating tapeworm infestation together with an antibiotic derivative can significantly enhance killing of breast cancer cells. Since these drugs are already approved for clinical use, it may be possible to repurpose them to treat breast cancer.
Breast cancer gene expression signatures are described herein include ET-29 (29 genes as shown in Tables 2 and 3) that are co-upregulated by HDAC1 and HDAC7 in metastatic TNBC and have a ZNF92 binding site in their promotor. Upregulation of HDAC1, HDAC7, and ZNF92 predicts sensitivity to a 3-drug combination including a Histone Deacetylase (HDAC inhibitor), combined with a heat shock protein 90 (HSP 90) inhibitor and a helminth such as the tape-worm drug Niclosamide. These 3 drugs are synergistic in killing breast cancer cells. Importantly, the two-drug combinations are not synergistic.
Described herein are methods that can include: a. assaying a biological sample from a subject for expression of HDAC1, HDAC7, and ZNF92 genes to determine one or more expression levels for the HDAC1, HDAC7, and ZNF92 genes; b. comparing the determined expression levels with one or more reference values to identify any altered expression levels in the subject's biological sample, wherein altered expression levels of the HDAC1, HDAC7, and ZNF92 genes in the biological sample relative to the reference value indicates that the subject has cancer with poor prognosis or the subject has malignant cancer, and absence of altered expression of the HDAC1, HDAC7, and ZNF92 genes relative to the reference value indicates that the subject does not have a cancer with poor prognosis or does not have malignant cancer; and optionally c. administering one or more histone deacetylase inhibitor(s) (HDAC), one or more inhibitor of heat shock protein 90 (HSP90), and one or more helminths to a subject determined to have a cancer with poor prognosis or a malignant cancer. In one embodiment, the biological sample from the subject is further assayed for expression of one or more genes encoding an amino acid sequence having at least 90% sequence identity to SEQ ID NOs: 3-31. In one embodiment, the sample is a breast cancer sample. In one embodiment, the sample is a cervical cancer sample, uterine cancer, or prostate cancer. In one embodiment, one or more of ML239, Tipifarnib, Tivantinib, CHIR-99021, Tubastatin-A, GDC-0879, SB-525334, PRIMA-1-met, or a combination thereof, is further administered to the subject. In one embodiment, the administering one or more histone deacetylase inhibitor(s), one or more heat shock factor inhibitor, and the one or more helminths are delivered simultaneously. In one embodiment, the sample is a physiological fluid sample, a tumor tissue sample, or a cancer cell sample. In one embodiment, the subject is a human. In one embodiment, the mammal has inflammatory breast cancer. In one embodiment, the mammal has triple negative breast cancer. In one embodiment, RNA expression is assayed. In one embodiment, protein expression is assayed. In one embodiment, the inhibitor of HSP90 comprises an inhibitor of HSP90a and HSP90s. In one embodiment, the inhibitor of HSP90 comprises an antibiotic. In one embodiment, the inhibitor of HSP90 is Pimitespib, Tanespimycin, or a combination thereof. In one embodiment, the helminth is niclosamide. In one embodiment, the at least one HDAC inhibitor is Belinostat, Entinostat, Vorinostat, or a combination thereof.
Described herein are methods that can include preventing, inhibiting or treating cancer in a mammal, comprising: administering to the mammal a composition comprising one or more histone deacetylase inhibitor(s), one or more heat shock factor inhibitor, and one or more helminths, wherein the cancer in the mammal is determined to have altered expression levels of HDAC1, HDAC7, and ZNF92 genes, relative to a reference value. In one embodiment, the biological sample from the subject is further assayed for expression of one or more genes encoding an amino acid sequence having at least 90% sequence identity to SEQ ID NOs: 3-31. In one embodiment, the mammal is a human. In one embodiment, the mammal has breast cancer, cervical cancer, uterine cancer, or prostate cancer. Methods described herein can include (a) contacting cancer cells expressing HDAC1, HDAC7, or ZNF92 genes or producing HDAC1, HDAC7, or ZNF92 proteins with a test agent; (b) measuring HDAC1, HDAC7, or ZNF92 RNA or protein expression levels in the cells or measuring HDAC1, HDAC7, or ZNF92 protein activity levels; and (c) determining that the test agent reduces the expression levels or activity levels of HDAC1, HDAC7, or ZNF92 genes, to thereby identifying a test agent as a candidate agent that reduces HDAC1, HDAC7, or ZNF92 genes expression levels or activity levels. Also described herein is a pharmaceutical composition comprising one or more histone deacetylase inhibitor(s), one or more heat shock factor inhibitor, and one or more helminths.
BRIEF DESCRIPTION OF THE FIGURES
The figures illustrate generally, by way of example, but not by way of limitation, various embodiments discussed herein.
FIGS. 1A-1C show the identification of metastatic TNBC cell-of-origin targets and drugs. FIG. 1A: The estrogen receptor negative normal breast cells cultures, BPE and HME, were generated from same donor [2]. Transformation of BPE and HME with the same genes gave rise metastatic BPLER and non-metastatic HMLER tumor cell lines with a TNBC phenotypes [2]. The metastatic TNBC BPLER cells are associated with higher expression of HDAC1, HDAC7 and ZNF92, compared to non-metastatic TNBC HMLER cells [25, 27]. FIG. 1B: HDAC1 upregulates HDAC7, which in turn upregulates 29 super-enhancer (SE) associated genes with ZNF92 binding sites (Table 1) [27]. This HDAC1-HDAC7-ZNF92 axis can be targeted by HDAC inhibitors (Entinostat, Belinostat, and Vorinostat). FIG. 1C: ZNF92 expression correlates only with 17-AAG sensitivity (arrow) among 265 small molecules in the Genomics of Drug Sensitivity in Cancer (GDSC) dataset, with IC50 in 860 cell lines and their corresponding mRNA gene expression (red circle, top left). All the other drugs have a negative correlation with ZNF92 (blue circles). GSCA only draws plot for the top 30 ranked drugs; blue filled circles represent negative correlation, red filled circles represent positive correlation, the color intensity reflects higher correlation, the circle size is positively correlated with the false discovery rate (FDR) significance, and the black outline border indicates FDR ≤0.05. See supplemental data for details. FIG. 1D summarizes the correlation between the HDAC1-HDAC7-ZNF92 axis targets including the 29 genes (shown in Table 2) expression signature and the sensitivity of CTRP drugs (top 30), including the IC50 of 481 small molecules in 1001 cell lines and corresponding mRNA gene expression from Genomics of Therapeutics Response Portal (CTRP). The mRNA expression data and drug sensitivity data were merged. Pearson correlation analysis was performed to get the correlation between gene mRNA expression and drug IC50. P-value was adjusted by FDR. Only the top 17 ranked drugs shown, Niclosamide is ranked #2 (arrow). The blue filled circles represent negative correlation, red filled circles represent positive correlation, the color intensity reflects higher correlation, the circle size is positively correlated with the false discovery rate (FDR) significance, and the black outline border indicates FDR ≤0.05.
FIG. 2 shows treatment of triple-negative breast carcinoma with HDAC-I inhibitor Entinostat (E), Niclosamide (N), and HSP90-I inhibitor Tanespimycin (T). The TNBC cell lines BT549, BT20, HCC38, CAL148, HCC 1143, HCC 1913, MDAMB231, MDAMB43, MFM223, Sum159 and Sum1315 are treated with Entinostat (E: 100 nM), Niclosamide (N: 100 nM), and Tanespimycin (T: 50 nM) alone and in combination (ENT) in 96-well plates for 7 days with indicated drugs. The inhibition of cell proliferation by E, N, T and ENT is measured by WST assay. The relative cell number is calculated compared to vehicle control (DMSO) and expressed as mean±SD (n=6) of percent viability. (*) Inhibition in cell proliferation greater than expected by additivity.
FIGS. 3A-3B shows treatment of triple-negative breast carcinoma with HDAC-I inhibitors Vorinostat (V) or Belinostat (B), with Niclosamide (N), and HSP90-I Pimitespib (P). The TNBC cell lines BT20, MDAMB231, MDAMB435 and Sum159 are cultured in 96-well plates for 7 days with indicated drugs. The inhibition of cell proliferation is measured by WST assay. The relative cell numbers are calculated compared to vehicle control (DMSO) and expressed as mean±SD (n=6) of percent viability. (A) Vorinostat (V: 1000 nM), Niclosamide (N: 100 nM), and Pimitespib (P: 600 nM), and (B) Belinostat (B: 1000 nM), Niclosamide (N: 100 nM) and Pimitespib (P: 400 nM). (*) Inhibition in cell proliferation greater than expected by additivity.
FIGS. 4A-4D show triple-negative breast carcinoma dose response and test combination of HDAC inhibitor Entinostat (E), Niclosamide (N), and HSP90 inhibitor Tanespimycin (T). All experiments were performed in 96-well plates for 7 days. DMSO was used as vehicle control. Inhibition of cell proliferation is measured by the WST assay. Each value is expressed as mean t SD (n=6) of percent viability. (*) Inhibition in cell proliferation greater than expected by additivity. FIG. 4A: Dose response plots of Entinostat (E), Niclosamide (N) and Tanespimycin (T) in BPLER-TNBC cells (0, 15, 32.5, 65, 125, 250, 500, 1000, 2500, 5000, 10000 nM). FIG. 4B: Dose response plots of Entinostat (E), Niclosamide (N) and Tanespimycin (T) in BT20, MDAMB231 and Sum159 TNBC cells lines (0, 15, 32.5, 65, 125, 250, 500, 1000, 2500, 5000, 10000 nM). Highlighted regions in the graphs show the range of doses used for three-drug synergy experiments. FIG. 4C: Three drug combination of Entinostat (E: 100 nM), Niclosamide (N: 100 nM) and Tanespimycin (T: 30 nM), abbreviated as “ENT”, in BLER cells. FIG. 4D: Three drug combination of Entinostat (250 nM: Sum159, 100 nM: MDA-MB231, 50 nM: BT20), Niclosamide (250 nM: Sum159, 100 nM: MDA-MB231, 50 nM: BT20), and Tanespimycin (50 nM: Sum 159 and MDA-MB231, 30 nM:BT20).
FIGS. 5A-5B. HSP90 inhibitor Pimitespib (P) and HDAC inhibitors Vorinostat (V) and Belinostat (B). FIG. 5A: Dose response plots of HSP90 inhibitor Pimitespib (P) and HDAC inhibitors Vorinostat (V) and Belinostat (B) in BPLER-TNBC cells (0, 15, 32.5, 65, 125, 250, 500, 1000, 2500, 5000, and 10000 nM). FIG. 5A: Three drug combination of Entinostat (50 nM for BT20 and 100 nM for all other cell lines), Niclosamide (N: 100 nM), and Pimitespib (P: 400 nM). FIG. 5B: The cells are cultured in 96-well plates for 7 days with drug. Inhibition of cell proliferation is measured by WST assay, the relative cell numbers are calculated compared to vehicle control (DMSO) and expressed as mean f SD (n=6) of percent viability. (*) Inhibition in cell proliferation greater than expected by additivity.
FIGS. 6A-6C. Triple-negative breast carcinoma triple-drug combinations of HDAC inhibitor Vorinostat (V), Belinostat (B) or Entinostat (E), with Niclosamide (N) and HSP90-I Pimitespib (P). The TNBC cell lines BT20, MDAMB231, MDAMB435 and Sum159 are cultured in 96-well plates for 7 days with drug. Inhibition of cell proliferation is measured by WST assay, the relative cell numbers are calculated compared to vehicle control (DMSO) and expressed as mean f SD (n=6) of percent viability. (*) Inhibition in cell proliferation greater than expected by additivity. See supplementary table 1 for details. FIG. 6A: Vorinostat (V: 1000 nM), Niclosamide (N. 100 nM), and Pimitespib (P: 200 nM). FIG. 6B: Belinostat (B: 1000 nM), Niclosamide (N: 100 nM), and Pimitespib (P: 200 nM). FIG. 6C: Entinostat (E: 100 nM), Niclosamide (N: 100 nM), and Pimitespib (P: 200 nM).
FIGS. 7A-7C. Inflammatory breast cancer triple-drug combinations. The combination of HDAC inhibitor Entinostat (E) and Belinostat (B) with Niclosamide (N) and HSP90 inhibitor Pimitespib (P) in inflammatory breast cancer (IBC) cell lines. The IBC03, SUM-149 and SUM-190 cell lines are cultured in F12 medium (Gibco 11765-054) and KPL-4 is cultured in Ham's F12 medium (Corning 10-080-CV) with the indicated drugs. The inhibition of cell proliferation is measured by WST assay, the relative cell numbers are calculated compared to vehicle control (DMSO) and expressed as mean±SD (n=6) of percent viability. (*) Inhibition in cell proliferation greater than expected by additivity. FIG. 7A: Dose response plots of HDAC inhibitor Entinostat (E), Niclosamide (N) and Pimitespib (P) in IBC cell lines (0, 15, 32.5, 65, 125, 250, 500, 1000, 2500, 5000, and 10000 nM). FIG. 7B: Entinostat (100 or 150 nM), alone or combined with Pimitespib (100 or 200 nM), and Niclosamide (100 nM). FIG. 7C: Belinostat (500 or 1000 nM), alone or combined with Pimitespib (100 or 200 nM), and Niclosamide (100 nM).
FIG. 8. Frequency of genomic alterations in the cell-of-origin signature. The image illustrates that frequency of mutations, structural variants and copy number changes in HDAC1, HDAC7, ZNF92 and the 29 genes with the ZNF92 binding sites (excluding alterations of unknown significance). The analysis is carried out in the cBioPortal combined TCGA and Metabric datasets with 3,593 breast cancer samples and shows that none of the genes in the cell-origin associated HDAC1-HDAC7-ZNF92 axis and their 29 downstream targets have bona fide driver mutations in more than 0.4% of breast cancer (https://www.cbioportal.org/).[1, 2]
DETAILED DESCRIPTION
As illustrated herein, breast cancer gene expression signatures referred to herein as ET-29 (29 genes as shown in Tables 2 and 3), together with HDAC1, HDAC7 and ZNF92 genes predicts sensitivity of triple-negative breast cancer (TNBC) to a 3-drug combination including a Histone Deacetylase (HDAC inhibitor), combined with a heat shock protein 90 (HSP 90) inhibitor and a tape-worm drug Niclosamide. These 3 drugs are synergistic in killing breast cancer cells. Expression of ET-29, together with HDAC1, HDAC7 and ZNF92 genes, are useful markers for detecting, diagnosing, and determining the prognosis of cancer, including breast cancer. Methods for detecting, diagnosing, and determining the prognosis of cancer, including breast cancer, are also described herein.
The markers are a cell-of-origin specific mRNA signature associated with the over-expression of histone deacetylases and zinc finger protein and metastasis and poor outcome in triple-negative breast cancer (TNBC). Based on this signature, it was discovered that the combination of three drugs: a histone deacetylase (HDAC) inhibitor, an anti-helminthic Niclosamide, and an antibiotic Tanespimycin that inhibits heat shock protein 90 (HSP90) synergistically reduces the proliferation of all twelve TNBC cell lines that were tested.
Additionally, it was discovered that four out of five inflammatory breast cancer (IBC) cells are sensitive to this combination. Significantly, the concentration of the drugs that are used in these experiments are within or below clinically achievable dose, and the synergistic activity only emerged when all three drugs were combined. The present results indicate that combining HDAC and HSP90 inhibitors with the tapeworm drug Niclosamide can achieve remarkably synergistic inhibition of TNBC and IBC. Since Niclosamide, HDAC and HSP90 inhibitors were approved for clinical use for other cancer types, it may be possible to repurpose their combination for TNBC and IBC.
Historically, three or more drugs have been routinely combined for many cancer treatments[49]. Partly due to the difficulty of screening high-order combinations, new drug candidates are often tested in combination with first-line treatments, sometimes without a compelling hypothesis[50-56]. As expected, most of the 54 breast clinical trials with HDAC and HSP90 inhibitors have been in combination with anti-estrogen, anti-HER2 and/or chemotherapy drugs, and we have not found any breast cancer trials for niclosamide (Table 1).
TABLE 1
|
|
Breast cancer clinical trials with HDAC and HSP90 inhibitors.
|
Drug 2
|
Rank
NCT Number
Drug 1
Drug 2
target
Phase
Status
|
|
1
NCT00413075
Belinostat
Phase 1
Completed
|
2
NCT00413322
Belinostat
5-FU
DNA
Phase 1
Completed
|
synthesis
|
3
NCT04315233
Belinostat
Ribociclib
Cyclin D1/
Phase 1
Recruiting
|
CDK4/6
|
4
NCT04703920
Belinostat
Talazoparib
PARP
Phase 1
Recruiting
|
5
NCT03432741
Belinostat
Phase 1
Recruiting
|
7
NCT00627627
Belinostat
Phase 1|2
Withdrawn
|
6
NCT00817362
Belinostat
Trastuzumab
HER2
Phase 2
Terminated
|
8
NCT02453620
Entinostat
Nivolumab
PD-L1/2
Phase 1
Active, not
|
recruiting
|
13
NCT02833155
Entinostat
Phase 1
Completed
|
14
NCT02820961
Entinostat
Exemestane
ER
Phase 1
Completed
|
15
NCT01594398
Entinostat
Phase 1
Completed
|
16
NCT01434303
Entinostat
Lapatinib
HER2
Phase 1
Completed
|
17
NCT02623751
Entinostat
Phase 1
Completed
|
18
NCT02897778
Entinostat
Phase 1
Completed
|
19
NCT00020579
Entinostat
Phase 1
Completed
|
24
NCT03473639
Entinostat
Phase 1
Recruiting
|
26
NCT04296942
Entinostat
Adotrastuzumab
HER2
Phase 1
Terminated
|
27
NCT00754312
Entinostat
Phase 1
Terminated
|
20
NCT02708680
Entinostat
Atezolizumab
PD-L1
Phase 1| 2
Completed
|
9
NCT03280563
Entinostat
Phase 1|2
Active, not
|
recruiting
|
10
NCT01349959
Entinostat
Azacitidine
DNA
Phase 2
Active, not
|
methyl-
recruiting
|
transferase
|
21
NCT00828854
Entinostat
Phase 2
Completed
|
22
NCT00676663
Entinostat
Exemestane
ER
Phase 2
Completed
|
23
NCT03291886
Entinostat
Phase 2
Completed
|
25
NCT03361800
Entinostat
Phase 2
Terminated
|
28
NCT01234532
Entinostat
Anastrozole
ER
Phase 2
Terminated
|
29
NCT02115594
Entinostat
Phase 2
Withdrawn
|
11
NCT03538171
Entinostat
Phase 3
Active, not
|
recruiting
|
12
NCT02115282
Entinostat
Phase 3
Active, not
|
recruiting
|
30
NCT00004065
Tanespimycin
Phase 1
Completed
|
31
NCT00773344
Tanespimycin
Trastuzumab
HER2
Phase 1|2
Completed
|
32
NCT00096109
Tanespimycin
Phase 2
Terminated
|
34
NCT01720602
Vorinostat
N/A
Completed
|
35
NCT01153672
Vorinostat
N/A
Completed
|
55
NCT01655004
Vorinostat
N/A
Unknown
|
status
|
57
NCT01695057
Vorinostat
N/A
Withdrawn
|
36
NCT00719875
Vorinostat
Capecitabine
DNA
Phase 1
Completed
|
synthesis
|
37
NCT01084057
Vorinostat
Ixabepilone
Microtubules
Phase 1
Completed
|
38
NCT00788112
Vorinostat
Phase 1
Completed
|
39
NCT00838929
Vorinostat
Radiation
Phase 1
Completed
|
40
NCT00045006
Vorinostat
Phase 1
Completed
|
46
NCT03742245
Vorinostat
Olaparib
PARP
Phase 1
Recruiting
|
47
NCT03878524
Vorinostat
Phase 1
Recruiting
|
49
NCT01249443
Vorinostat
Carboplatin
Phase 1
Terminated
|
41
NCT00574587
Vorinostat
Chemotherapy
Phase 1|2
Completed
|
42
NCT00368875
Vorinostat
Paclitaxel
Microtubules
Phase 1|2
Completed
|
43
NCT00258349
Vorinostat
Trastuzumab
HER2
Phase 1|2
Completed
|
50
NCT01118975
Vorinostat
Lapatinib
HER2
Phase 1|2
Terminated
|
56
NCT00416130
Vorinostat
Phase 1|2
Unknown
|
status
|
33
NCT00616967
Vorinostat
Carboplatin
Phase 2
Active, not
|
recruiting
|
44
NCT00365599
Vorinostat
Tamoxifen
ER
Phase 2
Completed
|
45
NCT00262834
Vorinostat
Phase 2
Completed
|
48
NCT04190056
Vorinostat
Pembrolizumab
PD-L1
Phase 2
Recruiting
|
51
NCT01194427
Vorinostat
Tamoxifen
ER
Phase 2
Terminated
|
52
NCT00132002
Vorinostat
Phase 2
Terminated
|
53
NCT02395627
Vorinostat
Phase 2
Terminated
|
54
NCT00126451
Vorinostat
Phase 2
Terminated
|
|
However, these combinations do not necessarily originate with high-order experimental synergy screens, which are currently unfeasible due to exponential increase in complexity to analyze the interaction of more than two drugs[57]. For example, the Gene Set Cancer Analysis (GSCA) we used in this study integrates >10,000 genomic data sets and 750 drugs from GDSC and CTRP, which represents more than 70 million possibilities for 3-drug combinations. Thus, a systematic three drug biological screen was not feasible even with 20 candidates, because it would require testing 1,140 three-drug combinations[57].
As a solution, it is thought that pairwise interaction scores can provide reliable estimates for three-drug interactions 11. However, the pairwise results of HDAC-L, HSP90-L, and niclosamide were not significantly additive. FIGS. 3A-3B. Described herein is the de novo discovery of the three-drug synergy of the HDAC-I, HSP90-I and niclosamide combination, which would not have been discovered empirically without a cell-of-origin hypothesis-based approach[59]. The three-drug synergy of HDAC-I inhibitor, HSP90-I inhibitor, and Niclosamide is demonstrated in FIGS. 2, 3A, 3B, 4C, 4Dd, 5B, 6A-6C, 7B, and 7C.
Samples
Breast cancer can be assessed through the evaluation of expression patterns, or profiles, of the HDAC1, HDAC7 and ZNF92 genes, and optionally one or more genes in the ET-29 gene mRNA signature shown in Tables 2 and 3, in one or more subject samples. The term subject, or subject sample, refers to an individual regardless of health and/or disease status. A subject can be a subject, a study participant, a control subject, a screening subject, or any other class of individual from whom a sample is obtained and assessed using the markers and/or methods described herein. Accordingly, a subject can be diagnosed with breast cancer, can present with one or more symptoms of breast cancer, or a predisposing factor, such as a family (genetic) or medical history (medical) factor, for breast cancer, can be undergoing treatment or therapy for breast cancer, or the like. Alternatively, a subject can be healthy with respect to any of the aforementioned factors or criteria. It will be appreciated that the term “healthy” as used herein, is relative to breast cancer status, as the term “healthy” cannot be defined to correspond to any absolute evaluation or status. Thus, an individual defined as healthy with reference to any specified disease or disease criterion, can in fact be diagnosed with any other one or more diseases, or exhibit any other one or more disease criterion, including one or more cancers other than breast cancer. However, the healthy controls are preferably free of any cancer.
In some cases, the methods for detecting, predicting, and/or assessing the prognosis of breast cancer include collecting a biological sample comprising a cell or tissue, such as a breast tissue sample or a primary breast tumor tissue sample. By “biological sample” is intended any sampling of cells, tissues, or bodily fluids in which expression of the ET-29, HDAC1, HDAC7 and ZNF92 genes can be detected. Examples of such biological samples include, but are not limited to, biopsies and smears. Bodily fluids useful in the present invention include blood, lymph, urine, saliva, nipple aspirates, gynecological fluids, or any other bodily secretion or derivative thereof. Blood can include whole blood, plasma, serum, or any derivative of blood. In some embodiments, the biological sample includes breast cells, particularly breast tissue from a biopsy, such as a breast tumor tissue sample. Biological samples may be obtained from a subject by a variety of techniques including, for example, by scraping or swabbing an area, by using a needle to aspirate cells or bodily fluids, or by removing a tissue sample (i.e., biopsy). In some embodiments, a breast tissue sample is obtained by, for example, fine needle aspiration biopsy, core needle biopsy, or excisional biopsy.
The samples can be stabilized for evaluating and/or quantifying ET-29, HDAC1, HDAC7 and ZNF92 genes expression levels.
In some cases, fixative and staining solutions may be applied to some of the cells or tissues for preserving the specimen and for facilitating examination. Biological samples, particularly breast tissue samples, may be transferred to a glass slide for viewing under magnification. In one embodiment, the biological sample is a formalin-fixed, paraffin-embedded breast tissue sample, particularly a primary breast tumor sample.
Gene Expression
Various methods can be used for evaluating and/or quantifying ET-29, HDAC1, HDAC7 and ZNF92 expression levels. “Evaluating and/or quantifying” is intended to determine the quantity or presence of an RNA transcript or its expression product of ET-29, HDAC1, HDAC7 and ZNF92 genes.
TABLE 2
|
|
The ET-29 gene mRNA signature downstream of the HDACI-HDAC7-ZNF92
|
axis.
|
Entrez ID
Gene Symbol
Gene Name
|
|
1
80325
ABTB1
ankyrin repeat and BTB domain containing 1
|
2
9289
ADGRG1
adhesion G protein-coupled receptor G1
|
3
113451
AZIN2
antizyme inhibitor 2
|
4
55653
BCAS4
breast carcinoma amplified sequence 4
|
5
79934
COQ8B
coenzyme Q8B
|
6
9696
CROCC
ciliary rootlet coiled-coil, rootletin
|
7
1523
CUX1
cut like homeobox 1
|
8
27122
DKK3
dickkopf WNT signaling pathway inhibitor 3
|
9
1891
ECH1
enoyl-CoA hydratase 1
|
10
2049
EPHB3
EPH receptor B3
|
11
23149
FCHO1
FCH and mu domain containing endocytic adaptor 1
|
12
84929
FIBCD1
fibrinogen C domain containing 1
|
13
81544
GDPD5
glycerophosphodiester phosphodiesterase domain
|
14
3855
KRT7
keratin 7
|
15
3985
LIMK2
LIM domain kinase 2
|
16
114783
LMTK3
lemur tyrosine kinase 3
|
17
4016
LOXL1
lysyl oxidase like 1
|
18
4430
MYO1B
myosin IB
|
19
5097
PCDH1
protocadherin 1
|
20
9124
PDLIM1
PDZ and LIM domain 1
|
21
8398
PLA2G6
phospholipase A2 group VI
|
22
5875
RABGGTA
Rab geranylgeranyltransferase subunit alpha
|
23
6337
SCNN1A
sodium channel epithelial 1 subunit alpha
|
24
92799
SHKBP1
SH3KBP1 binding protein 1
|
25
55315
SLC29A3
solute carrier family 29 member 3
|
26
56848
SPHK2
sphingosine kinase 2
|
27
79816
TLE6
TLE family member 6, subcortical maternal
|
28
79639
TMEM53
transmembrane protein 53
|
29
641649
TMEM91
transmembrane protein 91
|
|
Methods for detecting expression of the HDAC1, HDAC7, and ZNF92 genes, and optionally the ET-29 gene mRNA signature, including gene expression profiling, can involve methods based on hybridization analysis of polynucleotides, methods based on sequencing of polynucleotides, immunohistochemistry methods, and proteomics-based methods. The methods generally involve detecting expression products (e.g., mRNA or proteins) of the HDAC1, HDAC7, and ZNF92 genes, and optionally one or more of the ET-29 genes. The methods include detecting expression products of any number of the ET-29 genes including 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, or 29 of the ET-29 genes.
In some cases, PCR-based methods, which can include reverse transcription PCR (RT-PCR) (Weis et al., TIG 8:263-64, 1992), array-based methods such as microarray (Schena et al., Science 270:467-70, 1995), or combinations thereof are used. By “microarray” is intended an ordered arrangement of hybridizable array elements, such as, for example, polynucleotide probes, on a substrate. The term “probe” refers to any molecule that is capable of selectively binding to a specifically intended target biomolecule, for example, a nucleotide transcript or a protein encoded by or corresponding to the ET-29, HDAC1, HDAC7 and ZNF92 genes. Probes can be synthesized or obtained from the ET-29, HDAC1, HDAC7 and ZNF92 nucleic acids or they can be derived from appropriate biological preparations. Probes may be specifically designed to be labeled. Examples of molecules that can be utilized as probes include, but are not limited to, RNA, DNA, proteins, antibodies, and organic molecules.
Many expression detection methods use isolated RNA. The starting material is typically total RNA isolated from a biological sample, such as a cell or tissue sample, a tumor or tumor cell line, a corresponding normal tissue or cell line, or a combination thereof. If the source of RNA is a sample from a subject, RNA (e.g., mRNA) can be extracted, for example, from stabilized, frozen or archived paraffin-embedded, or fixed (e.g., formalin-fixed) tissue samples (e.g., pathologist-guided tissue core samples).
General methods for RNA extraction are available and are disclosed in standard textbooks of molecular biology, including Ausubel et al., ed., Current Protocols in Molecular Biology, John Wiley & Sons, New York 1987-1999. Methods for RNA extraction from paraffin embedded tissues are disclosed, for example, in Rupp and Locker (Lab Invest. 56:A67, 1987) and De Andres et al. (Biotechniques 18:42-44, 1995). In some cases, RNA isolation can be performed using a purification kit, a buffer set and protease from commercial manufacturers, such as Qiagen (Valencia, Calif.), according to the manufacturer's instructions. For example, total RNA from cells can be isolated using Qiagen RNeasy mini-columns. Other commercially available RNA isolation kits include MASTERPURE™ Complete DNA and RNA Purification Kit (Epicentre, Madison, Wis.) and Paraffin Block RNA Isolation Kit (Ambion, Austin, Tex.). Total RNA from tissue samples can be isolated, for example, using RNA Stat-60 (Tel-Test, Friendswood, Tex.). RNA prepared from tissue or cell samples (e.g. tumors) can be isolated, for example, by cesium chloride density gradient centrifugation. Additionally, large numbers of tissue samples can readily be processed using available techniques, such as, for example, the single-step RNA isolation process of Chomczynski (U.S. Pat. No. 4,843,155).
Isolated RNA can be used in hybridization or amplification assays that include, but are not limited to, PCR analyses and probe arrays. One method for the detection of RNA levels involves contacting the isolated RNA with a nucleic acid molecule (probe) that can hybridize to the mRNA encoded by the gene being detected. The nucleic acid probe can be, for example, a full-length cDNA, or a portion thereof, such as an oligonucleotide of at least 7, 15, 30, 60, 100, 250, or 500 nucleotides in length and sufficient to specifically hybridize under stringent conditions to any of the ET-29, HDAC1, HDAC7 and ZNF92 genes, or any derivative DNA or RNA. Hybridization of an mRNA with the probe indicates that the ET-29, HDAC1, HDAC7 and ZNF92 genes in question is being expressed.
In cases, the mRNA from the sample is immobilized on a solid surface and contacted with a probe, for example by running the isolated mRNA on an agarose gel and transferring the mRNA from the gel to a membrane, such as nitrocellulose. In other cases, the probes are immobilized on a solid surface and the mRNA is contacted with the probes, for example, in an Agilent gene chip array. A skilled artisan can readily adapt available mRNA detection methods for use in detecting the level of expression of the ET-29, HDAC1, HDAC7 and ZNF92 genes.
An alternative method for determining the level of the ET-29, HDAC1, HDAC7 and ZNF92 gene expression in a sample involves the process of nucleic acid amplification of the ET-29, HDAC1, HDAC7 and ZNF92 mRNA (or cDNA thereof), for example, by RT-PCR (U.S. Pat. No. 4,683,202), ligase chain reaction (Barany, Proc. Natl. Acad. Sci. USA 88:189-93, 1991), self-sustained sequence replication (Guatelli et al., Proc. Natl. Acad. Sci. USA 87:1874-78, 1990), transcriptional amplification system (Kwoh et al., Proc. Natl. Acad. Sci. USA 86:1173-77, 1989), Q-Beta Replicase (Lizardi et al., Bio/Technology 6:1197, 1988), rolling circle replication (U.S. Pat. No. 5,854,033), or any other nucleic acid amplification method, followed by the detection of the amplified molecules using available techniques. These detection schemes are especially useful for the detection of nucleic acid molecules if such molecules are present in very low numbers.
In some cases, the ET-29, HDAC1, HDAC7 and ZNF92 gene expression is assessed by quantitative RT-PCR. Numerous different PCR or QPCR protocols are available and can be directly applied or adapted for use using the ET-29, HDAC1, HDAC7 and ZNF92 genes. Generally, in PCR, a target polynucleotide sequence is amplified by reaction with at least one oligonucleotide primer or pair of oligonucleotide primers. The primer(s) hybridize to a complementary region of the target nucleic acid and a DNA polymerase extends the primer(s) to amplify the target sequence. Under conditions sufficient to provide polymerase-based nucleic acid amplification products, a nucleic acid fragment of one size dominates the reaction products (the target polynucleotide sequence which is the amplification product). The amplification cycle is repeated to increase the concentration of the single target polynucleotide sequence. The reaction can be performed in any thermocycler commonly used for PCR. However, preferred are cyclers with real-time fluorescence measurement capabilities, for example, SMARTCYCLER® (Cepheid, Sunnyvale, Calif.), ABI PRISM 7700® (Applied Biosystems, Foster City, Calif.), ROTOR-GENE™ (Corbett Research, Sydney, Australia), LIGHTCYCLER® (Roche Diagnostics Corp, Indianapolis, Ind.), ICYCLER® (Biorad Laboratories, Hercules, Calif.) and MX4000® (Stratagene, La Jolla, Calif.).
Quantitative PCR (QPCR) (also referred as real-time PCR) is preferred under some circumstances because it provides not only a quantitative measurement, but also reduced time and contamination. In some instances, the availability of full gene expression profiling techniques is limited due to requirements for fresh frozen tissue and specialized laboratory equipment, making the routine use of such technologies difficult in a clinical setting. However, QPCR gene measurement can be applied to standard formalin-fixed paraffin-embedded clinical tumor blocks, such as those used in archival tissue banks and routine surgical pathology specimens (Cronin et al. (2007) Clin Chem 53:1084-91)[Mullins 2007] [Paik 2004]. As used herein, “quantitative PCR (or “real time QPCR”) refers to the direct monitoring of the progress of PCR amplification as it is occurring without the need for repeated sampling of the reaction products. In quantitative PCR, the reaction products may be monitored via a signaling mechanism (e.g., fluorescence) as they are generated and are tracked after the signal rises above a background level but before the reaction reaches a plateau. The number of cycles required to achieve a detectable or “threshold” level of fluorescence varies directly with the concentration of amplifiable targets at the beginning of the PCR process, enabling a measure of signal intensity to provide a measure of the amount of target nucleic acid in a sample in real time.
In some cases, microarrays are used for expression profiling. Microarrays are particularly well suited for this purpose because of the reproducibility between different experiments. DNA microarrays provide one method for the simultaneous measurement of the expression levels of large numbers of genes. Each array consists of a reproducible pattern of capture probes attached to a solid support. Labeled RNA or DNA is hybridized to complementary probes on the array and then detected by laser scanning. Hybridization intensities for each probe on the array are determined and converted to a quantitative value representing relative gene expression levels. See, for example, U.S. Pat. Nos. 6,040,138, 5,800,992 and 6,020,135, 6,033,860, and 6,344,316. High-density oligonucleotide arrays are particularly useful for determining the gene expression profile for a large number of RNAs in a sample. Techniques for the synthesis of these arrays using mechanical synthesis methods are described in, for example, U.S. Pat. No. 5,384,261. Although a planar array surface can be used, the array can be fabricated on a surface of virtually any shape or even a multiplicity of surfaces. Arrays can be nucleic acids (or peptides) on beads, gels, polymeric surfaces, fibers (such as fiber optics), glass, or any other appropriate substrate. See, for example, U.S. Pat. Nos. 5,770,358, 5,789,162, 5,708,153, 6,040,193 and 5,800,992. Arrays can be packaged in such a manner as to allow for diagnostics or other manipulation of an all-inclusive device. See, for example, U.S. Pat. Nos. 5,856,174 and 5,922,591.
When using microarray techniques, PCR amplified inserts of cDNA clones can be applied to a substrate in a dense array. The microarrayed genes, immobilized on the microchip, are suitable for hybridization under stringent conditions. Fluorescently labeled cDNA probes can be generated through incorporation of fluorescent nucleotides by reverse transcription of RNA extracted from tissues of interest. Labeled cDNA probes applied to the chip hybridize with specificity to each spot of DNA on the array. After stringent washing to remove non-specifically bound probes, the chip is scanned by confocal laser microscopy or by another detection method, such as a CCD camera. Quantitation of hybridization of each arrayed element allows for assessment of corresponding mRNA abundance.
With dual color fluorescence, separately labeled cDNA probes generated from two sources of RNA can be hybridized pairwise to the array. The relative abundance of the transcripts from the two sources corresponding to each specified gene is thus determined simultaneously. A miniaturized scale can be used for the hybridization, which provides convenient and rapid evaluation of the expression pattern for large numbers of genes. Such methods have been shown to have the sensitivity required to detect rare transcripts, which are expressed at a few copies per cell, and to reproducibly detect at least approximately two-fold differences in the expression levels (Schena et al., Proc. Natl. Acad. Sci. USA 93:106-49, 1996). Microarray analysis can be performed by commercially available equipment, following manufacturer's protocols, such as by using the Affymetrix GenChip technology, or Agilent ink jet microarray technology. The development of microarray methods for large-scale analysis of gene expression makes it possible to search systematically for molecular markers of cancer classification and outcome prediction in a variety of tumor types.
As used herein “level”, refers to a measure of the amount of, or a concentration of a transcription product, for instance an mRNA, or a translation product, for instance a protein or polypeptide.
As used herein “activity” refers to a measure of the ability of a transcription product or a translation product to produce a biological effect or to a measure of a level of biologically active molecules.
As used herein “expression level” further refer to gene expression levels or gene activity. Gene expression can be defined as the utilization of the information contained in a gene by transcription and translation leading to the production of a gene product.
The terms “increased,” or “increase” in connection with expression of the biomarkers described herein generally means an increase by a statically significant amount. For the avoidance of any doubt, the terms “increased” or “increase” means an increase of at least 10% as compared to a reference value, for example an increase of at least about 20/6, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90% or up to and including a 100% increase or any increase between 10-100% as compared to a reference value or level, or at least about a 1.5-fold, at least about a 1.6-fold, at least about a 1.7-fold, at least about a 1.8-fold, at least about a 1.9-fold, at least about a 2-fold, at least about a 3-fold, or at least about a 4-fold, or at least about a 5-fold, at least about a 10-fold increase, any increase between 2-fold and 10-fold, at least about a 25-fold increase, or greater as compared to a reference level. In some embodiments, an increase is at least about 1.8-fold increase over a reference value.
Similarly, the terms “decrease,” or “reduced,” or “reduction,” or “inhibit” in connection with expression of the biomarkers described herein generally to refer to a decrease by a statistically significant amount. However, for avoidance of doubt, “reduced”, “reduction” or “decrease” or “inhibit” means a decrease by at least 10% as compared to a reference level, for example a decrease by at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90% or up to and including a 100% decrease (e.g. absent level or non-detectable level as compared to a reference sample), or any decrease between 10-100% as compared to a reference level.
A “reference value” is a predetermined reference level, such as an average or median of expression levels of each of the ET-29, HDAC1, HDAC7 and ZNF92 genes in, for example, biological samples from a population of healthy subjects. The reference value can be an average or median of expression levels of each of the ET-29, HDAC1, HDAC7 and ZNF92 genes in a chronological age group matched with the chronological age of the tested subject. In some embodiments, the reference biological samples can also be gender matched. In some embodiments, the reference biological samples can also be cancer containing tissue from a specific subgroup of patients, such as stage 1, stage 2, stage 3, or grade 1, grade 2, grade 3 cancers, non-metastatic cancers, untreated cancers, hormone treatment resistant cancers, HER2 amplified cancers, triple negative cancers, estrogen negative cancers, or other relevant biological or prognostic subsets. For example, as explained herein, malignancy associated response signature expression levels in a sample can be assessed relative to normal breast tissue from the same subject or from a sample from another subject or from a repository of normal subject samples. If the expression level of a biomarker is greater or less than that of the reference or the average expression level, the biomarker expression is said to be “increased” or “decreased,” respectively, as those terms are defined herein. Exemplary analytical methods for classifying expression of a biomarker, determining a malignancy associated response signature status, and scoring of a sample for expression of a malignancy associated response signature biomarker are explained in detail herein.
Treatment
Methods are described herein for treating cancer. Such methods can involve administering therapeutic agents that can treat cancers with poor prognosis. Examples of such therapeutic agents can include one or more histone deacetylase inhibitor (HDAC), ZNF92 inhibitor, histone demethylase inhibitor, mTOR inhibitor, polo-like kinase (PLK) inhibitor, heat shock factor inhibitor, and/or inhibitors of any of the ET-9 and/or ET-60 breast cancer cell-origin associated signature biomarkers described herein.
In some cases, the cancer includes breast cancer, ovarian cancer, colon cancer, brain cancer, pancreatic cancer, prostate cancer, lung cancer, or melanoma. In some embodiments, the cancer includes leukemia, myeloma, or lymphoma.
The methods include downregulating expression of the ET-29, HDAC1, HDAC7 and ZNF92 genes. Suitable methods for downregulating such expression can include: inhibiting transcription of mRNA; degrading mRNA by methods including, but not limited to, the use of interfering RNA (RNAi); blocking translation of mRNA by methods including, but not limited to, the use of antisense nucleic acids or ribozymes, or the like. In some embodiments, a suitable method for downregulating expression may include providing to the cancer a small interfering RNA (siRNA) targeted to the ET-29, HDAC1, HDAC7 and ZNF92 genes.
Suitable methods for down-regulating the function or activity of the ET-29, HDAC1, HDAC7 and ZNF92 genes, may include administering a small molecule inhibitor that inhibits the function or activity of any of these markers or factors.
In some cases, one or more histone deacetylase inhibitors can be administered to treat cancers with poor prognosis, such as cancers identified by measuring and/or monitoring the ET-29, HDAC1, HDAC7 and ZNF92 genes described herein. In some cases, histone deacetylase inhibitors are not administered to treat cancers with poor prognosis, such as cancers identified by measuring and/or monitoring the ET-29, HDAC1, HDAC7 and ZNF92 genes described herein.
As used herein a “Histone Deacetylase inhibitor” or “HDAC inhibitor” refers to inhibitors of Histone Deacetylase 1 (HDAC1), Histone Deacetylase 7 (HDAC7), and/or phosphorylated HDAC7, including agents that inhibit the level and/or activity of HDAC1 and/or HDAC7 and/or phosphorylated HDAC7, as well as agents that inhibit the phosphorylation of HDAC7 e.g., inhibitors of EMK protein kinase, C-TAK1 protein kinase, and/or CAMK protein kinase, and agents that activate or increase the level and/or activity of phosphatase activity to remove phosphoryl groups from HDAC7, e.g., activators of PP2A phosphatase and/or myosin phosphatase. In some cases, HDAC inhibitors include molecules that bind directly to a functional region of HDAC1 and/or HDAC7 and/or phosphorylated HDAC7 in a manner that interferes with the enzymatic activity of HDAC1 and/or HDAC7 and/or phosphorylated HDAC7 e.g., agents that interfere with substrate binding to HDAC1 and/or HDAC7 and/or phosphorylated HDAC7. In some embodiments, HDAC inhibitors include molecules that bind directly to HDAC7 in a manner that prevents the phosphorylation of HDAC7. HDAC inhibitors include agents that inhibit the activity of peptides, polypeptides, or proteins that modulate the activity of HDAC1 and/or HDAC7 e.g., inhibitors of EMK protein kinase, C-TAK1 kinase, CAMK protein kinase inhibitors of C-TAK1 protein kinase. Examples of suitable inhibitors include, but are not limited to antisense oligonucleotides, oligopeptides, interfering RNA e.g., small interfering RNA (siRNA), small hairpin RNA (shRNA), aptamers, ribozymes, small molecule inhibitors, or antibodies or fragments thereof, and combinations thereof.
In some cases, HDAC inhibitors are specific inhibitors or specifically inhibit the level and/or activity of HDAC1 and/or HDAC7 and/or phosphorylated HDAC7. As used herein, “specific inhibitor(s)” refers to inhibitors characterized by their ability to bind to with high affinity and high specificity to HDAC1 and/or HDAC7 and/or phosphorylated HDAC7 proteins or domains, motifs, or fragments thereof, or variants thereof, and preferably have little or no binding affinity for non-HDAC1 and/or non-HDAC7 and/or non-phosphorylated HDAC7 proteins. As used herein, “specifically inhibit(s)” refers to the ability of an HDAC inhibitor of the present invention to inhibit the level and/or activity of a target polypeptide, e.g., HDAC1, and/or HDAC7, and/or phosphorylated HDAC7, and/or EMK protein kinase, and/or C-TAK1 protein kinase and/or CAMK protein kinase and preferably have little or no inhibitory effect on non-target polypeptides. As used herein, “specifically activate(s)” and “specifically increase(s)” refers to the ability of an HDAC inhibitor of the present invention to stimulate (e.g., activate or increase) the level and/or activity of a target polypeptide, e.g., PP2A phosphatase and/or myosin phosphatase and preferably to have little or no stimulatory effect on non-target polypeptides.
Examples of HDAC inhibitors include Vorinostat (SAHA), Entinostat (MS-275), Panobinostat (LBH589), Trichostatin A (TSA), Mocetinostat (MGCD0103), 4-Phenylbutyric acid (4-PBA), ACY-775, Belinostat (PXD101), Romidepsin (FK228, Depsipeptide), MC1568, Tubastatin A HCl, Givinostat (ITF2357), Dacinostat (LAQ824), CUDC-101, Quisinostat (JNJ-26481585) 2HCl, Pracinostat (SB939), PCI-34051, Droxinostat, Abexinostat (PCI-24781), RGFP966, AR-42, Ricolinostat (ACY-1215), Valproic Acid (NSC 93819) sodium salt, Tacedinaline (CI994), Fimepinostat (CUDC-907), Sodium butyrate, Curcumin, M344, Tubacin, RG2833 (RGFP109), Resminostat, Divalproex Sodium, Scriptaid, Sodium Phenylbutyrate, Tubastatin A, Tubastatin A TFA, Sinapinic Acid, TMP269, Santacruzamate A (CAY10683), TMP195, Valproic acid (VPA), UF010, Tasquinimod, SKLB-23bb, Isoguanosine, NKL22, Sulforaphane, BRD73954, BG45, Domatinostat (4SC-202), Citarinostat (ACY-241), Suberohydroxamic acid, BRD3308, Splitomicin, HPOB, LMK-235, Biphenyl-4-sulfonyl chloride, Nexturastat A, BML-210 (CAY10433), TC-H 106, SR-4370, TH34, Tucidinostat (Chidamide), SIS17, (−)-Parthenolide, WT161, CAY10603, ACY-738, Raddeanin A, GSK3117391, Tinostamustine(EDO-S101), or combinations thereof. Such HDAC inhibitors are available from Selleckchem.com.
In some cases, one or more histone demethylase inhibitors can be administered to treat cancers with poor prognosis, such as cancers identified by measuring and/or monitoring the ET-29, HDAC1, HDAC7 and ZNF92 genes described herein.
In some cases, an inhibitor of histone demethylase can be administered to treat cancers with poor prognosis, such as cancers identified by measuring and/or monitoring the ET-29, HDAC1, HDAC7 and ZNF92 biomarkers described herein. Histone demethylase inhibitors can include GSK-J4, 2,4-Pyridinedicarboxylic Acid, AS8351, Clorgyline hydrochloride, CPI-455, Daminozide, GSK-2879552, GSK-J1, GSK-J2, GSK-J5, GSK-LSD1, IOX1, IOX2, JIB-04, ML-324, NCGC00244536, OG-L002, ORY-1001, SP-2509, TC-E 5002, UNC-926, β-Lapachone, or combinations thereof. Such inhibitors are available, e.g., from Selleckchem.com.
In some cases, one or more mTOR inhibitors can be administered to treat cancers with poor prognosis, such as cancers identified by measuring and/or monitoring the ET-29, HDAC1, HDAC7 and ZNF92 genes described herein. Examples of mTOR inhibitors include Rapamycin (AY-22989), Everolimus (RAD001), AZD8055, Temsirolimus (CCI-779), PI-103, NU7441 (KU-57788), KU-0063794, Torkinib (PP242), Ridaforolimus (Deforolimus, MK-8669), Sapanisertib (MLNO128), Voxtalisib (XL765) Analogue, Torin 1, Omipalisib (GSK2126458), OSI-027, PF-04691502, Apitolisib (GDC-0980), GSK1059615, WYE-354, Gedatolisib (PKI-587), Vistusertib (AZD2014), Torin 2, WYE-125132 (WYE-132), BGT226 (NVP-BGT226) maleate, Palomid 529 (P529), PP121, WYE-687, Clemastine (HS-592) fumarate, Nitazoxanide (NSC 697855), WAY-600, ETP-46464, GDC-0349, PI3K/Akt Inhibitor Library, 4EGI-1, XL388, MHY1485, 3-Hydroxyanthranilic acid, Bimiralisib (PQR309), Samotolisib (LY3023414), Lanatoside C, Rotundic acid, L-Leucine, Chrysophanic Acid, Voxtalisib (XL765), GNE-477, CZ415, Astragaloside IV, CC-115, Salidroside, Compound 401, 3BDO, Zotarolimus (ABT-578), GNE-493, Paxalisib (GDC-0084), Onatasertib (CC 223), ABTL-0812, PQR620, SF2523, Niclosamide, or combinations thereof. Such HDAC inhibitors are available from Selleckchem.com.
In some cases, one or more Polo-Like Kinase (PLK) inhibitors can be administered to treat cancers with poor prognosis, such as cancers identified by measuring and/or monitoring the ET-29, HDAC1, HDAC7 and ZNF92 genes described herein. Examples of PLK inhibitors include BI 2536, Volasertib (BI 6727), Wortmannin (KY 12420), Rigosertib (ON-01910), GSK461364, HMN-214, MLN0905, Ro3280, SBE 13 HCl, Centrinone (LCR-263), CFI-400945, HMN-176, Onvansertib (NMS-P937), or combinations thereof.
In some cases, one or more heat shock factor inhibitors can be administered to treat cancers with poor prognosis, such as cancers identified by measuring and/or monitoring the expression of the ET-29, HDAC1, HDAC7 and ZNF92 genes described herein. Examples of heat shock factor inhibitors include one or more of the following Tanespimycin (17-AAG), Pimitespib (TAS-116, Luminespib (NVP-AUY922), Alvespimycin (17-DMAG) HCl, Ganetespib (STA-9090), Onalespib (AT13387), Geldanamycin (NSC 122750), SNX-2112 (PF-04928473), PF-04929113 (SNX-5422), KW-2478, Cucurbitacin D, VER155008, VER-50589, CH5138303, VER-49009, NMS-E973, Zelavespib (PU-H71), HSP990 (NVP-HSP990), XL888 NVP-BEP800, BIIB021 or a combination thereof. Such heat shock factor inhibitors can be obtained from Tocris.com.
As used herein, “solid tumor” is intended to include, but not be limited to, the following sarcomas and carcinomas: fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon carcinoma, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinomas, cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatoma, bile duct carcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilms' tumor, cervical cancer, testicular tumor, lung carcinoma, small cell lung carcinoma, bladder carcinoma, epithelial carcinoma, glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, melanoma, neuroblastoma, and retinoblastoma. Solid tumor is also intended to encompass epithelial cancers.
In embodiments, the ET-29, HDAC1, HDAC7 and ZNF92 biomarkers comprise a nucleic acid sequence or amino acid sequence with at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or less than 100% nucleic acid sequence identity or amino acid sequence identity to a nucleic acid sequence or an amino acid sequence having any of SEQ ID NOs: 3-31.
Zinc Finger Protein (ZNF92) ZNF92 is a zinc finger protein that functions as transcription factor that binds nucleic acids and regulates transcription. The ZNF92 gene is located on chromosome 7 (Gene ID: 168374; location NC_000007.14 (65373855..65401136). An example of an amino acid sequence for ZNF92 isoform 1 is available as UNIPROT accession no. Q03936-1 and shown below as SEQ ID NO:1.
10 20 30 40
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MGPLTFRDVK IEFSLEEWQC LDTAQRNLYR DVMLENYRNL
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50 60 70 80
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VFLGIAVSKP DLITWLEQGK EPWNLKRHEM VDKTPVMCSH
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90 100 110 120
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FAQDVWPEHS IKDSFQKVIL RTYGKYGHEN LQLRKDHKSV
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130 140 150 160
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DACKVYKGGY NGLNQCLTTT DSKIFQCDKY VKVFHKFPNV
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170 180 190 200
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NRNKIRHTGK KPFKCKNRGK SFCMLSQLTQ HKKIHTREYS
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210 220 230 240
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YKCEECGKAF NWSSTLTKHK IIHTGEKPYK CEECGKAFNR
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250 260 270 280
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SSNLTKHKII HTGEKPYKCE ECGKAFNRSS TLTKHKRIHT
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290 300 310 320
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EEKPYKCEEC GKAFNQFSIL NKHKRIHMED KPYKCEECGK
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330 340 350 360
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AFRVFSILKK HKIIHTGEKP YKCEECGKAF NQFSNLTKHK
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370 380 390 400
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IIHTGEKPYK CDECGKAFNQ SSTLTKHKRI HTGEKPYKCE
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410 420 430 440
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ECGKAFKQSS TLTEHKIIHT GEKPYKCEKC GKAFSWSSAF
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450 460 470 480
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TKHKRNHMED KPYKCEECGK AFSVFSTLTK HKIIHTREKP
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490 500 510 520
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YKCEECGKAF NQSSIFTKHK IIHTEGKSYK CEKCGNAFNQ
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530 540 550 560
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SSNLTARKII YTGEKPYKYE ECDKAFNKFS TLITHQIIYT
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570 580
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GEKPCKHECG RAFNKSSNYT KEKLQT
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A cDNA sequence encoding the amino acid sequence of SEQ ID NO:1 ZNF92 protein is available as NCBI accession no. BC040594.1, shown below as SEQ ID NO:
1
CTCTCGCTGC AGCCGGCGCT CCACGTCTAG TCTTCACTGC
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41
TCTGCGTCCT GTGCTGATAA AGGCTCGCCG CTGTGACCCT
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81
GTTACCTGCA AGAACTTGGA GGTTCACAGC TAAGACGCCA
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121
GGACCCCCTG GAAGCCTAGA AATGGGACCA CTGACATTTA
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161
GGGATGTGAA AATAGAATTC TCTCTAGAGG AATGGCAATG
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201
CCTGGACACT GCGCAGCGGA ATTTATATAG AGATGTGATG
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241
TTAGAGAACT ACAGAAACCT GGTCTTCCTT GGTATTGCTG
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281
TCTCTAAGCC AGACCTGATC ACCTGGCTGG AGCAAGGAAA
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321
AGAGCCCTGG AATCTGAAGA GACATGAGAT GGTAGACAAA
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361
ACCCCAGTTA TGTGTTCTCA TTTTGCCCAA GATGTTTGGC
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401
CAGAGCACAG CATAAAAGAT TCTTTCCAAA AAGTGATACT
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441
GAGAACATAT GGAAAATATG GACATGAGAA TTTACAGCTA
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481
AGAAAAGACC ATAAAAGTGT GGATGCATGT AAGGTGTACA
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521
AAGGAGGTTA TAATGGACTT AACCAGTGTT TGACAACTAC
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|
561
TGACAGCAAG ATATTTCAGT GTGATAAATA TGTGAAAGTC
|
|
601
TTTCATAAAT TTCCAAATGT AAATAGAAAT AAGATAAGAC
|
|
641
ATACTGGAAA GAAACCTTTC AAATGTAAAA ACCGTGGCAA
|
|
681
ATCATTTTGC ATGCTTTCAC AATTAACTCA ACATAAGAAA
|
|
721
ATTCATACTA GAGAGTATTC TTACAAATGT GAAGAATGTG
|
|
761
GTAAAGCCTT TAACTGGTCC TCAACCCTTA CTAAACATAA
|
|
801
GATAATTCAT ACTGGAGAAA AACCCTACAA ATGTGAAGAA
|
|
841
TGTGGCAAAG CTTTTAACCG GTCCTCAAAT CTTACTAAAC
|
|
881
ATAAAATAAT TCATACTGGA GAGAAACCCT ACAAATGTGA
|
|
921
AGAATGTGGC AAAGCTTTTA ACCGGTCCTC AACCCTTACT
|
|
961
AAACATAAAA GAATTCATAC AGAAGAGAAA CCCTACAAAT
|
|
1001
GTGAAGAATG TGGCAAGGCC TTTAACCAGT TCTCGATTCT
|
|
1041
TAATAAACAT AAGAGAATTC ATATGGAAGA TAAACCCTAC
|
|
1081
AAATGTGAAG AATGTGGCAA AGCCTTTAGA GTATTCTCAA
|
|
1121
TTCTTAAAAA ACATAAGATA ATCCATACTG GGGAAAAACC
|
|
1161
ATACAAATGT GAAGAATGTG GCAAAGCCTT TAACCAGTTC
|
|
1201
TCAAACCTTA CTAAACATAA GATAATTCAT ACTGGAGAGA
|
|
1241
AACCCTACAA ATGTGATGAA TGTGGCAAAG CCTTTAACCA
|
|
1281
GTCCTCAACC CTTACTAAAC ATAAAAGAAT TCATACGGGA
|
|
1321
GAAAAACCCT ACAAATGTGA AGAATGTGGC AAAGCTTTTA
|
|
1361
AACAGTCCTC AACCCTTACT GAACATAAGA TAATTCATAC
|
|
1401
TGGAGAGAAA CCCTACAAAT GTGAAAAATG TGGCAAGGCC
|
|
1441
TTTAGCTGGT CCTCAGCTTT TACTAAACAT AAGAGAAATC
|
|
1481
ATATGGAAGA TAAACCCTAC AAATGTGAAG AATGTGGCAA
|
|
1521
AGCCTTTAGT GTATTCTCAA CCCTTACTAA ACATAAAATA
|
|
1561
ATTCATACTA GAGAAAAACC CTACAAATGT GAAGAATGTG
|
|
1601
GCAAAGCCTT TAACCAGTCC TCAATTTTTA CTAAACATAA
|
|
1641
GATAATTCAC ACTGAAGGGA AATCCTACAA ATGTGAAAAA
|
|
1681
TGTGGCAATG CTTTTAACCA GTCCTCAAAC CTTACTGCAC
|
|
1721
GTAAGATAAT TTATACTGGA GAGAAACCCT ACAAATATGA
|
|
1761
AGAATGTGAC AAAGCCTTTA ACAAGTTCTC AACCCTTATT
|
|
1801
ACACATCAGA TAATTTATAC TGGAGAGAAA CCCTGCAAAC
|
|
1841
ATGAATGTGG CAGAGCCTTT AACAAATCCT CAAATTATAC
|
|
1881
TAAAGAGAAA CTACAAACCT GAAAGATGTG ACAATGATTT
|
|
1921
TCACTACACC TCAAACTTTT CTAAACATAA ACCATATTGG
|
|
1961
TGCCCTAGAA ATGTGAGGAA TATGACAAGG ACTTTAAATG
|
|
2001
GTTGTCACGC TTGATTGTAG GTAAGATAAT TTATATTGGA
|
|
2041
GAAAAATCCT CCAAGTATGA AGAATGTGGC AAACTTTTAA
|
|
2081
CCAATCCTCA CACCTTATTG CACAGGAAAG CATTTATACT
|
|
2121
TGAGAAAAAT TGTATAAAGA ATATGGAAAA GCCATTTATA
|
|
2161
TCTGCTCACA TGTAAAAACA TCAGTTCATA CTTAATAAAA
|
|
2201
TGCAATTACC GTCAAATCTT TCAGAAAATA TAAGCCTTTA
|
|
2241
ATACGAGGAA GAGTATTCTT AAGATGAACA TTACAAATAG
|
|
2281
AAAGAGGGTT GTAGTACCTT TAGTTTTATG ATAGATCTTA
|
|
2321
TTGTACACAT TTTGTACCAG AGGAAAACCC TAAAGCATTA
|
|
2361
GTTGCTCAAA CTTTGTTCGA CATCAGGGAA TTTGTATTGG
|
|
2401
AGAAAAACCC TGCAAATGTA ATAAATATGG AAAAACATTT
|
|
2441
TTTCAAAAAC TACAGCTTGG AAAACATCAG AGAGTTCATA
|
|
2481
CTAAAATATA TTTTTGCAGA TGCAGTAAAT ATGAAAAATA
|
|
2521
TTTAATCCCA AATTAAGTCT ATGTAAATAT CAGAATTCAC
|
|
2561
AGTAGAAATC ATAAGGCATA AGGCACTGAT ACTTCAGACA
|
|
2601
TTACACTAAA TTAGAGTGTT GAGTATAGGA GATCCAAAAC
|
|
2641
TAAAATTGTT AGGTAAGTTA TTTATATATA ACTTTAAAAG
|
|
2681
AAGTAGAAGA TTTTTTGGAG ATTTATAATT ACATTCAAAG
|
|
2721
TATACTTTTT TCTTGAAAAA AATTACAGAT TTTTTGAAAA
|
|
2761
GCAATTGATG TAATTTAACT CTCAAATTCA TGTTTTTCTT
|
|
2801
CATTCCTATT ATATTCACAT GTGAAAGCAA GTGATCTGTT
|
|
2841
GTTGCTGAAT CAGAGATATG AGAGATTCTT TTTTATAGGT
|
|
2881
GGGCATTATT TATGCCCCTT TCTGTGGAAG AGTAAGAAAA
|
|
2921
TTAAAATACA AGATGCATGA GGAAAATGTA GAGATGCTCT
|
|
2961
TTGTGATTAA CTTAGAATAT TAAGTGCTAC TTGACGTACA
|
|
3001
TGTTCAGACT AACATTCTTT TGCAGTATAG TGAGAAAAAA
|
|
3041
ACATTTTAAA ATTAATTATC ATTTTGTTGA TTGTGCTTTT
|
|
3081
ATGTAATAAA ATGCAGTACT TTAAAACAAA AAAAAAAAAA
|
|
3121
AAA
|
The ET-29 signature (shown in Table 2) genes are listed below in Table 3 with UNIPROT accession numbers and examples of amino acid sequences.
Table 3: ET-29 Signature (Shown in Table 2)
TABLE 3
|
|
The ET-29 gene mRNA signature downstream of the HDAC1-HDAC7-ZNF92
|
axis.
|
Entrez
ET-29 subset Name &
|
ID
Example of Human Amino Acid Sequence
|
|
80325
ABTB1 (ankyrin repeat and BTB domain containing 1; SEQ ID NO: 3)
|
Uniprot
MDTSDLFASCRKGDVGRVRYLLEQRDVEVNVRDKWDSTPLYYACLCGHEELVLYLLANGA
|
Q969K4
RCEANTEDGERCLYGALSDPIRRALRDYKQVTASCRRRDYYDDFLQRLLEQGIHSDVVFV
|
VHGKPFRVHRCVLGARSAYFANMLDTKWKGKSVVVLRHPLINPVAFGALLQYLYTGRLDI
|
GVEHVSDCERLAKQCQLWDLLSDLEAKCEKVSEFVASKPGTCVKVITIEPPPADPRLRED
|
MALLADCALPPELRGDLWELPFPCPDGENSCPDICERVAGCSFLCHKAFFCGRSDYFRAL
|
LDDHFRESEEPATSGGPPAVILHGISPDVFTHVLYYMYSDHTELSPEAAYDVLSVADMYL
|
LPGLKRLCGRSLAQMLDEDTVVGVWRVAKLERLARLEDQCTEYMAKVIEKLVEREDEVEA
|
VKEEAAAVAARQETDSIPLVDDIRFHVASTVQTYSAIEEAQQRLRALEDLLVSIGLDC
|
|
9289
ADGRG1 (adhesion G protein-coupled receptor G1; SEQ ID NO: 4)
|
Uniprot
MTPQSLLQTTLFLLSLLELVQGAHGRGHREDFRFCSQRNQTHRSSLHYKPTPDLRISIEN
|
Q9Y653
SEEALTVHAPFPAAHPASRSFPDPRGLYHFCLYWNRHAGRLHLLYGKRDFLLSDKASSLL
|
CFQHQEESLAQGPPLLATSVTSWWSPQNISLPSAASFTFSFHSPPHTAAHNASVDMCELK
|
RDLQLLSQFLKHPQKASRRPSAAPASQQLQSLESKLTSVREMGDMVSFEEDRINATVWKL
|
QPTAGLQDLHIHSRQEEEQSEIMEYSVLLPRTLFQRTKGRSGEAEKRLLLVDESSQALFQ
|
DKNSSQVLGEKVLGIVVQNTKVANLTEPVVLTFQHQLQPKNVTLQCVFWVEDPTLSSPGH
|
WSSAGCETVRRETQTSCFCNHLTYFAVLMVSSVEVDAVHKHYLSLLSYVGCVVSALACLV
|
TIAAYLCSRVPLPCRRKPRDYTIKVHMNLLLAVELLDTSELLSEPVALTGSEAGCRASAI
|
FLHESLLTCLSWMGLEGYNLYRLVVEVFGTYVPGYLLKLSAMGWGFPIFLVTLVALVDVD
|
NYGPIILAVHRTPEGVIYPSMCWIRDSLVSYITNLGLFSLVELENMAMLATMVVQILRLR
|
PHTQKWSHVITLLGLSLVLGLPWALIFFSFASGTFQLVVLYLFSIITSFQGFLIFIWYWS
|
MRLQARGGPSPLKSNSDSARLPISSGSTSSSRI
|
|
113451
AZIN2 (antizyme inhibitor 2; SEQ ID NO: 5)
|
Uniprot
MAGYLSESDEVMVEEGESTRDLLKELTLGASQATTDEVAAFFVADLGAIVRKHFCFLKCL
|
Q96A70
PRVRPFYAVKCNSSPGVLKVLAQLGLGFSCANKAEMELVQHIGIPASKIICANPCKQIAQ
|
IKYAAKHGIQLLSEDNEMELAKVVKSHPSAKMVLCIATDDSHSLSCLSLKFGVSLKSCRH
|
LLENAKKHHVEVVGVSFHIGSGCPDPQAYAQSIADARLVFEMGTELGHKMHVLDLGGGFP
|
GTEGAKVRFEEIASVINSALDLYFPEGCGVDIFAELGRYYVTSAFTVAVSIIAKKEVLLD
|
QPGREEENGSTSKTIVYHLDEGVYGIFNSVLEDNICPTPILQKKPSTEQPLYSSSLWGPA
|
VDGCDCVAEGLWLPQLHVGDWLVFDNMGAYTVGMGSPFWGTQACHITYAMSRVAWEALRR
|
QLMAAEQEDDVEGVCKPLSCGWEITDTLCVGPVETPASIM
|
|
55653
BCAS4 (breast carcinoma amplified sequence 4; SEQ ID NO: 6)
|
Uniprot
MQRTGGGAPRPGRNHGLPGSLRQPDPVALLMLLVDADQPEPMRSGARELALFLTPEPGAE
|
Q8TDM0
AKEVEETIEGMLLRLEEFCSLADLIRSDTSQILEENIPVLKAKLTEMRGIYAKVDRLEAF
|
VKMVGHHVAFLEADVLQAERDHGAFPQALRRWLGSAGLPSERNVECSGTIPARCNLRLPG
|
SSDSPASASQVAGITEVTCTGARDVRAAHTV
|
|
79934
COQ8B (coenzyme Q8B; SEQ ID NO: 7)
|
Uniprot
MWLKVGGLLRGTGGQLGQTVGWPCGALGPGPHRWGPCGGSWAQKFYQDGPGRGLGEEDIR
|
Q96D53
RAREARPRKTPRPQLSDRSRERKVPASRISRLANFGGLAVGLGLGVLAEMAKKSMPGGRL
|
QSEGGSGLDSSPFLSEANAERIVQTLCTVRGAALKVGQMLSIQDNSFISPQLQHIFERVR
|
QSADFMPRWQMLRVLEEELGRDWQAKVASLEEVPFAAASIGQVHQGLLRDGTEVAVKIQY
|
PGIAQSIQSDVQNLLAVLKMSAALPAGLFAEQSLQALQQELAWECDYRREAACAQNERQL
|
LANDPFFRVPAVVKELCTTRVLGMELAGGVPLDQCQGLSQDLRNQICFQLLTLCLRELFE
|
FRFMQTDPNWANFLYDASSHQVILLDEGASREFGTEFTDHYIEVVKAAADGDRDCVLQKS
|
RDLKELTGFETKAFSDAHVEAVMILGEPFATQGPYDFGSGETARRIQDLIPVLLRHRLCP
|
PPEETYALHRKLAGAFLACAHLRAHIACRDLFQDTYHRYWASRQPDAATAGSLPTKGDSW
|
VDPS
|
|
9696
CROCC (ciliary rootlet coiled-coil, rootletin; SEQ ID NO: 8)
|
Uniprot
MSLGLAGAQEVELTLETVIQTLESSVLCQEKGLGARDLAQDAQITSLPALIREIVTRNLS
|
Q5TZA2
QPESPVLLPATEMASLLSLQEENQLLQQELSRVEDLLAQSRAERDELAIKYNAVSERLEQ
|
ALRLEPGELETQEPRGLVRQSVELRRQLQEEQASYRRKLQAYQEGQQRQAQLVQRLQGKI
|
LQYKKRCSELEQQLLERSGELEQQRLRDTEHSQDLESALIRLEEEQQRSASLAQVNAMLR
|
EQLDQAGSANQALSEDIRKVINDWTRCRKELEHREAAWRREEESFNAYFSNEHSRLLLLW
|
RQVVGFRRLVSEVKMETERDLLQLGGELARTSRAVQEAGLGLSTGLRLAESRAEAALEKQ
|
ALLQAQLEEQLRDKVLREKDLAQQQMQSDLDKADLSARVTELGLAVKRLEKQNLEKDQVN
|
KDLTEKLEALESLRLQEQAALETEDGEGLQQTLRDLAQAVLSDSESGVQLSGSERTADAS
|
NGSLRGLSGQRTPSPPRRSSPGRGRSPRRGPSPACSDSSTLALIHSALHKRQLQVQDMRG
|
RYEASQDLLGTLRKQLSDSESERRALEEQLQRLRDKTDGAMQAHEDAQREVQRLRSANEL
|
LSREKSNLAHSLQVAQQQAEELRQEREKLQAAQEELRRQRDRLEEEQEDAVQDGARVRRE
|
LERSHRQLEQLEGKRSVLAKELVEVREALSRATLQRDMLQAEKAEVAEALTKAEAGRVEL
|
ELSMTKLRAEEASLQDSLSKLSALNESLAQDKLDLNRLVAQLEEEKSALQGRQRQAEQEA
|
TVAREEQERLEELRLEQEVARQGLEGSLRVAEQAQEALEQQLPTLRHERSQLQEQLAQLS
|
RQLSGREQELEQARREAQRQVEALERAAREKEALAKEHAGLAVQLVAAEREGRTLSEEAT
|
RLRLEKEALEGSLFEVQRQLAQLEARREQLEAEGQALLLAKETLTGELAGLRQQIIATQE
|
KASLDKELMAQKLVQAEREAQASLREQRAAHEEDLQRLQREKEAAWRELEAERAQLQSQL
|
QREQEELLARLEAEKEELSEEIAALQQERDEGLLLAESEKQQALSLKESEKTALSEKLMG
|
TRHSLATISLEMERQKRDAQSRQEQDRSTVNALTSELRDLRAQREEAAAAHAQEVRRLQE
|
QARDLGKQRDSCIREAEELRTQLRLLEDARDGLRRELLEAQRKLRESQEGREVQRQEAGE
|
LRRSLGEGAKEREALRRSNEELRSAVKKAESERISLKLANEDKEQKLALLEEARTAVGKE
|
AGELRTGLQEVERSRLEARRELQELRRQMKMLDSENTRLGRELAELQGRLALGERAEKES
|
RRETLGLRQRLLKGEASLEVMRQELQVAQRKLQEQEGEFRTRERRLLGSLEEARGTEKQQ
|
LDHARGLELKLEAARAEAAELGIRLSAAEGRAQGLEAELARVEVQRRAAEAQLGGLRSAL
|
RRGLGLGRAPSPAPRPVPGSPARDAPAEGSGEGINSPSTLECSPGSQPPSPGPATSPASP
|
DLDPEAVRGALREFLQELRSAQRERDELRTQTSALNRQLAEMEAERDSATSRARQLQKAV
|
AESEEARRSVDGRLSGVQAELALQEESVRRSERERRATLDQVATLERSLQATESELRASQ
|
EKISKMKANETKLEGDKRRLKEVLDASESRTVKLELQRRSLEGELQRSRLGLSDREAQAQ
|
ALQDRVDSLQRQVADSEVKAGTLQLTVERINGALAKVEESEGALRDKVRGLTEALAQSSA
|
SLNSTRDKNLHLQKALTACEHDRQVLQERIDAARQALSEARKQSSSIGEQVQTLRGEVAD
|
LELQRVEAEGQLQQLREVLRQRQEGEAAALNTVQKLQDERRLLQERLGSLQRALAQLEAE
|
KREVERSALRLEKDRVALRRTLDKVEREKLRSHEDTVRLSAEKGRLDRTLTGAELELAEA
|
QRQIQQLEAQVVVLEQSHSPAQLEVDAQQQQLELQQEVERLRSAQAQTERTLEARERAHR
|
QRVRGLEEQVSTLKGQLQQELRRSSAPFSPPSGPPEK
|
|
1523
CUX1 (cut like homeobox 1; SEQ ID NO: 9)
|
Uniprot
MLCVAGARLKRELDATATVLANRQDESEQSRKRLIEQSREFKKNTPEDIRKQVAPLIKSE
|
P39880
QGEIDALSKRSKEAEAAFINVYKRLIDVPDPVPALDLGQQLQLKVQRLHDIETENQKLRE
|
TLEEYNKEFAEVKNQEVTIKALKEKIREYEQTLKNQAETIALEKEQKLQNDFAEKERKLQ
|
ETQMSTTSKLEEAEHKVQSLQTALEKTRTELFDLKTKYDEETTAKADEIEMIMTDLERAN
|
QRAEVAQREAETLREQLSSANHSLQLASQIQKAPDVEQAIEVLTRSSLEVELAAKEREIA
|
QLVEDVQRLQASLTKLRENSASQISQLEQQLSAKNSTLKQLEEKLKGQADYEEVKKELNI
|
LKSMEFAPSEGAGTQDAAKPLEVLLLEKNRSLQSENAALRISNSDLSGSARRKGKDQPES
|
RRPGSLPAPPPSQLPRNPGEQASNINGTHQFSPAGLSQDFFSSSLASPSLPLASTGKFAL
|
NSLLQRQLMQSFYSKAMQEAGSTSMIFSTGPYSTNSISSQSPLQQSPDVNGMAPSPSQSE
|
SAGSVSEGEEMDTAEIARQVKEQLIKHNIGQRIFGHYVLGLSQGSVSEILARPKPWNKLT
|
VRGKEPFHKMKQFLSDEQNILALRSIQGRQRENPGQSLNRLFQEVPKRRNGSEGNITTRI
|
RASETGSDEAIKSILEQAKRELQVQKTAEPAQPSSASGSGNSDDAIRSILQQARREMEAQ
|
QAALDPALKQAPLSQSDITILTPKLISTSPMPTVSSYPPLAISLKKPSAAPEAGASALPN
|
PPALKKEAQDAPGLDPQGAADCAQGVLRQVKNEVGRSGAWKDHWWSAVQPERRNAASSEE
|
AKAEETGGGKEKGSGGSGGGSQPRAERSQLQGPSSSEYWKEWPSAESPYSQSSELSLTGA
|
SRSETPQNSPLPSSPIVPMSKPTKPSVPPLTPEQYEVYMYQEVDTIELTRQVKEKLAKNG
|
ICQRIFGEKVLGLSQGSVSDMLSRPKPWSKITQKGREPFIRMQLWINGELGQGVLPVQGQ
|
QQGPVLHSVTSLQDPLQQGCVSSESTPKTSASCSPAPESPMSSSESVKSLTELVQQPCPP
|
IEASKDSKPPEPSDPPASDSQPTTPLPLSGHSALSIQELVAMSPELDTYGITKRVKEVLT
|
DNNLGQRLFGETILGLTQGSVSDLLARPKPWHKLSLKGREPFVRMQLWINDPNNVEKLMD
|
MKRMEKKAYMKRRHSSVSDSQPCEPPSVGTEYSQGASPQPQHQLKKPRVVLAPEEKEALK
|
RAYQQKPYPSPKTIEDLATQLNLKTSTVINWFHNYRSRIRRELFIEEIQAGSQGQAGASD
|
SPSARSGRAAPSSEGDSCDGVEATEGPGSADTEEPKSQGEAEREEVPRPAEQTEPPPSGT
|
PGPDDARDDDHEGGPVEGPGPLPSPASATATAAPAAPEDAATSAAAAPGEGPAAPSSAPP
|
PSNSSSSSAPRRPSSLQSLFGLPEAAGARDSRDNPLRKKKAANLNSIIHRLEKAASREEP
|
IEWEF
|
|
27122
DKK3 (dickkopf WNT signaling pathway inhibitor 3; SEQ ID NO: 10)
|
Uniprot
MQRLGATLLCLLLAAAVPTAPAPAPTATSAPVKPGPALSYPQEEATLNEMFREVEELMED
|
Q9UBP4
TQHKLRSAVEEMEAEEAAAKASSEVNLANLPPSYHNETNTDTKVGNNTIHVHREIHKITN
|
NQTGQMVESETVITSVGDEEGRRSHECIIDEDCGPSMYCQFASFQYTCQPCRGQRMLCTR
|
DSECCGDQLCVWGHCTKMATRGSNGTICDNQRDCQPGLCCAFQRGLLFPVCTPLPVEGEL
|
CHDPASRLLDLITWELEPDGALDRCPCASGLLCQPHSHSLVYVCKPTFVGSRDQDGEILL
|
PREVPDEYEVGSFMEEVRQELEDLERSLTEEMALREPAAAAAALLGGEEI
|
|
1891
ECHI (enoyl-CoA hydratase 1; SEQ ID NO: 11)
|
Uniprot
MAAGIVASRRLRDLLTRRLTGSNYPGLSISLRLTGSSAQEEASGVALGEAPDHSYESLRV
|
Q13011
TSAQKHVLHVQLNRPNKRNAMNKVFWREMVECENKISRDADCRAVVISGAGKMFTAGIDL
|
MDMASDILQPKGDDVARISWYLRDIITRYQETENVIERCPKPVIAAVHGGCIGGGVDLVT
|
ACDIRYCAQDAFFQVKEVDVGLAADVGTLQRLPKVIGNQSLVNELAFTARKMMADEALGS
|
GLVSRVFPDKEVMLDAALALAAEISSKSPVAVQSTKVNLLYSRDHSVAESLNYVASWNMS
|
MLQTQDLVKSVQATTENKELKTVTESKL
|
|
2049
EPHB3 (EPH receptor B3; SEQ ID NO: 12)
|
Uniprot
MARARPPPPPSPPPGLLPLLPPLLLLPLLLLPAGCRALEETLMDTKWVTSELAWTSHPES
|
P54753
GWEEVSGYDEAMNPIRTYQVCNVRESSQNNWLRTGFIWRRDVQRVYVELKFTVRDCNSIP
|
NIPGSCKETENLFYYEADSDVASASSPFWMENPYVKVDTIAPDESFSRLDAGRVNTKVRS
|
FGPLSKAGFYLAFQDQGACMSLISVRAFYKKCASTTAGFALFPETLTGAEPTSLVIAPGT
|
CIPNAVEVSVPLKLYCNGDGEWMVPVGACTCATGHEPAAKESQCRPCPPGSYKAKQGEGP
|
CLPCPPNSRTTSPAASICTCHNNFYRADSDSADSACTTVPSPPRGVISNVNETSLILEWS
|
EPRDLGGRDDLLYNVICKKCHGAGGASACSRCDDNVEFVPRQLGLTERRVHISHLLAHTR
|
YTFEVQAVNGVSGKSPLPPRYAAVNITTNQAAPSEVPTLRLHSSSGSSLTLSWAPPERPN
|
GVILDYEMKYFEKSEGIASTVTSQMNSVQLDGLRPDARYVVQVRARTVAGYGQYSRPAEF
|
ETTSERGSGAQQLQEQLPLIVGSATAGLVEVVAVVVIAIVCLRKQRHGSDSEYTEKLQQY
|
IAPGMKVYIDPFTYEDPNEAVREFAKEIDVSCVKIEEVIGAGEFGEVCRGRLKQPGRREV
|
FVAIKTLKVGYTERQRRDFLSEASIMGQFDHPNIIRLEGVVTKSRPVMILTEFMENCALD
|
SFLRENDGQFTVIQLVGMLRGIAAGMKYLSEMNYVHRDLAARNILVNSNLVCKVSDEGLS
|
RFLEDDPSDPTYTSSLGGKIPIRWTAPEAIAYRKFTSASDVWSYGIVMWEVMSYGERPYW
|
DMSNQDVINAVEQDYRLPPPMDCPTALHQLMLDCWVRDRNLRPKESQIVNTLDKLIRNAA
|
SLKVIASAQSGMSQPLLDRTVPDYTTFTTVGDWLDAIKMGRYKESFVSAGFASEDLVAQM
|
TAEDLLRIGVTLAGHQKKILSSIQDMRLQMNQTLPVQV
|
|
23149
FCHO1 (FCH and mu domain containing endocytic adaptor 1; SEQ ID
|
Uniprot
NO: 13)
|
O14526
MSYFGEHFWGEKNHGFEVLYHSVKQGPISTKELADFIRERATIEETYSKAMAKLSKLASN
|
GTPMGTFAPLWEVERVSSDKLALCHLELTRKLQDLIKDVLRYGEEQLKTHKKCKEEVVST
|
LDAVQVLSGVSQLLPKSRENYLNRCMDQERLRRESTSQKEMDKAETKTKKAAESLRRSVE
|
KYNSARADFEQKMLDSALRFQAMEETHLRHMKALLGSYAHSVEDTHVQIGQVHEEFKQNI
|
ENVSVEMLLRKFAESKGTGREKPGPLDFEAYSAAALQEAMKRLRGAKAFRLPGLSRRERE
|
PEPPAAVDFLEPDSGTCPEVDEEGFTVRPDVTQNSTAEPSRESSSDSDEDDEEPRKFYVH
|
IKPAPARAPACSPEAAAAQLRATAGSLILPPGPGGTMKRHSSRDAAGKPQRPRSAPRTSS
|
CAERLQSEEQVSKNLFGPPLESAFDHEDFTGSSSLGFTSSPSPFSSSSPENVEDSGLDSP
|
SHAAPGPSPDSWVPRPGTPQSPPSCRAPPPEARGIRAPPLPDSPQPLASSPGPWGLEALA
|
GGDLMPAPADPTAREGLAAPPRRLRSRKVSCPLTRSNGDLSRSLSPSPLGSSAASTALER
|
PSFLSQTGHGVSRGPSPVVLGSQDALPIATAFTEYVHAYFRGHSPSCLARVTGELTMTFP
|
AGIVRVFSGTPPPPVLSFRLVHTTAIEHFQPNADLLESDPSQSDPETKDEWLNMAALTEA
|
LQRQAEQNPTASYYNVVLLRYQFSRPGPQSVPLQLSAHWQCGATLTQVSVEYGYRPGATA
|
VPTPLINVQILLPVGEPVINVRLQPAATWNLEEKRLTWRLPDVSEAGGSGRLSASWEPLS
|
GPSTPSPVAAQFTSEGTTLSGVDLELVGSGYRMSLVKRRFATGMYLVSC
|
|
84929
FIBCD1 (fibrinogen C domain containing 1; SEQ ID NO: 14)
|
Uniprot
MVNDRWKTMGGAAQLEDRPRDKPQRPSCGYVLCTVLLALAVLLAVAVTGAVLFINHAHAP
|
Q8N539
GTAPPPVVSTGAASANSALVIVERADSSHLSILIDPRCPDLTDSFARLESAQASVLQALT
|
EHQAQPRLVGDQEQELLDTLADQLPRLLARASELQTECMGLRKGHGTLGQGLSALQSEQG
|
RLIQLLSESQGHMAHLVNSVSDILDALQRDRGLGRPRNKADLQRAPARGTRPRGCATGSR
|
PRDCLDVLLSGQQDDGVYSVFPTHYPAGFQVYCDMRTDGGGWTVFQRREDGSVNFFRGWD
|
AYRDGFGRITGEHWLGLKRIHALTTQAAYELHVDLEDFENGTAYARYGSFGVGLESVDPE
|
EDGYPLTVADYSGTAGDSLLKHSGMRFTTKDRDSDHSENNCAAFYRGAWWYRNCHTSNLN
|
GQYLRGAHASYADGVEWSSWTGWQYSLKFSEMKIRPVREDR
|
|
81544
GDPD5 (glycerophosphodiester phosphodiesterase domain; SEQ ID NO:
|
Uniprot
15)
|
Q8WTR4
MVRHQPLQYYEPQLCLSCLTGIYGCRWKRYQRSHDDTTPWERLWFLLLTFTFGLTLTWLY
|
FWWEVHNDYDEFNWYLYNRMGYWSDWPVPILVTTAAAFAYIAGLLVLALCHIAVGQQMNL
|
HWLHKIGLVVILASTVVAMSAVAQLWEDEWEVLLISLQGTAPFLHVGAVAAVTMLSWIVA
|
GQFARAERTSSQVTILCTFFTVVFALYLAPLTISSPCIMEKKDLGPKPALIGHRGAPMLA
|
PEHTLMSFRKALEQKLYGLQADITISLDGVPFLMHDTTLRRTINVEEEFPELARRPASML
|
NWTTLQRLNAGQWELKTDPFWTASSLSPSDHREAQNQSICSLAELLELAKGNATLLLNLR
|
DPPREHPYRSSFINVTLEAVLHSGFPQHQVMWLPSRQRPLVRKVAPGFQQTSGSKEAVAS
|
LRRGHIQRLNLRYTQVSRQELRDYASWNLSVNLYTVNAPWLESLLWCAGVPSVTSDNSHA
|
LSQVPSPLWIMPPDEYCLMWVTADLVSFTLIVGIFVLQKWRLGGIRSYNPEQIMLSAAVR
|
RTSRDVSIMKEKLIFSEISDGVEVSDVLSVCSDNSYDTYANSTATPVGPRGGGSHTKTLI
|
ERSGR
|
|
3855
KRT7 (keratin 7; SEQ ID NO: 16)
|
Uniprot
MSIHFSSPVFTSRSAAFSGRGAQVRESSARPGGLGSSSLYGLGASRPRVAVRSAYGGPVG
|
P08729
AGIREVTINQSLLAPLRLDADPSLQRVRQEESEQIKTINNKFASFIDKVRFLEQQNKLLE
|
TKWILLQEQKSAKSSRLPDIFEAQIAGLRGQLEALQVDGGRLEAELRSMQDVVEDEKNKY
|
EDEINHRTAAENEFVVLKKDVDAAYMSKVELEAKVDALNDEINFLRTLNETELTELQSQI
|
SDTSVVLSMDNSRSLDLDGIIAEVKAQYEEMAKCSRAEAEAWYQTKFETLQAQAGKHGDD
|
LRNTRNEISEMNRAIQRLQAEIDNIKNQRAKLEAAIAEAEERGELALKDARAKQEELEAA
|
LQRGKQDMARQLREYQELMSVKLALDIEIATYRKLLEGEESRLAGDGVGAVNISVMNSTG
|
GSSSGGGIGLILGGTMGSNALSFSSSAGPGLLKAYSIRTASASRRSARD
|
|
3985
LIMK2 (LIM domain kinase 2; SEQ ID NO: 17)
|
Uniprot
MSALAGEDVWRCPGCGDHIAPSQIWYRTVNETWHGSCFRCSECQDSLINWYYEKDGKLYC
|
P53671
PKDYWGKFGEFCHGCSLLMTGPFMVAGEFKYHPECFACMSCKVIIEDGDAYALVQHATLY
|
CGKCHNEVVLAPMFERLSTESVQEQLPYSVTLISMPATTEGRRGFSVSVESACSNYATTV
|
QVKEVNRMHISPNNRNAIHPGDRILEINGTPVRTLRVEEVEDAISQTSQTLQLLIEHDPV
|
SQRLDQLRLEARLAPHMQNAGHPHALSTLDTKENLEGTLRRRSLRRSNSISKSPGPSSPK
|
EPLLESRDISRSESLRCSSSYSQQIFRPCDLIHGEVLGKGFFGQAIKVTHKATGKVMVMK
|
ELIRCDEETQKTFLTEVKVMRSLDHPNVLKFIGVLYKDKKLNLLTEYIEGGTLKDFLRSM
|
DPFPWQQKVRFAKGIASGMAYLHSMCIIHRDLNSHNCLIKLDKTVVVADFGLSRLIVEER
|
KRAPMEKATTKKRTLRKNDRKKRYTVVGNPYWMAPEMLNGKSYDETVDIFSFGIVLCEII
|
GQVYADPDCLPRTLDEGLNVKLEWEKFVPTDCPPAFFPLAAICCRLEPESRPAFSKLEDS
|
FEALSLYLGELGIPLPAELEELDHTVSMQYGLTRDSPP
|
|
114783
LMTK3 (lemur tyrosine kinase 3; SEQ ID NO: 18)
|
Uniprot
MPAPGALILLAAVSASGCLASPAHPDGFALGRAPLAPPYAVVLISCSGLLAFIFLLLTCL
|
Q96Q04
CCKRGDVGFKEFENPEGEDCSGEYTPPAEETSSSQSLPDVYILPLAEVSLPMPAPQPSHS
|
DMTTPLGLSRQHLSYLQEIGSGWEGKVILGEIFSDYTPAQVVVKELRASAGPLEQRKFIS
|
EAQPYRSLQHPNVLQCLGLCVETLPFLLIMEFCQLGDLKRYLRAQRPPEGLSPELPPRDL
|
RTLQRMGLEIARGLAHLASHNYVHSDLALRNCLLTSDLTVRIGDYGLAHSNYKEDYYLTP
|
ERLWIPLRWAAPELLGELHGTEMVVDQSRESNIWSLGVTLWELFEFGAQPYRHLSDEEVL
|
AFVVRQQHVKLARPRIKLPYADYWYDILQSCWRPPAQRPSASDLQLQLTYLLSERPPRPP
|
PPPPPPRDGPFPWPWPPAHSAPRPGTLSSPFPLLDGFPGADPDDVLTVTESSRGLNLECL
|
WEKARRGAGRGGGAPAWQPASAPPAPHANPSNPFYEALSTPSVLPVISARSPSVSSEYYI
|
RLEEHGSPPEPLFPNDWDPLDPGVPAPQAPQAPSEVPQLVSETWASPLFPAPRPFPAQSS
|
ASGSFLLSGWDPEGRGAGETLAGDPAEVLGERGTAPWVEEEEEEEEGSSPGEDSSSLGGG
|
PSRRGPLPCPLCSREGACSCLPLERGDAVAGWGGHPALGCPHPPEDDSSLRAERGSLADL
|
PMAPPASAPPEFLDPLMGAAAPQYPGRGPPPAPPPPPPPPRAPADPAASPDPPSAVASPG
|
SGLSSPGPKPGDSGYETETPFSPEGAFPGGGAAEEEGVPRPRAPPEPPDPGAPRPPPDPG
|
PLPLPGPREKPTFVVQVSTEQLLMSLREDVTRNLLGEKGATARETGPRKAGRGPGNREKV
|
PGLNRDPTVLGNGKQAPSLSLPVNGVTVLENGDQRAPGIEEKAAENGALGSPEREEKVLE
|
NGELTPPRREEKALENGELRSPEAGEKVLVNGGLIPPKSEDKVSENGGLREPRNTERPPE
|
TGPWRAPGPWEKTPESWGPAPTIGEPAPETSLERAPAPSAVVSSRNGGETAPGPLGPAPK
|
NGTLEPGTERRAPETGGAPRAPGAGRLDLGSGGRAPVGTGTAPGGGPGSGVDAKAGWVDN
|
TRPQPPPPPLPPPPEAQPRRLEPAPPRARPEVAPEGEPGAPDSRAGGDTALSGDGDPPKP
|
ERKGPEMPRLFLDLGPPQGNSEQIKARLSRLSLALPPLTLTPFPGPGPRRPPWEGADAGA
|
AGGEAGGAGAPGPAEEDGEDEDEDEEEDEEAAAPGAAAGPRGPGRARAAPVPVVVSSADA
|
DAARPLRGLLKSPRGADEPEDSELERKRKMVSFHGDVTVYLEDQETPTNELSVQAPPEGD
|
TDPSTPPAPPTPPHPATPGDGEPSNDSGEGGSFEWAEDFPLLPPPGPPLCFSRFSVSPAL
|
ETPGPPARAPDARPAGPVEN
|
|
4016
LOXL1 (lysyl oxidase like 1; SEQ ID NO: 19)
|
Uniprot
MALARGSRQLGALVWGACLCVLVHGQQAQPGQGSDPARWRQLIQWENNGQVYSLLNSGSE
|
Q08397
YVPAGPQRSESSSRVLLAGAPQAQQRRSHGSPRRRQAPSLPLPGRVGSDTVRGQARHPFG
|
FGQVPDNWREVAVGDSTGMARARTSVSQQRHGGSASSVSASAFASTYRQQPSYPQQFPYP
|
QAPFVSQYENYDPASRTYDQGFVYYRPAGGGVGAGAAAVASAGVIYPYQPRARYEEYGGG
|
EELPEYPPQGFYPAPERPYVPPPPPPPDGLDRRYSHSLYSEGTPGFEQAYPDPGPEAAQA
|
HGGDPRLGWYPPYANPPPEAYGPPRALEPPYLPVRSSDTPPPGGERNGAQQGRLSVGSVY
|
RPNQNGRGLPDLVPDPNYVQASTYVQRAHLYSLRCAAEEKCLASTAYAPEATDYDVRVLL
|
RFPQRVKNQGTADFLPNRPRHTWEWHSCHQHYHSMDEFSHYDLLDAATGKKVAEGHKASF
|
CLEDSTCDEGNLKRYACTSHTQGLSPGCYDTYNADIDCQWIDITDVQPGNYILKVHVNPK
|
YIVLESDETNNVVRCNIHYTGRYVSATNCKIVQS
|
|
4430
MYO1B (myosin IB; SEQ ID NO: 20)
|
Uniprot
MAKMEVKTSLLDNMIGVGDMVLLEPLNEETFINNLKKRFDHSEIYTYIGSVVISVNPYRS
|
O43795
LPIYSPEKVEEYRNRNFYELSPHIFALSDEAYRSLRDQDKDQCILITGESGAGKTEASKL
|
VMSYVAAVCGKGAEVNQVKEQLLQSNPVLEAFGNAKTVRNDNSSREGKYMDIEFDFKGDP
|
LGGVISNYLLEKSRVVKQPRGERNFHVFYQLLSGASEELLNKLKLERDESRYNYLSLDSA
|
KVNGVDDAANFRTVRNAMQIVGFMDHEAESVLAVVAAVLKLGNIEFKPESRVNGLDESKI
|
KDKNELKEICELTGIDQSVLERAFSFRTVEAKQEKVSTTLNVAQAYYARDALAKNLYSRL
|
FSWLVNRINESIKAQTKVRKKVMGVLDIYGFEIFEDNSFEQFIINYCNEKLQQIFIELTL
|
KEEQEEYIREDIEWTHIDYENNAIICDLIENNINGILAMLDEECLRPGTVTDETFLEKLN
|
QVCATHQHFESRMSKCSRFINDTSLPHSCFRIQHYAGKVLYQVEGFVDKNNDLLYRDESQ
|
AMWKASHALIKSIFPEGNPAKINLKRPPTAGSQFKASVATLMKNLQTKNPNYIRCIKPND
|
KKAAHIFNEALVCHQIRYLGLLENVRVRRAGYAFRQAYEPCLERYKMLCKQTWPHWKGPA
|
RSGVEVLFNELEIPVEEYSFGRSKIFIRNPRTLFKLEDLRKQRLEDLATLIQKIYRGWKC
|
RTHFLLMKKSQIVIAAWYRRYAQQKRYQQTKSSALVIQSYIRGWKARKILRELKHQKRCK
|
EAVTTIAAYWHGTQARRELRRLKEEARNKHAIAVIWAYWLGSKARRELKRLKEEARRKHA
|
VAVIWAYWLGLKVRREYRKEFRANAGKKIYEFTLQRIVQKYFLEMKNKMPSLSPIDKNWP
|
SRPYIFLDSTHKELKRIFHLWRCKKYRDQFTDQQKLIYEEKLEASELFKDKKALYPSSVG
|
QPFQGAYLEINKNPKYKKLKDAIEEKIIIAEVVNKINRANGKSTSRIFLLTNNNLLLADQ
|
KSGQIKSEVPLVDVTKVSMSSQNDGFFAVHLKEGSEAASKGDELESSDHLIEMATKLYRT
|
TLSQTKQKLNIEISDEFLVQFRQDKVCVKFIQGNQKNGSVPTCKRKNNRLLEVAVP
|
|
5097
PCDH1 (protocadherin 1; SEQ ID NO: 21)
|
Uniprot
MDSGAGGRRCPEAALLILGPPRMEHLRHSPGPGGQRLLLPSMLLALLLLLAPSPGHATRV
|
Q08174
VYKVPEEQPPNTLIGSLAADYGFPDVGHLYKLEVGAPYLRVDGKTGDIFTTETSIDREGL
|
RECQNQLPGDPCILEFEVSITDLVQNGSPRLLEGQIEVQDINDNTPNFASPVITLAIPEN
|
TNIGSLFPIPLASDRDAGPNGVASYELQAGPEAQELFGLQVAEDQEEKQPQLIVMGNLDR
|
ERWDSYDLTIKVQDGGSPPRASSALLRVTVLDTNDNAPKFERPSYEAELSENSPIGHSVI
|
QVKANDSDQGANAEIEYTFHQAPEVVRRLLRLDRNTGLITVQGPVDREDLSTLRESVLAK
|
DRGTNPKSARAQVVVTVKDMNDNAPTIEIRGIGLVTHQDGMANISEDVAEETAVALVQVS
|
DRDEGENAAVTCVVAGDVPFQLRQASETGSDSKKKYFLQTTTPLDYEKVKDYTIEIVAVD
|
SGNPPLSSTNSLKVQVVDVNDNAPVFTQSVTEVAFPENNKPGEVIAEITASDADSGSNAE
|
LVYSLEPEPAAKGLFTISPETGEIQVKTSLDREQRESYELKVVAADRGSPSLQGTATVLV
|
NVLDCNDNDPKFMLSGYNFSVMENMPALSPVGMVTVIDGDKGENAQVQLSVEQDNGDEVI
|
QNGTGTILSSLSFDREQQSTYTFQLKAVDGGVPPRSAYVGVTINVLDENDNAPYITAPSN
|
TSHKLLTPQTRLGETVSQVAAEDFDSGVNAELIYSIAGGNPYGLFQIGSHSGAITLEKEI
|
ERRHHGLHRLVVKVSDRGKPPRYGTALVHLYVNETLANRTLLETLLGHSLDTPLDIDIAG
|
DPEYERSKQRGNILFGVVAGVVAVALLIALAVLVRYCRQREAKSGYQAGKKETKDLYAPK
|
PSGKASKGNKSKGKKSKSPKPVKPVEDEDEAGLQKSLKENLMSDAPGDSPRIHLPLNYPP
|
GSPDLGRHYRSNSPLPSIQLQPQSPSASKKHQVVQDLPPANTFVGTGDTTSTGSEQYSDY
|
SYRTNPPKYPSKQVGQPFQLSTPQPLPHPYHGAIWTEVWE
|
|
9124
PDLIM1 (PDZ and LIM domain 1; SEQ ID NO: 22)
|
Uniprot
MTTQQIDLQGPGPWGFRLVGGKDFEQPLAISRVTPGSKAALANLCIGDVITAIDGENTSN
|
O00151
MTHLEAQNRIKGCTDNLTLTVARSEHKVWSPLVTEEGKRHPYKMNLASEPQEVLHIGSAH
|
NRSAMPFTASPASSTTARVITNQYNNPAGLYSSENISNENNALESKTAASGVEANSRPLD
|
HAQPPSSLVIDKESEVYKMLQEKQELNEPPKQSTSELVLQEILESEEKGDPNKPSGERSV
|
KAPVTKVAASIGNAQKLPMCDKCGTGIVGVFVKLRDRHRHPECYVCTDCGTNLKQKGHFF
|
VEDQIYCEKHARERVTPPEGYEVVTVEPK
|
|
8398
PLA2G6 (phospholipase A2 group VI; SEQ ID NO: 23)
|
Uniprot
MQFFGRLVNTFSGVTNLFSNPFRVKEVAVADYTSSDRVREEGQLILFQNTPNRTWDCVLV
|
O60733
NPRNSQSGFRLFQLELEADALVNFHQYSSQLLPFYESSPQVLHTEVLQHLTDLIRNHPSW
|
SVAHLAVELGIRECFHHSRIISCANCAENEEGCTPLHLACRKGDGEILVELVQYCHTQMD
|
VTDYKGETVFHYAVQGDNSQVLQLLGRNAVAGLNQVNNQGLTPLHLACQLGKQEMVRVLL
|
LCNARCNIMGPNGYPIHSAMKFSQKGCAEMIISMDSSQIHSKDPRYGASPLHWAKNAEMA
|
RMLLKRGCNVNSTSSAGNTALHVAVMRNREDCAIVLLTHGANADARGEHGNTPLHLAMSK
|
DNVEMIKALIVFGAEVDTPNDFGETPTFLASKIGRLVTRKAILTLLRTVGAEYCFPPIHG
|
VPAEQGSAAPHHPFSLERAQPPPISLNNLELQDLMHISRARKPAFILGSMRDEKRTHDHL
|
LCLDGGGVKGLIIIQLLIAIEKASGVATKDLFDWVAGTSTGGILALAILHSKSMAYMRGM
|
YFRMKDEVERGSRPYESGPLEEFLKREFGEHTKMTDVRKPKVMLTGTLSDRQPAELHLER
|
NYDAPETVREPRENQNVNLRPPAQPSDQLVWRAARSSGAAPTYFRPNGRELDGGLLANNP
|
TLDAMTEIHEYNQDLIRKGQANKVKKLSIVVSLGTGRSPQVPVTCVDVERPSNPWELAKT
|
VFGAKELGKMVVDCCTDPDGRAVDRARAWCEMVGIQYFRLNPQLGTDIMLDEVSDTVLVN
|
ALWETEVYIYEHREEFQKLIQLLLSP
|
|
5875
RABGGTA (Rab geranylgeranyltransferase subunit alpha; SEQ ID NO:
|
Uniprot
24)
|
Q92696
MHGRLKVKTSEEQAEAKRLEREQKLKLYQSATQAVFQKRQAGELDESVLELTSQILGANP
|
DEATLWNCRREVLQQLETQKSPEELAALVKAELGFLESCLRVNPKSYGTWHHRCWLLGRL
|
PEPNWTRELELCARFLEVDERNFHCWDYRRFVATQAAVPPAEELAFTDSLITRNESNYSS
|
WHYRSCLLPQLHPQPDSGPQGRLPEDVLLKELELVQNAFFTDPNDQSAWFYHRWLLGRAD
|
PQDALRCLHVSRDEACLTVSFSRPLLVGSRMEILLLMVDDSPLIVEWRTPDGRNRPSHVW
|
LCDLPAASLNDQLPQHTFRVIWTAGDVQKECVLLKGRQEGWCRDSTTDEQLFRCELSVEK
|
STVLQSELESCKELQELEPENKWCLLTIILLMRALDPLLYEKETLQYFQTLKAVDPMRAT
|
YLDDIRSKELLENSVLKMEYAEVRVLHLAHKDLTVLCHLEQLLLVTHLDLSHNRLRTLPP
|
ALAALRCLEVLQASDNAIESLDGVTNLPRLQELLLCNNRLQQPAVLQPLASCPRLVLLNL
|
QGNPLCQAVGILEQLAELLPSVSSVLT
|
|
6337
SCNN1A (sodium channel epithelial 1 subunit alpha; SEQ ID NO: 25)
|
Uniprot
MEGNKLEEQDSSPPQSTPGLMKGNKREEQGLGPEPAAPQQPTAEEEALIEFHRSYRELFE
|
P37088
FFCNNTTIHGAIRLVCSQHNRMKTAFWAVLWLCTFGMMYWQFGLLFGEYFSYPVSLNINL
|
NSDKLVFPAVTICTLNPYRYPEIKEELEELDRITEQTLEDLYKYSSFTTIVAGSRSRRDL
|
RGTLPHPLQRLRVPPPPHGARRARSVASSLRDNNPQVDWKDWKIGFQLCNQNKSDCFYQT
|
YSSGVDAVREWYRFHYINILSRLPETLPSLEEDTLGNFIFACRENQVSCNQANYSHEHHP
|
MYGNCYTENDKNNSNLWMSSMPGINNGLSLMLRAEQNDFIPLLSTVTGARVMVHGQDEPA
|
FMDDGGFNLRPGVETSISMRKETLDRLGGDYGDCTKNGSDVPVENLYPSKYTQQVCIHSC
|
FQESMIKECGCAYIFYPRPQNVEYCDYRKHSSWGYCYYKLQVDFSSDHLGCFTKCRKPCS
|
VTSYQLSAGYSRWPSVTSQEWVFQMLSRQNNYTVNNKRNGVAKVNIFFKELNYKINSESP
|
SVTMVTLLSNLGSQWSLWFGSSVLSVVEMAELVEDLLVIMFLMLLRRERSRYWSPGRGGR
|
GAQEVASTLASSPPSHFCPHPMSLSLSQPGPAPSPALTAPPPAYATLGPRPSPGGSAGAS
|
SSTCPLGGP
|
|
92799
SHKBP1 (SH3KBP1 binding protein 1; SEQ ID NO: 26)
|
Uniprot
MAAAATAAEGVPSRGPPGEVIHLNVGGKRFSTSRQTLTWIPDSFFSSLLSGRISTLKDET
|
Q8TBC3
GAIFIDRDPTVFAPILNFLRTKELDPRGVHGSSLLHEAQFYGLTPLVRRLQLREELDRSS
|
CGNVLFNGYLPPPVFPVKRRNRHSLVGPQQLGGRPAPVRRSNTMPPNLGNAGLLGRMLDE
|
KTPPSPSGQPEEPGMVRLVCGHHNWIAVAYTQFLVCYRLKEASGWQLVESSPRLDWPIER
|
LALTARVHGGALGEHDKMVAAATGSEILLWALQAEGGGSEIGVFHLGVPVEALFFVGNQL
|
IATSHTGRIGVWNAVTKHWQVQEVQPITSYDAAGSFLLLGCNNGSIYYVDVQKFPLRMKD
|
NDLLVSELYRDPAEDGVTALSVYLTPKTSDSGNWIEIAYGTSSGGVRVIVQHPETVGSGP
|
QLFQTFTVHRSPVTKIMLSEKHLISVCADNNHVRTWSVTRFRGMISTQPGSTPLASFKIL
|
ALESADGHGGCSAGNDIGPYGERDDQQVFIQKVVPSASQLFVRLSSTGQRVCSVRSVDGS
|
PTTAFTVLECEGSRRLGSRPRRYLLTGQANGSLAMWDLTTAMDGLGQAPAGGLTEQELME
|
QLEHCELAPPAPSAPSWGCLPSPSPRISLTSLHSASSNTSLSGHRGSPSPPQAEARRRGG
|
GSFVERCQELVRSGPDLRRPPTPAPWPSSGLGTPLTPPKMKLNETSF
|
|
55315
SLC29A3 (solute carrier family 29 member 3; SEQ ID NO: 27)
|
Uniprot
MAVVSEDDFQHSSNSTYRTTSSSLRADQEALLEKLLDRPPPGLQRPEDRECGTYIIFFSL
|
Q9BZD2
GIGSLLPWNFFITAKEYWMFKLRNSSSPATGEDPEGSDILNYFESYLAVASTVPSMLCLV
|
ANFLLVNRVAVHIRVLASLTVILAIFMVITALVKVDTSSWTRGFFAVTIVCMVILSGAST
|
VFSSSIYGMTGSFPMRNSQALISGGAMGGTVSAVASLVDLAASSDVRNSALAFFLTATVE
|
LVLCMGLYLLLSRLEYARYYMRPVLAAHVESGEEELPQDSLSAPSVASRFIDSHTPPLRP
|
ILKKTASLGFCVTYVFFITSLIYPAICTNIESLNKGSGSLWTTKFFIPLTTFLLYNFADL
|
CGRQLTAWIQVPGPNSKALPGFVLLRTCLIPLFVLCNYQPRVHLKTVVFQSDVYPALLSS
|
LLGLSNGYLSTLALLYGPKIVPRELAEATGVVMSFYVCLGLTLGSACSTLLVHLI
|
|
56848
SPHK2 (sphingosine kinase 2; SEQ ID NO: 28)
|
Uniprot
MNGHLEAEEQQDQRPDQELTGSWGHGPRSTLVRAKAMAPPPPPLAASTPLLHGEFGSYPA
|
Q9NRA0
RGPRFALTLTSQALHIQRLRPKPEARPRGGLVPLAEVSGCCTLRSRSPSDSAAYFCIYTY
|
PRGRRGARRRATRTFRADGAATYEENRAEAQRWATALTCLLRGLPLPGDGEITPDLLPRP
|
PRLLLLVNPFGGRGLAWQWCKNHVLPMISEAGLSFNLIQTERQNHARELVQGLSLSEWDG
|
IVTVSGDGLLHEVINGLLDRPDWEEAVKMPVGILPCGSGNALAGAVNQHGGFEPALGLDL
|
LINCSLLLCRGGGHPLDLLSVTLASGSRCFSFLSVAWGFVSDVDIQSERFRALGSARFTL
|
GTVLGLATLHTYRGRLSYLPATVEPASPTPAHSIPRAKSELTLTPDPAPPMAHSPLHRSV
|
SDLPLPLPQPALASPGSPEPLPILSINGGGPELAGDWGGAGDAPLSPDPLLSSPPGSPKA
|
ALHSPVSEGAPVIPPSSGLPLPTPDARVGASTCGPPDHLLPPIGTPLPPDWVTLEGDEVL
|
MLAISPSHLGADLVAAPHARFDDGLVHLCWVRSGISRAALLRLFLAMERGSHFSLGCPQL
|
GYAAARAFRLEPLTPRGVLTVDGEQVEYGPLQAQMHPGIGTLLTGPPGCPGREP
|
|
79816
TLE6 (TLE family member 6, subcortical maternal complex member;
|
Uniprot
SEQ ID NO: 29)
|
Q9H808
MTSRDQPRPKGPPKSTSPCPGISNSESSPTLNYQGILNRLKQFPRESPHFAAELESIYYS
|
LHKIQQDVAEHHKQIGNVLQIVESCSQLQGFQSEEVSPAEPASPGTPQQVKDKTLQESSF
|
EDIMATRSSDWLRRPLGEDNQPETQLFWDKEPWFWHDTLTEQLWRIFAGVHDEKAKPRDR
|
QQAPGLGQESKAPGSCDPGTDPCPEDASTPRPPEASSSPPEGSQDRNTSWGVVQEPPGRA
|
SRFLQSISWDPEDFEDAWKRPDALPGQSKRLAVPCKLEKMRILAHGELVLATAISSFTRH
|
VFTCGRRGIKVWSLTGQVAEDRFPESHLPIQTPGAFLRTCLLSSNSRSLLTGGYNLASVS
|
VWDLAAPSLHVKEQLPCAGLNCQALDANLDANLAFASFTSGVVRIWDLRDQSVVRDLKGY
|
PDGVKSIVVKGYNIWTGGPDACLRCWDQRTIMKPLEYQFKSQIMSLSHSPQEDWVLLGMA
|
NGQQWLQSTSGSQRHMVGQKDSVILSVKFSPFGQWWASVGMDDFLGVYSMPAGTKVFEVP
|
EMS PVTCCDVSSNNRLVVTGSGEHASVYQITY
|
|
79639
TMEM53 (transmembrane protein 53; SEQ ID NO: 30)
|
Uniprot
MASAELDYTIEIPDQPCWSQKNSPSPGGKEAETRQPVVILLGWGGCKDKNLAKYSAIYKK
|
Q6P2H8
RGCIVIRYTAPWHMVFFSESLGIPSLRVLAQKLLELLEDYEIEKEPLLFHVFSNGGVMLY
|
RYVLELLQTRRECRLRVVGTIFDSAPGDSNLVGALRALAAILERRAAMLRLLLLVAFALV
|
VVLFHVLLAPITALFHTHEYDRLQDAGSRWPELYLYSRADEVVLARDIERMVEARLARRV
|
LARSVDFVSSAHVSHLRDYPTYYTSLCVDFMRNCVRC
|
|
641649
TMEM91 (transmembrane protein 91; SEQ ID NO: 31)
|
Uniprot
MDSPSLRELQQPLLEGTECETPAQKPGRHELGSPLREIAFAESIRGLQFLSPPLPSVSAG
|
Q6ZNR0
LGEPRPPDVEDMSSSDSDSDWDGGSRLSPFLPHDHLGLAVFSMLCCFWPVGIAAFCLAQK
|
TNKAWAKGDIQGAGAASRRAFLLGVLAVGLGVCTYAAALVTLAAYLASRDPP
|
|
Histone Deacetylase 1 (HDAC1) Protein
HDAC1 catalyzes the deacetylation of lysine residues on the N-terminal part of the core histones (H2A, H2B, H3 and H4). The HDAC1 gene is located on chromosome 1 (Gene ID: 3065; location NC_000001.11 (32292083 . . . 32333626). An example of an amino acid sequence for HDAC1 isoform 1 is available as UNIPROT accession no. Q13547 and shown below as SEQ ID NO:32. MAQTQGTRRKVCYYYDGDVGNYYYGQGHPMKPHRIRMTHNLLLNYGLYRKMEIYRPHKANAE EMTKYHSDDYIKFLRSIRPDNMSEYSKQMQRFNVGEDCPVFDGLFEFCQLSTGGSVASAVKL NKQQTDIAVNWAGGLHHAKKSEASGFCYVNDIVLAILELLKYHQRVLYIDIDIHHGDGVEEA FYTTDRVMTVSFHKYGEYFPGTGDLRDIGAGKGKYYAVNYPLRDGIDDESYEAI FKPVMSKV MEMFQPSAVVLQCGSDSLSGDRLGCFNLTIKGHAKCVEFVKSFNLPMLMLGGGGYTIRNVAR CWTYETAVALDTEIPNELPYNDYFEYFGPDFKLHISPSNMTNQNTNEYLEKIKQRLFENLRM LPHAPGVQMQAIPEDAIPEESGDEDEDDPDKRISICSSDKRIACEEEFSDSEEEGEGGRKNS SNFKKAKRVKTEDEKEKDPEEKKEVTEEEKTKEEKPEAKGVKEEVKLA
A cDNA sequence encoding the SEQ ID NO:32 HDAC1 protein is available as NCBI accession no. NM 004964.3, shown below as SEQ ID NO:33:
1
cccctccccc ctgggtcgga cgctgagcgg agccgcgggc gggagggcgg acggaccgac
|
|
61
tgacggtagg gacgggaggc gagcaagatg gcgcagacgc agggcacccg gaggaaagtc
|
|
121
tgttactact acgacgggga tgttggaaat tactattatg gacaaggcca cccaatgaag
|
|
181
cctcaccgaa tccgcatgac tcataatttg ctgctcaact atggtctcta ccgaaaaatg
|
|
241
gaaatctatc gccctcacaa agccaatgct gaggagatga ccaagtacca cagcgatgac
|
|
301
tacattaaat tcttgcgctc catccgtcca gataacatgt cggagtacag caagcagatg
|
|
361
cagagattca acgttggtga ggactgtcca gtattcgatg gcctgtttga gttctgtcag
|
|
421
ttgtctactg gtcgttctgt ggcaagtgct gtgaaactta ataagcagca gacggacatc
|
|
481
gctgtgaatt gggctggggg cctgcaccat gcaaagaagt ccgaggcatc tggcttctgt
|
|
541
tacgtcaatg atatcgtctt ggccatcctg gaactgctaa agtatcacca gagggtgctg
|
|
601
tacattgaca ttgatattca ccatggtgac ggcgtggaag aggccttcta caccacggac
|
|
661
cgggtcatga ctgtgtcctt tcataagtat ggagagtact tcccaggaac tggggaccta
|
|
721
cgggatatcg gggctggcaa aggcaagtat tatgctgtta actacccgct ccgagacggg
|
|
781
attgatgacg agtcctatga ggccattttc aagccggtca tgtccaaagt aatggagatg
|
|
841
ttccagccta gtgcggtggt cttacagtgt ggctcagact ccctatctgg ggatcggtta
|
|
901
ggttgcttca atctaactat caaaggacac gccaagtgtg tggaatttgt caagagcttt
|
|
961
aacctgccta tgctgatgct gggaggcggt ggttacacca ttcgtaacgt tgcccggtgc
|
|
1021
tggacatatg agacagctgt ggccctggat acggagatcc ctaatgagct tccatacaat
|
|
1081
gactactttg aatactttgg accagatttc aagctccaca tcagtccttc caatatgact
|
|
1141
aaccagaaca cgaatgagta cctggagaag atcaaacagc gactgtttga gaaccttaga
|
|
1201
atgctgccgc acgcacctgg ggtccaaatg caggcgattc ctgaggacgc catccctgag
|
|
1261
gagagtggcg atgaggacga agacgaccct gacaagcgca tctcgatctg ctcctctgac
|
|
1321
aaacgaattg cctgtgagga agagttctcc gattctgaag aggagggaga ggggggccgc
|
|
1381
aagaactctt ccaacttcaa aaaagccaag agagtcaaaa cagaggatga aaaagagaaa
|
|
1441
gacccagagg agaagaaaga agtcaccgaa gaggagaaaa ccaaggagga gaagccagaa
|
|
1501
gccaaagggg tcaaggagga ggtcaagttg gcctgaatgg acctctccag ctctggcttc
|
|
1561
ctgctgagtc cctcacgttt cttccccaac ccctcagatt ttatattttc tatttctctg
|
|
1621
tgtatttata taaaaattta ttaaatataa atatccccag ggacagaaac caaggccccg
|
|
1681
agctcagggc agctgtgctg ggtgagctct tccaggagcc accttgccac ccattcttcc
|
|
1741
cgttcttaac tttgaaccat aaagggtgcc aggtctgggt gaaagggata cttttatgca
|
|
1801
accataagac aaactcctga aatgccaagt gcctgcttag tagctttgga aaggtgccct
|
|
1861
tattgaacat tctagaaggg gtggctgggt cttcaaggat ctcctgtttt tttcaggctc
|
|
1921
ctaaagtaac atcagccatt tttagattgg ttctgttttc gtaccttccc actggcctca
|
|
1981
agtgagccaa gaaacactgc ctgccctctg tctgtcttct cctaattctg caggtggagg
|
|
2041
ttgctagtct agtttccttt ttgagatact attttcattt ttgtgagcct ctttgtaata
|
|
2101
aaatggtaca tttctata
|
Histone Deacetylase 7 (HDAC7)
HDAC7 catalyzes the deacetylation of lysine residues on the N-terminal part of the core histones (H2A, H2B, H3 and H4). The HDAC7 gene is located on chromosome 12 (Gene ID: 51564; location NC_000012.12 (47782722..47821344). An example of an amino acid sequence for HDAC7 is available as UNIPROT accession no. Q8WUI4 and shown below as SEQ ID NO:34.
MDLRVGQRPPVEPPPEPTLLALQRPQRLHHHLFLAGLQQQRSVEPMRLSMDTPMPELQVG
|
|
PQEQELRQLLHKDKSKRSAVASSVVKQKLAEVILKKQQAALERTVHPNSPGIPYRTLEPL
|
|
ETEGATRSMLSSFLPPVPSLPSDPPEHFPLRKTVSEPNLKLRYKPKKSLERRKNPLLRKE
|
|
SAPPSLRRRPAETLGDSSPSSSSTPASGCSSPNDSEHGPNPILGSEALLGQRLRLQETSV
|
|
APFALPTVSLLPAITLGLPAPARADSDRRTHPTLGPRGPILGSPHTPLFLPHGLEPEAGG
|
|
TLPSRLQPILLLDPSGSHAPLLTVPGLGPLPFHFAQSLMTTERLSGSGLHWPLSRTRSEP
|
|
LPPSATAPPPPGPMQPRLEQLKTHVQVIKRSAKPSEKPRLRQIPSAEDLETDGGGPGQVV
|
|
DDGLEHRELGHGQPEARGPAPLQQHPQVLLWEQQRLAGRLPRGSTGDTVLLPLAQGGHRP
|
|
LSRAQSSPAAPASLSAPEPASQARVLSSSETPARTLPFTTGLIYDSVMLKHQCSCGDNSR
|
|
HPEHAGRIQSIWSRLQERGLRSQCECLRGRKASLEELQSVHSERHVLLYGTNPLSRLKLD
|
|
NGKLAGLLAQRMFVMLPCGGVGVDTDTIWNELHSSNAARWAAGSVTDLAFKVASRELKNG
|
|
EAVVRPPGHHADHSTAMGECFENSVAIACRQLQQQSKASKILIVDWDVHHGNGTQQTEYQ
|
|
DPSVIYISLHRHDDGNFFPGSGAVDEVGAGSGEGENVNVAWAGGLDPPMGDPEYLAAFRI
|
|
VVMPIAREFSPDLVLVSAGFDAAEGHPAPLGGYHVSAKCFGYMTQQLMNLAGGAVVLALE
|
|
GGHDLTAICDASEACVAALLGNRVDPLSEEGWKQKPNLNAIRSLEAVIRVHSKYWGCMQR
|
|
LASCPDSWVPRVPGADKEEVEAVTALASLSVGILAEDRPSEQLVEEEEPMNL
|
A cDNA sequence encoding the amino acid sequence of SEQ ID NO:34 HDAC1 protein is available as NCBI accession no. NM_001098416.4, shown below as SEQ ID NO:35:
1
cttcctctcg ccgtctcaca gtcgctctgc agcctccggc gactgggggg atgtgaggcc
|
|
61
ggcgccccag ccccccgccc cgccatgagc cccccgctct gagggccccg gcccctggat
|
|
121
gcacagcccc ggcgctgatg ggacccaggt gagcccgggt gcccactact gcagccccac
|
|
181
tggcgcaggc tgccccaggc cctgtgcaga cacaccaggc cctcagccgc agcccatgga
|
|
241
cctgcgggtg ggccagcggc ccccagtgga gcccccacca gagcccacat tgctggccct
|
|
301
gcagcgtccc cagcgcctgc accaccacct cttcctagca ggcctgcagc agcagcgctc
|
|
361
ggtggagccc atgaggctct ccatggacac gccgatgccc gagttgcagg tgggacccca
|
|
421
ggaacaagag ctgcggcagc ttctccacaa ggacaagagc aagcgaagtg ctgtagccag
|
|
481
cagcgtggtc aagcagaagc tagcggaggt gattctgaaa aaacagcagg cggccctaga
|
|
541
aagaacagtc catcccaaca gccccggcat tccctacaga accctggagc ccctggagac
|
|
601
ggaaggagcc acccgctcca tgctcagcag ctttttgcct cctgttccca gcctgcccag
|
|
661
tgacccccca gagcacttcc ctctgcgcaa gacagtctct gagcccaacc tgaagctgcg
|
|
721
ctataagccc aagaagtccc tggagcggag gaagaatcca ctgctccgaa aggagagtgc
|
|
781
gccccccagc ctccggCggc ggcccgcaga gaccctcgga gactcctccc caagtagtag
|
|
841
cagcacgccc gcatcagggt gcagctcccc caatgacagc gagcacggcc ccaatcccat
|
|
901
cctgggctcg gaggctgaca gtgaccgcag gacccatccg actctgggcc ctcgggggcc
|
|
961
aatcctgggg agcccccaca ctcccctctt cctgccccat ggcttggagc ccgaggctgg
|
|
1021
gggcaccttg ccctctcgcc tgcagcccat tctcctcctg gacccctcag gctctcatgc
|
|
1081
cccgctgctg actgtgcccg ggcttgggcc cttgcccttc cactttgccc agtccttaat
|
|
1141
gaccaccgag cggctctctg ggtcaggcct ccactggcca ctgagccgga ctcgctcaga
|
|
1201
gcccctgccc cccagtgcca ccgctccccc accgccgggc cccatgcagc cccgcctgga
|
|
1261
gcagctcaaa actcacgtcc aggtgatcaa gaggtcagcc aagccgagtg agaagccccg
|
|
1321
gctgcggcag ataccctcgg ctgaagacct ggagacagat ggcgggggac cgggccaggt
|
|
1381
ggtggacgat ggcctggagc acagggagct gggccatggg cagcctgagg ccagaggccc
|
|
1441
cgctcctctc cagcagcacc ctcaggtgtt gctctgggaa cagcagcgac tggctgggcg
|
|
1501
gctcccccgg ggcagcaccg gggacactgt gctgcttcct ctggcccagg gtgggcaccc
|
|
1561
gcctctgtcc cgggctcagt cttccccagc cgcacctgcc tcactgtcag ccccagagcc
|
|
1621
tgccagccag gcccgagtcc tctccagctc agagacccct gccaggaccc tgcccttcac
|
|
1681
cacagggctg atctatgact cggtcatgct gaagcaccag tgctcctgcg gtgacaacag
|
|
1741
caggcacccg gagcacgccg gccgcatcca gagcatctgg tcccggctgc aggagcgggg
|
|
1801
gctccggagc cagtgtgagt gtctccgagg ccggaaggcc tccctggaag agctgcagtc
|
|
1861
ggtccactct gagcggcacg tgctcctcta cggcaccaac ccgctcagcc gcctcaaact
|
|
1921
ggacaacggg aagctggcag ggctcctggc acagcggatg tttgtgatgc tgccctgtgg
|
|
1981
tggggttggg gtggacactg acaccatctg gaatgagctt cattcctcca atgcagcccg
|
|
2041
ctgggccgct ggcagtgtca ctgacctcgc cttcaaagtg gcttctcgtg agctaaagaa
|
|
2101
tggtttcgct gtggtgcggc ccccaggaca ccatgcagat cattcaacag ccatgggctt
|
|
2161
ctgcttcttc aactcagtgg ccatcgcctg ccggcagctg caacagcaga gcaaggccag
|
|
2221
caagatcctc attgtagact gggacgtgca ccatggcaac ggcacccagc aaaccttcta
|
|
2281
ccaagacccc agtgtgctct acatctccct gcatcgccat gacgacggca acttcttccc
|
|
2341
ggggagtggg gctgtggatg aggtaggggc tggcagcggt gagggcttca atgtcaatgt
|
|
2401
ggcctgggct ggaggtctgg acccccccat gggggatcct gagtacctgg ctgctttcag
|
|
2461
gatagtCgtg atgcccatcg cccgagagtt ctctccagac ctagtcctgg tgtctgctgg
|
|
2521
atttgatgct gctgagggtc acccggcccc actgggtggc taccatgttt ctgccaaatg
|
|
2581
ttttggatac atgacgcagc aactgatgaa cctggcagga ggcgcagtgg tgctggcctt
|
|
2641
ggagggtggc catgacctca cagccatctg tgacgcctct gaggcctgtg tggctgctct
|
|
2701
tctgggtaac agggtggatc ccctttcaga agaaggctgg aaacagaaac ccaacctcaa
|
|
2761
tgccatccgc tctctggagg ccgtgatccg ggtgcacagt aaatactggg gctgcatgca
|
|
2821
gcgcctggcc tcctgtccag actcctgggt gcctagagtg ccaggggctg acaaagaaga
|
|
2881
agtggaggca gtgaccgcac tggcgtccct ctctgtgggc atcctggctg aagataggcc
|
|
2941
ctcggagcag ctggtggagg aggaagaacc tatgaatctc taaggctctg gaaccatctg
|
|
3001
cccgcccacc atgcccttgg gacctggttc tcttctaacc cctggcaata gcccccattc
|
|
3061
ctgggtcttt agagatcctg tgggcaagta gttggaacca gagaacagcc tgcctgcttt
|
|
3121
gacagttatc ccagggagcg tgagaaaatc cctgggtcta gaatgggaac tggagaggac
|
|
3181
cctgagagga gacgggctgg gCggcgaccc ccacagggct ctcgagaaca gattctcccc
|
|
3241
tccagtatgg gccctggctg tggcccccat tcctcaggac tgcacagagg aggactggct
|
|
3301
ccggctccgt cgggctcacc cttaaccact attcctggct ctgcaaaccc cagactttgc
|
|
3361
acacagcctc aggctccaca cagaaatgtg aacttggcct cagacaggct ggcccttcct
|
|
3421
aggctctagg ggctaggggg gagtggggag ccaagaggtc ccatattcct gagtgcaggg
|
|
3481
gtagtccctc tcacctgctt cctcagacga ctctggaagc ttccctctac cactgggcac
|
|
3541
tgagacgaag ctccctgaca gccgagactg gcagccctcc atctggtccg taccctcgcc
|
|
3601
agaggccccc ctacatcaac ctcctggcga tgccctggtg gagcagatgg gtgctctggg
|
|
3661
agtcctgtgc ttcctgatcc aatggtgcca aacccttcat ctccccaaga agcgcagcat
|
|
3721
acccctggga cccctcggcc actgcccact cggggagcct tctctgtttc tggggcctcc
|
|
3781
cccaccatag ctctgattcc caccccacat aggagtagcc tgactgaggg ggaaggggtg
|
|
3841
ggagagaaga tacagacatg gaggagggga ggctgctctg gcaaagtctt caaggctttt
|
|
3901
gggggtccag gcctggggtc aagaaggaaa atgtgtgtga gcatgtgtgt gagtgaggcg
|
|
3961
tgtgtgtgag cgtgtgtgtg agtgaggcgt gtgtgtgtgt ctttcctagg acccaccata
|
|
4021
ccctgtgtat gtatgcatgt ttttgtaaaa aggaagaaaa tggaaaaaaa tctgaacaat
|
|
4081
aaatgtttta tttgctttaa aa
|
Isoforms and variants of the ET-29, HDAC1, HDAC7 and ZNF92 genes and gene products can be present in subjects and can be detected, measured, evaluated, and the subjects with such isoforms and variants can be treated by the methods and compositions described herein. Such isoforms and variants can have sequences with between 65-100% sequence identity to a reference sequence, for example with at least at least 65%1, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97;% sequence, at least 98%, at least 99%, or at least 99.5% identity to a sequence described herein or a reference sequence (such as one described in the NCBI or Uniprot databases) over a specified comparison window. Optimal alignment may be ascertained or conducted using the homology alignment algorithm of Needleman and Wunsch, J. Mol. Biol. 48:443-53 (1970).
Definitions
The “absolute amplitude” of correlation expressions means the distance, either positive or negative, from a zero value; i.e., both correlation coefficients −0.35 and 0.35 have an absolute amplitude of 0.35 ET-29, HDAC1, HDAC7 and ZNF92.
“Status” means a state of gene expression of a set of genetic markers whose expression is strongly correlated with a particular phenotype. For example, “ZNF92 status” means a state of gene expression of a set of genetic markers (e.g., HDAC1, HDAC7 markers) whose expression is strongly correlated with that of the ZNF92 gene, wherein the expression pattern of these (e.g. HDAC1, HDAC7,) can differ detectably between tumors expressing the ZNF92 and tumors not expressing ZNF92.
“Good prognosis” means that a patient is expected to have longer overall survival (OS), or progression-free survival (PFS), or disease-specific survival (DSS) or recurrence-free survival (RFS) compared to “poor prognosis” patients. These metrics are typically described by National Cancer Institute (NCI) as overall survival (OS), or progression-free survival (PFS) which is the length of time during and after the treatment of cancer, that a patient lives with the disease but it does not get worse, or disease-specific survival (DSS) that is the percentage of people in a treatment group who have not died from their cancer in a defined period of time, or recurrence-free survival (RFS) that is length of time after primary treatment for a cancer ends that the patient survives without any signs or symptoms of that cancer, also called as disease-free survival (DFS), or relapse-free survival (see website at cancer.gov/publications/dictionaries/cancer-terms/def/rfs).
“Poor prognosis” means that a patient is expected to have a shorter overall survival (OS), or progression-free survival (PFS), or disease-specific survival (DSS) or recurrence-free survival (RFS) compared to “good prognosis” patients.
“Marker” means an entire gene, mRNA, EST, or a protein product derived from that gene, where the expression or level of expression changes under different conditions, where the expression of the gene (or combination of genes) correlates with a certain condition, the gene or combination of genes is a marker for that condition.
“Marker-derived polynucleotides” means the RNA transcribed from a marker gene, any cDNA, or cRNA produced therefrom, and any nucleic acid derived therefrom, such as synthetic nucleic acid having a sequence derived from the gene corresponding to the marker gene.
A “similarity value” is a number that represents the degree of similarity between two things being compared. For example, a similarity value may be a number that indicates the overall similarity between a patient's expression profile using specific phenotype-related markers and a control specific to that phenotype (for instance, the similarity to a “good prognosis” template, where the phenotype is a good prognosis). The similarity value may be expressed as a similarity metric, such as a correlation coefficient, or may simply be expressed as the expression level difference, or the aggregate of the expression level differences, between a patient sample and a template.
The present description is further illustrated by the following examples, which should not be construed as limiting in any way.
EXAMPLES
Example 1: Materials and Methods
Reagents: Entinostat (Active motif 14043), Vorinostat (Abcam-ab1444480), Belinostat (Tokyo Chemical Industry-B5888), Niclosamide (Bio-Vision-1826),[25-27] Tanespimycin/17AAG (Bio-Vision-1774) and Pimitespib (Tas1116, Cayman Chemical Company-33779) were reconstituted in DMSO (Sigma Aldrich D-8418). Cell Proliferation Reagent WST-1 (11644807001, Sigma Aldrich).
Cell culture: The ATCC sourced breast cancer cell lines were previously acquired and extensively validated including STR profiling in our previous studies[11, 25, 27, 29]. All the ATCC cell lines including HCC38, HCC202, HCC1187, HCC 1937, HCC 1143, HCC 1954, MFM223, CAL148, MDAMB231, SKBR3, ZR75-1, ZR75-B, MCF7, MDAMB231, MDAMB435, BT549, BT20, and T47D were maintained in their respective media as recommended by ATCC (Gibco, Thermo-Fisher, NY, USA). The inflammatory breast cancer cell lines IBC02 (FC-IBC02) IBC03, KPL4, Sum149, and Sum190 were generously provided by Dr. Sandra Fernandez and cultured in F-12 medium (Gibco 11765-054). All the drug sensitivity experiments were carried out in medium supplemented with 2% heat-inactivated fetal bovine serum. The BPLER and HMLER cells were previously characterized, deposited to the American Type Culture Collection and the European Collection of Authenticated Cell Cultures (ATCC #CRL-3546 and ECACC #20012032 and 20012041) and cultured in BMI-T medium (US Biological, cat #506387.500)[27]. All the cells were cultured at 37° C. and 5% CO2 in a humidified incubator.
WST cell proliferation assay: The changes in cell proliferation and viability due to treatment with Entinostat, Vorinostat, Belinostat, Niclosamide, Tanespimycin and Pimitespib alone or in combination was determined by WST assay following manufacturer's protocol. Briefly, ˜3000 of cells per well were plated in 96-well plates and treated with various drugs for 7 days. At the end of the specified time following drug treatment, the WST reagent (10 μL) was added into each well, incubated at 37° C. for 2 h and the color absorbance of each well was recorded at 450 nm with a Thermo Labsystems Multiskan Ascent microplate reader with a blank reference.
Drug screening: The Gene Set Cancer Analysis (GSCA) and the Cancer Therapeutics Response Portal (CTRP) online analysis tools were used to identify drugs that may target ZNF92 and its 29 gene signature. These platforms integrate more than 10,000 comprehensive genomic data set of 33 diverse types of cancers from TCGA with 750 drug molecules[30-32]. Graphical representation and Statistical analyses: GraphPad Prism (V9) software was used for statistical analysis and graph generation. All data are expressed as the mean±SD of six independent experiments. The differences between the control and the treatment groups were determined by one-way ANOVA, and significance was determined by using Dunnett's multiple comparison test (P<0.01).
Example 2: The Intrinsic Properties of the Normal Cell-Origin is Involved in Shaping Tumor Biology in Subgroups of Breast Cancer Including TNBC
Based on clinical observations, it was hypothesized that the intrinsic properties of the normal cell-origin might be involved in shaping tumor biology in other subgroups of breast cancer such as TNBC[2]. To test this hypothesis, identical genetic elements were used to transform two different ER-negative normal breast cell-origins isolated from the same donor[2]. While one normal cell-origin developed invasive and metastatic TNBC, the other normal cell-origin developed non-metastatic indolent TNBC, confirming our hypothesis[2] (FIG. 1A). Since then, other investigators independently verified this hypothesis in numerous cancers[3-15]. However, these cell-origin signatures have been difficult to translate into mechanistic insights because they involve thousands of genes[16-18]. As such, we had a similar challenge having identified more than 6,000 breast cell-origin associated mRNAs in our metastatic TNBC model.
It was reasoned that examining histone modifications could uncover a mechanism that may propagate the normal cell-origin signature in TNBC[19]. After exploring several candidates[20-24], HDAC7 and HDAC1 were found to be specifically upregulated in the metastatic TNBC cell lineage that was developed, but not in the isogenic non-metastatic cell lineage[25]. Next, it was determined that in these cells HDAC1 is upstream of HDAC7, and these two HDACs co-regulate 1,243 mRNAs in the metastatic TNBC cells[26]. Finally, it was found that approximately 10% of the HDAC1&7 upregulated (n=125) mRNAs are increased via super-enhancers in metastatic TNBC[27], but not in non-metastatic TNBC[25]. Further, a 60 gene mRNA subset of this cell-origin signature predicted remarkably shorter breast cancer survival, 8.7 years overall and 6.2 years relapse free respectively[26].
Next, it was discovered that twenty-nine genes that are co-upregulated by HDAC1&7 in metastatic TNBC have a ZNF92 binding site in their promoter[26]. ZNF92 is a primate-specific member of the KRAB-ZNF (Kruppel-associated box domain zinc finger) family with more than 700 genes encoding tissue-specific transcription suppressors. ZNFs have been an understudied protein family (534 papers in PubMed for 700 genes). Even among this family, ZNF92 is perhaps an exceptionally unexplored protein since its cloning in 1991, as it was only mentioned once among 121 genes that are altered with cholesterol-lowering drug atorvastatin in the liver cell line HepG2[28]. Previous to our study, ZNF92 had not been examined in any cancer.
It was inferred that the metastatic TNBC cell-origin related HDAC1-HDAC7-ZNF92 axis could be targeted with a combination of small molecules. As a proof-of-concept, a combination of an anti-worm drug, an antibiotic that inhibits HSP90, and a histone deacetylase inhibitor can synergistically inhibit proliferation of sixteen out of seventeen TNBC and IBC cell lines (FIG. 1B). The repurposing of these clinically approved drugs provides an opportunity to develop a cell-targeted therapy for TNBC and IBC.
Example 3: Three-Drug Synergy
To illustrate the three-drug synergy, low concentrations of HDAC-I inhibitor, HSP90-I inhibitor, and Niclosamide were used in the experiments. Nevertheless, up to 98% reduction in TNBC cell proliferation was observed, suggesting that it might be possible to achieve meaningful results with clinically recommended doses of Niclosamide (2000 mg/day, 220 mg/m2)[35, 60], Vorinostat (400 mg)[61], Pimitespib (160 mg/day)[62], and Belinostat (1,000 mg/m2/day)[44, 53]. At these doses, the side effects of HDAC-1 include diarrhea, fatigue, pyrexia, nausea, thrombocytopenia, and anorexia. While Niclosamide side effects are generally non-overlapping (itching, drowsiness, dizziness, and skin rash); the side effects of HSP90 inhibitor Pimitespib partially overlap (diarrhea, decreased appetite, malaise, renal impairment, and anemia). Whether any of these toxicities would be exacerbated or other toxicities would emerge with a three-drug combination remains to be seen. However, in the present experiments Entinostat (100 nM), Niclosamide (100 nM), Tanespimycin (50 nM), Belinostat (1 uM), Vorinostat (1 uM), and Pimitespib (0.2-0.6 uM) were used at doses that are 10 to 1000-fold lower than the peak serum concentrations achieved in different clinical trials for Entinostat (1 uM), Niclosamide (2 uM), Tanespimycin (15 uM), Belinostat (200 uM), Vorinostat (40 M), and Pimitespib (7 uM). Therefore, it may be possible to manage these side effects by lowering the dose of some drugs when used in combination. Moreover, two recent studies reported development of novel dual HDAC and HSP90 inhibitors, where a single small molecule inhibited both HDAC and HSP90 that were tested on age-related macular degeneration [63] and leukemia [64].
The concept of targeted therapy has been often mentioned for a class of drugs that target tumor specific gene alterations. While these gene-targeted therapies have been transformative for HER2+ breast cancer, other breast cancer subtypes do not necessarily present such a straightforward gene-targeted therapy opportunity due to heterogeneity and clonal evolution. It was recently reported that the cell-of-origin patterns dominate the molecular classification of 10,000 tumors from 33 types of cancer [65]. The concept of cell-of-origin was expanded to targeted therapy because a substantial portion of the normal cell-origin mRNA, protein and DNA methylation signatures are retained in the breast cancer [2, 11, 12, 66, 67], and these cell-origin signatures can be associated with clinically relevant tumor features such as metastasis [2, 26]. Thus, it was postulated that such a persistent cell-origin signature can be targeted with drug combinations. It is worth mentioning that HDAC1-HDAC7-ZNF92 axis and their 29 downstream targets (ET-29) are rarely mutated in human breast cancers, consistent with a cell-of-origin signature (FIG. 8 and Table 4). As a proof-of-concept, this metastatic breast cancer cell-of-origin signature was shown to be synergistically targeted with HDAC and HSP90 inhibitors combined with Niclosamide in sixteen out of seventeen breast cancer cell lines.
TABLE 4
|
|
The frequency of genomic alterations in the cell-of-origin signature.
|
Frequency of genetic alterations in the cell-of-origin
|
signature genes
|
Gene
Number of
|
Symbol
Samples Altered
Percent Samples Altered
|
|
1
CUX1
14
0.40%
|
2
HDAC1
10
0.30%
|
3
CROCC
10
0.30%
|
4
EPHB3
10
0.30%
|
5
PCDH1
10
0.30%
|
6
FCHO1
9
0.30%
|
7
PLA2G6
7
0.20%
|
8
MYO1B
5
0.20%
|
9
LIMK2
4
0.10%
|
10
SCNN1A
4
0.10%
|
11
DKK3
3
0.10%
|
12
FIBCD1
3
0.10%
|
13
GDPD5
3
0.10%
|
14
KRT7
3
0.10%
|
15
SHKBP1
3
0.10%
|
16
ZNF92
2
0.10%
|
17
ABTB1
2
0.10%
|
18
PDLIM1
2
0.10%
|
19
RABGGTA
2
0.10%
|
20
TMEM53
2
0.10%
|
21
HDAC7
1
0.00%
|
22
ECH1
1
0.00%
|
23
LOXL1
1
0.00%
|
24
BCAS4
0
0.00%
|
|
Despite their expression ubiquitously in all tissues, hereditary mutations cause tissue-specific cancers [68]. It seems that rather than being a blank canvas the tissue origin is an active partner in the development of familial tumors[69]. Conversely, because the mutational spectrum of sporadic tumors seems less tissue-specific, it has been proposed that sporadic tumor should be classified according molecular alterations regardless of their tissue origin[69]. However, recent basket trials suggest that the response to targeted anti-cancer drug response often depends on the tissue type[69]. Consistent with these observations, the present work suggests that the cell-of-origin is involved in shaping breast tumor phenotype and drug response[70]. Further, it was demonstrate that the cell-of-origin signature can be leveraged to develop cell-targeted therapies.
Example 4: Therapies
The Gene Set Cancer Analysis (GSCA) online platformn[30] was utilized to discover that ZNF92 expression is uniquely associated with sensitivity to the 90-kDa heat shock protein (Hsp90) inhibitor Tanespimycin (17-AAG), a synthetic analog of the antibiotic Geldanamycin[33]. HSP90 is a molecular chaperone that regulates protein hemostasis by regulating the folding, stabilization, activation, and degradation of over 400 proteins[34].
The GSCA includes the Genomics of Drug Sensitivity in Cancer (GDSC) dataset that contains IC50 of 265 small molecules in 860 cell lines and their corresponding mRNA expression[31]. Significantly, 17-AAG was the only hit for ZNF92 in the GDSC, as all the other top 30 hits are anti-correlated, meaning that ZNF92 expression predicted resistance to these drugs (FIG. 1C).
Using the Genomics of Therapeutics Response Portal (CTRP)[30, 32], the 29 gene mRNA signature downstream of the HDAC1-HDAC7-ZNF92 axis (Table 2, reproduced below) was found to correlate with net sensitivity to 9/481 small molecules in 1001 cell lines.
TABLE 2
|
|
The 29 gene mRNA signature downstream of the HDACI-HDAC7-ZNF92 axis.
|
Entrez ID
Gene Symbol
Gene Name
|
|
1
80325
ABTB1
ankyrin repeat and BTB domain containing 1
|
2
9289
ADGRG1
adhesion G protein-coupled receptor G1
|
3
113451
AZIN2
antizyme inhibitor 2
|
4
55653
BCAS4
breast carcinoma amplified sequence 4
|
5
79934
COQ8B
coenzyme Q8B
|
6
9696
CROCC
ciliary rootlet coiled-coil, rootletin
|
7
1523
CUX1
cut like homeobox 1
|
8
27122
DKK3
dickkopf WNT signaling pathway inhibitor 3
|
9
1891
ECH1
enoyl-CoA hydratase 1
|
10
2049
EPHB3
EPH receptor B3
|
11
23149
FCHO1
FCH and mu domain containing endocytic adaptor 1
|
12
84929
FIBCD1
fibrinogen C domain containing 1
|
13
81544
GDPD5
glycerophosphodiester phosphodiesterase domain
|
14
3855
KRT7
keratin 7
|
15
3985
LIMK2
LIM domain kinase 2
|
16
114783
LMTK3
lemur tyrosine kinase 3
|
17
4016
LOXL1
lysyl oxidase like 1
|
18
4430
MYO1B
myosin IB
|
19
5097
PCDH1
protocadherin 1
|
20
9124
PDLIM1
PDZ and LIM domain 1
|
21
8398
PLA2G6
phospholipase A2 group VI
|
22
5875
RABGGTA
Rab geranylgeranyltransferase subunit alpha
|
23
6337
SCNN1A
sodium channel epithelial 1 subunit alpha
|
24
92799
SHKBP1
SH3KBP1 binding protein 1
|
25
S5315
SLC29A3
solute carrier family 29 member 3
|
26
56848
SPHK2
sphingosine kinase 2
|
27
79816
TLE6
TLE family member 6, subcortical maternal
|
28
79639
TMEM53
transmembrane protein 53
|
29
641649
TMEM91
transmembrane protein 91
|
|
The top nine Clinical Trials Reporting Program (CTRP) hits in rank order include ML239 (breast cancer stem cell inhibitor), Niclosamide (anti tapeworm drug), Tipifamib (H-Ras inhibitor), Tivantinib (MET inhibitor), CHIR-99021 (GSK-3α/β inhibitor), Tubastatin-A (HDAC6 inhibitor), GDC-0879 (B-Raf inhibitor), SB-525334 (transforming growth factor β1 receptor (ALK5) inhibitor, PRIMA-1-met (p53 re-activation and induction of massive apoptosis) (FIG. 11D). Among these, only two were approved by the U.S. Food and Drug Administration (FDA); Niclosamide was approved by the FDA to treat tapeworm infection in 1815, and Tipifarmib recently received a breakthrough therapy designation by the FDA for the treatment of patients with recurrent or metastatic HRAS mutant head and neck squamous cell carcinoma (HNSCC)[36]. Since Niclosamide was the highest ranked FDA approved drug it was selected for the combination experiments.
As was previously shown that Entinostat (MS-275) inhibits both HDAC1 and HDAC7[37], it was reasoned that combining Entinostat (E), with Tanespimycin (T) and Niclosamide (N) may target all the components of the HDAC1-HDAC7-ZNF92 axis. First, dose-response experiments were carried out in several TNBC cell lines and the drug concentrations that have minimal effect on cell proliferation (FIG. 4A-4C) were determined. Encouragingly, while these low doses reduced cell proliferation only by 16% individually, the three-drug combination resulted in over 84% average inhibition of proliferation, consistent with a synergistic activity (FIG. 4D). These results were confirmed in multiple TNBC cell lines, where the observed average inhibition was significantly greater than expected by additive activity (FIG. 2). In several TNBC cell lines, there was exceptional sensitivity to Niclosamide (HCC1913, MFM223) or Tanespimycin (Cal148) (FIG. 2).
These results provided a proof-of-concept that HDAC and HSP90 inhibitors can be combined with a tape-worm drug Niclosamide to achieve synergistic effects in TNBC. Entinostat is still undergoing Phase H-III trials and has not been FDA approved yet. Moreover, even though Tanespimycin concentrations required for activity in preclinical models could be safely achieved in patient plasma in phase 1/2 clinical trials, it was reported that the manufacturer of Tanespimycin declined advancing it to phase III trials.
To potentially translate the findings to the clinic more rapidly, clinically approved alternatives for Entinostat and Tanespimycin were determined. Currently, there are five FDA approved HDAC inhibitors (HDAC-I) available for cancer treatment: Vorinostat (SAHA), Belinostat (PXD-101), Romidepsin (FK-228), Epidaza (HBI-8000) and Panobinostat (LBH589)[38]. Furthermore, a new orally available HSP90 inhibitor TAS-116 (Pimitespib), was recently approved to treat gastrointestinal stromal tumors that have progressed after chemotherapy in Japan[39].
By way of single drug dose-response experiments the minimally effective doses of Pimitespib (P), Vorinostat (V), and Belinostat (B) (FIG. 5A) were determined. It was found that it was possible to replace one HSP90 inhibitor (HSP90-I) Tanespimycin with another HSP90-L, Pimitespib, in combination with Entinostat and Niclosamide with similar synergistic results (FIG. 5B). Next it was showed that HDAC inhibitor Entinostat could be replaced by two different HDAC-I Vorinostat (V) and Belinostat (B) in combination with Niclosamide (N) and Pimitespib (P) in five out of five TNBC cell lines tested. In these experiments it was expected approximately 20% to 30% inhibition of cell proliferation if these three drugs acted additively. In contrast, approximately 70 to 80% inhibition was observed consistent with synergistic action (FIG. 6A-6C). The ability to swap different small molecules for the same target suggests that the observed synergies are probably due to bonafide target synergies and not the result of an idiosyncratic drug interaction.
Next, it was determined the lowest combined doses that can produce a near complete inhibition of TNBC proliferation, and determined that 400-600 nM Pimitespib combined with 100 nM Niclosamide and 1,000 nM Vorinostat produced 96-98% inhibition of proliferation in three out of four TNBC cell lines (FIG. 3A), which was replicated with another HDAC inhibitor Belinostat (FIG. 3B). It is worth pointing out that 10 to 200-fold higher concentrations of Vorinostat, Belinostat, Pimitespib and Niclosamide are achievable in the clinic (Table 5)[39-46]. Therefore, it may be possible to eliminate tumor cells with a three-drug combination at clinically relevant doses.
TABLE 5
|
|
Comparison of the peak serum concentrations achieved
|
for HDAC inhibitors, HSP90 inhibitors and Niclosamide,
|
and the drug concentrations used in this study.
|
Phase I-II
Experimental
|
Cmax
three-drug
|
Drug
(nM)
(nM)
Ratio
|
|
Entinostat
1,000
100
10
|
Vorinostat
40,000
1,000
40
|
Belinostat
200,000
1,000
200
|
Niclosamide
2,000
100
20
|
Tanespimycin
15,000
50
300
|
Pimitespib
7,000
200-600
11-35
|
|
Lastly, it was previously showed that HDAC inhibitors inhibited self-renewal of IBC tumor spheroids and tumor emboli[47, 48]. Significantly, it was found that four out of five IBC cell lines (IBC02, IBC03, KPL4 and SUM-149) are sensitive to both EPN and BPN triple-drug combinations (Table 6).
TABLE 6
|
|
Survival of inflammatory breast cancer cells with HDAC-I,
|
HSP90-I and niclosamide.
|
IBC02
IBC03
KPL4
SUM149
SUM190
|
|
DMSO
101
100
100
103
100
|
Entinostat 100
21
61
45
99
159
|
Entinonostat150
12
35
20
102
223
|
Niclosamide100
20
46
91
113
135
|
Pimitespib100
107
89
89
115
114
|
Pimitespib200
114
81
118
172
102
|
E100N100P100
0
32
32
86
208
|
Expected Additive
4
25
36
128
244
|
E100N100P200
0
12
18
103
139
|
Expected Additive
3
14
48
192
218
|
E150N100P100
0
13
7
78
262
|
Expected Additive
5
23
16
132
342
|
E150N100P200
0
9
5
128
177
|
Expected Additive
3
13
22
197
306
|
DMSO
100
99
100
105
100
|
Belinostat500
14
91
91
71
95
|
Belinostat1000
0
45
23
21
107
|
Niclosamide100
17
35
104
74
133
|
Pimitespib100
114
89
102
111
112
|
Pimitespib200
105
81
136
158
100
|
B500N100P100
0
40
58
37
91
|
Expected Additive
3
28
97
59
142
|
B1000N100P100
0
12
42
37
62
|
Expected Additive
0
14
25
17
160
|
B500N100P200
0
7
17
6
79
|
Expected Additive
3
26
130
83
126
|
B1000N100P200
0
2
15
8
60
|
Expected Additive
0
13
33
24
143
|
|
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All patents and publications referenced or mentioned herein are indicative of the levels of skill of those skilled in the art to which the invention pertains, and each such referenced patent or publication is hereby specifically incorporated by reference to the same extent as if it had been incorporated by reference in its entirety individually or set forth herein in its entirety. Applicants reserve the right to physically incorporate into this specification any and all materials and information from any such cited patents or publications. The following statements are intended to describe and summarize various features of the invention according to the foregoing description provided in the specification and figures.
Statements
1. A method comprising:
- a. assaying a biological sample from a subject for expression of HDAC1, HDAC7, and ZNF92 genes to determine one or more expression levels for the HDAC1, HDAC7, and ZNF92 genes;
- b. comparing the determined expression levels with one or more reference values to identify any altered expression levels in the subject's biological sample, wherein altered expression levels of the HDAC1, HDAC7, and ZNF92 genes in the biological sample relative to the reference value indicates that the subject has cancer with poor prognosis or the subject has malignant cancer, and absence of altered expression of the HDAC1, HDAC7, and ZNF92 genes relative to the reference value indicates that the subject does not have a cancer with poor prognosis or does not have malignant cancer; and optionally
- c. administering one or more histone deacetylase inhibitor(s) (HDAC), one or more inhibitor of heat shock protein 90 (HSP90), and one or more helminths to a subject determined to have a cancer with poor prognosis or a malignant cancer.
2. The method of statement 1, wherein the biological sample from the subject is further assayed for expression of one or more genes encoding an amino acid sequence having at least 90% sequence identity to SEQ ID NOs: 3-31.
3. The method of statement 1, wherein the sample is a breast cancer sample.
4. The method of statement 1, wherein the sample is a cervical cancer sample, uterine cancer, or prostate cancer.
5. The method of statement 1, further administering one or more of ML239, Tipifarnib, Tivantinib, CHIR-99021, Tubastatin-A, GDC-0879, SB-525334, PRIMA-1-met, or a combination thereof, to the subject.
6. The method of statement 1, wherein the administering one or more histone deacetylase inhibitor(s), one or more heat shock factor inhibitor, and the one or more helminths are delivered simultaneously.
7. The method of statement 1, wherein the sample is a physiological fluid sample, a tumor tissue sample, or a cancer cell sample.
8. The method of statement 1, wherein the subject is a human.
9. The method of statement 8, wherein the mammal has inflammatory breast cancer.
10. The method of statement 8, wherein the mammal has triple negative breast cancer.
11. The method of statement 1, wherein RNA expression is assayed.
12. The method of statement 1, wherein protein expression is assayed.
13. The method of statement 1, wherein the inhibitor of HSP90 comprises an inhibitor of HSP90a and HSP90s.
14. The method of statement 1, wherein the inhibitor of HSP90 comprises an antibiotic.
15. The method of statement 1, wherein the inhibitor of HSP90 is Pimitespib, Tanespimycin, or a combination thereof.
16. The method of statement 1, wherein the helminth is niclosamide.
17. The method of statement 1, wherein the at least one HDAC inhibitor is Belinostat, Entinostat, Vorinostat, or a combination thereof.
18. A method to prevent, inhibit or treat cancer in a mammal, comprising: administering to the mammal a composition comprising one or more histone deacetylase inhibitor(s), one or more heat shock factor inhibitor, and one or more helminths, wherein the cancer in the mammal is determined to have altered expression levels of HDAC1, HDAC7, and ZNF92 genes, relative to a reference value.
19. The method of statement 18, wherein the biological sample from the subject is further assayed for expression of one or more genes encoding an amino acid sequence having at least 90% sequence identity to SEQ ID NOs: 3-31.
20. The method of statement 18, wherein the mammal is a human.
21. The method of statement 18, wherein the mammal has breast cancer, cervical cancer, uterine cancer, or prostate cancer.
22. A method comprising: (a) contacting cancer cells expressing HDAC1, HDAC7, or ZNF92 genes or producing HDAC1, HDAC7, or ZNF92 proteins with a test agent; (b) measuring HDAC1, HDAC7, or ZNF92 RNA or protein expression levels in the cells or measuring HDAC1, HDAC7, or ZNF92 protein activity levels; and (c) determining that the test agent reduces the expression levels or activity levels of HDAC1, HDAC7, or ZNF92 genes, to thereby identifying a test agent as a candidate agent that reduces HDAC1, HDAC7, or ZNF92 genes expression levels or activity levels.
23. A pharmaceutical composition comprising one or more histone deacetylase inhibitor(s), one or more heat shock factor inhibitor, and one or more helminths.
The specific methods, devices and compositions described herein are representative of preferred embodiments and are exemplary and not intended as limitations on the scope of the invention. Other objects, aspects, and embodiments will occur to those skilled in the art upon consideration of this specification and are encompassed within the spirit of the invention as defined by the scope of the claims. It will be readily apparent to one skilled in the art that varying substitutions and modifications may be made to the invention disclosed herein without departing from the scope and spirit of the invention.
The invention illustratively described herein suitably may be practiced in the absence of any element or elements, or limitation or limitations, which is not specifically disclosed herein as essential. The methods and processes illustratively described herein suitably may be practiced in differing orders of steps, and the methods and processes are not necessarily restricted to the orders of steps indicated herein or in the claims.
Under no circumstances may the patent be interpreted to be limited to the specific examples or embodiments or methods specifically disclosed herein. Under no circumstances may the patent be interpreted to be limited by any statement made by any Examiner or any other official or employee of the Patent and Trademark Office unless such statement is specifically and without qualification or reservation expressly adopted in a responsive writing by Applicants.
The terms and expressions that have been employed are used as terms of description and not of limitation, and there is no intent in the use of such terms and expressions to exclude any equivalent of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope of the invention as claimed. Thus, it will be understood that although the present invention has been specifically disclosed by preferred embodiments and optional features, modification and variation of the concepts herein disclosed may be resorted to by those skilled in the art, and that such modifications and variations are considered to be within the scope of this invention as defined by the appended claims and statements of the invention.
The invention has been described broadly and generically herein. Each of the narrower species and subgeneric groupings falling within the generic disclosure also forms part of the invention. This includes the generic description of the invention with a proviso or negative limitation removing any subject matter from the genus, regardless of whether or not the excised material is specifically recited herein. In addition, where features or aspects of the invention are described in terms of Markush groups, those skilled in the art will recognize that the invention is also thereby described in terms of any individual member or subgroup of members of the Markush group.