Patent Application
20230265524
The invention relates to immunogenic retroelements and more particularly to their use in cancer therapy.
Aging and disease are generally associated with epigenetic reprogramming and aberrant gene expression. Cancer therapeutics, such as DNA methyltransferase inhibitors (DNMTi), are often designed to restore the expression of inappropriately silenced genes, such as tumour suppressors. Many of these ‘epigenetic therapies’ are currently in clinical use or in pre-clinical development16. Intriguingly, epigenetic therapies targeting DNMTs, HDACs, LSD1, EZH2, G9a and SETDB1 can mediate their anti-tumour effects by activating retroelements1,2,4,6-10, which represent about 40% of the human genome. Retroelement activation can also contribute to the therapeutic efficacy of other classes of drugs, such as cyclin-dependent kinase inhibitors3,5.
Active retroelements generate double stranded RNA (dsRNA), which in turn stimulates the pattern recognition receptor MDA51,2,4,6-10. This “viral mimicry” state is characterized by a type I/III interferon response, loss of cancer cell fitness and activation of both innate and adaptive immune responses11,12. However, the identity of these immunogenic retroelements are unknown. We and others have previously shown that inhibition of repressive epigenetic machinery induces the expression of endogenous retroviruses (ERVs), a type of long-terminal repeat (LTR) retroelement1,2,4,6-9. Sense and anti-sense transcription of ERVs has been proposed to generate the immunogenic dsRNA that activate MDA5 after epigenetic therapies6, but direct evidence is lacking. Identifying the precise nature of immunogenic retroelements can have several implications, including understanding their physiological roles, developing biomarkers for predicting and monitoring response to epigenetic therapies and to identify vulnerabilities that could act in synergy with epigenetic therapies.
In an aspect, there is provided a method of assessing a subject's responsiveness to cancer therapy, comprising: providing a sample from the subject comprising cancers cells or suspected cancer cells; measuring or estimating the expression levels of inverted repeats (IR) Alus in the cells; and determining that the subject would be responsive to cancer therapy if the subject cells exhibit expression levels of inverted repeats (IR) Alus with reference to expression levels in control samples.
In an aspect, there is provided a method of cancer therapy in a subject in need thereof, the method comprising inhibiting ADAR1 in a patient.
In an aspect, there is provided an ADAR1 inhibitor for use in the treatment of cancer in a subject.
In an aspect, there is provided a use of an ADAR1 inhibitor in the preparation of a medicament for the treatment of cancer in a subject.
These and other features of the preferred embodiments of the invention will become more apparent in the following detailed description in which reference is made to the appended drawings wherein:
A, Effect of 5-AZA-CdR on repeat element expression in patient-derived xenograft colorectal cancer cells after 5 days of treatment. Plots show log (fold change) for 5-AZA-CdR treated cells over mock-treated cells for anti-sense (y-axis) and sense (x-axis) transcripts of each SINE, LINE and ERV repeat element (CPM). Triangle shape dots in the upper-right quadrant represent repeats that are significantly upregulated in 5-AZA-CdR treated condition at both the antisense and sense strands as compared to the mock. Square shape dots in the upper-right quadrant represent repeats that have baseline expression (CPM≥1) at the sense strand and are significantly upregulated at the antisense strand upon 5-AZA-CdR treatment. Diamond shape dots in the upper-right quadrant represent the repeats that have baseline expression (CPM≥1) at the antisense strand and are significantly upregulated at the sense strand upon 5-AZA-CdR treatment. The significance was determined as p≤0.05 and |log FC|≥1 at each strand.
A-C, Percentage of non-repeats and several families of Repetitive Elements in the total cytoplasmic fraction (‘Total CytoRNA’) and in the MDA5-protected fraction (‘MDA5-protected’) at the mock-treated baseline condition (A) and after 5-AZA-CdR treatment at 300 nM for 5 days, p<2.2e-16 (B). Treatment-induced immunogenic RNAs' refers to transcripts present in 5-AZA-CdR treated condition but not present in the baseline mock-treated condition (i.e. induced by the treatment), p<2.2e-16 (C). Each plot is the average percentage of two replicates. P-values represent the Fisher Exact test for enrichment of SINEs and depletion of non-repeats in immunogenic RNAs (5-AZA-CdR) and Treatment-induced immunogenic RNAs as compared to the MDA5-protected baseline immunogenic RNAs.
D-E, Counts of the expressed Alus in terms of the Log10 Fold change of their MDA5-protected expression over their Total CytoRNA expression. IR-Alus that are MDA5 dsRNA agonists have positive values on the x-axis. The histogram on the left shows the count of the IR-Alus from the Mock-treated cells while the histogram on the right shows the counts from the 5-AZA-CdR treated cells. In Mock-treated cells, most IR-Alus we identified as treatment-induced do not produce dsRNA and therefore are exactly at the 0 position. In 5-AZA-CdR treated cells, the distribution is skewed to the right and shows that after treatment these IR-Alus are able to make dsRNA and activate MDA5. The color code represents Alus making Inverted Repeats pairs (gray) and Non-IR pairs (black) for Baseline immunogenic RNAs (n=187 Non-IR Alus and 1,602 IR-Alus) (D) and for the Treatment-induced immunogenic RNAs (n=992 Non-IR Alus and 5,199 IR-Alus) (E).
F-G, Scatterplots showing the Log10 Fold change of MDA5-protected over Total CytoRNA for each repeat in the pair of Alus (y-axis represent the fold change in the first repeat, x-axis represent the fold change in the second repeat in the pair) identified as an immunogenic RNA at the baseline condition (n=1,040 IR-Alu pairs) (F) and as Treatment-induced immunogenic RNA (n=3,687 IR-Alu pairs) (G).
H-I, Transcriptional orientation for each pair of IR-Alus identified as an immunogenic RNA at the baseline condition (n=1,040 IR-Alu pairs) (H) and as Treatment-induced immunogenic RNA (n=3,687 IR-Alu pairs) (I). Each bar shows the count of IR-pairs where both repeats are in the sense strand (+/+), both repeats are in the antisense strand (−/+), one repeat is in the sense strand and the other is in the antisense strand (Discordant (+/−) or (−/+)). The plot on the left shows the counts in the RNA-seq data from the mock-treated cells and the plot on the right shows the counts in the RNA-seq data from the 5-AZA-CdR-treated cells.
A, Venn-diagram showing the IR-Alu pairs at the Baseline (n=1,040), Immunogenic (5-AZA-CdR) (n=4,482) and Treatment-induced (n=3,687) conditions.
B, Number of Baseline-immunogenic RNAs (IR-Alu pairs=1,040, Non-IR Alu pairs=97).
C, Number of Treatment-induced immunogenic RNAs (IR-Alu pairs=3,687, Non-IR Alu pairs=547).
D-E, Average RPM profile of the baseline MDA5-protedcted RNA transcripts (n=1,176) that include Baseline IR-Alus (D) and the average RPM profile of the Treatment-induced MDA5-protected transcripts (n=3,895) that include treatment-induced IR-Alus (E). The boxplots include the RPM scores of these transcripts. Wilcoxon signed-rank test.
A, Genomic distribution of the baseline immunogenic IR-Alu pairs (n=1,040), treatment-induced immunogenic IR-Alu pairs (n=3,687), and of IR-Alu pairs present in the human genome (n=746,470). Odd-Ratio shows depletion or enrichment for each genomic category (Intron, Exon, 3′UTR, 5′UTR and Intergenic) at baseline immunogenic IR-Alu pairs or treatment-induced immunogenic IR-Alu pairs as compared to the background genomic distribution of all IR-Alu pairs present in the human genome. **** p<0.0001; Fisher Exact test.
B-C, Average CpG density (B) or bona-fide CpG Island (CGI) intersection density (C) flanking (−50 kb/+50 kb; Upstream/downstream) the Treatment-induced IR-Alu pairs (n=3,687, Black line or light gray line respectively) and all existing IR-Alu pairs in the human genome (n=746,470, Gray line or dash line respectively). For Treatment-induced IR-Alu pairs, the orientation was based on the transcriptional orientation in the MDA5-protected RNA-seq data and shows a higher CpG density and CpG Island frequency immediately upstream to the first repeat.
D, Heatmaps and average CUT & RUN profiles of H3K4me3 (left) and H3K27ac (right) signal at −50 kb/+50 kb (Upstream/downstream) of the Treatment-induced IR-Alu pairs regions. The orientation was based on the transcriptional orientation in the MDA5-protected RNA-seq data
E-G, Illustrative genome tracks of Treatment-induced IR-Alu pair located at an intronic (E) and intergenic region (F) and a baseline IR-Alu pair located at a 3′UTR region (G).
H, Schematic representation of Baseline and Treatment-induced immunogenic IR-Alu pairs.
A-B, Genomic distribution of the IR Alu pairs based on their genomic orientation for the Baseline immunogenic RNAs (A) and for the Treatment-induced immunogenic RNAs (B). Arrows represent the genomic orientation (5′ to 3′) and arrow color represent the transcriptional orientation (Black for sense and White for antisense).
C, Alu subfamily distribution of the baseline immunogenic IR-Alus (n=1,602), treatment-induced immunogenic IR-Alus (n=5,199), and of IR-Alus present in the human genome (n=641,262). Odd-Ratio shows depletion or enrichment for each Alu subfamily (AluS, AluJ and AluY) at baseline immunogenic IR-Alus or treatment-induced immunogenic IR-Alus as compared to Alu subfamily distribution of all IR-Alus present in the human genome **** p<0.0001; Fisher Exact test.
D, Average CpG density 50 kb upstream and 50 kb downstream of the Baseline IR-Alu pairs and all the existing IR-Alu pairs in the hg19 genome.
E, Average intersection of IR-Alu pairs region (Baseline and all IR-Alu pairs) with the CpG islands.
F, Genome track plot of a Treatment-induced intronic IR-Alu pair, Genome track plot of a Treatment-induced intergenic IR-Alu pair and Genome track plot of a Treatment-induced intergenic IR-Alu pairs.
A-B, Percentage of CGIs (n=1,338) directly upstream of 5-AZA-CdR induced immunogenic IR-Alus with at least one fully methylated (light gray), partially methylated (dark gray), or fully unmethylated (black) CpG site in (A) colorectal cancer cell lines (n=51) or (B) pan-cancer cell lines (n=988) from the GDSC project.
C-D, Regions that following epigenetic therapy become H3K4me3 marked directly upstream of induced immunogenic IR-Alus (n=991 peaks) with at least one fully methylated (light gray), partially methylated (dark gray), or fully unmethylated (black) CpG site in (C) colorectal cancer cell lines or (D) pan-cancer cell lines from the GDSC project.
E, Scatter plot showing inverse correlation (r=−0.23, p=7.58-13) between the DNA methylation score of regulatory regions that following epigenetic therapy become H3K4me3 marked directly upstream to the induced immunogenic IR-Alus (n=991 H3K4me3 peaks) and viral mimicry ISG signature score (n=22 ISGs). Each dot is one pan-cancer cell line (n=988) from the GDSC project.
A-B, Distribution of each known human polyA signal (PAS) motif with respect to the distance from the end of the downstream Alu of each IR-Alu pair in the set of (A) baseline immunogenic IR-Alus (n=1,040 pairs) and the set of (B) treatment-induced immunogenic IR-Alus (n=3,687 pairs). The y-axis is the counts of the IR-Alu pairs that include the motif in the MDA5-protected RNA-seq, and the x-axis is the distance in bp from the end of the downstream Alu in an IR-Alu pair.
C-D, Heatmap and average profile of the MDA5-protected RNA-seq signal centered at the PAS locations detected in (A-B). Each row represents the downstream Alu for each IR-Alu pair. The orientation and the strand are based on the MDA5-protected transcriptional orientation.
E, Percentage of non-repeats and several families of repetitive elements in total RNA-seq (nuclear and cytoplasmic RNA) and in RNA-seq from the cytoplasmic fraction (‘Total CytoRNA-seq’) after 5-AZA-CdR treatment at 300 nM for 5 days. The Total CytoRNA-Seq donut plot (on right) is the same as in
F, Confocal microscopy of Mock-treated and 5-AZA-CdR treated ADAR1WT patient-derived xenograft CRCs. DNA was stained by DAPI (left) and dsRNA was staining using the J2 antibody (middle).
A, Knock-down efficiency of ADAR1 using two distinct shRNAs (ADAR1KDA and ADAR1KDB) at mRNA (left) and protein (right) levels in patient-derived xenograft colorectal cancer cells (CRCs). The mRNA quantification was performed by qRT-PCR. Results were normalized to RPLP0. The protein quantification was performed by western-blot and the two ADAR1 isoforms (p110 and p150) are marked by an arrowhead. Alpha-Tubulin served as a loading control.
B, dsRNA quantification based on J2/DAPI staining measured by ImageJ. Data are mean±s.d. from n=20 randomly sampled regions of two independent experiments. ***P<0.001, ****P<0.0001, Dunnett-corrected ordinary one-way ANOVA.
C, qPCR analysis of total ADAR1 mRNA level after treatment with 5-AZA-CdR (300 nM, for 5 days) or transfection with 100 ng ml-1 poly(I:C) over a course of 24 days. Cells were washed out at day 5 and seeded in drug-free medium. Data are mean±s.e.m.****P<0.0001, Sidak-corrected two-way ANOVA.
D-F, Percentage of non-repeats and several families of Repetitive Elements in the total cytoplasmic fraction (‘Total CytoRNA’) and in the MDA5-protected fraction (‘MDA5-protected’) at the baseline condition (ADAR1WT) (D) and after ADAR1 knockdown (‘ADAR1KD-baseline immunogenic RNAs’), p<2.2e-16 E). ‘ADAR1KD-induced immunogenic RNAs’ refers to transcripts present in ADAR1KD condition but not present in the baseline ADAR1WT condition (i.e. induced by the knockdown), p<2.2e-16 F). Each plot is the average percentage of two replicates. P-values represent the Fisher Exact test for enrichment of SINEs and depletion of non-repeats in ADAR1KD-baseline immunogenic RNAs and ADAR1KD-induced immunogenic RNAs as compared to the MDA5-protected ADAR1WT baseline immunogenic RNAs.
G, Genome-wide A-to-I editing loci counts in Alus in the RNA-seq data performed in the total cytoplasmic fraction (‘Total CytoRNA’) and in the MDA5-protected fraction (‘MDA5-protected’) at the mock-treated (MT) ADAR1WT, 5-AZA-CdR-treated ADAR1WT, mock-treated ADAR1KD, and 5-AZA-CdR-treated ADAR1KD conditions.
H, Geneset Enrichment Analysis showing an enrichment of the geneset ‘Hallmark_Interferon_Alpha_Response’ in ADAR1KD 5-AZA-treated cells compared to ADAR1WT 5-AZA-CdR treated cells.
I, Interferon-responsive geneset enrichment score in mock-treated ADAR1WT, 5-AZA-CdR-treated ADAR1WT, mock-treated ADAR1KD, and 5-AZA-CdR-treated ADAR1KD conditions. Samples were collected 5, 14, and 24 days post-treatment.
J, MAVS aggregation analyzed by SDS-AGE and MAVS protein level analyzed by SDS-PAGE. VDAC served as a loading control in SDS-PAGE.
A, dsRNA quantification based on J2/DAPI staining measured by ImageJ. Data are mean±s.d. from n=20 randomly sampled regions of two independent experiments. ***P<0.001, ****P<0.0001, Dunnett-corrected ordinary one-way.
B, Venn-diagram showing the number of IR-Alu pairs at the baseline (n=1,040), ADAR1KD-baseline immunogenic (n=9,030) and ADAR1KD-induced immunogenic conditions.
C-D, Log10 Fold change of MDA5-protected to Total CytoRNA enriched inverted (IR)-Alus and Non-IR Alus for Baseline immunogenic RNAs (Top) and for the ADAR1KD-induced immunogenic RNAs (Bottom).
E-F, Correlation in the enrichment score of each pair of IR-Alus which make dsRNA in ADAR1WT (baseline) and ADAR1KD patient-derived colorectal cancer cells.
G-H, Strand specific transcription (+/+ or −/−) of repeat element pairs which form immunogenic dsRNA in ADAR1WT(LACZ as baseline) and ADAR1KD patient-derived xenograft colorectal cancer cells versus pairs appeared with discordant transcription strandness (+/− or −/+).
I-J, Average RPM profile of the baseline MDA5-protedcted RNA transcripts (n=1,176) that include Baseline IR-Alus (I) and the average RPM profile of the ADAR1KD-induced MDA5-protected transcripts that include Treatment-induced IR-Alus (J). The boxplots indicate the RPM scores of these transcripts. Wilcoxon signed-rank test.
A, ADAR1 isoforms (p110 and p150) relative expression analysed by qPCR. LACZ denotes ADAR1WT; PIC denotes 100 ng/ml poly (I:C) for 5 days; AZA denotes 300 nM 5-AZA-CdR for 5 days). Data are mean±s.e.m. (n=3 from three independent experiments). ***P<0.001, ****P<0.0001, Tukey-corrected two-way ANOVA.
B, Kinetics of interferon stimulated genes (ISG15, DDX58 and IRF7) relative expression in ADAR1WT (Black) and ADAR1KD (Gray) patient-derived colorectal cancer cells (CRCs) treated with 5-AZA-CdR analyzed by qRT-PCR. Data are mean±s.e.m. (n=3 from three independent experiments). *P<0.05, **P<0.01, ****P<0.0001, Sidak-corrected two-way ANOVA.
C, Interferon-responsive gene expression in ADAR1WT mock-treated (MT), 5-AZA-CdR treated, ADAR1KD NT and ADAR1KD p(I:C) transfected samples collected 5 days, 14 days and 24 days post-treatment, measured by RNA-seq.
D, Kinetics of interferon stimulated genes (ISG15, DDX58 and IRF7) relative expression in ADAR1WT (Black) and ADAR1KD (Gray) patient-derived CRCs transfected with p(I:C), analyzed by qRT-PCR. Data are mean±s.e.m. **P<0.01, ****P<0.0001, Sidak-corrected two-way ANOVA.
E, Overexpression of ADAR1 p150 isoform at mRNA (analyzed by qRT-PCR) and protein levels in patient-derived CRCs (analyzed by western-blot). Data are mean±s.e.m. (n=3 from three independent experiments). P=0.0144, Wilcoxon test.
F, Interferon stimulated genes (ISG15, DDX58 and IRF7) relative expression in CTRL (LUC) (Black) and ADAR1p150CE (Gray) patient-derived CRCs treated with 5-AZA-CdR, analyzed by qRT-PCR. Data are mean±s.e.m. **P<0.01, ***P<0.001, Sidak-corrected two-way ANOVA.
G, IFN enrichment score in CTRL(LUC) Mock-treated, 5-AZA-CdR treated, ADAR1-p150 overexpressing NT and ADAR1-p150 overexpressing 5-AZA-CdR treated samples collected 5 days post-treatment and measured by RNA-seq.
H, dsRNA quantification by ImageJ in CTRL (LUC) and ADAR1p150OE cells Mock-treated (MT) or treated with 5-AZA-CdR. Data are mean±s.d. from n=20 randomly sampled regions of two independent experiments. ****P<0.0001, Tukey-corrected ordinary one-way ANOVA. ns, non significant.
A) ADAR1 p150 and p110 isoforms relative expression in LIM1215 CRC cells.
B) Interferon-stimulated genes (MDA5, IRF7 and DDX58) relative expression in ADAR1WT and ADAR1KD LIM1215 colorectal cancer cell lines treated with 300 nM 5-AZA-CdR for 5 days.
C) ADAR1 p150 and p110 isoforms relative expression in HT29 CRC cells.
D) Interferon-stimulated genes (MDA5, ISG15 and DDX58) relative expression in ADAR1WT and ADAR1KD HT29 colorectal cancer cell lines treated with 300 nM 5-AZA-CdR for 5 days. Data are mean±s.e.m. (n=3 from three independent experiments). *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001, Tukey-corrected two-way ANOVA.
A, Survival of wild-type ADAR1 (black) and ADAR1-knockdown (gray) patient-derived CRC cells after treatment with 5-AZA-CdR. Luminescence signal was normalized, and dose-response curves and EC50 values were calculated using a nonlinear regression curve fit.
B, Schematic representation of in vivo experimental scheme of non-infected, ADAR1WT, ADAR1KDA and ADAR1KDB patient-derived xenograft CRC cells injected subcutaneously into flanks of NSG mice (n=10 mice per group; 2 tumours per mouse). Arrows represent days of treatment (0.5 mg kg-1 5-AZA-CdR by intraperitoneal injection or vehicle).
C, Tumour volume measured at the indicated time points. Arrows represent days of treatment. ADAR1KDA 5-AZA-CdR (n=20 tumours) and ADAR1KDB 5-AZA-CdR (n=18 tumours at day 12 and n=14 tumours at day15) compared with non-infected (NI) 5-AZA-CdR (n=19 tumours) cohort. Data are mean±s.e.m. ****P<0.0001, Tukey-corrected two-way ANOVA
D, Colorectal cancer tumours dissected from Vehicle and 5-AZA-CdR cohorts at day 14 post-treatment are depicted for each condition (n=5) (NI=non-infected)
E, Tumour weight for each group at the time of dissection (day 14 after treatment). Data are mean±s.d. ****P<0.0001, Tukey-corrected ordinary-one-way ANOVA.
F Schematic representation of in vivo LDA measuring tumour-initiation potential. NSG mice were injected with 10,000 cells (n=8 tumours), 1,000 cells (n=8 tumours), 100 cells (n=8 tumours) and 10 cells (n=16 tumours).
G, Tumour-initiating cell frequency from the in vivo LDA of patient-derived CRC cells for each condition. ADAR1WT 5-AZA-CdR vs ADAR1KDA 5-AZA-CdR P=0.00862; ADAR1WT 5-AZA-CdR vs ADAR1KDB 5-AZA-CdR P=0.009; chi-square test.
A, Change in mean body weight of NSG mice transplanted with non-infected, ADAR1WT and ADAR1KDA/B patient-derived CRCs treated with vehicle or 5-AZA-CdR (0.5 mg kg-1 through intraperitoneal injection, for two cycles of 4 days with a 3 day break).
B, The normalized percentage of reduction in sphere forming ability was calculated in each condition compared to Mock-treated.
C, Representative confocal microscopy images from two independent experiments of ADAR1WT and ADAR1KDA cells treated with CDK4/6 inhibitor (250 nM palbociclib) for 7 days. DNA was stained with DAPI, and dsRNA was stained using the J2 antibody.
D, dsRNA quantification using ImageJ. Data are mean±s.d. from n=15 randomly sampled regions of two independent experiments. **P<0.01, ***P<0.001, Tukey-corrected ordinary one-way ANOVA
E, qPCR analysis of relative expression of ISGs (ISG15, DDX58 and IRF7) in cells treated with 250 nM palbociclib. Data are mean±s.e.m. ***P<0.001, ****P<0.0001, Tukey-corrected twoway ANOVA.
A, A-to-I editing loci counts in IR-Alus in the total RNA-seq data of three CRC cell lines. NCIH716 and COLO678 cell linsea are classified as resistant to ADAR1 inhibitors, while NCIH747 is classified as sensitive to ADAR1 inhibitors.
B, Scatter plot of the ADAR1 decency of the CRC cell lines. ADAR1 dependent cell lines have more negative CERES scores.
A suitable configured computer device, and associated communications networks, devices, software and firmware to provide a platform for enabling one or more embodiments as described herein.
In the following description, numerous specific details are set forth to provide a thorough understanding of the invention. However, it is understood that the invention may be practiced without these specific details.
Cancer therapies targeting epigenetic repressors can mediate their effects by activating retroelements within the human genome. Retroelement transcripts can form double-stranded RNAs (dsRNAs) that activate the pattern recognition receptor MDA51-10. This viral mimicry state leads to loss of cancer cell fitness and stimulates innate and adaptive immune responses11,12. Nevertheless, the clinical efficacy of epigenetic therapies has been limited. To uncover targets that would synergize with the viral mimicry response, we set out to identify the immunogenic retroelements that are activated by epigenetic therapies. We discovered that intronic and intergenic SINE elements, specifically inverted repeats (IR) Alus, are the major source of drug-induced immunogenic dsRNAs. These IR-Alus are frequently located downstream of ‘orphan’ CpG Islands (CGIs)13. We show that orphan CGIs acquire active histone modifications after drug treatment, suggesting that they are evolutionarily conserved cryptic promoters. In mammals, the enzyme Adenosine Deaminases Acting on RNA (ADAR1) targets and destabilizes IR-Alu dsRNAs14, which prevents activation of MDA515. We found that ADAR1 establishes a negative feedback loop, restricting the viral mimicry response to epigenetic therapy. Depletion of ADAR1 in patient-derived cancer cells potentiates the efficacy of epigenetic therapy, restraining tumor growth and reducing cancer initiation. Thus, epigenetic therapies trigger viral mimicry by inducing a subset of IR-Alus, which is enhanced by depleting ADAR1. Our findings suggest that combining epigenetic therapies with ADAR1 inhibitors represents a promising new strategy for cancer treatment.
In an aspect, there is provided a method of assessing a subject's responsiveness to cancer therapy, comprising: providing a sample from the subject comprising cancers cells or suspected cancer cells; measuring or estimating the expression levels of inverted repeats (IR) Alus in the cells; and determining that the subject would be responsive to cancer therapy if the subject cells exhibit expression levels of inverted repeats (IR) Alus with reference to expression levels in control samples.
The term “level of expression” or “expression level” as used herein refers to a measurable level of expression of the products of biomarkers, such as, without limitation, the level of messenger RNA transcript expressed or of a specific exon or other portion of a transcript, or in the context estimating expression, whether promoters of that biomarker are activated, such as through the methylation of CpG islands in the case of the IR Alus.
As used herein, the term “control” refers to a specific value or dataset that can be used to prognose or classify the value e.g. expression level or reference expression profile obtained from the test sample associated with an outcome class. A person skilled in the art will appreciate that the comparison between the expression of the biomarkers in the test sample and the expression of the biomarkers in the control will depend on the control used.
The term “differentially expressed” or “differential expression” as used herein refers to a difference in the level of expression of the biomarkers that can be assayed by measuring the level of expression of the products of the biomarkers, such as the difference in level of messenger RNA transcript or a portion thereof expressed or of proteins expressed of the biomarkers. In a preferred embodiment, the difference is statistically significant. The term “difference in the level of expression” refers to an increase or decrease in the measurable expression level of a given biomarker, for example as measured by the amount of messenger RNA transcript in a sample as compared with the measurable expression level of a given biomarker in a control.
The term “responsiveness” as used herein in respect of cancer therapy refers to a subject's relative response to a therapy compared to a general or control population.
The term “sample” as used herein refers to any fluid, cell or tissue sample from a subject that can be assayed for biomarker expression products and/or a reference expression profile, e.g. genes differentially expressed in subjects.
In some embodiments, the method comprises measuring the expression levels of IR Alus by measuring the transcription product of IR Alus.
In some embodiments, the IR Alus to be measured are a majority of the IR Alus shown in Table B. In a preferred embodiment, the IR Alus to be measured are at least 60%, 70%, 80%, 90%, or 95% of the IR Alus shown in Table B. In a further preferred embodiment, the IR Alus to be measured are all or substantially all of the IR Alus shown in Table B.
In some embodiments, the measuring comprises use of RNA sequencing or PCR/digital PCR.
In some embodiments, the method comprises estimating expression levels of IR Alus by detecting the presence of methylated CpG islands upstream of IR Alus.
In some embodiments, the methylated CpG islands upstream to be detected are a majority of the CpG islands shown in Table A. In a preferred embodiment, the methylated CpG islands upstream to be detected are at least 60%, 70%, 80%, 90%, or 95% of the CpG islands shown in Table A. In a further preferred embodiment, the methylated CpG islands upstream to be detected are all or substantially all of the CpG islands shown in Table A.
In some embodiments, the method further comprises the treating the patient with the cancer therapy. The cancer therapy is preferably epigenetic therapy.
In some embodiments, the epigenetic therapy targets at least one of DNMTs, HDACs, LSD1, EZH2, G9a and SETDB1.
In some embodiments, the epigenetic therapy is a DNA methyltransferase inhibitor (DNMTi).
In some embodiments, the treatment further comprises decreasing, knocking or inhibiting Adenosine Deaminases Acting on RNA, preferably ADAR1.
In some embodiments, the measured level of A to I editing in treatment-induced IR Alus in the subject are high when compared to a reference level of A to I editing in treatment-induced IR Alus.
In an aspect, there is provided a method of cancer therapy in a subject in need thereof, the method comprising inhibiting ADAR1 in a patient.
In some embodiments, the inhibiting comprises administration of an ADAR1 inhibitor.
In some embodiments, the subject had been determined to be responsive to cancer therapy according to a method described herein.
In some embodiments, said cancer therapy comprises combination therapy with at least one other cancer therapy. Preferably, the at least one other cancer therapy is any of epigenetic therapy, surgery, endocrine therapy, chemotherapy, radiotherapy, hormone therapy, gene therapy, thermal therapy, or ultrasound therapy.
In an aspect, there is provided an ADAR1 inhibitor for use in the treatment of cancer in a subject.
In an aspect, there is provided a use of an ADAR1 inhibitor in the preparation of a medicament for the treatment of cancer in a subject.
As used herein, “pharmaceutically acceptable carrier” means any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like that are physiologically compatible. Examples of pharmaceutically acceptable carriers include one or more of water, saline, phosphate buffered saline, dextrose, glycerol, ethanol and the like, as well as combinations thereof. In many cases, it will be preferable to include isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, or sodium chloride in the composition. Pharmaceutically acceptable carriers may further comprise minor amounts of auxiliary substances such as wetting or emulsifying agents, preservatives or buffers, which enhance the shelf life or effectiveness of the pharmacological agent.
As used herein, “therapeutically effective amount” refers to an amount effective, at dosages and for a particular period of time necessary, to achieve the desired therapeutic result. A therapeutically effective amount of the pharmacological agent may vary according to factors such as the disease state, age, sex, and weight of the individual, and the ability of the pharmacological agent to elicit a desired response in the individual. A therapeutically effective amount is also one in which any toxic or detrimental effects of the pharmacological agent are outweighed by the therapeutically beneficial effects.
The present system and method may be practiced in various embodiments. A suitably configured computer device, and associated communications networks, devices, software and firmware may provide a platform for enabling one or more embodiments as described above. By way of example,
The present system and method may be practiced on virtually any manner of computer device including a desktop computer, laptop computer, tablet computer or wireless handheld. The present system and method may also be implemented as a computer-readable/useable medium that includes computer program code to enable one or more computer devices to implement each of the various process steps in a method in accordance with the present invention. In case of more than computer devices performing the entire operation, the computer devices are networked to distribute the various steps of the operation. It is understood that the terms computer-readable medium or computer useable medium comprises one or more of any type of physical embodiment of the program code. In particular, the computer-readable/useable medium can comprise program code embodied on one or more portable storage articles of manufacture (e.g. an optical disc, a magnetic disk, a tape, etc.), on one or more data storage portioned of a computing device, such as memory associated with a computer and/or a storage system.
The advantages of the present invention are further illustrated by the following examples. The examples and their particular details set forth herein are presented for illustration only and should not be construed as a limitation on the claims of the present invention.
DNMTi Induces Immunogenic Inverted Alus
To identify drug-induced immunogenic retroelements on a global scale, we examined the endogenous dsRNA formation in patient-derived colorectal cancer cells (CRCs) that were treated with low doses of 5-AZA-CdR, an FDA-approved DNMTi, or mock-treated. To this end, we performed total stranded RNA-seq and assessed repetitive elements transcribed in both strands, therefore likely to produce dsRNA species. We observed increased sense and/or antisense transcription of long interspersed nuclear elements (LINEs) and ERVs, but not short interspersed nuclear elements (SINEs), after DNMTi treatment (
Viral mimicry response to epigenetic therapy is dependent on activation of the MDA5 pattern recognition receptor by endogenous dsRNAs1,6,8. Therefore, to directly identify drug-induced immunogenic retroelements, we performed an MDA5-protection assay19, followed by RNA sequencing. Surprisingly, SINEs, specifically Alu retroelements, were remarkably enriched in the MDA5-protected RNA compared to total cytosolic RNA (
Then, to identify the immunogenic retroelements induced only by DNMTi treatment (“Treatment-Induced immunogenic RNAs”), we excluded those that were expressed in both the mock- and DNMTi-treated samples (
To uncover the molecular mechanisms of how repetitive elements generate immunogenic dsRNA, we examined all repeats in MDA5-protected transcripts and searched for repeat pairs within a 3 Kb window. Interestingly, we found that the majority of aligned pairs in both baseline and treatment-induced RNA were inverted repeat (IR) Alus (
DNMTi Induces Cryptic Transcription of Intronic and Intergenic Inverted Alus
Next, we investigated how DNMTi treatment induces expression of IR-Alu elements. We detected 746,470 IR-Alu pairs in the human genome, with about 55% originating from introns and 43% from intergenic regions (
In addition, we mapped the average CpG density flanking IR-Alu pairs. Notably, CpG density was high directly upstream of IR-Alu pairs induced by DNMTi treatment (
Active promoters and enhancers are often marked by H3K4me3 and H3K27ac, respectively. We performed genome-wide CUT&RUN (cleavage under targets and release using nuclease)28 analysis to assess these modifications at treatment-induced IR-Alus, and found that DNMTi treatment led to elevated levels of H3K4me3, but not H3K27ac, directly upstream of treatment-induced immunogenic IR-Alus repeats (
Overall, our data suggest that orphan CGIs located upstream of treatment-induced IR-Alu regions in intronic and intergenic regions are frequently methylated and act as sites of cryptic transcription initiation upon treatment with DNA demethylating drugs, mediating the formation of immunogenic dsRNA (
To further characterize how these immunogenic IR-Alus can reach the cytoplasm, where they can be recognized by MDA5, we assessed the presence of poly(A) signals (PAS) within immunogenic IR-Alu pairs. It is well established that PAS play an important role in pre-mRNA cleavage and polyadenylation, leading to RNA stabilization and nuclear export29. We searched for known poly(A) signal motifs29 in the second half (3′ end) of the downstream Alu for each pair using the MDA5-protected RNA-seq data and observed that most baseline and treatment-induced IR-Alu pairs have at least one PAS, with motifs AAUACA and ACUAAA being the most frequently represented (
ADAR1 Restricts Viral Mimicry Response to DNMTi
After identifying the precise nature of immunogenic IR-Alus induced by epigenetic therapy, we sought to investigate whether they create vulnerabilities that could act in synergy with epigenetic therapies. Interestingly, the RNA stem-loops formed by IR-Alu elements are a main substrate for the enzyme ADAR1 (Adenosine Deaminases Acting on RNA)14. ADAR1 regulates dsRNA through adenosine-to-inosine (A-to-I) editing and subsequent destabilization of RNA duplexes15,30. This mechanism normally prevents baseline Alu sequences from activating MDA515,22 and the downstream interferon response in mammals. Moreover, ADAR1 is itself an interferon-stimulated gene22,31,32. Therefore, we hypothesized that ADAR1 acts as a negative feedback loop after epigenetic therapies, where the interferon response would increase ADAR1 expression and then limit the formation of immunogenic dsRNA and, in turn, the downstream anti-tumour response.
To test this hypothesis, we first determined if ADAR1 depletion affects dsRNA formation by IR-Alus in untreated patient-derived CRCs. We generated ADAR1 knock-down CRCs using two different hairpin RNAs (ADAR1KDA/B) and confirmed depletion of both the constitutively expressed (p110) and interferon-inducible (p150) ADAR1 isoforms (
Next, we tested whether epigenetic therapy can induce and activate ADAR1 editing activity. As a positive control, we verified that CRC cells transiently transfected with synthetic dsRNA (Poly(I:C)) displayed acute up-regulation of the interferon-inducible isoform of ADAR1 (p150) (
Based on these findings, we hypothesized that ADAR1 depletion would synergize with the viral mimicry response induced by DNMTi treatment. To assess how ADAR1 depletion affects 5-AZA-CdR treated samples, we performed RNA-seq analyses in patient-derived CRC cells. Gene Set Enrichment Analysis (GSEA) revealed that the type I interferon response was the most enriched pathway between wild-type and ADAR1-depleted samples treated with 5-AZA-CdR (
ADAR1 Depletion Synergizes with DNMTi Treatment
Finally, we sought to investigate whether ADAR1 inhibition would synergize with epigenetic therapy to increase the anti-tumour response. Indeed, ADAR1 knock-down causes four to six-fold reduction on 5-AZA-CdR EC50 in vitro (
One of the major challenges facing conventional chemotherapy is the persistence of cancer initiating cells (CICs), often resulting in relapse38-40. We and others have previously shown that 5-AZA-CdR treatment can target colorectal cancer CICs by inducing the viral mimicry response1,41. We therefore tested whether ADAR1KD by itself or in combination with 5-AZA-CdR can decrease tumour initiation. To this end, we first employed an in vitro sphere initiation assay followed by limiting dilution analysis (LDA) using patient-derived CRC cells. Depletion of ADAR1 alone had minimal effects, but reduced the spheroid forming potential of CRC cells treated with 5-AZA-CdR (
Finally, to investigate the potential of ADAR1 inhibition to synergize with other anti-cancer therapies by enhancing viral mimicry response, we combined ADAR1 depletion to CDK4/6 inhibition. CDK4/6 inhibitors were recently shown to up-regulate dsRNA expression, by reducing DNMT1 levels, leading to an interferon response3. Indeed, we observed a small accumulation of dsRNA in wild-type CRC cells treated with the CDK4/6 inhibitor plabociclib (
Further, we analyzed the A to I editing in the RNAseq of the cell lines that are different in the response to ADAR1 inhibition. We quantified the A to I editing in the treatment-induced IR Aus in the RNA-seq of three cell lines which are NCIH716, COLO678 and NCIH747. Cell lines that have lower level of A to I editing in treatment-induced IR-Alus are resistant to ADAR1 inhibition, while cell lines that have higher level of A to I editing are sensitive to the ADAR1 inhibition (
In summary, our study identifies IR-Alus, rather than bi-directional ERVs transcripts, as the main immunogenic dsRNA induced by DNA hypomethylating agents. Molecularly, our data suggest that these treatment-induced IR pairs form stem-loops to act as endogenous, immunogenic dsRNA. We provide evidence that most of the immunogenic dsRNA are induced from cryptic promoters within intronic or intergenic regions, which are enriched for orphan CpG islands and acquire active epigenetic marks upon DNMTi treatment. The presence of CpG Islands suggest that treatment-induced immunogenic repeats have physiological roles, in line with our previous hypothesis that viral mimicry represents an ‘epigenetic checkpoint’ to reduce cell fitness in response to a genome-wide loss of heterochromatin, for instance due to environmental or genetic factors42,43. Moreover, our study reveals that ADAR1 induction establishes a negative feedback loop that limits the ability of epigenetic therapy to induce a robust and sustained immunogenic dsRNA response. Indeed, inhibition of ADAR1 in combination with DNMTi treatment profoundly restrained colorectal cancer tumour growth in vitro and in vivo and reduced the frequency of cancer initiating cells that have self-renewal potential. Thus, our findings point to the potential use of immunogenic IR-Alus as biomarkers for epigenetic therapy response and suggest that combination of ADAR1 inhibition with epigenetic therapy represents a novel and potent therapeutic strategy. Finally, our findings show that measuring A to I editing in IR-Alus is predictive of ADAR1 dependency and, therefore, provide prognostic information regarding sensitivity to ADAR1 inhibitors.
Although preferred embodiments of the invention have been described herein, it will be understood by those skilled in the art that variations may be made thereto without departing from the spirit of the invention or the scope of the appended claims. All documents disclosed herein, including those in the following reference list, are incorporated by reference.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/CA2021/050765 | 6/3/2021 | WO |
| Number | Date | Country | |
|---|---|---|---|
| 63035081 | Jun 2020 | US |
