Patent Application
20180313843
The present invention pertains to novel treatments for cancer diseases. Treatment of cancers with nucleoside analogs (NA), which specifically inhibit rapidly dividing cells, may face preexisting NA resistance or development of resistant cancer cells resulting in poor clinical prognosis. Treatment of cancers with oncolytic herpes simplex viruses (HSV) may face preexisting resistance or development of resistant cancer cells resulting in poor clinical prognosis. The invention overcomes chemotherapy resistance or resistance to oncolytic HSV by providing methods for detecting the resistance in a cancer disease based on the expression of SAM domain and HD domain-containing protein 1 (SAMHD1) in cancer cells. Furthermore provided are treatment options addressing the chemotherapy resistance such as a combination of a SAMHD1 inhibitor with a NA. Moreover, provided are treatment options addressing the resistance to oncolytic HSV such as a combination of a SAMHD1 inhibitor or depletion of SAMHD1 with an oncolytic HSV. The combination in some embodiments may furthermore include an inhibitor of CTP-synthetase. The invention provides new medicines and companion diagnostics supporting clinical treatment decisions.
The use of chemical drugs as cytostatics for cancer therapy constitutes one of the choices as first-line treatment for the majority of solid and hematopoietic tumors. The most commonly used chemical drugs for cancer therapy are: platin agents, topoisomerase inhibitors, vinca alkaloids, taxanes and nucleoside analogs (NA) that tackle specifically rapidly dividing cells, however, via different mechanisms. Often first line treatments are insufficient to destroy all tumor cells from the organism or have significant adverse effects. Also drug resistance is recognized as the primary cause of failure of cancer therapy when chemotherapeutic agents are applied. In spite of that, sub-optimal drug concentration on the tumor milieu could influence the drug resistance; other factors like cellular origin plays an essential role for chemo-resistance of many tumors. Drug resistance is a multifactorial phenomenon depending on multiple independent mechanisms which involve intracellular detoxification, changes of the cellular response, tolerance to stress and defects in apoptosis signaling pathways. The glyco-protein-P and the glutathione S-transferase are the major proteins that mediate the intracellular detoxification process linked to well established modes of drug resistance in cancer. Other proteins like beta-tubulins have been reported to be involved in the drug resistance phenomenon and whose levels directly correlate with the tumor resistance to Paclitaxel. Moreover, the cisplatin resistance has been reported to be influenced by the over-expression of different proteins like T-plastin, the heat shock protein (HSP70) and (HSP90) and the transcription factor YB1. Reports from different groups have indicated the existence of a set of proteins which either inhibit apoptosis or increase cell survival on tumor cells thus contributing to the chemo-resistance phenomenon of tumors.
NAs are therapeutically inactive molecules, which have to be activated in tumor cells through different cellular enzymes into the corresponding potent nucleoside triphosphate (NTP) and deoxy-nucleoside triphosphate (dNTP) analogs. Consequently, resistance of tumor cells to NAs may be associated with changes in expression or gene mutations of the enzymes involved in the activation of NAs to NTP analogs.
There are considerable efforts to increase therapeutic efficacy of nucleoside analogs used for treatment of leukemia or glioma patients especially those with recurrent disease. Clinically tested approaches involve combinations of NAs with one or more other NAs or with inhibitors of signaling pathways relevant for leukemogenesis. These treatment strategies combine drugs which exert cytotoxic effects and mostly influence more than one target in human cells. Consequently, unprecedented toxicity in treated patients has frequently occurred and synergistic activities are poorly defined.
SAM domain and HD domain-containing protein 1 (SAMHD1) is a cellular enzyme activated by GTP or dGTP and which has a triphosphohydrolase activity (dNTPase). The enzyme catalyzes the hydrolysis of dNTPs into component nucleosides and an inorganic triphosphate. Therefore the enzyme is involved in the depletion of cellular dNTP pools and counteracts cell division processes. By reducing the dNTP substrate levels required for reverse transcription of the HIV RNA into a complementary DNA, the enzyme is also responsible for blocking replication of HIV in dendritic cells, macrophages, monocytes, and resting CD4 T-cells. Different experimental tools and naturally evolved mechanisms exist to deplete SAMHD1 from cells including siRNA/shRNA, CRISP/CAS9, as well as Vpx proteins from HIV-2 and SIV and Vpr proteins from SIV (Baldauf, 2012). Methods for quantitation of endogenous levels of NTPs and dNTPs in human cells, e.g. by liquid chromatography tandem mass spectrometry, were established (Thomas, 2015). Enzymatic assays for the triphosphohydrolase activity of SAMHD1 to determine the mode of interaction of NTPAs and NTPAs with SAMHD1 were also developed (Arnold 2014). A number of monoclonal and polyclonal antibodies for quantitative detection of SAMHD1 in leukemic and other cells are commercially available. Technologies for targeting of leukemic cells and other cancer cells using delivery systems and/or coupling to antibodies specific for leukemia or the respective cancer cell have been described and tested in clinical trials (Row 2013).
In view of the state of the art the present invention provides a novel approach for overcoming chemotherapy resistance that naturally exists or develops in response to a first line treatment with NAs, in particular in the context of a treatment of leukemia or glioma, but is not restricted to these types of cancer.
The above problem is solved in a first aspect by a method for determining resistance of a cancer patient to a cancer therapy, comprising the steps of
In another aspect the problem of the invention is solved by an in-vitro method for stratifying a cancer patient as responder or non-responder to a cancer therapy, comprising the steps of
A “different level” in context of the invention shall refer to either an increased (higher) or decreased (lower) level compared to a control or reference. For example in some embodiments an increased level may be indicative for the presence of a chemotherapy resistance or resistance against an oncolytic virus. Then a decreased or equal level would be indicative for the absence of the chemotherapy resistance or resistance against an oncolytic virus. In other embodiments an equal or increased level may be indicative for the presence of a chemotherapy resistance or resistance against an oncolytic virus. Then a decrease of the level would be indicative for the absence of the chemotherapy resistance or resistance against an oncolytic virus.
The term “sample” or “biological sample” means any biological sample derived from a subject or patient. Examples of such samples include tissues, cell samples, cell lysates, biopsies, etc. Biological samples may be selected from a tumor sample or a biopsy such as a sample from a solid glioma. Other biological samples are whole blood, serum or plasma. Preferably, the sample is a whole blood sample. Most preferred in context of the present invention is that the biological sample is a blood sample, a bone marrow sample, or a biopsy sample. The nature of the sample will depend on the cancer disease diagnosed or treated in context with the herein described invention.
Preferred for the present invention is that the sample of the cancer disease is a sample containing cancer cells of the cancer disease, for example from a resected tumor.
The term “determining” as used herein includes qualitative and/or quantitative detection (i.e. detecting and/or measuring expression level) with or without reference to a control or a predetermined value. “Determining the level” shall refer to a quantitative detection of a biomarker as disclosed herein. For determining the level of SAMHD1 in the biological sample and/or control sample any method can be used that allows the quantification of SAMHD1 concentrations. In a preferred embodiment the content of SAMHD1 in a sample to be analyzed is determined immunologically by using a SAMHD1-specific antibody or mass spectrometry (protein detection) or by quantitative real time-PCR (qPCR) (mRNA detection). In particular “determining the level of SAMHD1” in context of the present invention may include both direct determination of SAMHD1 protein or mRNA expression, as well as indirect determination of the level of SAMHD1 as for example detection of negative or positive regulators of SAMHD1 expression and/or function. Such a negative regulator of SAMHD1 may be an inhibitory nucleic acid, such as a microRNA, for example miRNA 181a or 181b. Cytokines or other bioactives may function as positive or negative regulators of SAMHD1 expression and/or function.
The term “subject,” as used herein, describes a mammal including, but not limited to, humans, apes, chimpanzees, orangutans, monkeys, dogs, cats, horses, pigs, sheep, goats, mice, rats, and guinea pigs. However, in some embodiments a subject is preferably a human. In certain embodiments, where the methods of the present invention are used for determining the presence of a chemotherapy resistance, the subject is a subject suffering from cancer disease, such as a recurrent cancer disease.
The term “level” in conjunction with one or more biomarkers of the present disclosure shall refer preferably to the concentration of the respective biomarker.
The term “control” as used in the context of the present invention may refer to various reference values depending on the diagnostic context for which the present method is used. A control level may therefore be any reference value of the respective biomarker which allows for a meaningful interpretation of the status or development of a chemotherapy resistance or resistance against an oncolytic virus in a patient having a cancer disease. In general, a control level may correspond to a level of the biomarker in cancer cells having different levels of SAMHD1 expression (high, medium, low) not having or displaying the malignancy. Alternatively, a control level may correspond to a level of SAMHD1 in a biological sample of the subject at an earlier time point, for example, before said subject underwent chemotherapy or other medical treatments. In certain embodiments of the invention the control level is a cut-off level, and an increased level or equal level of SAMHD1 in the biological sample compared to the cut-off level is indicative for the presence of the chemotherapy resistance or resistance against an oncolytic virus. Most preferably, an increased level or equal level of SAMHD1 in the biological sample compared to the cut-off level is indicative for the presence of chemotherapy resistance or resistance against an oncolytic virus in the subject.
In certain embodiments of the invention the cancer therapy is a chemotherapy, such as a therapy with a nucleoside analog (NA), or a therapy with an oncolytic virus, such as an oncolytic Herpes simplex virus (HSV).
The term “cancer disease” disclosed herein comprises lung cancer, arseniccellular lung cancer, liver cancer, colon cancer, bone cancer, pancreatic cancer, skin cancer, cephalic or cervical cancer, skin or endophthalmic melanoma, hysterocarcinoma, ovarian cancer, rectal cancer, stomach cancer, perianal cancer, colonic cancer, breast cancer, endometrioma, cervical carcinoma, vaginal carcinoma, vulval carcinoma, Hodgkin's disease, esophageal cancer, enteric cancer, endocrine gland cancer, thyroid cancer, parathyroid cancer, adrenal cancer, smooth tissue sarcoma, urethral cancer, penile cancer, prostatic cancer, chronic or acute leukemia, lymphocytoma, cystic cancer, nephritic or hydrouretic cancer, renal cell carcinoma, renal pelvic carcinoma, CNS tumor, primary CNS lymphoma, spinal medulla tumor, brain stem neuroglioma, hypophyseal adenomatosis and the like, preferably, lung cancer, liver cancer, skin cancer or endophthalmic melanoma. Preferred cancers are glioma, glioblastoma, urothelial carcinoma, or a leukemia, such as AML, ALL, CLL, or CML, and/or wherein the cancer disease is a recurrent cancer disease.
In context of the present invention a chemotherapy or a chemotherapeutic is an NA, for example selected from didanosine, vidarabine, BCX4430, cytarabine, emtricitabine, lamivudine, zalcitabine, abacavir, aciclovir, entecavir, stavudine, telbivudine, zidovudine, idoxuridine, nelarabine, fludarabine, or trifluridine. The most preferred NA is cytarabine.
The term “oncolytic virus” is meant to comprise any virus that infects/enters and lyses cancer cells. The ideal oncolytic virus efficiently kills a clinically relevant fraction of the patient's cancer cells by direct cytolysis with a minimal destruction of non-neoplastic tissue. Targeted tumor cell entry and specificity of replication are desirable. Furthermore, the virus should be safe and apathogenic when applied in patients. Oncolytic viruses derived from many different types of viruses have been described by Liu et al. (Liu et al., Nature Clinical Practice Oncology 4: (2) 101-117, 2007). Among these enveloped viruses such as herpes simplex virus (HSV), vaccinia virus (VV) and paramyxoviruses such as measles virus (MeV), Newcastle disease virus (NDV) or rhabdoviruses like vesicular stomatitis virus (VSV), are most prominent. Besides applying unmodified wildtype virus, genetic engineering can further improve safety and efficacy of oncolytic viruses. Engineering the envelope proteins can restrict virus infection to tumor cells and insertion of suicide genes can enhance therapeutic effects (Nakamura et al., Expert Opin. Bio. Ther. 4: (10): 1685-1692, 2004); Liu et al., Nature Clinical Practice Oncology 4: (2) 101-117, 2007). Most preferred is a HSV virus.
Yet another aspect of the invention provides a method of selecting a cancer therapy for treating a cancer patient, comprising the steps of
Another aspect also relates to a method for monitoring a cancer therapy of a cancer disease, wherein during the cancer therapy, chemotherapy resistance of the cancer disease is determined according to any of the aforementioned methods for detecting resistance to cancer therapy or for stratifying patients.
It is in some embodiments preferably that chemotherapy resistance of the cancer disease is determined at least twice during the chemotherapy, more preferably regularly during the therapy, e.g. monthly.
All of the methods disclosed herein may comprise an additional first step of providing a biological sample from the subject/patient.
Any of the herein above or below described methods are in preferred embodiments methods excluding any steps performed at the human or animal body. In some preferred embodiments the herein disclosed methods are strictly performed in vitro or ex vivo. In particular the term “providing a biological sample from the subject” should not be misinterpreted to explicitly or implicitly include an invasive method step of obtaining the biological sample from a living subject (animal or human). The term “providing a biological sample” shall explicitly exclude any steps of obtaining a biological sample from a subject.
Another aspect of the invention then pertains to an NA or oncolytic virus for use in the treatment of a cancer disease in a subject, wherein the treatment comprises, obtaining from the subject a sample from the cancer disease, and determining chemotherapy resistance of the cancer disease in the subject according to the method described herein above using the sample from the cancer disease, wherein if a chemotherapy resistance is indicated for the cancer disease, the treatment is terminated, and wherein if no chemotherapy-resistance is indicated, the treatment is continued.
If a chemotherapy resistance is indicated for the cancer disease, the treatment may be terminated and a second treatment is commenced comprising the administration of a combination of a NA and a SAMHD1 inhibitor and/or inhibitor of CTPS. Preferred combinations of the invention therefore include NA and a SAMHD1 inhibitor, NA and a CTPS inhibitor, and NA, a SAMHD1 inhibitor together with a CTPS inhibitor.
Another aspect of the invention then pertains to a SAMHD1 inhibitor or antagonist for use in the treatment of a cancer disease, wherein the treatment comprises the concomitant or sequential administration of an SAMHD1 inhibitor and a second therapeutic selected from an NA or oncolytic virus.
As used herein, the terms “SAMHD1-antagonist” or “SAMHD1-inhibitor” are used synonymously. The term refers to a substance that affects a decrease in the amount or rate of SAMHD1 expression or activity. Such a substance can act directly, for example, by binding to SAMHD1 and decreasing the amount or rate of SAMHD1 expression or activity. A SAMHD1-antagonist can also decrease the amount or rate of SAMHD1 expression or activity, for example, by binding to SAMHD1 in such a way as to reduce or prevent SAMHD1 enzymatic activity; by binding to SAMHD1 and modifying it, such as by removal or addition of a moiety; and by binding to SAMHD1 and reducing its stability. A SAMHD1-antagonist can also act indirectly, for example, by binding to a regulatory molecule in the cell or at the cell surface, such as a cytokine receptor, or a gene region so as to modulate regulatory protein or gene region function and affect a decrease in the amount or rate of SAMHD1 expression or activity. Such activities may transiently or permanently affect SAMHD1 expression or function in a cancer cell. Thus, a SAMHD1-antagonist can act by any mechanisms that result in decrease in the amount or rate of SAMHD1 expression or activity.
A SAMHD1-antagonist can be, for example, a naturally or non-naturally occurring macromolecule, such as a polypeptide, peptide, peptidomimetic, nucleic acid, carbohydrate or lipid. A SAMHD1-antagonist further can be an antibody, or antigen-binding fragment thereof, such as a mono-clonal antibody, humanized antibody, chimeric antibody, minibody, bifunctional antibody, single chain antibody (scFv), variable region fragment (Fv or Fd), Fab or F(ab)2. A SAMHD1-antagonist can also be polyclonal antibodies specific for SAMHD1. A SAMHD1-antagonist further can be a partially or completely synthetic derivative, analog or mimetic of a naturally occurring macromolecule, or a small organic or inorganic molecule.
A SAMHD1-antagonist that is an antibody can be, for example, an antibody that binds to SAMHD1 and inhibits its catalytic center, or alters the activity of a molecule that regulates SAMHD1 expression or activity, such that the amount or rate of SAMHD1 expression or activity is decreased. An antibody useful in a method of the invention can be a naturally occurring antibody, including a monoclonal or polyclonal antibody or fragment thereof, or a non-naturally occurring antibody, including but not limited to a single chain antibody, chimeric antibody, bifunctional antibody, complementarity determining region-grafted (CDR-grafted) antibody and humanized antibody or an antigen-binding fragment thereof. In addition to antibodies the present invention may also relate to SAMHD1-specific and -inhibitory T-cell receptors.
A SAMHD1-antagonist that is a nucleic acid can be, for example, an anti-sense nucleotide sequence, an RNA molecule, or an aptamer sequence. An anti-sense nucleotide sequence can bind to a nucleotide sequence within a cell and modulate the level of expression of SAMHD1, or modulate expression of another gene that controls the expression or activity of SAMHD1. Similarly, an RNA molecule, such as a catalytic ribozyme, can bind to and alter the expression of the SAMHD1 gene, or other gene that controls the expression or activity of SAMHD1. An aptamer is a nucleic acid sequence that has a three dimensional structure capable of binding to a molecular target. Specifically preferred in context of the present invention are expression constructs of miR181a or 181b, which were shown to be a negative regulator for SAMHD1 expression. Also included are vector constructs for targeted knock out or mutation of SAMHD1 for example by the CRISPR-Cas9 technology or similar targeted gene editing approaches.
A SAMHD1-antagonist that is a nucleic acid also can be a double-stranded RNA molecule for use in RNA interference methods. RNA interference (RNAi) is a process of sequence-specific gene silencing by post-transcriptional RNA degradation, which is initiated by double-stranded RNA (dsRNA) homologous in sequence to the silenced gene. A suitable double-stranded RNA (dsRNA) for RNAi contains sense and antisense strands of about 21 contiguous nucleotides corresponding to the gene to be targeted that form 19 RNA base pairs, leaving overhangs of two nucleotides at each 3′ end (Elbashir et al., Nature 411:494-498 (2001); Bass, Nature 411:428-429 (2001); Zamore, Nat. Struct. Biol. 8:746-750 (2001)). dsRNAs of about 25-30 nucleotides have also been used successfully for RNAi (Karabinos et al., Proc. Natl. Acad. Sci. USA 98:7863-7868 (2001)). dsRNA can be synthesized in vitro and introduced into a cell by methods known in the art.
Other preferred embodiments pertain to SAMHD1 inhibitory viral proteins. In this case proteins such as lentiviral protein X (Vpx) or viral protein R (Vpr) are preferred examples. For instance the accessory protein Vpx is encoded by HIV-2, SIVSM, or SIVMAC, and/or wherein the accessory protein Vpr is encoded by SIVmus and SIVdeb.
In certain embodiments the treatment aspect described by the present invention relates to the treatment of a cancer disease which is a solid cancer disease, such as glioma, or leukemia, such as AML, and/or a recurrent cancer disease.
Another aspect of the invention relates to a combination of a SAMHD1 inhibitor and a second therapeutic agent selected from an oncolytic virus, for use in the treatment of a cancer disease.
In this context, the term “combination” refers to an active substance combination of two or more active substances in a formulation and also as a combination in the sense of individual formulations of the active substances administered at specified intervals from one another in a therapeutic treatment. Thus the term “combination” shall include the clinical reality of a co-administration of two therapeutically effective compounds, as is described in context of the present invention.
Co-administration: In the context of the present application, co-administration of two compounds is defined as administration of the two compounds to the patient within one year, including separate administration of two medicaments each containing one of the compounds as well as simultaneous administration whether or not the two compounds are combined in one formulation or whether they are in two separate formulations.
The combination of the invention in one embodiment includes that the SAMHD1 inhibitor and the second therapeutic agent are combined by sequential or concomitant administration to a subject during said prevention or treatment, preferably wherein the antagonists and chemotherapeutics are concomitantly administered during said prevention or treatment.
The second therapeutic agent is preferably selected from an oncolytic virus, an NA, or an inhibitor of CTPS as described herein before/below.
The combination of the invention in some embodiments may furthermore include as second or third therapeutic agent an inhibitor of cytidine triphosphate synthetase (CTPS). Therefore, the combination of the invention may include an NA as described herein before and an inhibitor of CTPS, and/or an inhibitor of SAMHD1, as described herein before.
In context of the invention the “inhibitor of CTPS” refers to any compound natural or not which has the ability of reducing or suppressing the activity or expression of CTPS, an enzyme that catalyzes the formation of CTP from UTP and glutamine. Typically the CTPS inhibitor can act directly on the activity by binding to the protein, or can act indirectly on the activity by reducing or inhibiting the expression of the enzyme. Thus CTPS inhibitors encompass inhibitor of CTPS expression. For example, CTPS inhibitors also include any compound that can compete with the substrate of CTPS (e.g. CTP or glutamine) to the corresponding catalytic domains. Typically, said inhibitor is a small organic molecule or a biological molecule (peptides, lipid, aptamer).
In some embodiments, the CTPS inhibitor is any functional analogue, derivative, substitution product, isomer, or homologue of the amino acid glutamine, which retain the property of glutamine to bind CTPS.
In some embodiments, the CTPS inhibitor is a norleucine derivative, such as 6-diazo-5-oxo-L-norleucine (DON). DON is a glutamine analogue that inhibits a wide range of glutamine requiring reactions although the main effect seems to be on de novo purine biosynthesis and CTP synthetase in mammalian cells (Lyons, S. D., Sant, M. E., Christopherson, R. I. (1990) J. Biol. Chem. 265, 11377-11381). It blocks proliferation and has gone through extensive clinical trials as a cancer drug (reviewed in Catane, R., Von Hoff, D. D., Glaubiger, D. L. and Muggia, F. M. (1979) Cancer Treat. Rep. 63, 1033-1038; and Ahluwalia, G. S., Grem, J. L., Hao, Z., and Cooney, D. A. (1990) Pharmacol. Ther. 46, 243-271). U.S. Pat. No. 2,965,634 relates to norleucine derivatives, such as DON, and a process for the production thereof. In some embodiments, the CTPS inhibitor is acivicin. Acivicin has been described in U.S. Pat. No. 5,489,562. In some embodiments, the CTPS inhibitor is an analogue of UTP. Example of such an analogue is deazuridine (CAS Number 23205-42-7). Other examples include Cyclopentenyl cytosine (CPEC), Gemcitabine (2′,2′-difluorodeoxycytidine, dFdC), actinomycin D, cycloheximide, dibutyryl cyclic AMP, and 6-azauridine. A preferred example of an inhibitor of CTPS is 3-deazuridine (3-DU).
Furthermore provided are pharmaceutical compositions comprising the combinations of the present invention together with a pharmaceutically acceptable excipient and/or carrier. The pharmaceutical compositions of the invention are preferably for use in the aforementioned medical treatments.
Another aspect of the invention then pertains to a diagnostic kit, comprising means for determining the presence or absence of SAMHD1 in a sample from a cancer disease. Such means for determining the presence, or absence, or level, of SAMHD1 may be selected from polynucleotide probes and/or primer, or antibodies.
Nonwithstanding that the numerical ranges and parameters setting forth the broad scope of the invention are approximations, the numerical values set forth in the specific examples are reported as precisely as possible. Any numerical value, however, inherently contains certain errors necessarily resulting from the standard deviation found in the respective testing measurements. Also, as used herein, the term “about” generally means within 10%, 5%, 1%, or 0.5% of a given value or range. Alternatively, the term “about” means within an acceptable standard error of the mean when considered by one of ordinary skill in the art. Unless indicated to the contrary, the numerical parameters set forth in the present disclosure and attached claims are approximations that can vary as desired. At the very least, each numerical parameter should at least be construed in light of the number of reported significant digits and by applying ordinary rounding techniques.
The present invention will now be further described in the following examples with reference to the accompanying figures and sequences, nevertheless, without being limited thereto. For the purposes of the present invention, all references as cited herein are incorporated by reference in their entireties. In the Figures:
Ethics statement. Whole blood and bone marrow aspirates of AML patients were obtained and collected pre- and post-treatment. All patients gave informed consent according to the Declaration of Helsinki to participate in the collection of samples. The use of whole blood and bone marrow aspirates was approved by the Ethics Committee of Frankfurt University Hospital (approval no. SPO-01-2015).
Cells and reagents. Human leukemic cell lines from AML were obtained from DSMZ (Deutsche Sammlung von Mikroorganismen and Zellkulturen GmbH, Braunschweig, Germany) THP-1, OCI-AML2, OCI-AML3, Molm13, PL-21, HL-60, MV4-11, SIG-M5, ML2, NB4, KG1, MonoMac6, and HEL and cultured as previously reported (reference 7).
Ara-C resistant cell lines THP-1, HEL, HL-60, Molm13, and Molt4 were established by continuous exposure to increasing drug concentrations and are part of the Resistant Cancer Cell Line (RCCL) collection (http://www.kent.ac.uk/stms/cmp/RCCL/RCCLabout). The generation of resistant cell lines required 1 to 2 years, depending on the cell line used. All cell lines were maintained in IMDM supplemented with 10% FCS (SIG-M5 20% FCS), 200 mM/ml L-Glutamine, 100 IU/ml penicillin, and 100 mg/ml streptomycin at 37° C. in a humidified 5% CO2 incubator. 293T cells were maintained as previously described (Keppler et al).
THP-1 CRISP/Cas9 control and SAMHD1 knock-out cells were provided by Prof. Veit Hornung and cultivated in RPMI supplemented with 10% FCS, 100 IU/ml penicillin, and 100 ml/ml streptomycin. PDX cells were thawed and cultivated as previously described (Vick, 2015).
Peripheral blood mononuclear cells (PBMCs) from AML patients were purified by Ficoll-Hypaque gradient centrifugation (Keppler, 2001). Leukemic cells were isolated from PBMCs by negative selection with a combination of CD3-, CD19- and CD235a-microbeads (Miltenyi Biotec) and separated by the autoMACS™ Pro Separator.
All primary cells were cultivated in X-vivo 10 medium (Lonza) supplemented with 10% Hy-Clone FCS (Perbio), 200 mM/ml L-Glutamine, 25 ng/ml hTPO, 50 ng/ml hSCF, 50 ng/m1 hFlt3-Ligand and 20 ng/ml hIL3 (all from Miltenyi Biotec) at 37° C. in a humidified 5% CO2 incubator.
The following fluorochrome-conjugated antibodies were used: CD33-PE, CD34-FITC (both from Miltenyi Biotech) and CD45-V450 (from BD Pharmingen). Additional antibodies were: rabbit anti-SAMHD1 (Proteintech), MHC-1 (W6-32 from Thermo Scientific), b-actin (Bio-Vision via BioCat), dCK (Santa Cruz), phospho-SAMHD1 (T592P-antibody, generated by Eurogentec), Alexa-Fluor-488 and Alexa-Fluor-660 (both from Invitrogen, Life technologies).
Plasmids. The SIVmac251-based gag-pol expression constructs pSIV3+R- (Vpr-deficient) and pSIV3+X- (Vpx-deficient) were previously reported (Gramberg, 2010). pLK0.1-puro-control-shRNA and pLK0.1-puro-SAMHD1-shRNA#1-3 (reference 1) for shRNA-mediated silencing of SAMHD1 were previously described (reference 12). pHR-based transfer vectors expressing SAMHD1 wt and D137N, D207N, D311A, Q548A, T592A and T592D mutants were generated by site-directed mutagenesis in human codon-optimized SAMHD1 expression constructs (kindly provided by Dr. Thomas Gramberg) and subcloned into pHR-luc transfer vectors. pPAX2 was purchased from Addgene and pVSV-G has been previously described (reference 10).
Cell viability assay. Cell viability of the AML cell lines and primary AML cultures were determined by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) dye reduction assay after 96 hour incubation as described previously (reference 7). Due to lower cell proliferation frequencies of the primary cells the CellTiter-Glo® Luminescent Cell Viability Assay (Promega) was used according to manufacturer's instruction. Here, the number of viable cells was determined 96 hours post treatment based on the quantification of the ATP levels.
Immunoblotting. Cells were lysed in Triton X-100 sample buffer and separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Proteins were blotted on a nitrocellulose membrane (Thermo Scientific), detected by specific antibodies: rabbit anti-SAMHD1 (Proteintech), b-actin (BioVision via BioCat), dCK (Santa Cruz), and phospho-SAMHD1 (T592P-antibody, generated by Eurogentec) and fluorescently labeled secondary antibodies (LI-COR) and visualized and quantified by infrared fluorescence protein detection (Odyssey LI-COR).
dNTP measurement. 5×105 cells were treated with 10 μg/ml Ara-C 13C3 (Santa Cruz) and incubated at 37° C. in a humidified 5% CO2 incubator for 6 hours. Subsequently, cells were washed twice in 1×PBS, pelleted and stored at −20° C. until measurement. The samples were analyzed at the Institute of Clinical Pharmacology (Goethe University Frankfurt) by liquid chromatography-electrospray ionization-tandem mass spectrometry as previously described (reference 8).
Briefly, the analytes were extracted by protein precipitation with methanol. An anion exchange HPLC column (BioBasic AX, Thermo) was used for the chromatographic separation and a 5500 QTrap (Sciex, Darmstadt, Germany) was used as analyzer, operating as triple quadrupole in positive multiple reaction monitoring (MRM) mode. Additional to the analytes reported in the publication, 13C3-Ara-CMP, 13C3-Ara-CDP and 13C3-Ara-CTP were quantified using the validated method and cytidine-13C9, 15N3-5′-triphosphate as internal standard (IS). The precursor-to-product ion transitions used as quantifiers were m/z 327.0→115.1 for 13C3-Ara-CMP, m/z 407.0→115.1 for 13C3-Ara-CDP and m/z 487.0→115.1 for 13C3-Ara-CTP. A calibration curve was constructed for the quantitation of 13C3-Ara-CMP while no calibration standards were commercially available for 13C3-Ara-CDP and 13C3-Ara-CTP. For this reason, these analytes were quantified semiquantitatively by comparing the peak area ratios (analyte/IS) determined in the differently treated samples.
Flow cytometry. Detection of intracellular SAMHD1 was performed as previously described (reference 1). Additional stainings for surface markers (MHC-I, CD33, CD34, CD45) were applied prior to fixation. A FACSVerse or FACSCanto II flow cytometer (BD Biosciences) and FlowJo software (TreeStar) were used for analyses.
RT-Q-PCR analyses. RNA extraction and TaqMan-based mRNA quantification of SAMHD1 (Applied Biosystems: assay no. Hs00210019_ml) and RNaseP (Applied Biosystems: TaqMan® RNase P Control Reagents Kit (4316844), endogenous reference control) were performed in principle as reported (reference 13).
miRNA extractions were performed according to manufacturer's instruction using Ambion PureLink® miRNA isolation kits (Thermo Fisher Scientific). cDNA conversions were performed as recommended using Applied Biosystems® TaqMan® MicroRNA Reverse Transcription kits and miRNA-specific primers. Following miRNAs were analyzed by quantitative PCR based on their significance for AML and predictive binding sites in the SAMHD1 genomic sequence based on targetscan.org, microcosm and microRNA.org: hsa-miRNA-181a (assay-ID: 000480); hsa-miRNA-181b (assay-ID: 001098); hsa-miRNA124a (assay-ID: 000446); hsa-miRNA150 (assay-ID: 000473); hsa-miRNA155 (assay-ID: 002623); hsa-miRNA30c (assay-ID: 000419); hsa-miRNA30d (assay-ID: 000420); hsa-miRNA142-5p (assay-ID:002248); RNU6B (reference control; assay-ID: 001093). Relative mRNA and miRNA levels were calculated as ΔCt and are presented relative to results obtained for THP-1 cells.
Immunohistochemistry. Paraffinized bone marrow aspirates were provided by the hematology/oncology department (University Hospital Frankfurt, Germany). SAMHD1 together with other hematopoietic markers was immunohistochemically stained and the expression of SAMHD1 in leukemic blasts was rated by board certified pathologist.
Production of lentiviral expression vectors and VLPs. Lentiviral vectors expressing SAMHD1 wt or SAMHD1 mutants were generated by co-transfection of packaging vector pPAX2, either pHR-SAMHD1 wt or pHR-mutant SAMHD1 and a plasmid encoding VSV.G. VLP stocks carrying either Vpx or Vpr from SIVmac251 were produced by co-transfection of 293T cells with pSIV3+ gag pol expression plasmids and a plasmid encoding VSV-G. The SAMHD1 degradation capacity of Vpx VLPs was determined on THP-1 cells 1 day post transduction by intracellular SAMHD1 staining.
Manipulation of intracellular SAMHD1 levels. For shRNA-mediated silencing of SAMHD1, OCI-AML3 cells were transduced by spinoculation with VSV-G pseudotyped lentiviral vectors carrying either pLK0.1-puro-control-shRNA or pLK0.1-puro-SAMHD1-shRNA#1-3 (reference 12). On day 30, transduced cells were cultivated in the presence of puromycin (7.5 μg/ml). Knockdown of SAMHD1 levels was monitored by intracellular SAMHD1 staining and Western blotting.
For re-constitution of SAMHD1, THP-1 CRISP/Cas9 SAMHD1 knock-out cells as well as HEL cells were transduced by spinoculation with VSV-G pseudotyped lentiviral vectors carrying SAMHD1 wt or mutants. Expression of SAMHD1 was monitored by intracellular SAMHD1 staining and Western blotting.
Cell lines and primary patient samples were transduced by spinoculation with VSV.G pseudo-typed Vpx or Vpr VLPs. SAMHD1 levels were monitored by intracellular SAMHD1 staining.
HSV-1 YFP. HSV-1 VP26-YFP virus (based on strain SC16, HSV-1 YFP) was produced on RITA cells as follows: a confluent monolayer of RITA cells was infected with HSV-1 YFP for 2-3 days, until a cytopathic effect was observed. After three freeze and thaw cycles, cellular debris was removed by centrifugation and virion-containing supernatants were collected and stored at −80° C.
SAMHD1 expression levels in 13 cell lines from AMLs were examined by Western Blot, flow cytometry and qPCR. Quantification of SAMHD1 protein levels by Western Blot or flow cytometry showed considerable differences in SAMHD1 expression in different cell lines (
Next, it was explored whether SAMHD1 expression levels correlate with cytotoxic activity of cytarabine in different cell lines. As shown in Table 1, IC50 values (concentrations inhibiting 50% of cell growth) of cytarabine ranged for AML cell lines from 7.8 to 560 ng/ml. Importantly, both protein and mRNA levels of SAMHD1 positively correlated with high statistical significance with IC50 values of cytarabine (
To further elucidate the role of SAMHD1 in anti-leukemic activity of cytarabine, SAMHD1 was depleted in cell lines which express high endogenous levels of SAMHD1 (OCI-AML3, SIG-M5 and THP-1) using stable expression of SAMHD1 shRNA or disruption of the SAMHD1 gene using the CRISP/Cas9 technology. Expression of SAMHD1 shRNA in OCI-AML3 and SIG-M5 cells strongly reduced SAMHD1 protein levels, and in THP-1 CRISP/Cas9 knock-out cells SAMHD1 was undetectable (
SAMHD1 hydrolyzes intracellular dNTPs resulting in a reduction of intracellular dNTP pools. It was therefore tested whether SAMHD1 also affects cytarabine triphosphate levels in AML cells treated with cytarabine. To this end, THP-1 (−/−) and THP-1 (+/+) cells were incubated with 10 μg/ml cytarabine for 6 h, and cytarabine triphosphate was measured by liquid chromatography tandem mass spectrometry. About 20-fold higher cytarabine triphosphate amounts accumulated in THP-1(−/−) cells when compared with THP-1(+/+) cells suggesting that SAMHD1 decreases cytarabine triphosphate levels and, as a direct consequence, the anti-leukemic activity of cytarabine (
HIV-2 and some SIV strains encode for the accessory viral protein X (Vpx), which has the ability to target SAMHD1 to the proteasome for degradation. Virus-like particles pseudotyped with VSV-G that carry Vpx from SIVmac251 or other SIV and HIV-2 strains (Vpx-VLPs) were shown to reduce intracellular levels of SAMHD1. In macrophages and resting CD4 T-cells delivery or expression of Vpx resulted in a decrease of dNTP levels. Here, it was tested whether Vpx-VLPs may increase anti-leukemic activity of cytarabine through SAMHD1 degradation in THP-1 cells. As shown in
Several microRNAs (miRNAs) were shown to target SAMHD1. For example, expression of miRNA181a was shown to decrease SAMHD1 expression in leukemic cells. Here, steady-state levels of miRNA181a and miRNA181b in different leukemic cell lines were tested. Both miRNAs were expressed to different extent in the panel of AML cell lines presented in
AML patients frequently display at the start of therapy or develop resistance to therapy during treatment with cytarabine. Therefore, it was investigated whether AML cell lines with experimentally acquired resistance to cytarabine exert changes in SAMHD1 expression. For this aim, three AML cell lines, HEL, HL-60 and MOLM-13, with low endogenous SAMHD1 expression and high sensitivity to cytarabine were cultivated with increasing concentrations of cytarabine (up 2 μg/ml). Highly resistant cell sublines with IC50 values of cytarabine ranging from 24 to 94 μg/ml were obtained. Remarkably, expression levels of SAMHD1 were increased in resistant cells relative to the parental cell lines (
In a next step, it was tested whether SAMHD1 affects anti-leukemic activity of other clinically relevant NAs. Cladribine, thioguanine, and gemcitabine as well as topoisomerase II inhibitor daunorubicin inhibited proliferation of leukemic cells independently of SAMHD1 as demonstrated by similar IC50 values in THP-1 (−/−) and THP-1 (+/+) cells (
Next, it was tested whether SAMHD1 may influence anti-tumoral activity of cytarabine in cultured cells from a malignant disease other than leukemia. As a solid tumor model we used glioma because SAMHD1 was previously shown to be expressed in glioma cells and cytarabine therapy was already tested in glioma patients. As shown
To determine the clinical relevance of SAMHD1 for AML therapy, a blinded SAMHD1 immunostaining of leukemic blasts in bone marrow samples of 72 AML patients taken prior to the start of standard cytarabine and daunorubicin-based combination therapy was performed by pathologists (examples shown in
To address whether SAMHD1 expression levels can modulate cancer cells' sensitivity to cytopathic and oncolytic effects of herpes viruses, THP-1 (+/+) and THP-1 SAMHD1 (−/−) cells were challenged with different volumes of an HSV-1 YFP reporter virus and assessed for morphology, cell viability and infection levels at different time points. THP-1 (−/−) cells lacking SAMHD1 were highly susceptible to HSV-1 infection, indicated by high percentages of productively infected cells (
The inventors further tested the effect of a combination of the uridine nucleoside analog 3-deazauridin (3-DU) in combination with cytarabine in SAMHD1 expressing cells, and cells in which SAMHD1 was inhibited. To this end, first the correlation of 3-DU IC50 values and SAMHD1 expression was determined.
However, interestingly, combination of 3-DU with cytarabine and/or SAMHD1 inhibition significantly and synergistically increased cytotoxicity of cytarabine as shown in table 1 below.
Concentration of Ara C IC50 was significantly reduced when combined with 3-DU in SAMHD1-expressing cells as well as to a lower extent in SAMHD1-depleted cells. The effect is 3-DU concentration-dependent (see Table 1). Shown is the combination on SAMHD1 CRISPR/Cas9 knock outs (THP-1 −/−) compared to SAMHD1-positive control cells (THP-1+/+), in shRNA knock-downs (OCI-AML3 shSAMHD1) compared to control cells (OCI-AML3 CTRL), and SAMHD1 wt-expressing HEL cells compared to HEL cells expressing a triphosphohydrolase-defective mutant (HEL SAMHD1-D311A).
3-DU is an inhibitor of cytidine-triphosphate synthetase, an enzyme which reduces intracellular cytidine-triphosphate levels. The effect of 3-DU and Ara-C on triphosphate levels was thus tested. Indeed, 3-DU treatment in combination with Ara-C strongly increased intracellular Ara-CTP levels (
Protein atlas staining demonstrated patient-dependent expression of SAMHD1 in urothelial carcinoma. Three patients with low-grade urothelial carcinoma showed no or weak expression of SAMHD1, while 50% of high grade patients (n=8) were positive for SAMHD1 (
In gliomas from 10 patients heterogeneity in SAMHD1 expression was observed. In SAMHD1 stainings deposited in the protein atlas, glioma patients showed a tendency for stronger SAMHD1 expression and an increased quantity of SAMHD1-positive malignant cells in patients with high grade glioma relative to patients with low grade glioma (
SAMHD1 protein expression was furthermore observed in various urothelial carcinoma cell lines (
The inventors then intended to test whether the combination therapy of SAMHD1 inhibition and NA treatment results also in increased cytotoxic effects compared to the single treatment. To this end, Ara-C and Vpx-VLPs as SAMHD1 inhibitor were used alone and in combination to treat solid tumor cell lines. The results for urothelial carcinoma are provided in table 2 below:
Cytotoxic effects of Ara-C in urothelial carcinoma cells treated HIV-2/Vpxor non-degrading HIV-1/VprVLPs (Vpx-VLPs and or Vpx-VLPs, respectively) or untreated cells. The combination of SAMHD1 inhibition and Ara-C significantly increased cytotoxicity of Ara-C. A similar result was observed for the combination treatment in glioma cells (
| Number | Date | Country | Kind |
|---|---|---|---|
| 15192494.1 | Nov 2015 | EP | regional |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/EP2016/076388 | 11/2/2016 | WO | 00 |
