Compositions and methods for the treatment of cancers by triggering Anti-viral response

Abstract

Compositions and methods for the treatment of cancers through activation of an anti-viral are provided herein.

Description

[0001] COMPOSITIONS AND METHODS FOR THE TREATMENT OF CANCERS BY TRIGGERING ANTI-VIRAL RESPONSE

[0002] By

[0003] Israel Canadas

[0004] Takahiko Murayama

[0005] Cross-reference to Related Application

[0006] This patent application claims the benefit of U.S. Provisional Patent Application No. 63 / 510,001, filed June 23, 2023. The entire contents of the foregoing application are incorporated herein by reference, including all text, tables, drawings, and sequences.

[0007] Incorporation-by-Reference of Material Submitted in Electronic Form

[0008] The Contents of the electronic sequence listing (FCCC-109-PCT.xml; Size: 77,619 bytes; and Date of Creation: June 21, 2024) is herein incorporated by reference in its entirety.

[0009] Field of the Invention

[0010] The present invention relates to the fields of oncology and the treatment of various cancers. More specifically, the invention provides anti-cancer compositions and methods for administration of at least one viral-mimicry inducing agent or factor which triggers an antiviral response, thereby converting immunologically “cold” tumors into “hot” tumors, rendering them significantly more susceptible to chemotherapeutic agents.

[0011] Background of the Invention

[0012] Several publications and patent documents are cited throughout the specification in order to describe the state of the art to which this invention pertains. Each of these citations is incorporated by reference herein as though set forth in full.

[0013] Activating innate immunity in cancer cells is a potent means by which cold tumors can be turned hot, enhancing responsiveness to immune checkpoint blockade (ICB) therapies. One promising strategy to trigger an innate immune response within tumors and boost cancer immunotherapy is by inducing the intracellular accumulation of endogenous “virus-mimetic” nucleic acids, including double-stranded RNAs (dsRNA) and double- stranded DNAs (dsDNA). These nucleic acids are sensed by the host innate immune system as evidence of viral replication and provoke an immediate and robust interferon (IFN)-drivcn antiviral response. Such an antiviral response is potently immunogenic, and viral mimicry inducing therapies have shown very promising results in pre-clinical models and in clinical trials.

[0014] Small cell lung cancer (SCLC), the most lethal type of lung cancer, is a classic example of a cold tumor. While having one of the highest mutational burdens because of its strong association with tobacco smoking, SCLC is characterized by a reduced antigen presentation and an immunologically desert tumor microenvironment (TME). Despite the addition of ICB therapy to standard platinum-based chemotherapy in first-line treatment, SCLC remains a devastating disease and only a minority of SCLC patients derive lasting benefit from these treatments. Recent studies analyzing murine and human SCLC tumors suggest the existence of an “inflamed” SCLC subtype characterized by high expression of immune-related genes and human leukocyte antigens (HLAs) and exhibits the greatest benefit from ICB therapy among all the SCLC subtypes.

[0015] Although SCLCs have been uniformly treated with DNA damaging platinum-based chemotherapy, recent studies have demonstrated the potential of replication stress inducers as a therapeutic strategy for SCLC tumors, including inhibitors of poly (ADP-ribose) polymerase (PARP) and checkpoint kinase 1 (CHK1). These inhibitors block the DNA damage response (DDR) pathway to induce replication stress and cell death in cancer cells whose genome is unstable due to mutations in DDR pathway genes and / or dysregulated cell cycle progression. Recently, PARP, CHK1 and WEE1 inhibitors have been reported to work in synergy with PD- 1 / PD-L1 blockade in SCLC (24, 25), as well as triple negative breast cancer and ovarian cancer (27). PARP or CHK1 inhibition induces cyclic GMP-AMP synthase (cGAS)-stimulator of interferon genes (STING) pathway activation, which senses cytoplasmic DNA generated by DNA damage, activating tumor-intrinsic innate immunity. However, other critical regulators of dsRNA in cold cancers have not been identified.

[0016] Clearly there is an urgent need to identify effective therapeutic targets able to enhance antitumor immunity and ultimately sensitize these cold tumors to effective immunotherapies, thereby providing effective therapeutic strategies to eliminate resistance in certain deadly cancers. Summary of the Invention

[0017] In accordance with the present invention, methods for treating a subject having tumors comprising administering to said subject at least one viral-mimicry-inducing agent (VMIA) which increases cellular dsRNA levels, thereby provoking an antiviral response and rendering said tumor more sensitive to anti-cancer agents, which exceeds that observed in tumor cells not treated with the VMIA, are provided. Also provided herein are methods of inducing an antiviral response in a tumor cell, comprising contacting the tumor cell with at least one viral-mimicry- inducing agent (VMIA) which increases cellular levels of dsRNA, R-loops, and / or dsDNA, thereby increasing the sensitivity of said tumor cells to cancer cell killing agents which exceeds that observed in tumor cells not treated with the VMIA. Methods of enhancing an immune checkpoint blockade (ICB) therapy in a subject having tumors comprising administering to said subject at least one viral-mimicry-inducing agent (VMIA) which provokes an antiviral response and induces tumor cell killing exceeding that observed in tumor cells not treated with the VMIA are also provided herein.

[0018] In certain embodiments, the VMIA is a DHX9 modulator, In certain embodiments, the DHX9 modulator is an inhibitor of EWS-FLI1 binding to DHX9. In certain embodiments, the DHX9 modulator is TK216 or YK-4-279.

[0019] In another embodiment, said VMIA is an inhibitory nucleic acid which represses DHX9 expression and function in a cancer cell, selected from an siRNA, an antisense oligonucleotide, an shRNA, and a ribozyme having sufficient sequence homology to a DHX9 encoding nucleic acid to reduce expression of thereof in a target cell. In certain embodiments, said inhibitory nucleic acid comprises one or more modified nucleotides or nucleosides. In certain embodiments, said VMIA is administered in a pharmaceutically acceptable carrier via route selected from systemic, oral, intraperitoneal, intravenous, intracerebral, intratumoral and topical administration.

[0020] In certain aspects of the invention, the methods above further comprise administering at least one additional anti-cancer therapy. In certain embodiments, the anti-cancer therapy is selected from Cisplatin, Etoposide, PARP inhibitors, CHK1 inhibitors, WEE1 inhibitors, docetaxol, carboplatin, vincristine, vinblastine, methotrexate, cyclophosphamide, CPT-11, 5- fhiorouracil (5-FU), gemcitabine, estramustine, carmustine, adriamycin (doxorubicin), rsenic trioxide, irinotecan, and epothilone derivatives. In another embodiment, the at least one additional anti-cancer therapy is an immune checkpoint blockade (TCB) therapy. In certain embodiments, the ICB therapy is selected from at least one PD-1 inhibitor, PD-L1 inhibitor, CTLA-4 inhibitor, and / or LAG-3 inhibitor. In certain embodiments, the at least one PD-1 inhibitor, PD-L1 inhibitor, CTLA-4 inhibitor, and / or LAG-3 inhibitor is selected from Pembrolizumab (Keytruda), Nivolumab (Opdivo), Cemiplimab (Libtayo), Atezolizumab (Tecentriq), Avelumab (Bavencio), Durvalumab (Imfinzi), Ipilimumab (Yervoy) tremelimumab (Imjuno), and Relatlimab.

[0021] In certain embodiments of the above methods, the tumor is a recalcitrant tumor or a COLD tumor. In certain embodiments, the tumor is selected from a lung cancer, a brain cancer, an ovarian cancer, a kidney cancer, an esophageal cancer, a sarcoma, a liver cancer, a bone cancer, a stomach cancer, a bladder cancer, head and neck cancer and thyroid cancer. In certain embodiments, the cancer is Small Cell Lung Cancer (SCLC).

[0022] In another aspect of the invention, a pharmaceutical composition comprising at least one viral-mimicry-inducing agent (VMIA) and a pharmaceutically acceptable carrier is provided. In certain embodiments, the VMIA is a DHX9 modulator. In certain embodiments, the DHX9 modulator is an inhibitor of EWS-FLI1 binding to DHX9. In certain embodiments, the DHX9 modulator is TK216 or YK-4-279.

[0023] In another embodiment, said VMIA is an inhibitory nucleic acid which represses DHX9 expression and function in a cancer cell, selected from an siRNA, an antisense oligonucleotide, an shRNA, and a ribozyme having sufficient sequence homology to a DHX9 encoding nucleic acid to reduce expression of thereof in a target cell. In certain embodiments, said inhibitory nucleic acid comprises one or more modified nucleotides or nucleosides. In certain embodiments, said composition is formulated for administration via a route selected from systemic, oral, intraperitoneal, intravenous, intracerebral, intratumoral and topical administration.

[0024] In certain aspects of the invention, the compositions above further comprise at least one additional anti-cancer therapy. In certain embodiments, the anti-cancer therapy is selected from Cisplatin, Etoposide, PARP inhibitors, CHK1 inhibitors, WEE1 inhibitors, docetaxol, carboplatin, vincristine, vinblastine, methotrexate, cyclophosphamide, CPT-11, 5 -fluorouracil (5- FU), gemcitabine, estramustine, carmustine, adriamycin (doxorubicin), rsenic trioxide, irinotecan, and epothilone derivatives. In another embodiment, the at least one additional anticancer therapy is an immune checkpoint blockade (ICB) therapy. In certain embodiments, the ICB therapy is selected from at least one PD-1 inhibitor, PD-L1 inhibitor, CTLA-4 inhibitor, and / or LAG-3 inhibitor. In certain embodiments, the at least one PD-1 inhibitor, PD-L1 inhibitor, CTLA-4 inhibitor, and / or LAG-3 inhibitor is selected from Pembrolizumab (Keytruda), Nivolumab (Opdivo), Cemiplimab (Libtayo), Atezolizumab (Tecentriq), Avelumab (Bavencio), Durvalumab (Imfinzi), Ipilimumab (Yervoy) tremelimumab (Imjuno), and Relatlimab.

[0025] Still other aspects and advantages of these compositions and methods are readily apparent and described further in the following detailed description of the invention.

[0026] Brief Description of the Drawings

[0027] FIG. 1A-1I: DHX9 suppresses double-stranded RNA (dsRNA) accumulation in SCLCs FIG. 1A. Schematic of the screen to identify critical regulators of dsRNA. Created with BioRender.com. FIG. IB. Result of the dsRNA regulator screen. Relative mean fluorescence intensity (MFI) of dsRNA level in H446 cells depleted of RNA helicase genes was compared. FIG. 1C. DHX9 mRNA expression was profiled in 28 cancer types. Cancer Cell Line Encyclopedia (CCLE) data were downloaded from cBioPortal. FIG. ID. Analysis of DHX9 expression in indicated lung cancer subtypes and normal lung. Data were downloaded from GEO database (GSE30219). Normal lung tissue (N = 14), LU AD (N = 85), LUSC (N = 61), LCNE (N = 56), SCLC (N = 20). Bars indicate the min and max values. FIG. IE. Survival curve analysis of lung tumor patients. Data were downloaded from GEO database (GSE30219). FIG. IF. Immunoblot (IB) of DHX9 protein in Scramble, sgDHX9 and sgDHX9 #2 H446 cells. FIG. 1G. Immunofluorescence images of dsRNA (red) staining of Scramble or sgDHX9 cells (treated w / wo RNase III). Nuclei were counterstained with DAPI. Scale bar = 10 pm. FIG. 1H. Schematic (left) and result (right) of RIP- I2-RIP-seq analysis. Expression levels of specific retrotransposon classes (SINE, LINE, LTR) in Scramble or sgDHX9 cells are summarized (n = 3). CPM: Counts Per Million. FIG. II, Result of RIP-qRT-PCR analysis of the indicated retrotransposon elements (n = 3). 36B4 gene was used as a reference. Data represent mean ± SEM. ns, not significant; *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 by unpaired Student’s t test (FIG. ID and 1H), log-rank test (FIG. IE), two-way ANOVA followed by Tukey’s multiple comparisons test (FIG. II). FIG. 2A-2F: FIG. 2A. Flow cytometry analysis of intracellular dsRNA levels in H446 cells, treated with DMSO or 100 nM Dccitabinc. Data arc representative of three independent experiments. FIG. 2B. Immunoblot (IB) of DHX9 protein in the indicated SCLC cell lines. FIG. 2C. Survival curve analysis of lung, ovarian and breast tumor patients. Data were downloaded from GEO database. FIG. 2D. Immunoblot (IB) of DHX9 protein in H196, H82, and DMS 114 cells (Scramble and sgDHX9). FIG. 2E. Flow cytometry analysis of intracellular dsRNA levels in H446, H196, H82, and DMS 114 cells (Scramble and sgDHX9). Data are representative of three independent experiments (top). Mean fluorescence intensity (MFI) was quantified by FlowJo (bottom) (n = 3). FIG. 2F. Heatmap of J2-RIP-seq results comparing Scramble and sgDHX9 (n = 3). Data represent mean ± SEM. ns, not significant; *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 by log-rank test (FIG. 2C) and unpaired Student’s t test (FIG. 2E).

[0028] FIG. 3A-3H: DHX9 depletion induces IFN response. FIG. 3A, Gene sets significantly upregulated and downregulated are shown, based on Gene Set Enrichment Analysis (GSEA) result. Immune response related gene sets are in red, DNA damage related gene sets in pink and DNA replication / cell cycle related gene sets in blue. FIG. 3B. GSEA analysis with C5 (ontology) gene sets, based on RNA-seq results of sgDHX9 versus Scramble cells. FIG. 3C. qRT-PCR analysis of the immune-related genes comparing Scramble and sgDHX9 Hl 96 cells (n = 3). 36B4 gene was used as a reference. FIG. 3D. Immunoblot (IB) of the indicated proteins in Scramble and sgDHX9 H196 cells. FIG. 3E. ELISA of human IFN-P protein in conditioned medium from Scramble and sgDHX9 Hl 96 cells. FIG. 3F. Log2 fold change (FC) of cytokine / chemokine differences of sgDHX9 Hl 96 compared to Scramble. The cytokine / chemokine levels were quantified with Proteome Profiler Human Cytokine Array Kit. FIG. 3G and FIG. 3H. Flow cytometry analysis of HLA-A.B.C (FIG. 3G) or PD-L1 (FIG. 3H) expression on the cell surface of Scramble and sgDHX9 Hl 96 cells. Data are representative of three independent experiments (left). Mean fluorescence intensity (MFI) was quantified by FlowJo (right) (n = 3). Data represent mean ± SEM. ns, not significant; *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 by unpaired Student’s t test (FIG. 3C, 3E, 3G and 3H).

[0029] FIG. 4A-4E: FIG. 4A. Heat map of RNA-seq results comparing Scramble and sgDHX9 Hl 96 cells. FIG. 4B. qRT-PCR analysis of the immune-related genes comparing Scramble and sgDHX9 of H446 and H82 cells (n = 3). 36B4 gene was used as a reference. FIG. 4C. Log2 fold change (FC) of cytokinc / chcmokinc differences of sgDHX9 compared to Scramble, of H446 and H82 cells. The cytokine / chemokine levels were quantified with Proteome Profiler Human Cytokine Array Kit. FIG. 4D and 4E. Flow cytometry analysis of HLA-A.B.C (FIG. 4D) or PD- L1 (FIG. 4E) expression on the cell surface of H446 and H82 cells (Scramble and sgDHX9). Data are representative of three independent experiments (left). Mean fluorescence intensity (MFI) was quantified by FlowJo (right) (n = 3). Data represent mean ± SEM. ns, not significant; *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 by unpaired Student’s t test (FIG. 4B, 4D and 4E).

[0030] FIG. 5A-5I: DHX9 depletion causes R-loop accumulation, DNA damage and cGAS- STING pathway activation. FIG. 5A. GSEA analysis with C2 (curated) gene sets, based on RNA-seq results of sgDHX9 versus Scramble cells. FIG. 5B. qRT-PCR analysis of the direct irradiation response and replication-related genes comparing Scramble and sgDHX9 Hl 96 cells (n = 3). 36B4 gene was used as a reference. FIG. 5C. Immunofluorescence images of p-H2AX (red) staining of Scramble and sgDHX9 H196 cells. Scale bar = 50 pm. FIG. 5D. Flow cytometry analysis of intracellular p-H2AX levels in Scramble and sgDHX9 Hl 96 cells. Data are representative of three independent experiments (left). Mean fluorescence intensity (MFI) was quantified by FlowJo (right) (n = 3). FIG. 5E. Immunofluorescence images of DNA / RNA hybrid (red) staining of Scramble and sgDHX9 Hl 96 cells (left) and quantification of fluorescence intensity (right) (150 cells were counted per group, n = 3). Scale bar = 50 pm. FIG. 5F. Immunoblot (IB) of the indicated proteins in Scramble and sgDHX9 Hl 96 cells. FIG. 5G. DNA fiber assay of Scramble and sgDHX9 Hl 96 cells. The percentage of stalled forks over the total number of different replication structures was measured (>150 labeled forks were counted per group, n = 3). FIG. 5H. Immunofluorescence images of dsDNA (green) and cGAS (red) staining of Scramble and sgDHX9 Hl 96 cells (left) and quantification of cells with cGAS-i- micronuclei (150 cells were counted per group, n = 3). Scale bar = 25 pm. FIG. 51. ELISA of human cGAMP protein in Scramble and sgDHX9 H196 cells. Data represent mean ± SEM. ns, not significant; *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 by unpaired Student’s t test (FIG. 5B, 5D, 5E, 5G, 5H and 51). FIG. 6A-6D: FIG. 6A. qRT-PCR analysis of the direct irradiation response and replication-related genes comparing Scramble and sgDHX9 of H446 and H82 cells (n = 3). 36B4 gene was used as a reference. FIG. 6B. Immunofluorescence images of p-H2AX (red) staining of Scramble and sgDHX9 H446 cells. Scale bar = 50 pm. FIG. 6C. Immunofluorescence images of DNA / RNA hybrid (red) staining of Scramble and sgDHX9 H196 cells, treated with or without RNase H. Scale bar = 25 pm. FIG. 6D. Immunoblot (IB) of the indicated proteins in Scramble and sgDHX9 H82 cells, w / wo overexpression of RNase H1-V5. Data represent mean ± SEM. ns, not significant; *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 by unpaired Student’s t test (FIG. 6A).

[0031] FIG. 7A-7H: DHX9 loss triggers dsRNA and dsDNA antiviral sensing pathways and IFN signaling in SCLC cells. FIG. 7A. Growth curves of the indicated SCLC cell lines (n = 3). FIG. 7B. Flow cytometry analysis of apoptotic (annexin V+) cells in Scramble and sgDHX9 H196 and H446 cells. Data are representative of three independent experiments (left). Quantification of apoptotic cells is shown (right) (n = 3). FIG. 7C. Growth curves of the indicated normal cell lines (n = 3). FIG. 7D. Flow cytometry analysis of apoptotic (annexin V+) cells in Scramble and sgDHX9 FC1010 and RPE cells. Data are representative of three independent experiments (left). Quantification of apoptotic cells is shown (right) (n = 3). FIG. 7E. qRT-PCR analysis of the immune-related genes comparing Scramble and sgSTING+ sgMAVS H196 cells, transfected with siCtrl or siDHX9 (n = 3). 36B4 gene was used as a reference. FIG. 7F. ELISA of human IFNP protein in conditioned medium from Scramble and sgSTING+ sgMAVS H196 cells, transfected with siCtrl or siDHX9 (n = 3). SM: sgSTING+ sgMAVS. FIG. 7G. Immunoblot (IB) of the indicated proteins in Scramble and sgSTING+ sgMAVS H196 cells, transfected with siCtrl or siDHX9. FIG. 7H. Growth curves of Scramble and sgSTING+ sgMAVS Hl 96 cells, transfected with siCtrl or siDHX9 (n = 3). Data represent mean ± SEM. ns, not significant; *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 by unpaired Student’s t test (FIG. 7A, 7B, 7C, and 7D), two-way ANOVA followed by Tukey’s multiple comparisons test (FIG. 7E, 7F and 7H).

[0032] FIG. 8A-8C: FIG. 8A. Relative cell number of the indicated SCLC cell lines. Luminescence of CellTiter-Glo was detected on Day 4 after seeding (n = 3). FIG. 8B. Immunofluorescence images of DNA / RNA hybrid (red) staining of Scramble and sgDHX9 FC 1010 cells (left) and quantification of fluorescence intensity (right) (60 cells were counted per group, n = 3). Scale bar = 50 pm. FIG. 8C. Immunoblot (IB) of the indicated proteins in FC1010 and H446 cells (Scramble and sgDHX9). Data represent mean ± SEM. ns, not significant; *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 by unpaired Student’s t test (FIG. 8A and 8B).

[0033] FIG. 9A-9H: CRISPR screen identifies modulators of sensitivity and resistance to DHX9 loss. FIG. 9A. Schematic of the genome-wide CRISPR screening method to reveal regulators of DHX9 loss-related cell death. Created with BioRender.com. FIG. 9B. Top-rated enriched and depleted sgRNAs from the genome-wide CRISPR screening are summarized. FIG. 9C and 9D. Gene ontology analysis of sgRNA targeted genes of depleted (FIG. 9C) and enriched (FIG. 9D) in sgDHX9 population. FIG. 9E. Relative cell number of Scramble and sgDHX9 H82 cells treated with DMSO or 0.5 pM BAY- 1143572. Luminescence of CellTiter-Glo was detected on Day 5 after seeding (n = 3). FIG. 9F. Immunoblot (IB) of the indicated proteins in Scramble and sgDHX9 H82 cells treated with DMSO or 0.5 pM BAY- 1143572. FIG. 9G. DNA fiber assay of Scramble and sgDHX9 H82 cells treated with DMSO or 0.5 pM BAY- 1143572. The percentage of stalled forks over the total number of different replication structures was measured (> 150 labeled forks were counted per group, n = 3). FIG. 9H. Schematic model of growth rescue effect by CDK9 inhibition in DHX9 depleted cells. Created with BioRender.com. Data represent mean ± SEM. ns, not significant; *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 by two-way ANOVA followed by Tukey’s multiple comparisons test (FIG. 9E and 9G).

[0034] FIG. 10A-10D: FIG. 10A and 10B. Relative cell number of H82 cells, transfected with the indicated siRNAs, targeting genes depleted (FIG. 10A) and enriched (FIG. 10B) in the CRISPR screen. Luminescence of CellTiter-Glo was detected on Day 4 after seeding (n = 3). FIG. 10C. Relative cell number of Scramble and sgDHX9 FC1010 cells, treated with DMSO or 0.5 pM BAY- 1143572. Luminescence of CellTiter-Glo was detected on Day 4 after seeding (n = 3). FIG. 10D. Relative cell number of H82 cells, treated with 0.01, 0.1, 0.5, 1.0, 2.0, 5.0 pM BAY- 1143572. DMSO-treated control was used as a reference. Luminescence of CellTiter-Glo was detected on Day 4 after seeding (n = 3). Data represent mean ± SEM. ns, not significant; *p < 0.05, **p < 0.01 , ***p < 0.001 , ****p < 0.0001 by two-way ANOVA followed by Tukey’s multiple comparisons test (FIG. 10A, 10B and IOC).

[0035] FIG. 11A-11I: DHX9 depletion decreases tumor growth, induces immune cell infiltration and enhances response to immune checkpoint blockade therapy. FIG. 11 A. Schematic of in vivo tumor growth assay. RPP cells, which were transduced with DOX- inducible shCtrl or shDhx9 vector, were transplanted into C57BL / 6 mice. Created with BioRender.com. FIG. 11B and 11C, Immunoblot (IB) (FIG. 11B) and qRT-PCR analysis (FIG. 11C) of DHX9 expression in shCtrl and shDhx9 RPP cells treated w / wo DOX. FIG. 11D. Tumor growth curves of shCtrl and shDhx9 RPP tumors (n = 6). FIG. HE. Flow cytometry quantification of the indicated infiltrating immune cells in shCtrl and shDhx9 RPP tumors. Each population was analyzed by Flowlo (n = 4). FIG. HF. Flow cytometry quantification of infiltrating CD8+ T cells and CD4+ T cells of CD45+CD3+ cells in shCtrl and shDhx9 RPP tumors (n = 6). FIG. 11G. Representative IHC images of indicated infiltrating immune cells in shCtrl and shDhx9 RPP tumors (left) and quantification (n = 6) (right). Scale bar = 100 pm. FIG. 11H. Tumor growth curves of shCtrl and shDhx9 RPP tumors treated with isotype control or anti-PD-1 antibody (n = 9). FIG. HI. Survival curves for mice in (FIG. HH). Data represent mean ± SEM. ns, not significant; *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 by unpaired Student’s t test (FIG. HC, HD, HE, 11F and 11G), two-way ANOVA followed by Tukey’s multiple comparisons test (FIG. 11H) and log-rank test (FIG. HI).

[0036] FIG. 12A-12B: FIG. 12A. Tumor weight of shCtrl and shDhx9 RPP tumors (n = 6), collected on day 32 post-inoculation. FIG. 12B. Body weight changes of mice, injected with shCtrl and shDhx9 RPP tumors (n = 6). FIG. 12C. Gating strategy used to analyze CD8+ and CD4+ T cells, infiltrated in shCtrl and shDhx9 RPP tumors. Data represent mean ± SEM. ns, not significant; *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 by unpaired Student’s t test (FIG. 12A and FIG. 12B).

[0037] FIG. 13A-13G: DHX9 is associated with poor clinical outcomes in cancer patient datasets. FIG. 13A. Representative IHC images of DHX9 expression in SCLC tumors and normal lung tissue. FIG. 13B. Quantification of IHC images of DHX9 in (FIG. 13A). Normal lung tissue (N = 10), Stage I (N = 9), Stage II (N = 23), Stage III (N = 8). FIG. 13C. GSEA analysis with H (hallmark) gene sets, based on RNA-scq results of 81 SCLC tumors (N = 40 DHX9lowversus N = 41 DHX9lllgh). Data were downloaded from cBioPortal (U Cologne, Nature 2015 (47)). FIG. 13D. Gene ontology analysis of genes upregulated in DHX9lowlung tumors. Data were downloaded from The Cancer Genome Atlas (TCGA). FIG. 13E. Correlation analysis between DHX9 expression level and z- scores of the indicated gene sets in different tumor types of TCGA. FIG. 13F. Boxplots of DHX9-depleted signature z-scores in non-responder and responder of patients treated with anti-PD-1 Ab. FIG. 13G. Schematic model of anti-tumor effects caused by DHX9 inhibition. Created with BioRender.com.Data represent mean ± SEM. ns, not significant; *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 by one-way ANOVA (FIG. 13B) and unpaired Student’s t test (FIG. 13F).

[0038] FIG. 14A-14C: FIG. 14A. Expression of immune-related genes in DHX9lowSCLC and DHX9hlghSCLC. Data were downloaded from cBioPortal. FIG. 14B. GSEA analysis with H (hallmark) gene sets, based on RNA-seq results of pan-cancer tumors (N = 100 DHX9lowversus N = 100 DHX9high). FIG. 14C. Estimated immune score of tumor microenvironment in DHX9lowand DHX9hlg11melanoma tumors. Datasets were downloaded from cBioPortal.

[0039] Data represent mean ± SEM. ns, not significant; *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 by Mann-Whitney test (FIG. 14A), unpaired Student’s t test (FIG. 14C) and log-rank test (FIG. 14B).

[0040] FIG. 15: Quantitative analysis of H82 cells, H446 cells, H69ARcells, H196 cells, RPP cells, and RPP-A cells after treatment with TK216 for 72 hours.

[0041] FIG. 16: Western Blot Analysis of Hl 96 cells after 48-hour treatment with DMSO, IpM TK216, or 5 pM cisplatin.

[0042] FIG. 17: Western Blot Analysis of RPP cells expressing shRNA targeting either control (shCt) or DHX9 (shDHX9), and RPP cells where the DHX9 was depleted by sgRNA sequences (sgDHX9) and Wild Type RPP cells treated DMSO, 2pM TK216, or 5 pM cisplatin for 48 hours. Detailed Description of the Invention

[0043] Small cell lung cancer (SCLC) is a rapidly growing, highly metastatic, and relatively immune-cold lung cancer subtype. Historically viewed in the laboratory and clinic as a single disease, new discoveries suggest that SCLC comprises multiple molecular subsets. Described herein is a discovery effort to identify critical regulators of dsRNA in SCLC cells through a curated CRISPR-based screen of RNA helicases. We identified the DExD / H-box helicase 9 (DHX9) as a promising target in therapeutic strategies employing viral mimicry which can be effectively convert immunologically cold tumors into “hot tumors” exhibiting increased sensitivity to cell killing by chemotherapeutic and immunotherapeutic agents.

[0044] DHX9 is an abundant RNA / DNA helicase capable of unwinding both RNA and DNA duplexes, as well as more complex nucleic acid structures. Its functions include regulation of transcription, RNA processing and transport, and maintenance of genomic stability (30). We demonstrate that DHX9 suppresses the accumulation of dsRNAs as well as R-loops (DNA / RNA hybrids). Its depletion leads not only to an antiviral immune response, but also to DNA replication stress and DNA damage in SCLC cells. The data presented show that DHX9 deletion causes a dramatic decrease in cancer cell viability in vitro. More importantly, DHX9 reduction promotes increased immunogenicity in mouse models of SCLC, significantly enhancing ICB responsiveness.

[0045] These findings provide the first description of the role of DHX9 tumor immunity and genomic instability, and the identification of an effective viral mimicry-inducing strategy to enhance anti-tumor inflammatory reactions, thereby increasing efficacy of anti-cancer immunotherapies in immunologically cold tumors, such as SCLC.

[0046] Definitions

[0047] Unless otherwise defined herein, scientific and technical terms used in connection with the present application shall have the meanings that are commonly understood by those of ordinary skill in the art. In addition to definitions included in this sub-section, further definitions of terms are interspersed throughout the text.

[0048] In this invention, “a” or “an” means “at least one” or “one or more,” etc., unless clearly indicated otherwise by context. The term “or” means “and / or” unless stated otherwise. In the case of a multiple-dependent claim, however, use of the term “or” refers back to more than one preceding claim in the alternative only.

[0049] The terms “about” or “approximately” in the context of numerical values and ranges refers to values or ranges that approximate or are close to the recited values or ranges such that the embodiment may perform as intended, such as having a desired amount of nucleic acids or polypeptides in a reaction mixture, as is apparent to the skilled person from the teachings contained herein. In some embodiments, about means plus or minus 10% of a numerical amount.

[0050] Furthermore, a compound "selected from the group consisting of" refers to one or more of the compounds in the list that follows, including mixtures (i.e. combinations) of two or more of the compounds. According to the present invention, an isolated, or biologically pure molecule is a compound that has been removed from its natural milieu. As such, "isolated" and "biologically pure" do not necessarily reflect the extent to which the compound has been purified. An isolated compound of the present invention can be obtained from its natural source, can be produced using laboratory synthetic techniques or can be produced by any such chemical synthetic route.

[0051] The terms “agent” and “test compound” denote a chemical compound, a mixture of chemical compounds, a biological macromolecule, or an extract made from biological materials such as bacteria, plants, fungi, or animal (particularly mammalian) cells or tissues. Biological macromolecules include siRNA, shRNA, antisense oligonucleotides, peptides, peptide / DNA complexes, and any nucleic acid-based molecule which encoded the proteins described herein.

[0052] It is also contemplated that the term “compound” or “compounds” refers to the compounds discussed herein and includes precursors and derivatives of the compounds, and pharmaceutically acceptable salts of the compounds, precursors, and derivatives.

[0053] The phrase "consisting essentially of" when referring to a particular nucleotide or amino acid means a sequence having the properties of a given SEQ ID NO. For example, when used in reference to an amino acid sequence, the phrase includes the sequence per se and molecular modifications that would not affect the functional and novel characteristics of the sequence.

[0054] The term "delivery" as used herein refers to the introduction of foreign molecule (i.e., miRNA encoding the polypeptide of interest) into cells. The term "administration" as used herein means the introduction of a foreign molecule into a cell. The term is intended to be synonymous with the term "delivery". Viral-Mimicry-Inducing Agents and Factors

[0055] The phrases “virus-mimetic nucleic acid” or “virus-mimetic” refer to nucleic acids that are sensed by the host’s innate immune system as evidence of viral replication. Once identified, these virus-mimetics provoke an immediate and robust interferon (INF)-driven antiviral response that causes the death of the cell. Such virus-mimetics include, without limitation, double stranded RNAs (dsRNA) and double-stranded DNAs (dsDNA). The antiviral response by the cell is potently immunogenic and useful in the treatment of cancer.

[0056] The term “viral-mimicry-inducing agent” “viral-mimicry-inducing factor” or “VMIA” refers to any compound or composition which acts to increase the intracellular accumulation of endogenous vims mimetics e.g., dsRNA molecules etc., and induces an antiviral response directed to targeted cancer cells. In certain embodiments, the VMIA can act on specific cells or tissues. In one embodiment, the VMIA is specific to tumor cells. In a preferred embodiment, the tumor cells are SCLC tumor cells. In certain embodiments, the VMIA is a small molecule inhibitor or siRNA that targets a specific gene. An exemplary VMIA is a DHX9 modulator.

[0057] The term “tumor specific viral-mimicry-inducing agent” or “tumor specific VMIA” refers to a VMIA that is specific to tumor cells.

[0058] DHX9 is a multifunctional DEAH-box ATP-independent RNA helicase which has been reported to play important roles in replication, transcription, translation, RNA splicing and RNA processing which contribute to DHX9’s role in maintenance of genomic stability. Functionally, DHX9’s role involves binding to, as well as unwinding and / or resolving double-stranded and single- stranded DNA / RNA, DNA / RNA hybrids (R-loops), circular RNA and DNA / RNA G quadraplexes. Modulation of DHX9 can produce virus-mimetics.

[0059] As used herein, the terms “DHX9 modulator” refer to compounds that have anti-cancer activity by altering the expression of the gene encoding for DHX9 and / or the expression of DHX9 and / or the biological activity of DHX9. In some embodiments, a DHX9 modulator is a DHX9 inhibitor. Exemplary DHX9 modulators include, without limitation, TK216, YK-4-279 and derivatives thereof. In certain embodiments, the DHX9 modulator functions in several ways. These include, without limitation, promotion of a decrease in tumor growth, inducing a more immunogenic tumor microenvironment (TME), and / or enhancing responsiveness to other anticancer therapies, such as immune checkpoint blockade (ICB) therapies and other chemotherapeutic agents. For administration of a DHX9 modulator, the dosage will depend on the mode of administration and the age, weight, and general health of the individual being treated. Dosage amounts may also be selected depending upon the combination partner (i.e., the other active components). If the drug is formulated for or delivered by a non-oral route, it may be desirable to decrease the unit or daily dose amounts delivered. In view of these factors, the skilled artisan would adjust the particular dose so as to obtain an effective dose for treating an individual.

[0060] The exact amount required will vary from subject to subject, depending on the species, age, and general condition of the subject, the severity of the disease that is being treated, the particular compound used, its mode of administration, and the like. However, an appropriate effective amount can be determined by one of ordinary skill in the art using only routine experimentation. The dosages or amounts of the compounds described herein are large enough to produce the desired effect in the method by which delivery occurs. The dosage should not be so large as to cause adverse side effects, such as unwanted cross-reactions, anaphylactic reactions, and the like. Generally, the dosage will vary with the age, condition, sex and extent of the disease in the subject and can be determined by one of skill in the ail. The dosage can be adjusted by the individual physician based on the clinical condition of the subject involved. The dose, schedule of doses and route of administration can be varied.

[0061] The compositions are administered in an effective amount and for a period of time effect to reduce one or more symptoms associated with the disease to be treated. It should be understood that the “effective amount” for a composition having anti-cancer cell proliferation properties may vary. In one embodiment an effective amount includes without limitation about 0.001 to about 25 mg / kg subject body weight. In one embodiment, the range of effective amount is 0.001 to 0.01 mg / kg body weight. In another embodiment, the range of effective amount is 0.001 to 0.1 mg / kg body weight. In another embodiment, the range of effective amount is 0.001 to 1 mg / kg body weight. In another embodiment, the range of effective amount is 0.001 to 10 mg / kg body weight. In another embodiment, the range of effective amount is 0.001 to 20 mg / kg body weight. In another embodiment, the range of effective amount is 0.01 to 25 mg / kg body weight. In another embodiment, the range of effective amount is 0.01 to 0.1 mg / kg body weight. In another embodiment, the range of effective amount is 0.01 to 1 mg / kg body weight. In another embodiment, the range of effective amount is 0.01 to 10 mg / kg body weight. In another embodiment, the range of effective amount is 0.01 to 20 mg / kg body weight. In another embodiment, the range of effective amount is 0.1 to 25 mg / kg body weight. In another embodiment, the range of effective amount is 0.1 to 1 mg / kg body weight. In another embodiment, the range of effective amount is 0.1 to 10 mg / kg body weight. In another embodiment, the range of effective amount is 0.1 to 20 mg / kg body weight. In another embodiment, the range of effective amount is 1 to 25 mg / kg body weight. In another embodiment, the range of effective amount is 1 to 5 mg / kg body weight. In another embodiment, the range of effective amount is 1 to 10 mg / kg body weight. In another embodiment, the range of effective amount is 10 to 20 mg / kg body weight. In another embodiment, the range of effective amount is 20 to 30 mg / kg body weight. In another embodiment, the range of effective amount is 30 to 40 mg / kg body weight. In another embodiment, the range of effective amount is 40 to 50 mg / kg body weight. In another embodiment, the range of effective amount is 1 to 50 mg / kg body weight. Still other doses falling within these ranges are expected to be useful.

[0062] In another embodiment, the range of effective amount is O.OOlmg to 10g. In another embodiment, the range of effective amount is 0.01 mg to 1 g. In another embodiment, the range of effective amount is 0.01 mg to 100 mg. In another embodiment, the range of effective amount is 0.1 mg to 100 mg. In another embodiment, the range of effective amount is 0.1 mg to 500 mg. In another embodiment, the range of effective amount is 1 mg to 100 mg. In another embodiment, the range of effective amount is 10 mg to 500 mg. In another embodiment, the range of effective amount is 10 mg to 750 mg. In another embodiment, the range of effective amount is 0.01 mg to 100 mg. In another embodiment, the range of effective amount is 1 mg to 500 mg.

[0063] The terms “inhibition” or “inhibit” refer to a decrease or cessation of any event (such as protein ligand binding) or to a decrease or cessation of any phenotypic characteristic or to the decrease or cessation in the incidence, degree, or likelihood of that characteristic. To “reduce” or “inhibit” is to decrease, reduce or arrest an activity, function, and / or amount as compared to a reference. It is not necessary that the inhibition or reduction be complete. For example, in certain embodiments, “reduce” or “inhibit” refers to the ability to cause an overall decrease of 20% or greater. In another embodiment, “reduce” or “inhibit” refers to the ability to cause an overall decrease of 50% or greater. In yet another embodiment, “reduce” or “inhibit” refers to the ability to cause an overall decrease of 75%, 85%, 90%, 95%, or greater. Inhibition can be 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, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49, 50, 51 , 52, 53, 54, 55, 56, 57, 58, 59,

[0064] 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85,

[0065] 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100%.

[0066] The term “inhibitor” refers to an agent that slows down or prevents a particular chemical reaction, signaling pathway or other process, or that reduces the activity of a particular reactant, catalyst, or enzyme.

[0067] The term “modulate” as used herein refers to the ability of a compound to change an activity in some measurable way as compared to an appropriate control. As a result of the presence of compounds in the assays, activities can increase or decrease as compared to controls in the absence of these compounds. Preferably, an increase in activity is at least 25%, more preferably at least 50%, most preferably at least 100% compared to the level of activity in the absence of the compound. Similarly, a decrease in activity is preferably at least 25%, more preferably at least 50%, most preferably at least 100% compared to the level of activity in the absence of the compound. A compound that increases a known activity is an “agonist”. One that decreases, or prevents, a known activity is an “antagonist”.

[0068] The term “apoptosis”, or “programmed cellular death”, refers to an active process requiring new protein synthesis. Typically, the process requires ATP, involves new RNA and protein synthesis, and culminates in the activation of endogenous endonucleases that degrade the DNA of the cell, thereby destroying the genetic template required for cellular hemostasis. Apoptosis is observed in controlled deletion of cells during metamorphosis, differentiation, and general cell turnover and appears normally to be regulated by receptor-coupled events. For these reasons, apoptosis has been called “programmed cell death” or “cell suicide.” While every cell likely has the genetic program to commit suicide, it is usually suppressed. Under normal circumstances, only those cells no longer required by the organism activate this self-destruction program.

[0069] Apoptotic cell death is characterized by plasma membrane bleeding, cell volume loss, nuclear condensation, and endonucleolytic degradation of DNA at nucleosome intervals. Loss of plasma membrane integrity is a relatively late event in apoptosis, unlike the form of cell death termed necrosis, which can be caused by hypoxia and exposure to certain toxins, and which is typically characterized early-on by increased membrane permeability and cell rupture. “Necroptosis,” as used herein, refers to a regulated, caspase-independent cell death, that can be an alternative way to eliminate apoptosis-resistant cancer cells. The core nccroptotic pathway consisting of a receptor- interacting protein kinase 1 (RIP1 or RIPK1) — receptorinteracting protein kinase 3 (RIP3 or RIPK3) — mixed lineage kinase domain-like protein (MLKL) complex, also called the ‘necrosome’. The necrosome initiates downstream effector functions such as generation of a reactive oxygen species (ROS) burst, plasma membrane permeabilization, and cytosolic ATP reduction that further drives irreversible necroptosisexecuting mechanisms. Provided herein are methods of treating patients by modulating necroptosis comprising administering to a patient a disclosed antisense oligonucleotide.

[0070] The compounds described herein can be formulated for parenteral or systemic administration. For example, parenteral administration may include administration to a patient intravenously, intradermally, intraarterially, intraperitoneally, intralesionally, intracranially, intraarticularly, intraprostatically, intrapleurally, intratracheally, intravitreally, intratumorally, intramuscularly, subcutaneously, subconjunctivally, intravesicularly, intrapericardially, intraumbilically, by injection, and by infusion. The compounds can be combined with one or more pharmaceutically acceptable carriers and / or excipients that are considered safe and effective and may be administered to an individual without causing undesirable biological side effects or unwanted interactions. The carrier is all components present in the pharmaceutical formulation other than the active ingredient or ingredients. Typical carriers and conventional methods of preparing pharmaceutical compositions that can be used in conjunction with the preparation of formulations of the compounds are known by those skilled in the art. These compositions can take the form of solutions, suspensions, emulsion, tablets, pills, capsules, powders, sustained-release formulations and the like.

[0071] Parenteral formulations can be prepared as aqueous compositions using techniques known in the art. Typically, such compositions can be prepared as injectable formulations, for example, solutions or suspensions; solid forms suitable for using to prepare solutions or suspensions upon the addition of a reconstitution medium prior to injection; emulsions, such as water-in-oil (w / o) emulsions, oil-in-water (o / w) emulsions, and microemulsions thereof, liposomes, or emulsomes.

[0072] For intravenous administration, the compositions may be packaged in solutions of sterile isotonic aqueous buffer. Where necessary, the composition may also include a solubilizing agent. The components of the composition are supplied either separately or mixed together in unit dosage form, for example, as a dry lyophilized powder or concentrated solution in a hermetically sealed container such as an ampoule or sachet indicating the amount of active agent. If the composition is to be administered by infusion, it can be dispensed with an infusion bottle containing sterile pharmaceutical grade water or saline. Where the composition is administered by injection, an ampoule of sterile water or saline can be provided so that the ingredients may be mixed prior to injection.

[0073] The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, one or more polyols (e.g., glycerol, propylene glycol, and liquid polyethylene glycol), oils, such as vegetable oils (e.g., peanut oil, corn oil, sesame oil, etc.), and combinations thereof. The proper fluidity can be maintained, for example, by the use of a coating, such as lecithin, by the maintenance of the required particle size in the case of dispersion and / or by the use of surfactants. In many cases, it will be preferable to include isotonic agents, for example, sugars or sodium chloride.

[0074] Solutions and dispersions of the active compounds as the free acid or base or pharmacologically acceptable salts thereof can be prepared in water or another solvent or dispersing medium suitably mixed with one or more pharmaceutically acceptable excipients including, but not limited to, surfactants, dispersants, emulsifiers, pH modifying agents, viscosity modifying agents, and combination thereof.

[0075] Suitable surfactants may be anionic, cationic, amphoteric or nonionic surface-active agents. Suitable anionic surfactants include, but are not limited to, those containing carboxylate, sulfonate and sulfate ions.

[0076] The formulation can contain a preservative to prevent the growth of microorganisms. Suitable preservatives include, but are not limited to, parabens, chlorobutanol, phenol, sorbic acid, and thimerosal. The formulation may also contain an antioxidant to prevent degradation of the active agent(s).

[0077] The formulation is typically buffered to a pH of 3-8 for parenteral administration upon reconstitution. Suitable buffers include, but are not limited to, phosphate buffers, acetate buffers, and citrate buffers.

[0078] Sterile injectable solutions can be prepared by incorporating the active compounds in the required amount in the appropriate solvent or dispersion medium with one or more of the excipients listed above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those listed above.

[0079] The compounds described herein can be administered in an effective amount to a subject that is in need of alleviation or amelioration from one or more symptoms associated with cancer cell growth and proliferation.

[0080] With respect to the small nucleic acid inhibitors, the polynucleotides may be delivered through any known method, such as through one or more vectors (e.g., encoding siRNA, antisense oligonucleotides or other type of inhibitory nucleic acid), to a host cell. In some aspects, the invention further provides cells produced with said vectors, and organisms or cells comprising or produced from such cells. Conventional viral and non-viral based gene transfer methods can be used to introduce nucleic acids in cells or target tissues. Such methods can be used to administer nucleic acids encoding inhibitory compounds to cells in culture, or in a host organism. Non-viral vector delivery systems include DNA plasmids, RNA (e.g. a transcript of a vector described herein), naked nucleic acid, and nucleic acid complexed with a delivery vehicle, such as a liposome. Viral vector delivery systems include DNA and RNA viruses, which have either episomal or integrated genomes after delivery to the cell. For a review of gene therapy procedures, see Anderson, Science 256:808-813 (1992); Nabel & Feigner, TIBTECH 11:211- 217 (1993); Mitani & Caskey, TIBTECH 11:162-166 (1993); Dillon, TIBTECH 11:167-175 (1993); Miller, Nature 357:455-460 (1992); Van Brunt, Biotechnology 6( 10): 1149- 1154 (1988); Vigne, Restorative Neurology and Neuroscience 8:35-36 (1995); Kremer & Perricaudet, British Medical Bulletin 51 (1 ) :31 -44 (1995); Haddada et al., in Current Topics in Microbiology and Immunology Doerfler and Bihm (eds) (1995); and Yu et al., Gene Therapy 1:13-26 (1994).

[0081] Methods of non-viral delivery of nucleic acids include lipofection, nucleofection, microinjection, biolistics, virosomes, liposomes, immunoliposomes, polycation or lipidmucleic acid conjugates, naked DNA, artificial virions, and agent-enhanced uptake of DNA. Lipofection is described in e.g., U.S. Pat. Nos. 5,049,386, 4,946,787; and 4,897,355) and lipofection reagents are sold commercially (e.g., TransfectamTM and LipofectinTM). Cationic and neutral lipids that are suitable for efficient receptor-recognition lipofection of polynucleotides include those of Feigner, WO 91 / 17424; WO 91 / 16024. Delivery can be to cells (e.g. in vitro or ex vivo administration) or target tissues (e.g. in vivo administration). The preparation of lipid:nucleic acid complexes, including targeted liposomes such as immunolipid complexes, is well known to one of skill in the ail (sec, c.g., Crystal, Science 270:404-410 (1995); Blaese et al., Cancer Gene Ther. 2:291-297 (1995); Behr et al., Bioconjugate Chem. 5:382-389 (1994); Remy et al., Bioconjugate Chem. 5:647-654 (1994); Gao et al., Gene Therapy 2:710-722 (1995); Ahmad et al., Cancer Res. 52:4817-4820 (1992); U.S. Pat. Nos. 4,186,183, 4,217,344, 4,235,871, 4,261,975, 4,485,054, 4,501,728, 4,774,085, 4,837,028, and 4,946,787).

[0082] The use of RNA or DNA viral based systems for the delivery of nucleic acids take advantage of highly evolved processes for targeting a virus to specific cells in the body and trafficking the viral payload to the nucleus. Viral vectors can be administered directly to patients (in vivo) or they can be used to treat cells in vitro, and the modified cells may optionally be administered to patients (ex vivo). Conventional viral based systems could include retroviral, lentivirus, adenoviral, adeno-associated and herpes simplex virus vectors for gene transfer. Integration in the host genome is possible with the retrovirus, lentivirus, and adeno-associated virus gene transfer methods, often resulting in long term expression of the inserted transgene. Additionally, high transduction efficiencies have been observed in many different cell types and target tissues.

[0083] The tropism of a retrovirus can be altered by incorporating foreign envelope proteins, expanding the potential target population of target cells. Lentiviral vectors are retroviral vectors that are able to transduce or infect non-dividing cells and typically produce high viral titers. Selection of a retroviral gene transfer system would therefore depend on the target tissue. Retroviral vectors are comprised of cis-acting long terminal repeats with packaging capacity for up to 6-10 kb of foreign sequence. The minimum cis-acting LTRs are sufficient for replication and packaging of the vectors, which are then used to integrate the therapeutic gene into the target cell to provide permanent transgene expression. Widely used retroviral vectors include those based upon murine leukemia virus (MuLV), gibbon ape leukemia virus (GaLV), Simian Immuno deficiency virus (SIV), human immuno deficiency virus (HIV), and combinations thereof (see, e.g., Buchscher et al., J. Virol. 66:2731-2739 (1992); Johann et al., J. Virol. 66:1635-1640 (1992); Sommnerfelt et al., Virol. 176:58-59 (1990); Wilson et al., J. Virol. 63:2374-2378 (1989); Miller et al., J. Virol. 65:2220-2224 (1991); PCT / US94 / 05700). In applications where transient expression is preferred, adenoviral based systems may be used. Adenoviral based vectors are capable of very high transduction efficiency in many cell types and do not require cell division. With such vectors, high titer and levels of expression have been obtained. This vector can be produced in large quantities in a relatively simple system. Adeno-associated virus ("AAV") vectors may also be used to transduce cells with target nucleic acids, e.g., in the in vitro production of nucleic acids and peptides, and for in vivo and ex vivo gene therapy procedures (see, e.g., West et al., Virology 160:38-47 (1987); U.S. Pat. No. 4,797,368; WO 93 / 24641; Kotin, Human Gene Therapy 5:793-801 (1994); Muzyczka, J. Clin. Invest. 94:1351 (1994). Construction of recombinant AAV vectors are described in a number of publications, including U.S. Pat. No. 5,173,414; Tratschin et al., Mol. Cell. Biol. 5:3251-3260 (1985); Tratschin, et al., Mol. Cell. Biol. 4:2072-2081 (1984); Hermonat & Muzyczka, PNAS 81:6466-6470 (1984); and Samulski et al., J. Virol. 63:03822-3828 (1989).

[0084] Packaging cells are typically used to form virus particles that mediate infection of a host cell. Such cells include 293 cells, which package adenovirus, and q / 2 cells or PA317 cells, which package retrovirus. Viral vectors used in gene therapy are usually generated by producing a cell line that packages a nucleic acid vector into a viral particle. The vectors typically contain the minimal viral sequences required for packaging and subsequent integration into a host, other viral sequences being replaced by an expression cassette for the polynucleotide(s) to be expressed. The missing viral functions are typically supplied in trans by the packaging cell line. For example, AAV vectors used in gene therapy typically only possess ITR sequences from the AAV genome which are required for packaging and integration into the host genome. Viral DNA is packaged in a cell line, which contains a helper plasmid encoding the other AAV genes, namely rep and cap, but lacking ITR sequences. The cell line may also be infected with adenovirus as a helper. The helper virus promotes replication of the AAV vector and expression of AAV genes from the helper plasmid. The helper plasmid is not packaged in significant amounts due to a lack of ITR sequences. Contamination with adenovirus can be reduced by, e.g., heat treatment to which adenovirus is more sensitive than AAV.

[0085] In certain embodiments, the inhibitory nucleic acids, e.g., siRNAs described above comprise “modified nucleotides”. These are nucleotides comprising non-naturally occurring moieties that confer increased nuclease resistance or thermodynamic stability during hybridization as compared with a polynucleotide or polyribonucleotide that differs from the inhibitory nucleic acid only by having a natural nucleotide in place of the modified nucleotide. In certain embodiments, the ribose moiety of a nucleotide is modified with an extra bridge connecting the 2' oxygen and 4' carbon. Numerous chemical modifications arc commonly used for the synthesis of oligonucleotides for a variety of reasons. For example, to increase the phosphate backbone's stability, adjust duplex stability, change the oligonucleotide's conformation, or increase its ability to penetrate a lipid bilayer. Modified sugar' moieties are also being incorporated into therapeutic oligonucleotides. Changing the sugar moiety generally increases nuclease resistance and binding affinity to a complementary target.

[0086] “Bridged nucleic acid” (“BNA”) refers to 2'-O,4'-C-methylene-modified nucleic acids. In preferred embodiments, BNA, where the 2' oxygen and 4' carbon are bridged by a methylene group are used. In other approaches, 2'-O,4'-C-ethylene-bridged nucleic acids (ENA), the 2' oxygen and 4' carbon are bridged by an ethylene group. Other examples of BNA can include, but are not limited to, 2',4'-BNANC[NH], 2',4'-BNANC[NMe], and 2',4'-BNANC[NBn], (s)-cEt (S- constrained Ethyl). tcDNA (tricycloDNA) modifications can also be used to constrain nucleotides.

[0087] “Locked nucleic acid nucleotide” (“LNA nucleotide”) as used herein, refers to a modified RNA nucleotide that provides the polynucleotide with greater thermodynamic stability during hybridization as compared with a polynucleotide that differs from the LNA only by having a natural ribonucleotide in place of the modified RNA nucleotide. In certain embodiments, the ribose moiety of a modified RNA nucleotide is modified with an extra bridge connecting the 2' oxygen and 4' carbon. LNA nucleotides can comprise any type of extra bridge between the 2'-0 and 4'-C of the RNA that increases the thermodynamic stability of the duplex between the LNA and its complement.

[0088] Other 2'-O-modified nucleotides, such as 2'-0-Me, demonstrate greater stability, as well. Oligonucleotide backbone configurations that demonstrate particularly high binding affinities to the target (measured by melting temperature or Tm) are preferred for implementing the steric hindrance mechanism. BNA, LNA, FANA, 2'-fluoro, 2’-O-methoxyethyl (2’-M0E), 2’-NH2, 2’-F-RNA, morpholino and piperazine containing backbones are particularly well suited for this purpose.

[0089] Other modifications on the oligonucleotide ribose include, are not limited to, FHNA (Fluoro Hexitol Nucleic Acid), (s)-5’-C-methyl, UNA (Unlocked Nucleic Acid), 4’-thio-RNA, cyclohexene nucleic acid. Modified backbone linkages are sometimes used instead of phosphodiester linkage to minimize oligonucleotide degradation by nucleases. Some examples include, arc not limited to, phosphorothioate, boranophosphonate, phosphoramidate, methyl phosphonate, (SC5’ Rp)-a,P- CNA (Dioxaphosphorinane-Constrained Nucleic Acid), PNA (Peptide Nucleic Acid), PMO (Phosphorodiamidate Morpholino Oligonucleotide), phosphoryl guanidine. 5’ modifications to increase phosphate stability include, are not limited to, E-VP ((E)-VinylPhosphonate), 5’ methyl phosphonate, 5’ -phosphorothioate, (s)-5’-methyl with phosphate, 5’-methoxy. 3’ modifications to increase phosphate stability include, are not limited to, 2-hydroxyethylphosphate AND, 3’-ddc (dideoxyCytosine), 3’-amino. Base modifications to improve 3’ stability include, are not limited to, 2’-thio-dT.

[0090] The generation of oligonucleotides with mixed linkages such as boranophosphate and phosphate linkages has been accomplished by several solid phase methods including one involving the use of bis(trimethylsiloxy)cyclododecyloxysilyl as the 5'-0-protecting group (Brummel and Caruthers, Tetrahedron Lett 43: 749, 2002). In another example the 5'-hydroxyl is initially protected with a benzhydroxybis-(trimethylsilyloxy)silyl group and then deblocked by EtaNiHF before the next cycle (McCuen et al., J Am Chem Soc 128: 8138, 2006). This method can result in a 99% coupling yield and can be applied to the synthesis of oligos with pure boranophosphate linkages or boranophosphate mixed with phosphodiester, phosphorothioate, phosphorodithioate or methyl phosphonate linkages.

[0091] The boranophosphorylating reagent 2-(4-nitrophenyl)ethyl ester of boranophosphoramidate can be used to produce boranophosphate linked oligoribonucleotides This reagent readily reacts with a hydroxyl group on the nucleosides in the presence of 1H- tetrazole as a catalyst. The 2-(4-nitrophenyl)ethyl group can be removed by 1,4- diazabicyclo[5.4.0]undec-7-ene (DBU) through beta-elimination, producing the corresponding nucleoside boranomonophosphates (NMPB) in good yield.

[0092] Nucleobase modifications to increase binding affinity include, are not limited to, 5’- methylcytidine, 5 -methyluridine (ribothymidine), and abasic RNA.

[0093] By means of non-limiting examples, the compounds and any other active drugs of interest may be formulated as separate pharmaceutical preparations, as a single pharmaceutical preparation, or mixtures thereof. Any suitable form may be selected, e.g., in a form suitable for oral administration, for parenteral administration (such as by intravenous, intramuscular or subcutaneous injection or intravenous infusion), for topical administration (including ocular), for administration by inhalation, by a skin patch, by an implant, by a suppository, etc. Such suitable administration forms — which may be solid, semi-solid or liquid, depending on the manner of administration — as well as methods and carriers, diluents and excipients for use in the preparation thereof, will be clear to the skilled person; reference is made to the latest edition of Remington's Pharmaceutical Sciences.

[0094] Some preferred, but non-limiting examples of such preparations include tablets, pills, powders, lozenges, sachets, cachets, elixirs, suspensions, emulsions, solutions, syrups, aerosols, ointments, cremes, lotions, soft and hard gelatin capsules, suppositories, drops, sterile injectable solutions and sterile packaged powders (which are usually reconstituted prior to use) for administration as a bolus and / or for continuous administration, which may be formulated with carriers, excipients, and diluents that are suitable per se for such formulations, such as lactose, dextrose, sucrose, sorbitol, mannitol, starches, gum acacia, calcium phosphate, alginates, tragacanth, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, polyethylene glycol, cellulose, (sterile) water, methylcellulose, methyl- and propylhydroxybenzoates, talc, magnesium stearate, edible oils, vegetable oils and mineral oils or suitable mixtures thereof. The formulations can optionally contain other substances that are commonly used in pharmaceutical formulations, such as lubricating agents, wetting agents, emulsifying and suspending agents, dispersing agents, disintegrants, bulking agents, fillers, preserving agents, sweetening agents, flavoring agents, flow regulators, release agents, etc. The compositions may also be formulated so as to provide rapid, sustained or delayed release of the active compound(s) contained therein.

[0095] The pharmaceutical preparations are preferably in a unit dosage form, and may be suitably packaged, for example in a box, blister, vial, bottle, sachet, ampoule or in any other suitable single-dose or multi-dose holder or container (which may be properly labeled); optionally in a kit which also comprises with one or more leaflets containing product information and / or instructions for use.

[0096] Depending on the condition to be prevented or treated and the route of administration, each of the different active compounds may be independently administered as a single daily dose, divided over one or more daily doses, or essentially continuously, e.g. using a drip infusion. Solutions and dispersions of the active compounds as the free acid or base or pharmacologically acceptable salts thereof can be prepared in water or another solvent or dispersing medium suitably mixed with one or more pharmaceutically acceptable excipients including, but not limited to, surfactants, dispersants, emulsifiers, pH modifying agents, viscosity modifying agents, and combination thereof.

[0097] Suitable surfactants may be anionic, cationic, amphoteric or nonionic surface-active agents. Suitable anionic surfactants include, but are not limited to, those containing carboxylate, sulfonate and sulfate ions.

[0098] The formulation can contain a preservative to prevent the growth of microorganisms. Suitable preservatives include, but are not limited to, parabens, chlorobutanol, phenol, sorbic acid, and thimerosal. The formulation may also contain an antioxidant to prevent degradation of the active agent(s).

[0099] The formulation is typically buffered to a pH of 3-8 for parenteral administration upon reconstitution. Suitable buffers include, but are not limited to, phosphate buffers, acetate buffers, and citrate buffers.

[0100] Sterile injectable solutions can be prepared by incorporating the active compounds in the required amount in the appropriate solvent or dispersion medium with one or more of the excipients listed above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those listed above.

[0101] The term “solvate” is used herein to describe a compound that contains stoichiometric or sub- stoichiometric amounts of one or more pharmaceutically acceptable solvent molecule such as ethanol. The term “hydrate” refers to when the said solvent is water.

[0102] The term “prodrug” as used herein means the pharmacologically acceptable derivatives of the compounds, such as for example amides, whose in vivo biotransformation product generates the biologically active drug. Prodrugs are generally characterized by increased bioavailability and are readily metabolized into biologically active compounds in vivo.

[0103] The term “predrug”, as used herein, means any compound that will be modified to form a drug species, wherein the modification may take place either inside or outside of the body, and either before or after the predrug reaches the area of the body where administration of the drag is indicated. Methods of Treatment and Administration

[0104] The term “preventing” or “inhibiting “as used herein refers to administering a compound prior to the onset of clinical symptoms of a disease or conditions so as to prevent or inhibit a physical manifestation of aberrations associated with the disease or condition.

[0105] The term “in need of treatment” as used herein refers to a judgment made by a caregiver (e.g. physician, nurse, nurse practitioner, or individual in the case of humans; veterinarian in the case of animals, including non-human mammals) that a subject requires or will benefit from treatment. This judgment is made based on a variety of factors that are in the realm of a care giver's expertise, but that includes the knowledge that the subject is ill, or will be ill, as the result of a condition that is treatable by the disclosed compounds.

[0106] As used herein, “subject” includes, but is not limited to, vertebrate animals, more specifically a mammal (e.g., a human, horse, pig, rabbit, dog, sheep, goat, non-human primate, cow, cat, guinea pig or rodent), a fish, a bird or a reptile or an amphibian. The term does not denote a particular age or sex. Thus, adult and newborn subjects, as well as fetuses, whether male or female, are intended to be covered. A patient refers to a subject afflicted with a disease or disorder. The term “patient” includes human and veterinary subjects.

[0107] By “treatment” and “treating” is meant the medical management of a subject with the intent to cure, ameliorate, or stabilize, a pathological condition or disorder. This term includes active treatment, that is, treatment directed specifically toward the improvement of a disease, pathological condition, or disorder, and also includes causal treatment, that is, treatment directed toward removal of the cause of the associated disease, pathological condition, or disorder. In addition, this term includes palliative treatment, that is, treatment designed for the relief of symptoms rather than the curing of the disease, pathological condition, or disorder; and supportive treatment, that is, treatment employed to supplement another specific therapy directed toward the improvement of the associated disease, pathological condition, or disorder. It is understood that treatment, while intended to cure, ameliorate, or stabilize, a disease, pathological condition, or disorder, need not actually result in the cure, ameliorization, or stabilization. The effects of treatment can be measured or assessed as described herein and as known in the art as is suitable for the disease, pathological condition, or disorder involved. Such measurements and assessments can be made in qualitative and / or quantitative terms. Thus, for example, characteristics or features of a disease, pathological condition, or disorder and / or symptoms of a disease, pathological condition, or disorder can be reduced to any effect or to any amount.

[0108] The cancers to be treated using the compositions of the invention may be metastatic or non-metastatic. The cancer may be familial or sporadic.

[0109] As used herein, the terms "tumor", "tumor growth" or "tumor tissue" can be used interchangeably and refer to an abnormal growth of tissue resulting from uncontrolled progressive multiplication of cells and serving no physiological function.

[0110] A solid tumor can be malignant, e.g., tending to metastasize and being life threatening, or benign. Examples of solid tumors that can be treated or prevented according to a method of the present invention include sarcomas and carcinomas such as, but not limited to: fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma, endothelio sarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon carcinoma, colorectal cancer, gastic cancer, 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, liver metastases, bile duct carcinoma, choriocarcinoma, seminoma, embryonal carcinoma, thyroid carcinoma such as anaplastic thyroid cancer, Wilms' tumor, cervical cancer, testicular tumor, lung carcinoma such as small cell lung carcinoma and non- small cell lung carcinoma, bladder carcinoma, epithelial carcinoma, glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, melanoma, neuroblastoma, glioblastoma, and retinoblastoma.

[0111] Additional cancers that can be treated using the methods provided herein include, for example, benign and malignant solid tumors and benign and malignant non-solid tumors. In one embodiment, the cancer is benign solid tumors. In one embodiment, the cancer is malignant solid tumors. In one embodiment, the cancer is benign non-solid tumors. In one embodiment, the cancer is malignant non-solid tumors.

[0112] In some embodiments, the cancer is lymphoma, leukemia, or chronic lymphocytic leukemia (CLL), a non- Hodgkin’s lymphoma (NHL) selected from small lymphocytic lymphoma (SLL), follicular lymphoma (FL), marginal zone lymphoma (MZL), mantle cell lymphoma (MCL), diffuse large B-cell lymphoma (DLBCL), or other types of B-cell lymphoma or a T-ccll lymphoma selected from peripheral T-cell lymphoma and T-prolymphocytic lymphoma.

[0113] The phrase “cold cancer” refers to a cancer or tumor in a patient categorized by low immune infiltrates and evasion from the host immune responses. The phrase “tumor mutational burden” or “TMB” refers to the number of genetic changes or mutations in a cancer cell. The immune system can identify cancer cells and activate an immune response by detecting these mutations. Accordingly, cancers with low TMB have fewer mutations and a decreased chance of activating the immune system. Conversely, cancers with high TMB have more mutations and an increased chance of activating the immune system. Treatment of these difficult to treat cancers is considered herein.

[0114] Anti-cancer therapies may be used in combination with the VMIA. The phrase “anticancer therapy” refers to a therapy useful in treating cancer. Types of anti-cancer therapies include, but are not limited to, chemotherapy, immunotherapy, targeted therapies, surgery, and radiation therapies. Examples of anticancer therapeutic agents include, but are limited to, e.g., chemotherapeutic agents, growth inhibitory agents, cytotoxic agents, agents used in radiation therapy, anti-angiogenesis agents, apoptotic agents, anti-tubulin agents, and other agents to treat cancer, anti-CD20 antibodies, platelet derived growth factor inhibitors (e.g.. Gleevee™ (Imatinib Mesylate)), a COX-2 inhibitor (e.g., celecoxib), interferons, cytokines, antagonists (e.g., neutralizing antibodies) that bind to one or more of the following targets PDGFR-beta, BlyS. APRIL. BCMA receptor(s), TRAIL / Apo2, and other bioactive and organic chemical agents, etc. Combinations thereof are also included in the invention.

[0115] Chemotherapeutic agents are compounds that exhibit anticancer activity and / or are detrimental to a cell (e.g., a toxin). Suitable chemotherapeutic agents for use in the methods disclosed herein include, but are not limited to: toxins (e.g., saporin, ricin, abrin, ethidium bromide, diptheria toxin, Pseudomonas exotoxin, and others listed above); alkylating agents (e.g., nitrogen mustards such as chlorambucil, cyclophosphamide, isofamide, mechlorethamine, melphalan, and uracil mustard; aziridines such as thiotepa; methanesulphonate esters such as busulfan; nitroso ureas such as carmustine, lomustine, and streptozocin; platinum complexes such as cisplatin and carboplatin; bioreductive alkylators such as mitomycin, procarbazine, dacarbazine and altretamine); DNA strand-breakage agents (e.g., bleomycin); topoisomerase II inhibitors (e.g., amsacrine, dactinomycin, daunorubicin, idarubicin, mitoxantrone, doxorubicin, etoposide, and teniposide); DNA minor groove binding agents (e.g., plicamydin); antimetabolites (e.g., folate antagonists such as methotrexate and trimetrexate; pyrimidine antagonists such as fluorouracil, fluorodeoxyuridine, CB3717, azacitidine, cytarabine, and floxuridine; purine antagonists such as mercaptopurine, 6-thioguanine, fludarabine, pentostatin; asparginase; and ribonucleotide reductase inhibitors such as hydroxyurea); tubulin interactive agents (e.g., vincristine, vinblastine, and paclitaxel (Taxol)); hormonal agents (e.g., estrogens; conjugated estrogens; ethinyl estradiol; diethylstilbesterol; chlortrianisen; idenestrol; progestins such as hydroxyprogesterone caproate, medroxyprogesterone, and megestrol; and androgens such as testosterone, testosterone propionate, fluoxymesterone, and methyltestosterone); adrenal corticosteroids (e.g., prednisone, dexamethasone, methylprednisolone, and prednisolone); leutinizing hormone releasing agents or gonadotropin-releasing hormone antagonists (e.g., leuprolide acetate and goserelin acetate); and antihormonal antigens (e.g., tamoxifen, antiandrogen agents such as flutamide; and anti-adrenal agents such as mitotane and aminoglutethimide). In a particular embodiment, the chemotherapeutic agent is selected from the group consisting of: placitaxel (Taxol®), cisplatin, docetaxol, carboplatin, vincristine, vinblastine, methotrexate, cyclophosphamide, CPT-11, 5-fluorouracil (5-FU), gemcitabine, estramustine, carmustine, adriamycin (doxorubicin), etoposide, arsenic trioxide, irinotecan, and epothilone derivatives.

[0116] The term “immunotherapy” refers to the treatment of cancer by activating or suppressing the immune system. Immunotherapies designed to elicit or amplify an immune response may be referred to as activation immunotherapies or immune activators, whereas immunotherapies that reduce or suppress such response may referred to as suppression immunotherapies or immune suppressors. As used herein, the term “cancer immunotherapy” refers to an immunotherapy used for the treatment of a cancer, said immunotherapy modulating the immune response of a subject with the aim of inducing and / or stimulating the immune response of the subject towards cancer cells. Common antibodies used for the treatment of cancer include without limitation, trastuzumab (Herceptin), pertuzumab (Perjeta), bevacizumab (Avastin) rituximab (Mabthera) and obinutuzumab.

[0117] In certain embodiments, the immunotherapy is an immune checkpoint blockade therapy. The phrase “immune checkpoint blockade therapy” or “ICB therapy” refers to treatment with a drug that blocks proteins, called checkpoints, that are made by types of immune system cells, such as T cells, and some cancer cells. These drugs arc called immune checkpoint inhibitors.

[0118] An immune checkpoint inhibitor may be any molecule that inhibits an immune checkpoint. Immune checkpoints are well known in the art and include, without limitation, PD-1, PD-L1, PD-L2, CTLA4, B7-H3, B7-H4, BTLA, IDO, KIR, LAG3, A2AR, TIM-3, and VISTA. In some embodiments, the inhibitor is an antibody against the immune checkpoint protein. In certain embodiments, the immune checkpoint inhibitor is an inhibitor of PD-1 or PD-L1, e.g., Pembrolizumab (Keytruda), Nivolumab (Opdivo), Cemiplimab (Libtayo), Atezolizumab (Tecentriq), Avelumab (Bavencio), Durvalumab (Imfinzi) and an antibody that specifically binds PD-1 or PD-L1. In certain embodiments, the immune checkpoint inhibitor is an inhibitor of CTLA-4, e.g., Ipilimumab (Yervoy), tremelimumab (Imjuno) and an antibody that specifically binds CTLA-4. In certain embodiments, the immune checkpoint inhibitor is an inhibitor of LAG- 3, e.g., Relatlimab and an antibody that specifically binds LAG-3. In some embodiments, the cancer immunotherapy comprises, or consists of, the adoptive transfer of immune cells (ACT), in particular of T cells (such as alpha beta (aP) T cells or gamma delta (y5) T cells), NK cells or NK T cells.

[0119] The phrase “targeted therapy” or “targeted molecular therapy” refers to treatment using any molecule which aims at one or more particular target molecules (such as, e.g., proteins) involved in tumor genesis, tumor progression, tumor metastasis, tumor cell proliferation, cell repair, and the like. Targeted therapy agents include, for example, monoclonal antibodies and small molecule drugs. Non-limiting examples of targeted therapy agents include signal transduction inhibitors, growth factor inhibitors, tyrosine kinase inhibitors, EGFR inhibitors, histone deacetylase (HD AC) inhibitors, proteasome inhibitors, cell-cycle inhibitors, angiogenesis inhibitors, matrix-metalloproteinase (MMP) inhibitors, hepatocyte growth factor inhibitors, TOR inhibitors, KDR inhibitors, VEGF inhibitors, fibroblast growth factors (FGF) inhibitors, MEK inhibitors, ERK inhibitors, PI3K inhibitors, AKT inhibitors, MCL-1 inhibitors, BCL-2 inhibitors, SHP2 inhibitors, HER-2 inhibitors, BRAF-inhibitors, gene expression modulators, autophagy inhibitors, apoptosis inducers, antiproliferative agents, and glycolysis inhibitors.

[0120] In certain embodiments, the at least one VMIA and at least one additional anti-cancer therapy are administered together or sequentially. By sequential administration, the VMIA may be delivered to a subject before or after administration of the at least one additional anti-cancer therapy. In further embodiments, the VMIA may be delivered to a subject at least 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 days, or 1 week, 2 weeks, 3 weeks, 4 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, or 12 months before or after administration of the anti-cancer therapy. In still further embodiments, the VMIA may be delivered to a subject in any combination of months, days, hours, minutes, and seconds within these ranges. In further embodiments, the administration of anti-cancer therapy and VMIA may be repeated multiple times.

[0121] The term "drug response" as used herein, means any biological response in an organism that is the result of exposure to the drug. Drug responses can be favorable, such as when a patient's disease is eradicated by treatment with the drug, or unfavorable, such as when a patient enters a coma upon treatment with a drug.

[0122] The term “synergy” or “synergistic” refers to the interaction or cooperation of two or more substances, or other agents to produce a combined effect greater than the sum of their separate effects.

[0123] Provided herein are methods of treatment of cancer including lung cancers. In certain embodiments, the cancer is a COLD cancer or a cancer with a low tumor mutated burden (TMB). In certain embodiments, the cancer is small cell lung cancer (SCLC). The methods include administration of an effective amount of at least one VMIA to a subject in need thereof. In certain embodiments, the VMIA is selected from a small molecule inhibitor. In some embodiments, the symptoms of the cancer are reduced, as compared to a control.

[0124] In certain embodiments, the method of treatment effectively suppresses symptoms associated with cancer. Symptoms of malignancy vary according to the location and type of cancer being treated. In certain embodiments, symptoms of cancer include, fatigue, weight loss, lumps, swelling, pain, coughing, wheezing, new or unusual growth, discoloration, and no symptoms at all. In certain embodiments, the treatment reduces the risk of relapse. In the context of a cancer, treatment or inhibition may be assessed by inhibition of disease progression, inhibition of tumor growth, reduction of primary tumor, relief of tumor-related symptoms, inhibition of tumor secreted factors, delayed appearance of primary or secondary tumors, slowed development of primary or secondary tumors, decreased occurrence of primary or secondary tumors, slowed or decreased severity of secondary effects of disease, arrested tumor growth and regression of tumors, increased Time To Progression (TTP), increased Progression Free Survival (PFS), increased Overall Survival (OS), among others. OS as used herein means the time from treatment onset until death from any cause. TTP as used herein means the time from treatment onset until tumor progression; TTP does not include deaths. Time to Remission (TTR) as used herein means the time from treatment onset until remisison, for example, complete or partial remission. As used herein, PFS means the time from treatment onset until tumor progression or death. In one embodiment, PFS rates will be computed using the Kaplan-Meier estimates. Event- free survival (EFS) means the time from study entry until any treatment failure, including disease progression, treatment discontinuation for any reason, or death. Relapse-free survival (RFS) means the length of time after the treatment ends that the patient survives without any signs or symptoms of that cancer. Overall response rate (ORR) means the sum of the percentage of patients who achieve complete and partial responses. Complete remission rate (CRR) refers to the percentage of patients achieving complete remission (CR). Duration of response (DoR) is the time from achieving a response until relapse or disease progression. Duration of remission is the time from achieving remission, for example, complete or partial remission, until relapse. In the extreme, complete inhibition, is referred to herein as prevention or chemoprevention. In this context, the term “prevention” includes either preventing the onset of clinically evident cancer altogether or preventing the onset of a preclinically evident stage of a cancer. Also intended to be encompassed by this definition is the prevention of transformation into malignant cells or to arrest or reverse the progression of premalignant cells to malignant cells. This includes prophylactic treatment of those at risk of developing a cancer.

[0125] The compounds described herein can be administered in an effective amount to a subject that is in need of alleviation or amelioration from one or more symptoms associated with tumor growth.

[0126] Kits and Articles of Manufacture

[0127] Any of the aforementioned products can be incorporated into a kit which may contain a viral-mimicry inducing agent or factor, an optional immunotherapeutic or chemotherapeutic agent, a pharmaceutically acceptable carrier, instructions for use, a container, a vessel for administration, or any combination thereof. MATERIALS AND METHODS

[0128] The following materials and methods arc provided to facilitate the practice of the present invention.

[0129] CELL LINES AND CELL CULTURE

[0130] The human SCLC cell lines NCI-H69, NCI-H69M, NCI-H69AR, NCI-H841, SHP-77, NCI-H187, NCI-H345, NCI-H524 and NCI-H82 were obtained from the laboratory of Dr. Joan Albanell and were authenticated following short tandem repeat (STR) genotyping. Retinal Pigment Epithelium (RPE) cells were obtained from the laboratory of Dr. Johnathan R. Whetstine, FC 1010 primary fibroblast cells were isolated in the FCCC tissue culture facility (from Dr. Hossein Borghaei) and were authenticated following STR genotyping. NCLH196, NCI-H446, NCI-H1436, NCI-H2081, C0I0668, NCI-H1694, NCI-H841, DMS 114 and HEK293T cells were obtained from the American Type Culture Collection and used for all experiments before reaching 10 passages. All cells were routinely tested for mycoplasma and found to be free of contamination.

[0131] H69, H69M, H69AR, H82, H446, H196, SHP-77, H187, H345, H524, H1436, H2081, C0I0668, H1694, H841 and DMS 114 were cultured in RPMI-1640 containing 10% fetal bovine serum (FBS, Hyclone), 2.5 g / L Glucose and IX penicillin / streptomycin (10,000 U / mL; Thermo Fisher Scientific). HEK293T was maintained in Dulbecco’s Modified Eagles Medium (DMEM) containing 10% FBS and IX penicillin / streptomycin. RPP-631 (RPP) SCLC mouse cell line was cultured in RPMI-1640 containing 10% FBS, IX penicillin / streptomycin, and HITES (IX insulin-transferrin-selenium, 10 nM 0-estradiol, and 10 nM hydrocortisone). RPE was cultured in DMEM / F-12, 10% FBS, 2.5 mM L-glutamine, 15 mM HEPES, 0.5 mM Sodium pyruvate and 0.01 mg / ml hygromycin. FC1010 was cultured in RPMI- 1640 with 15% FBS, 2 mM L- glutaminc, 1 mM Sodium pyruvate and IX penicillin / streptomycin. All cells were maintained in a humidified incubator with 5% CO2 at 37°C.

[0132] CRISPR-CAS9 GENE EDITING VECTORS

[0133] Target sequences of DHX9, MAVS, STING and RNA helicases (for screen) for CRISPR interference were designed using the single-guide RNA (sgRNA) designer (found on the world wide web at: portals.broadinstitute.org / gpp / public / analysis-tools / sgma-design).

[0134] Sequences of sgRNA for human DHX9 were 5’-GGGGTAGAATCTGATACCGA-3’ (sgDHX9)(SEQ ID NO: 1), and 5’-CAAAACATTATACTGGCATG-3’ (sgDHX9 #2) (SEQ ID NO: 2). sgRNA sequences of human MAVS and STING were 5’- CACCGACTGGAGCAGATGATAGGCT-3’(SEQ ID NO: 3) and 5’- GGTACCGGGGCAGCTACTGG-3’(SEQ ID NO: 4), respectively. sgRNA sequences of RNA helicase genes are listed in Table 1. sgRNA from the Gecko library v2 was used as a dummy sgRNA (5’-ATCGTTTCCGCTTAACGGCG-3’) (SEQ ID NO: 5) for Scramble control (Scr).

[0135] LentiCRISPR v2 vectors were cloned as previously described (68,69). Lentiviral plasmids were transduced into HEK293T cells along with pMD2.G and psPAX2 using X-treme Gene 9 DNA Transfection Reagent (Roche; no. XTG9-R0) according to the manufacturer’s instructions. The 48-hour supernatant of transduced HEK293T cells was collected and filtered with a 0.45 pm filter.

[0136] TRANSDUCTION OF SCLC CELLS WITH LENTIVIRAL VECTORS

[0137] Culture supernatant from HEK293T cells containing vims particles was applied to SCLC cell lines, with 8 pg / mL polybrene (Santa Cruz Biotechnology; no. sc-134220). Cells were centrifuged with virus at 2000 rpm at 37 °C for 2 hours. The cells were incubated at 37 °C in 5% CO2 for 24 hours, and then the media were replaced with complete RPMI-1640. Virus-infected cells were selected for 48 hours using 1.0 pg / mL puromycin (Gibco) or 5.0 pg / mL blasticidin (Gibco), from 48 hours post-infection. siRNA TRANSFECTION

[0138] DHX9 siRNAs (no. s4020, s4021) were purchased from Thermo Fisher Scientific, with s4021 used in most experiments. A nonspecific control siRNA duplex (siCtrl) was purchased from Thermo Fisher Scientific (Silencer Select Negative Control No. 1 siRNA, no. 4390844). Other siRNAs used in this study included AURKA (no. sl97), NMT1 (no. s9602), RHOQ (no. s23825), CDK9 (no. s2835) and CDK7 (no. s2829) (Thermo Fisher Scientific). siRNAs were transfected using Lipofectamine RNAiMAX Transfection Reagent (Thermo Fisher Scientific; no. 13778500) according to the manufacturer’s instructions.

[0139] IMMUNOHISTOCHEMICAL (IHC) STAINING

[0140] Mouse tumor tissues were collected and fixed in 10% neutral buffered formalin for 24 hours, dehydrated, and embedded in paraffin. Hematoxylin and eosin (H&E)-stained sections were used for morphologic evaluation purposes and 5 -pm unstained sections for IHC studies.

[0141] IHC staining was earned out on a VENTANA Discovery XT automated staining instrument (Ventana Medical Systems) using VENTANA reagents according to the manufacturer's instructions. Briefly, slides were de-paraffinized using EZ Prep solution (no. 950- 102) for 16 min at 72 °C. Epitope retrieval was accomplished with CC1 solution (EDTA, pH 9.0.; no. 950-224) at high temperature (eg, 95-100 °C) for 32 min.

[0142] Rabbit primary antibodies: anti-mouse CD8 (1:50, Cell Signaling Technology; no. 98941), anti-mouse CD45 (1:50, Cell Signaling Technology; no. 70257) and F4 / 80 (1:800, Cell Signaling Technology; no. 70076) tittered with a TBS antibody diluent into user fillable dispensers for use on the automated Stainer. Immune complex was detected using the Ventana OmniMap anti-Rabbit detection kit (no. 760-4311) and developed using the VENTANA ChromMap DAB detection kit (no. 760-159) according to the manufacturer’s instructions. Slides were then counterstained with hematoxylin II (no. 790-2208) for 8 min, followed by Bluing reagent (no. 760-2037) for 4 min.

[0143] The slides were then dehydrated with ethanol series, cleared in xylene, and mounted. As a negative control, the primary antibody was replaced with normal rabbit IgG to confirm absence of specific staining.

[0144] QUANTITATIVE IMAGE ANALYSIS

[0145] Immuno-stained slides were scanned using an Aperio ScanScope CS 5 slide scanner (Aperio). Scanned images were then viewed with Aperio's image viewer software (ImageScope, version 11.1.2.760, Aperio). Selected regions of interest were outlined manually by a pathologist. The positive percentage score for CD8, CD45 and F4 / 80 was quantified using the Aperio V9 algorithm.

[0146] IMMUNOBLOTTING

[0147] Protein was extracted from cell lines with Pierce RIPA Buffer (Thermo Fisher Scientific; no. 89900) and quantified by Pierce BCA Protein Assay Kit (Thermo Fisher Scientific; no. 23225). Protein extracts were subjected to polyacrylamide gel electrophoresis using the 4%-12% NuPAGE gel system (Invitrogen), transferred to PVDF membranes (Millipore). Transferred protein was immunoblotted using antibodies against TBK1 (no. 3013), S172 pTBKl (no. 5483), IRF3 (no. 4302), S396 pIRF3 (no. 4947), S345 pCHKl (no. 2348), T68 pCHK2 (no. 2197), S139 pH2AX (no. 9718), Cleaved PARP (no. 5625), STING (no. 13647), MAVS (no. 3993) (Cell Signaling Technology), mouse DHX9 (no. ab26271), V5 tag (no. ab27671), -actin (no. abl85228) (Abeam) and human DHX9 (no. sc-137232) (Santa Cruz Biotechnology) after blocking with LICOR Blocking Buffer (LICOR; no. 927-60001). Secondary antibodies were purchased from LICOR Biosciences: IRDye 800CW Goat anti-Mousc IgG (H+L) (no. 926-32210) and IRDye 800CW Goat anti-Rabbit IgG (H+L) (no. 926-32211). LICOR Antibody Diluent (LICOR; no. 927-65001) was used to dilute primary and secondary antibodies. Pho sho- specific antibodies were diluted in CanGet Signal Immunoreaction Enhancer Solutions (TOYOBO; no. NKB-101) 1 (for primary) and 2 (for secondary). Imaging of blots was performed using the LICOR Odyssey system.

[0148] IMMUNOCYTOCHEMISTRY

[0149] Cells were plated on BioCoat Culture Slide (Corning; no. 354630) after trypsinization, and incubated overnight. To detect expression of proteins in nuclei and micronuclei, cells were fixed with 4% paraformaldehyde (PFA) for 10 minutes and permeabilized with 0.5% Triton X- 100 for 15 minutes. After blocking with MAXblock Blocking Medium (Active Motif; no. 15252) for 1 hour at 37 °C, cells were stained overnight at 4°C with primary antibodies and for 1 hour at room temperature with secondary antibodies. Coverslips were mounted with ProLong Gold Antifade reagent with DAPI (Invitrogen; no. P36935), and immunofluorescent visualization of nuclei was counterstained with DAPI in the reagent. Immunofluorescence was detected using Leica SP8 confocal microscope and analyzed with ImageJ software. Antibody against dsRNA (J2; 1:200, no. 10010200) was purchased from SCICONS, S139 pH2AX (1:1000, no. 9718) and cGAS (1:100, no. 79978) were purchased from Cell Signaling Technology, dsDNA antibody (1:100, no. ab27156) was from Abeam. For RNase III treatment, cells were treated with 20 U / mL of RNase III for 30 minutes at 37°C before fixation.

[0150] To detect DNA / RNA hybrid, cells were fixed with ice-cold, 100% methanol for 20 minutes at -20°C, and permeabilized with 0.5% Triton X-100 for 15 minutes. Cells were incubated with antibody against DNA / RNA hybrid (Sigma- Aldrich; no. MABE1095, clone S9.6) at 1:50 dilution overnight at 4°C, followed by secondary donkey anti-mouse IgG (H+L) conjugated with Alexa Fluor 594 at 1:500 dilution for 1 hour at room temperature. For RNase H treatment, cells were incubated with 120 U RNase H (Takara Bio; no. 2150A) for 4 hours in RNase H buffer (40 mM Tris-HCl pH8.0, 4 mM MgCh, 1 mM dithiothreitol, 4% glycerol, and 0.003% BSA) before immunocytochemistry assay.

[0151] ELISA

[0152] IFN-P ELISA (R&D Systems; no. DIFNB0) and 2’,3’-Cyclic GAMP (cGAMP) ELISA (Arbor Assays; no. K067-H1) kits were used according to the manufacturer’s instructions. Conditioned media from cells cultured for 72 hours after seeding (for IFN-0), and cell lysates (for cGAMP) were collected and analyzed. For cells treated with siRNAs, 72 hours-culturc conditioned media from the cells were collected at Day 6 after transfection.

[0153] For cytokine array assay, Proteome Profiler Human Cytokine Array Kit (R&D Systems; no. ARY005B) was used according to the manufacturer’s instructions. Conditioned media was collected 72 hours after seeding the cells.

[0154] FLOW CYTOMETRY ANALYSIS

[0155] For detecting proteins on cell membrane, collected cells were washed with PBS and stained with anti-PD-Ll (BioLegend; no. 329718, isotype control: no. 400232), anti-HLA-A, B, C (Biolegend; no. 311410, isotype control: no. 400220) antibodies diluted with PBS containing 2% FBS at 2 p.g / mL. Stained cells were analyzed on a BD LSR II Flow Cytometer, fluorescence levels were compared with isotype control antibodies. Data were analyzed using the Flowlo software (TreeStar). Dead cells were excluded by staining with PI or Zombie NIR Fixable Viability Kit (BioLegend; no. 423106).

[0156] For intracellular flow cytometry, collected cells were fixed with 4% formaldehyde in PBS and permeabilized with 0.1% Triton X-100 in PBS. The cells were then incubated with primary antibodies, followed by secondary antibodies. Primary antibody against dsRNA (12; 1:200, no. 10010200) was purchased from SCICONS, S139 pH2AX (1:1000, no. 9718) was from Cell Signaling Technology. Isotype controls, normal mouse IgG2a (no. abl8413) and rabbit IgG (no. abl72730) were purchased from Abeam. Secondary donkey anti-mouse IgG (H+L) conjugated with Alexa Fluor 488 (no. A32766) and donkey anti-rabbit IgG (H+L) conjugated with Alexa Fluor 488 (no. A32790) were purchased from Thermo Fisher Scientific. Dead cells were excluded by pre-fixation staining with Zombie NIR Fixable Viability Kit.

[0157] MURINE SCLC GEMM, CELL LINE DERIVATION, AND TUMOR IMPLANTATION STUDIES

[0158] The RPP-631 (RPP) SCLC mouse cell lines were established in the laboratory of Dr. Matthew G. Oser (Dana-Farber Cancer Institute, Boston, MA, USA), which were originally derived from SCLC tumors that were generated in LSL-Cas9 C57BL / 6 mice that were intratracheally injected with AAV that encode Cre -recombinase and sgRNAs targeting Trp53, Rbl, and Rbl2 (RPP) genes (54). Histopathology of the tumor from which the cell lines were derived showed small cell lung cancer. To generate the syngeneic mouse tumor model, 8.0 x 106RPP cells, which stably expressing SMARTvcctor tct-induciblc Dhx9 shRNA (horizon; no. V3SM 11253) or control shRNA (horizon; no. VSC11652), were subcutaneously implanted into the flank of C57BL / 6 mice, after mixing with Matrigel (Corning; no. 354234) at 1:1 ratio. Doxycycline water (5% sucrose with 2 mg / mL of doxycycline; Sigma) was provided to all the groups to induce knockdown of Dhx9 gene, once palpable tumor formation was confirmed. The doxycycline water was changed every other day. Tumor size was measured every 2-3 days by digital caliper. Tumor volumes were calculated using the formula: volume = (length x width2) / 2. Both Female and male C57BL / 6 mice (Jackson Laboratories) of 6-8 weeks old were used for the transplantation studies.

[0159] TUMOR COLLECTION AND SURVIVAL ANALYSIS

[0160] Mice were euthanized with CO? and their tumors were quickly extracted, washed in PBS, and minced using a sterilized razor blade. For tumor IHC staining of CD8, CD45 and F4 / 80, and flow cytometry analysis of infiltrated immune cells, all mice were euthanized on day 32 postinoculation of RPP cells. Collected tumor tissues were fixed in 10% phosphate-buffered formaldehyde for IHC and were processed for flow cytometry analysis using the Tumor Dissociation Kit, mouse (Miltenyi; no. 130-096-730) and GentleMACS Dissociator (Miltenyi; no. 130-093-235), according to manufacturer’s instructions, followed by washing and filtering with a 70 pm cell strainer.

[0161] For combination treatments, mice were administered 200 pg rat IgG2a isotype control (BioXCell; no. BP0089) or anti-PD-1 antibody (BioXCell; no. BP0146) via i.p. injection on days 17, 19, 21, 23 and 25 post-inoculation of RPP cells. Doxycycline water was provided once palpable tumor formation was confirmed, to induce knockdown of Dhx9 gene. Tumor size was measured every 2-3 days by digital caliper, and mouse survival was monitored with tumor volume exceeding 1000 mm3, weight loss >15%, and decreasing behavioral conditions considered as endpoints.

[0162] QUANTITATIVE PCR WITH REVERSE TRANSCRIPTION (QRT-PCR)

[0163] Total RNAs were extracted using the RNeasy Mini Kit (Qiagen; no. 74106) according to the manufacturer’s instructions. 1 pg of extracted RNA was used to generate cDNA with the SuperScript III First-Strand Synthesis SuperMix for RT-qPCR kit (Thermo Fisher Scientific; no. 18080-044). qRT-PCR of the indicated genes (Table 2) was performed using Power SYBR Green PCR Master Mix (Applied Biosystems; no. 4367659) and the Applied Biosystems QuantStudio 6 Pro Real-Time PCR System and software. The relative expression was normalized with the expression of the housekeeping genes 36B4 (for human cells) or Actb (for mouse cells), and analyzed with the -AACt relative quantification method.

[0164] RNA-SEQ

[0165] Total RNAs were extracted and purified using the RNeasy Mini Kit (Qiagen; no. 74106) from NC1-H196, NC1-H446 and NC1-H82 cells, which were infected with lentivirus containing Scramble or sgDHX9 vector, at day 7 post- selection. Using a 2100 Bioanalyzer RNA 6000 Nano assay (Agilent), quality of RNA was assessed. RNA concentration was measured using a Qubit 2.0 Fluorometer (Life Technologies). Illumina sequencing libraries were constructed using the NEBNext Ultra II Directional RNA Library Prep Kit for Illumina (NEB), and sequenced on Illumina NovaSeq 6000 by pair-end sequencing with a read length of 2 x 150 bp, by Novogene.

[0166] Expression levels for each gene were quantified from the sequencing data using Kallisto (70). The data were then summarized using the tximport package (ver. 1.18.0) of R software (ver. 4.0.3) and RStudio (RStudio), and scaledTPM counts were used for further analysis as expression values. GSEA was performed to identify gene signatures which are upregulated and downregulated in sgDHX9 cells compared to Scramble, or DHX9lowtumors compared to DHX9hlghtumors (for data from database).

[0167] RNA IMMUNOPRECIPITATION AND SEQUENCING (RIP-SEQ)

[0168] Scramble or sgDHX9 H446 cells (2.0 x 107) were harvested, and RNA immunoprecipitation (RIP) was conducted using a Magna RIP RNA-Binding Protein Immunoprecipitation kit (Sigma- Aldrich; no. 17-700), according to the manufacturer’s instructions. Briefly, cell pellets were lysed in RIP lysis buffer, followed by incubation with RIP buffer containing magnetic beads conjugated with J2 (SCICONS; no. 10010200) or isotype control (abeam; no. abl8413) antibody at 4°C overnight. Samples were then incubated with proteinase K, and immunoprecipitated RNAs were recovered by phenol:chloroform:isoamyl alcohol purification. RNA was quantified using a QuantiFluor RNA System (Promega; no. E3310) and assessed for quality with the 2100 Bioanalyzer RNA 6000 Nano assay (Agilent) before library generation. Ribosomal RNA (rRNA) was removed using NEBNext rRNA Depletion Kit v2 (NEB; no. E7400L) according to the manufacturer’s instructions.

[0169] Illumina sequencing libraries were prepared by Novogene, with the NEBNext Ultra II RNA Library Prep Kit for Illumina (NEB), according to the manufacturer’s instructions. Libraries were analyzed for insert size distribution using the 2100 Bioanalyzer RNA 6000 Nano assay (Agilent). Libraries were quantified using the Qubit 2.0 Fluorometer (Life Technologies), and sequenced on Illumina NovaSeq 6000 by pair-end sequencing with a read length of 2 x 150 bp, by Novogene.

[0170] For analysis of RE expression from RIP-seq data, the trimmed FASTQ reads by TrimGalore (version 0.6.4) were mapped against H. sapiens UCSC hgl9 using the Bowtie 2 (version 2.3.5) alignment software (71). The counts of RE were calculated by RepEnrich2 (72) using default settings, and were normalized by one million reads as CPM (counts per million). The heatmap was visualized using the “ggplot2” package of R software (version 4.0.5). RIP-qRT-PCR

[0171] Scramble or sgDHX9 H446 cells (5.0 x 106) were harvested, and cytoplasmic fractions were extracted using the Nuclear Extract Kit (Active Motif; no. 40010), according to the manufacturer’s instructions. To isolate RNA, an equal volume of 70% ethanol was added to the cytoplasmic fractions, and then, according to the manufacturer’s instructions, purification was performed using the RNeasy Plus Mini Kit (QIAGEN; no. 74106). The total RNA was dissolved with 38 pL RNase-free H2O. Then 2 pL total RNA was used as input and the remainder divided into 2 tubes. 2 pg of J2 antibody (SCICONS; no. 10010200) and mouse control IgG2a (abeam; no. abl8413) were conjugated to 20 pL protein G agarose (Millipore; no. 16-266) per pulldown, by rotation overnight at 4°C. To digest single stranded RNA, 1 pL of RNase A (Sigma- Aldrich; no. R6513) was added to each tube and then mixed with 1 mL IP buffer (50 mM Tris-HCl [pH 7.4], 125 mM NaCl, 1 mM EDTA, 0.1% Triton X-100). The RNA samples were incubated with antibody-conjugated protein G agarose beads overnight at 4°C. Beads were washed with IP buffer three times, and then incubated in 50 pL proteinase K digestion solution (IX TE, 100 mM NaCl, 1% SDS, and 1 pL of 20 mg / mL Proteinase K solution (Thermo Fisher Scientific; no. AM2546)) for 20 min at 45°C to isolate RNA. After centrifugation, 50 pL of the supernatant was added to 300 pL Buffer RLT Plus from the RNeasy Plus Mini Kit (QIAGEN; no. 74106) to purify RNA. The final product containing dsRNA was denatured for 5 min at 95°C, followed by reverse transcription using qScript cDNA SuperMix (Quantabio; no. 95048), which contains both random primers and oligo(dT) primer. qRT-PCR was performed using the primers listed in Table 2, by Applied Biosystems QuantStudio 6 Pro Real-Time PCR System and software. DNA FIBER ASSAY

[0172] Cells were pulsed-labeled with 25 pM IdU (Sigma-Aldrich; no. 17125) for the first 30 minutes, followed by 250 pM CIdU (Sigma-Aldrich; no. C6891) for 30 minutes. The cells were trypsinized and resuspended in PBS, then diluted to the concentration of 1.0 x 105- 1.0 x 106cells / mL. At the end of an APS-coated glass slide (Matsunami; no. SUAPS1190), 2 pL of cell suspension was placed. After air drying for 8 minutes, 7 pL of fiber lysis solution (200 mM Tris- HC1 [pH 7.5], 50 mM EDTA, 0.5% SDS) was pipetted on top of the cell suspension and mixed gently. Cell lysis proceeded for 5 minutes. The slides were tilted at 15° to allow the DNA spread down to the bottom of the slide. Slides were air-dried for 15 minutes and fixed in methanol / acetic acid (3:1). After washing with distilled water, DNA was denatured in 2.5 M HC1 for 80 minutes. The slides were washed with PBS three times and blocked in 5% BSA in PBS for 1 hour. After blocking, the slides were incubated with primary antibodies, anti-IdU (BD; no. 347580) and anti-CldU (Abeam; no. ab6326) and followed by secondary antibodies, donkey antimouse IgG (H+L) conjugated with Alexa Fluor 488 (Thermo Fisher Scientific; no. A32766) and donkey anti-rat IgG (H+L) conjugated with Alexa Fluor 594 (Thermo Fisher Scientific; no. A21209).

[0173] PROLIFERATION ASSAY

[0174] Cells were seeded in 12-well plates at low density (2,500-10,000 cells / well), cultured in complete RPMI-1640 medium, in a humidified incubator with 5% CO2 at 37°C. After 2, 4, 6 and 8 days, cells were harvested and counted. VIABILITY ASSAY

[0175] Cells were plated in 96-well plates at low density (2,000-8,000 cells / well), cultured in complete RPMI-1640 medium, in a humidified incubator with 5% CO2 at 37 °C for 96 hours. Luminescent values of CellTiter-Glo Cell Viability assay (Promega; no. G7571) were obtained by the CLARIOstar Plus Microplatc Reader and software (BMG Labtcch).

[0176] CRISPR SCREEN AND ANALYSIS

[0177] On day 0 (day of infection), ~7.0 x 107(~1000 cells / sgRNA) H82 cells, which stably express Cas9, were resuspended in complete media with 10% FBS, 8 pg / mL polybrene at a concentration of 1.0 x 106cells / mL in 50 mL conical tubes, and the Human Brunello CRISPR knockout pooled library was added at an MOI of 0.3. The cells were then distributed onto low- adherence 6-well plates at a density of 2.0 x 106cells per well, and the plates were centrifuged at 2000 rpm for 2 hours. The following day (day 1), the virus was removed by changing media, and the cells were transferred to low-adherence 10 cm plates at a concentration of 0.4 x 106cells / mL. On day 4, the cells were plated in fresh media in the presence of 1 pg / mL puromycin and were selected for 72 hours.

[0178] Following completion of puromycin selection (day 7), resistant cells were replated with fresh media and grown in complete media until day 13. On day 13, 2.5 x 107cells were mixed with lentivirus containing Scramble or sgDHX9, with 8 pg / mL polybrene. The cells were then distributed onto low-adherence 6-well plates at a density of 2.0 x 106cells per well, and the plates were centrifuged at 2000 rpm for 2 hours. The following day (day 14), the virus was washed away, and the cells were transferred to low-adherence 10 cm plates. Until Day 23, the cells were grown in complete media, keeping a minimum of 2.5 x 107cells per group. The screen was performed in 3 biological replicates.

[0179] After completion of the screen, genomic DNA (gDNA) was isolated using Blood & Cell Culture DNA Midi Kit (Qiagen; no. 13343) according to the manufacturer’s protocol. To attach sequencing adaptors and barcode samples, PCR of gDNA was performed using Ex Taq DNA Polymerase (TaKara; no. RR001), as previously described (48). Samples were purified with Agencourt AMPure XP SPRI beads according to the manufacturer’s instructions (Beckman Coulter; no. A63880). Purified DNA samples were sequenced on a Nextseq2000 (Illumina). The sgRNA read count and hits calling were analyzed by MAGeCK vO.5.7 algorithm (73). Each gene symbol of enriched or depleted sgRNAs in DHX9-loss population was mapped to Gene Ontology resources (found on the world wide web at: geneontology.org / ) to rank gene ontology terms. Human Brunello CRISPR knockout pooled library (48) was a gift from Drs. David Root and John Docnch (Addgcnc; no. 73178).

[0180] PAN-CANCER ANALYSIS OF TCGA DATASET

[0181] TCGA pan-cancer gene expression with patient annotation datasets were retrieved from the Genomic Data Commons (GDC) of the National Cancer Institute (found on the world wide web at:gdc.cancer.gov / about-data / publications / pancanatlas). Signature scores of different tumor types were calculated in GSVA using the “z-score” method (74). Spearman correlation and multiple testing corrections were done in R software (ver. 4.0.3). STATISTICAL ANALYSES

[0182] All graphs depict mean ± SEM unless otherwise indicated. Tests for differences between two groups were performed using two-tailed unpaired Student’s t-test or Mann-Whitney test. Two-way analysis of variance (ANOVA) was performed where applicable using Tukey’s multiple comparison test. Values of p < 0.01-0.05 (*), p < 0.001-0.01 (**), p < 0.001-0.0001 (***), or p < 0.0001 (****) were considered significant. GraphPad Prism7 was used for statistical analysis of experiments, data processing, and presentation.

[0183] The following examples are provided to illustrate certain embodiments of the invention. They are not intended to limit the invention in any way.

[0184] Example I: Identification of DHX9 as a Therapeutic Target

[0185] Activating innate immunity in cancer cells through cytoplasmic nucleic acid sensing pathways, a phenomenon known as “viral mimicry”, has emerged as an effective strategy to convert immunologically “cold” tumors into “hot” tumors with increased sensitivity to anticancer therapies. Through a curated CRISPR-based screen of RNA Helicases, we identified DExD / H-box helicase 9 (DHX9) as a potent repressor of double-stranded RNA (dsRNA) accumulation in cold tumors, including small cell lung cancer (SCLC) cells. Depletion of DHX9 induced an accumulation of cytoplasmic dsRNA, which mainly derived from repetitive sequences of short interspersed nuclear elements (SINEs) and long interspersed nuclear elements (LINEs), and triggered a tumor-intrinsic type I IFN response in SCLC cells. Intriguingly, ablating DHX9 also induced the aberrant accumulation of R-loops (DNA / RNA hybrids), which resulted in an increase of DNA damage-derived cytoplasmic DNA and replication stress in tumor cells, selectively killing them. In vivo, DHX9 deletion in SCLC tumor cells promoted a decrease in tumor growth while inducing a more immunogenic tumor microenvironment (TME), which exhibited enhanced responsiveness to immune checkpoint blockade (ICB) therapies. These findings indicate that DHX9 is a crucial repressor of tumor-intrinsic innate immunity and replication stress and represents an unexplored target for SCLC and other “cold” tumor types where replication stress and genomic instability contribute to pathology.

[0186] The data demonstrate that DHX9 can be suppressed resulting in increased levels of viral mimicry-inducing agents and factors, e.g., double- stranded RNAs and R-loops in cancer cells, thereby providing a novel target for enhancing antitumor immunity and boosting immunotherapy in “cold” tumors, such as SCLC.

[0187] DHX9 suppresses double-stranded RNA (dsRNA) accumulation in SCLCs

[0188] Some RNA helicases have the ability of unwinding dsRNA structures. Thus, we hypothesized that targeting RNA helicases would be an effective and efficient strategy to induce innate immune response through dsRNA accumulation in cold tumors.

[0189] To identify RNA helicases whose function are critical for dsRNA unwinding in SCLC cells, we performed a curated flow cytometry-based CRISPR screen that detected levels of intracellular dsRNA in SCLC cancer cells (Fig. 1A and IB). We included sgRNAs targeting 32 different genes (Table 1) which have been reported to work as RNA helicases and to contribute to RNA-related functions including splicing, nuclear export, cytoplasmic transport, translation regulation and mRNA degradation. The H446 SCLC cell line was used because it showed the potential to strongly increase endogenous dsRNA after treatment with Decitabine, a DNA- methyltransferase inhibitor reported to induce dsRNA accumulation in cancer cells (Fig. 2A).

[0190] Among the top hit candidates of the screen (Fig. IB), sgRNA-mediated depletion of the DExD / H-box helicase 9 (DHX9) significantly increased dsRNA. Intriguingly, the tumor type with higher expression of DHX9 levels among all the tumor types registered in Cancer Cell Line Encyclopedia (CCLE) database was SCLC (Fig. 1C). Indeed, most SCLC cell lines exhibited high expression of DHX9 at protein level (Fig. 2B). To investigate the relevance of DHX9 in human SCLC and other tumor types, we interrogated transcriptomic data from public cancer datasets, including The Cancer Genome Atlas (TCGA). Importantly, DHX9 overexpression was observed in many human lung cancer types compared to normal lung, and SCLC showed the strongest DHX9 expression among all the lung cancer subtypes (Fig. ID), consistent with the CCLE analysis (Fig. 1C). Together, these results indicated that DHX9 would be a promising target of SCLC.

[0191] Next, we sought to investigate the relevance of DHX9 expression on patient survival. Kaplan-Meier analysis of lung tumor patients, as well as other tumor types including ovarian and breast cancers, revealed that high DHX9 expression was associated with poor patient prognosis (Fig. IE; Fig. 2C).

[0192] DHX9 binds directly to short interspersed nuclear element (SINE) Alu elements, which could be a source of dsRNA and circular RNA (circRNA). This implicates DHX9 as important regulator of cellular dsRNA levels. To validate the finding of the screen, we depleted DHX9 by two different sgRNA sequences (sgDHX9 and sgDHX9 #2) in H446 cells (Fig. IF) and tested dsRNA accumulation by immunofluorescence (IF) staining using a dsRNA-specific antibody (J2 antibody). Cells lacking DHX9 showed an accumulation of cytoplasmic dsRNA, and the J2 signal was efficiently diminished by dsRNA-specific RNase III treatment, suggesting that the J2 antibody is specifically recognizing dsRNAs (Fig. 1G). We depleted DHX9 in additional SCLC cell lines (H196, H82 and DMS-114) and confirmed accumulation of dsRNA by intracellular’ flow cytometry in all the SCLC cell lines tested (Fig. 2D and 2E).

[0193] The source of immunogenic dsRNAs generated in cancer cells treated with certain epigenetic inhibitors appear to be endogenous retroviral elements (EREs) subtypes, including SINEs, long interspersed nuclear elements (LINEs) and long terminal repeats (LTRs). To identify the genomic sources of dsRNA accumulated in DHX9 depleted cells, we used J2 antibody to immunoprecipitate dsRNAs in these SCLC cells and performed RNA Immunoprecipitation Sequencing (RIP-seq). Sequencing J2-enriched RNAs showed that the dsRNAs derived from LINE family were the most strongly increased in sgDHX9 cells (Fig. 1H), indicating that DHX9 plays an important role on unwinding LINE-derived dsRNA structures in SCLC cells.

[0194] Also, dsRNAs from SINE and LTR families were significantly increased in sgDHX9 cells (Fig. 1H; Fig. 2F). Furthermore, I2-RIP experiments followed by quantitative RT-PCR (qRT-PCR) analysis confirmed that DHX9 depletion increased the dsRNAs derived from various EREs subfamilies, including SINEs (Alu), LTR-ERVs (HERV-K) and satellite repeats (SAT III), as well as LINEs (LI ORFs) (Fig. IT). Together, these results demonstrate that DHX9 suppresses EREs-dcrivcd dsRNA accumulation by unwinding dsRNA in SCLC cells.

[0195] DHX9 depletion induces IFN response in SCLCs

[0196] To investigate the biological impact of dsRNA accumulation in SCLC cells, we performed RNA sequencing (RNA-seq) on Scramble versus sgDHX9 SCLC cells. Consistently, Gene Set Enrichment Analysis (GSEA) revealed that DHX9 depletion induced the upregulation of gene expression pathways associated with immune and inflammatory responses and cytokine activity (Fig. 3A and 3B). Indeed, sgDHX9 cells showed increased expression of many Interferon Stimulated Genes (ISGs) by RNA-seq (Fig. 4A). We validated this finding using qRT- PCR, and confirmed that multiple ISGs (IFNB, CXCL10, CXCL11, CCL2), as well as nuclear factor kappa B (NF-kB)-responsive genes (TNFA, IL1B, RELB) were strongly induced by DHX9 loss, enhancing the activation of an antiviral transcription program in response to DHX9 depletion (Fig. 3C; Fig. 4B; Table 2). sgDHX9 cells also showed increased levels of phospho (p-)IRF3 and p-TBKl (Fig. 3D), markers of innate immunity which are able to activate a type I IFN response. Indeed, we were able to readily detect a significant increase in the secretion of IFN-P into the culture medium after DHX9 depletion (Fig. 3E), as well as the secretion of multiple other cytokines and chemokines detected by Luminex multiplex assay (Fig. 3F; Fig. 4C).

[0197] To further explore the potential impact of DHX9 loss on anti-tumor immunity, we analyzed cell surface expression of HLA-A, B, C on SCLC cells by flow cytometry, revealing a strong induction of these MHC class I molecules after DHX9 depletion (Fig. 3G; Fig. 4D).

[0198] Furthermore, DHX9 loss also induced cell surface PD-L1 expression (Fig. 3H; Fig. 4E) although it has been reported that more than 80% of SCLC tumors maintain expression of PD-L1 at low levels at steady state. Together, these results indicate that DHX9 depletion activates dsRNA-sensing pathways and demonstrate that targeting DHX9 provides heretofore unexplored strategies to activate anti-tumor immune pathways which promote cell killing in SCLC. DHX9 depletion causes R-Ioop accumulation, DNA damage and cGAS-STING pathway activation

[0199] Intriguingly, we found that DHX9 depletion led not only to an increase in dsRNAs and an IFN response, but also to an upregulation of DNA damage-associated gene signatures and downregulation of cancer-associated pathways, when examined by analysis of RNA-seq data from DHX9 depleted SCLC cells (Fig. 3A and 5A). We validated by qRT-PCR that genes involved in DNA damage were upregulated in DHX9 depleted cells, while cancer-associated genes, such as genes related to DNA replication, were downregulated (Fig. 5B; Fig. 6A), indicating that conflicts in completing DNA replication in sgDHX9 cells might result in DNA damage. Indeed, IF and flow cytometry analysis showed that the DNA double- stranded breaks (DSBs) marker p-H2AX was strongly induced in DHX9 depleted cells (Fig. 5C and 5D; Fig. 6B)

[0200] DHX9 mediated unwinding of R-loops, or DNA / RNA hybrid structures, and excessive R-loop formation can trigger genomic instability and replication stress by impairing replication fork progression and inducing DSBs. This suggests that aberrant R-loop accumulation might be a source of DNA damage in DHX9 depleted cells. To test this hypothesis, we first compared distribution and strength of R-loops (stained with S9.6 DNA / RNA hybrid antibody) in Scramble versus sgDHX9 cells. We observed an increased accumulation of R-loop structures in sgDHX9 cells, which were efficiently degraded by RNase H treatment (Fig. 5E; Fig. 6C). As we expected, loss of DHX9 also activated the DNA replication checkpoint pathway, as indicated by increased p-CHKl and p-CHK2, and apoptosis, as indicated by cleaved PARP, as well as p- H2AX (Fig. 5F).

[0201] To test whether accumulation of R-loops is directly responsible for genomic instability and DNA damage, SCLC cells were transfected with a vector expressing RNase Hl, which preferentially degrades the RNA portion of the R-loops. Exogenous RNase Hl expression suppressed the increase of p-CHK2 and cleaved PARP in sgDHX9 cells, supporting the finding that genomic instability and DNA damage in cells deficient for DHX9 was caused by accumulation of R-loops (Fig. 6D). Next, we sought to determine the impact of DHX9 depletion on DNA replication stress by performing the DNA fiber assay, which enables to analyze replication fork progression and processivity on single DNA molecules visualized by immunofluorescence. We found that the proportion of stalled forks significantly increased in DHX9 depleted cells (Fig. 5G), indicating that R-loop accumulation in cells deficient for DHX9 triggers replication stress contributes to genomic instability.

[0202] Replication stress inducers including PARP and CHK1 inhibitors have been reported to activate innate immune responses through cGAS-STING pathway in SCLC cells. Thus, we next interrogated whether DHX9 loss-induced replication stress contributed to the activation of cGAS-STING pathway and found that DHX9 depletion increased the number of cells with micronuclei that stained positive for cGAS and dsDNA, compared to Scramble cells (Fig. 5H). To measure cGAS activation, we then quantified by ELISA the levels of the second messenger protein, 2’3’-cyclic GMP-AMP (cGAMP). This assay revealed that DHX9 depletion also elicited substantial increase in cGAMP production (Fig. 51), indicating that the cGAS-STING pathway was activated. Altogether, these data indicate that the R-loops generated after DHX9 loss in SCLC cells promotes DNA replication stress and DSBs, which contributes to the activation of an innate immune response.

[0203] DHX9 loss triggers IFN signaling through dsRNA and dsDNA antiviral sensing pathways in SCLC cells

[0204] Based on the data above and that both innate immune responses and genomic instability can promote growth arrest and cell death, we evaluated the effects of DHX9 depletion on SCLC cell proliferation and viability. We found a dramatic decrease in cell proliferation in all SCLC cells tested, including chemo-resistant SCLC cells, as well as increased apoptosis (Fig. 7A and 7B; Fig. 8A). Notably, DHX9 depletion had minimal effects on the proliferation and apoptosis of normal cells (Fig. 7C and 7D), and did not induce R-loop accumulation, nor increased the expression of p-CHK2, p-H2AX or cleaved PARP, supporting the conclusion that DHX9 depletion has cancer cell-specific effects (Fig. 8B and 8C). Most SCLCs lack functional TP53 and RBI, which are known to be drivers of genomic instability and replication stress. Accordingly, other targets of genomic instability, such as DHX9, can be targeted to selectively accelerate replication stress-induced DNA damage in those tumors.

[0205] DHX9 depletion caused dsRNA and dsDNA accumulation in SCLC cells (Fig. 1G and Fig. 2E; Fig. 5H), which induces activation of dsRNA-MDA5-MAVS and dsDNA-cGAS- STING pathways. Thus, to determine whether dsRNA and dsDNA sensing pathways are responsible for inducing antiviral innate immunity after DHX9 depletion, we performed CRISPR-mediated depletion of MAVS and STING combined with transfection of siCtrl or siDHX9. Notably, double- KO (dKO) of MAVS and STING was able to abrogate the induction of multiple ISGs (Fig. 7E) and IFN-0 protein secretion (Fig. 7F) after DHX9 depletion. Additionally, it completely abrogated the phosphorylation of TBK1 and IRF3 induced by DHX9 loss (Fig. 7G). Furthermore, dKO of MAVS and STING partially rescued the effect of DHX9 depletion on cell proliferation (Fig. 7H). Together, these data display the role of dsRNA and dsDNA sensing pathways on the antiviral immune response and cell death induced by DHX9 depletion. However, the observed partial rescue of cell proliferation also indicates that there may be redundancy in dsRNA / dsDNA-recognition pathways or that other pathways might contribute to DHX9 depletion-induced cell death.

[0206] CRISPR screen identifies modulators of sensitivity and resistance to DHX9 loss

[0207] To interrogate the mechanisms by which DHX9 loss induces cell death in SCLC, we performed a genome- wide, in vitro pooled CRISPR screen in SCLC cells to identify the genes whose deletion would rescue or increase the lethality observed following DHX9 depletion. Briefly, cells were transduced with the Brunello Human CRISPR Knockout Pooled Library (48) and selected in the presence of puromycin for efficient lentivirus transduction. Then, cells were transduced with either Scramble control or sgDHX9 vectors. Cells were further propagated, and genomic DNA was extracted to identify sgRNAs enriched and depleted in DHX9 depleted cells compared to control (Fig. 9A). This CRISPR screen identified multiple genes whose ablation resulted in resistance or sensitivity to DHX9 loss-induced lethality, and consequent enriched or depleted sgRNAs representation in sgDHX9 cells (Fig. 9B).

[0208] As expected, gene ontology (GO) analysis of the list of genes targeted by sgRNAs depleted in sgDHX9 cells revealed gene signatures related to cellular stress, DNA damage and DNA repair, indicating that loss of these genes increased the sensitivity of SCLC cells to DHX9 loss -dependent cell death (Fig. 9C). Indeed, siRNA knockdown (KD) of AURKA, NMT1 and RHOQ genes, included in the top hits, accelerated cell death caused by siDHX9 (Fig. 10A). Conversely, the screen also identified genes whose ablation resulted in resistance to DHX9 loss- induced lethality (sgRNAs enriched in DHX9 depleted population). Functional annotation of these genes revealed a striking enrichment for factors that are involved in RNA metabolic process, transcription, ribosome biogenesis and cell cycle checkpoint signaling (Fig. 9D). One of the top sgRNAs enriched in DHX9 depleted cells were sgRNAs targeting CDK9 (Fig. 9B), a kinase essential for transcriptional elongation. sgRNAs targeting CDK7, also involved in transcription elongation by phosphorylating CDK9, were also enriched in DHX9 depleted population (Fig. 9B). Importantly, conflicts between transcription and DNA replication represent a significant cause of replication stress. These conflicts may result from the co-transcriptional occurrence of R-loops, which could impede the progression of DNA replication forks. Conflicts with transcription cause replication stress when DHX9 is depleted. This explains why sgRNAs targeting CDK9 were able to rescue DHX9 depiction-induced lethality in our screen. Thus, ongoing transcription and perhaps the resulting R-loop formation represents a prerequisite for impaired DNA replication upon DHX9 depletion leading to replication stress, DNA damage and cell death. Importantly, this also explains why SCLC cells are particularly vulnerable to DHX9 deletion. Almost all SCLC tumors have loss of function mutations in TP53 and RBI genes, which are linked with replication stress and genomic instability. A major source of replication stress in SCLC cells might arises from conflicts in transcription, as has been shown to occur in p53-deficient cells, indicating that therapeutic strategies that exacerbate this stress could selectively kill SCLC cells by replicative damage.

[0209] Importantly, we found that CDK9 inhibition with a potent and highly selective CDK9 inhibitor (BAY-1143572) rescued the decrease on cell proliferation and diminished the induction of p-CHK2, p-H2AX and cleaved-PARP observed after DHX9 depletion (Fig. 9E and 9F). Consistent with this, siRNAs targeting CDK9, as well as CDK7, recovered cell growth of DHX9 KD H82 cells (Fig. 10B), although CDK9 inhibition rather decreased cell growth of FC1010 normal fibroblast cells (Fig. IOC). Higher concentration of BAY- 1143572 inhibited the SCLC cell growth as well (Fig. 10D).

[0210] By performing the DNA fiber assay, we found that the proportion of stalled forks significantly increased in DHX9 depleted cells, and that CDK9 inhibition greatly reduced the proportion of stalled forks both in sgDHX9 and control cells (Fig. 9G), indicating that a major source of replication stress in SCLC cells arises from transcription-associated damage, which is further induced by DHX9 depletion (Fig. 9H). Together, these results illuminate a novel mechanism of DHX9 loss-induced cell death in cancer cells and implicate DHX9 as a therapeutic target for SCLC and other tumor types where genomic instability and replication stress contribute to pathology.

[0211] DHX9 depletion decreases tumor growth, induces immune cell infiltration and enhances responses to immune checkpoint blockade therapy

[0212] The induction of immune transcriptional programs by DHX9 loss (Figs. 6A-6C) suggests that such immune signaling may also impact the tumor microenvironment and provoke antitumor immune responses. To test this hypothesis, we generated RPP cells (a murine SCLC cell line generated by triple knockout of Tp53, Rbl, Rbl2 genes) stably expressing shRNA targeting either control (shCtrl) or Dhx9 (shDhx9) under control of a Tet-inducible promoter, into the flanks of C57BL / 6 mice (Fig. 11A). When tumors became palpable, the mice were fed with doxycycline water to induce DHX9 KD (Fig. 11B and 11C). We found that DHX9 knock down (KD) significantly decreased tumor volume and weight (Fig. 11D; Fig. 12A), while body weight of mice was not affected by the treatment (Fig. 12B). To better understand the in vivo immune consequences of DHX9 depletion in SCLC, we evaluated tumor infiltration of immune cells by flow cytometry analysis and observed a striking increase of immune cells expressing NK-1.1, CDl lb and F4 / 80 (Fig. HE). In addition, intratumoral cytotoxic CD8+ T cells were significantly increased in shDhx9 tumors, indicating a functional immune response, while CD4+ T cells did not change significantly (Fig. HF; Fig. 12C). Immunohistochemical (IHC) analysis also confirmed enhanced infiltration of CD8+ T cells, as well as F4 / 80+ macrophages in DHX9 depleted tumors (Fig. 11G). Thus, these data demonstrate that DHX9 KD enhances immunogenicity in vivo via bypassing a major pathway of cancer cell immune evasion.

[0213] As DHX9 loss induced IFN response and increased intratumoral infiltration of cytotoxic CD8+ T cells in SCLC tumors, we next sought to determine whether DHX9 depletion potentiates ICB-based immunotherapy in the SCLC syngeneic model. To accomplish this, we treated immunocompetent C57BL / 6 bearing RPP cells expressing DOX-inducible shCtrl or shDhx9 with either anti-PD-1 or isotype IgG control antibodies. While control RPP tumors were not very sensitive to PD-1 / PD-L1 blockade, DHX9 depletion dramatically enhanced the sensitivity of RPP tumors to anti-PD-1 therapy, resulting in increased mouse survival (Fig. 11H and HI). Taken together, these data indicate that DHX9 loss in SCLC tumors induces a robust antitumor immunity which ultimately leads to improved ICB treatment outcomes in immunologically cold tumors.

[0214] DHX9 is negatively correlated with immune signatures and associated with poor clinical outcomes in cancer patient datasets

[0215] To investigate whether DHX9 is associated with human tumorigenesis, we tested DHX9 protein expression by immunohistochemistry (IHC) in a human SCLC tissue microarray (TMA), including 40 SCLC tumor samples and normal lung controls (Fig. 13A). SCLC tumors exhibited higher staining intensity of DHX9 than normal counterparts (Fig. 13B). Notably, DHX9 expression increased in higher tumor stages, supporting the association of high DHX9 expression and poor patient prognosis in SCLC patients. We next interrogated transcriptomic data from a human SCLC dataset (47) to determine whether DHX9 expression is associated with the tumor immune microenvironment. GSEA analysis revealed that DHX9 expression inversely correlated with gene signatures involved in IFN-alpha response, as well as inflammatory response (Fig. 13C), confirming our results from in vitro and in vivo models. Indeed, many of the IFN-stimulated genes and NF-kB-responsive genes were tended to be highly expressed in DHX9lowgroup (Fig. 14A). We also interrogated TCGA to explore the broader relevance of DHX9 across human lung cancers and GO analysis revealed that gene signatures involved in IFN-related pathways were strongly upregulated in DHX9 low- expressing lung cancer tumors when compared to DHX9 high-expressing tumors (Fig. 13D), indicating that DHX9 is a crucial repressor of tumor-intrinsic innate immunity in human lung tumors. DHX9 expression was also inversely correlated with DNA damage response, consistent with activation of apoptosis and DNA replication checkpoint pathway in sgDHX9 cells in vitro (Fig. 5F). In addition, pan-cancer analysis exhibited a strong negative correlation between DHX9 expression and gene signatures of inflammatory response and innate immune response in a variety of tumor types (Fig. 13E). Consistent with this, GSEA analysis of pan-cancer RNA-seq data also showed an upregulation of interferon-alpha response and inflammatory response in DHX91OWtumors (Fig. 14B).

[0216] To anticipate the clinical relevance of targeting DHX9 for ICB therapy, we created a DHX9-depleted gene signature (Table 3), including genes related to DNA damage response and immune response and assessed its predictive value across public datasets of patients treated with anti-PD-1 -based or anti-CTLA-4- based ICB therapy (55-57). Notably, we found that high levels of the DHX9-depleted gene signature were associated with ICB response across most of the datasets, indicating that DHX9 inhibition sensitizes cancers to immunotherapy (Fig. 14F). In addition, estimated immune- score of tumor microenvironment in DHX9lowtumors tended to be higher than DHX9hlgh(Fig. 14C). Thus, these data provide evidence that DHX9 inhibition would sensitize tumors to ICB therapies, including immunologically cold tumors such as SCLC (Fig. 13G).

[0217] Discussion

[0218] Induction of innate immune responses in tumor cells has been gathering attention as a promising strategy to enhance ICB therapy response, especially in immunologically cold tumors. Here, we demonstrate that the RNA helicase DHX9 functions as a repressor of innate immune signaling and replication stress in cancer cells through unwinding of dsRNA and R-loop structures.

[0219] Our study revealed that depletion of DHX9 induces immunogenic dsRNAs, causing tumor cell-intrinsic antiviral signaling, and alters transcription and DNA replication in cancer cells to induce formation of R-loops, compromising genome stability and cell viability. Notably, triggering these tumor-intrinsic events greatly improves ICB treatment outcomes in immunologically cold tumors, such as SCLC. While prior studies have shown the potential of DHX9 to unwind dsRNA, DNA / RNA hybrids and other more complex nucleic acid structures, our findings indicate that dsRNAs and R-loops derived from DHX9 depletion directly contribute to induction of innate immune responses and replication stress in cancer cells, highlighting it as a novel target to enhance antitumor immunity and boost cancer immunotherapy. RNA-DNA hybrids derived from R-loops are sensed by cGAS and TLR3 and activate an innate immune response and apoptosis. The data presented here supports this finding, indicating that R-loop- inducing therapies arc a therapeutic strategy to effectively enhance antitumor immunity while concurrently induce DNA damage and compromise cell viability in cancer cells.

[0220] Almost all SCLCs exhibit inactivation of TP53 and RBI, both of which are key regulators of DNA damage and cell cycle checkpoint. Intriguingly, while genomic instability in SCLC is among the highest in all the cancer types, the tumor microenvironment tends to be immune desert, which explains why SCLCs are relatively unresponsive to ICB therapies and remains as a recalcitrant disease. The present invention addresses the urgent need to identify novel targets to improve outcomes of SCLC patients, as SCLC is considered the most lethal type of lung cancer.

[0221] Increasing evidence has now emerged indicating that targeting components of the DNA Damage Response (DDR) pathway, including PARP, CHK1 or WEE1, might be an effective strategy with significant antitumor effects in SCLC preclinical models. Importantly, DDR inhibitors potentiate ICB therapies in SCLC preclinical models by evoking beneficial IFN responses in the tumor microenvironment through accumulating cytosolic DNA and activating cGAS / STING and STAT1 pathways. Intriguingly, we found that DHX9 depletion in SCLC cells triggers innate immune signaling and DNA damage through dsRNA and R-loop production, at least partially generated from various EREs and genomic instability, representing a more robust viral mimicry-inducing agent or factor and a vulnerability preferentially in cancer cells under replication stress.

[0222] A major reason explaining the poor response of cold tumors to immunotherapy is the lack of an immunogenic tumor microenvironment, often linked to decreased CD8+ T cell abundance and a defective IFN signaling. Our data in immunocompetent SCLC mouse models indicate that the tumor-intrinsic effects of DHX9 depletion not only induced a significant decrease in tumor growth, but also led to the recruitment of multiple immune cells into the TME, including CD8+ T cells, dramatically sensitizing SCLC tumors to anti-PD-1 immunotherapy. These findings indicate that targeting DHX9 could convert immunologically cold tumors to hot, while concurrently promote cancer cell death.

[0223] Results from this study support efforts aimed at designing DHX9 inhibitors to exploit its therapeutic potential as anti-cancer therapy and to improve responsiveness to immunotherapy in immunologically cold tumors. While DHX9 is reported to be essential during embryonic development, reduced levels of DHX9 in adult mice did not cause any deleterious effects at the organismal level, although DHX9 loss was lethal to tumor cells. Our findings indicate that DHX9 depletion has minimal or no effects on the proliferation of normal cells, which also emphasize the contribution of intrinsic replication stress to DHX9 loss-related cell death in SCLC cells and indicates that targeting DHX9 provides an effective and realistic therapeutic strategy to treat cancers.

[0224] We have shown that DHX9 is overexpressed in certain cancers, including SCLC (Fig. ID), and that rapidly dividing tumor cells which are under replication stress are heavily vulnerable to DHX9 depletion compared to normal cells, highlighting a therapeutic window in which tumor growth control could be achieved with limited toxicity. Importantly, the fact that DHX9 depletion not only impact cancer cell survival but also potentiates antitumor immune responses to ICB therapy in SCLC models, opens a new range of therapeutic possibilities that will expand the breadth and depth of response to cancer immunotherapies.

[0225] In summary, our data identifies DHX9 as a novel vulnerability in immunologically cold tumors, especially in those whose genome is unstable, such as SCLC. We show that DHX9 is a crucial suppressor of dsRNA and R-loop accumulation, and the genetic perturbation of DHX9 leads to innate immune response activation, as well as DNA replication stress and DNA damage. Moreover, we found that these tumor-intrinsic effects can turn cold tumors into hot, dramatically enhancing ICB responsiveness and highlighting DHX9 as a therapeutic target to boost cancer immunotherapy in immunologically cold tumors.

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[0300] 73. Li W, Xu H, Xiao T, Cong L, Love MI, Zhang F, et al. MAGeCK enables robust identification of essential genes from genome-scale CRISPR / Cas9 knockout screens. Genome Biol 2014;15(12):554 doi 10.1186 / sl3059-014-0554-4.

[0301] 74. Hanzelmann S, Castelo R, Guinney J. GSVA: gene set variation analysis for microarray and RNA-seq data. BMC Bioinformatics 2013;14:7 doi 10.1186 / 1471-2105-14-7.

[0302] Example II - Small Molecule DHX9 modulators

[0303] EWS-FLI1 is an oncogenic protein that is present in several cancers, such as Ewing sarcoma. By binding to DHX9, EWS-FLI1 plays a key role in tumorigenesis by interacting with RNA polymerase. This interaction suggests that blocking EWS-FLIl’s binding to DHX9 would prevent tumorigenesis and induce replicative stress and tumor cell death. To test this hypothesis, H82 cells, H446 cells, H6AR cells, H196 cells, RPP cells and RPP-A cells were treated with TK216, a small molecule inhibitor of DHX9 + EWS-FLI1 binding, for 72 hours. After treatment, all cells showed a decrease in the relative number of surviving cells, however, human SCLC cells showed higher sensitivity to TK216. (FIG. 15)

[0304] Additionally, H196 cells were treated with DMSO, IpM TK216, or 5 pM cisplatin for 48 hours. After treatment, a western blot analysis was performed. (FIG. 16) Treatment with TK216 induced DNA damage and innate immune response in the SCLCs.

[0305] Lastly, RPP cells expressing shRNA targeting either control (shCt) or DHX9 (shDHX9), and RPP cells where the DHX9 was depleted by sgRNA sequences (sgDHX9) were compared by western blot analysis to wild type RPP cells treated with DMSO, 2pM TK216, or 5 pM cisplatin for 48 hours. Treatment with TK216 induced DNA damage in the mouse SCLC cells. (FIG. 17)

[0306] Example III - Method for Ameliorating Symptoms Associated with Cancer

[0307] The information herein above can be applied clinically to patients for therapeutic intervention. A preferred embodiment of the invention comprises clinical application of the information described herein to a patient. This can occur after a patient arrives in the clinic and presents with cancer-associated diseases or symptoms. The derived therapeutic doses of at least one VMIA for a human patient could be by those skilled in the art based on response rate. VMIAs, such as DHX9 modulators, have been shown to be well tolerated and the symptoms can be assessed using clinical scores criteria.

[0308] While certain features of the invention have been described herein, many modifications, substitutions, changes, and equivalents will now occur to those of ordinary skill in the art. It is, therefore, to be understood that the appended claims are intended to cover all such modifications and changes as fall within the scope of the invention.

Claims

What is claimed is:

1. A method for treating a subject having tumors comprising administering to said subject at least one viral-mimicry-inducing agent (VMIA) which increases cellular dsRNA levels, thereby provoking an antiviral response and rendering said tumor more sensitive to anti-cancer agents, which exceeds that observed in tumor cells not treated with the VMIA.

2. The method of claim 1, wherein said at least one VMIA is a DHX9 modulator.

3. The method of claim 2, wherein the DHX9 modulator is an inhibitor of EWS-FLI1 binding to DHX9.

4. The method of claim 2, wherein the DHX9 modulator is TK216 or YK-4-279.

5. The method of claim 1, wherein said VMIA is an inhibitory nucleic acid which repressesDHX9 expression and function in a cancer cell, selected from an siRNA, an antisense oligonucleotide, an shRNA, and a ribozyme having sufficient sequence homology to a DHX9 encoding nucleic acid to reduce expression of thereof in a target cell.

6. The method of claim 5, wherein said inhibitory nucleic acid comprises one or more modified nucleotides or nucleosides.

7. The method of any one of the preceding claims, further comprising administering at least one additional anti-cancer therapy.

8. The method of claim 7, wherein the anti-cancer therapy is selected from Cisplatin, Etoposide, PARP inhibitors, CHK1 inhibitors, WEE1 inhibitors, docetaxol, carboplatin, vincristine, vinblastine, methotrexate, cyclophosphamide, CPT-11, 5 -fluorouracil (5-FU), gemcitabine, estramustine, carmustine, adriamycin (doxorubicin), rsenic trioxide, irinotecan, and epothilone derivatives.

9. The method of claim 5, wherein the at least one additional anti-cancer therapy is an immune checkpoint blockade (ICB) therapy.

10. The method of claim 9, wherein the ICB therapy is selected from at least one PD-1 inhibitor, PD-L1 inhibitor, CTLA-4 inhibitor, and / or LAG-3 inhibitor.

11. The method of claim 10, wherein the at least one PD-1 inhibitor, PD-L1 inhibitor, CTLA- 4 inhibitor, and / or LAG-3 inhibitor is selected from Pembrolizumab (Keytruda), Nivolumab (Opdivo), Cemiplimab (Libtayo), Atezolizumab (Tecentriq), Avelumab (Bavencio), Durvalumab (Imfinzi), Ipilimumab (Yervoy) tremelimumab (Imjuno), and Relatlimab.

12. A method of inducing an antiviral response in a tumor cell, the method comprising contacting the tumor cell with at least one viral-mimicry-inducing agent (VMIA) which increases cellular levels of dsRNA, R-loops, and / or dsDNA, thereby increasing the sensitivity of said tumor cells to cancer cell killing agents which exceeds that observed in tumor cells not treated with the VMIA.

13. A method of enhancing an immune checkpoint blockade therapy a subject having tumors, the method comprising administering to said subject at least one viral-mimicry-inducing agent (VMIA), thereby inducing an antiviral response and inducing tumor cell killing which exceeds that observed in tumor cells not treated with the VMIA.

14. The method of claim 12 or 13, wherein said at least one VMIA is a DHX9 modulator.

15. The method of claim 14, wherein the DHX9 modulator is an inhibitor of EWS-FLI1 binding to DHX9.

16. The method of claim 14, wherein the DHX9 modulator is TK216 or YK-4-279.

17. The method of any one of claims 12-16, wherein said VMIA is an inhibitory nucleic acid which represses DHX9 expression and function in a cancer cell, selected from an siRNA, anantisense oligonucleotide, an shRNA, and a ribozyme having sufficient sequence homology to a DHX9 encoding nucleic acid to reduce expression of thereof in a target cell.

18. The method of claim 17, wherein said inhibitory nucleic acid comprises one or more modified nucleotides or nucleosides.

19. The method of claim 12, further comprising administering at least one additional anticancer therapy.

20. The method of any one of claims 14 or 19, wherein the anti-cancer therapy is selected from Cisplatin, Etoposide, PARP inhibitors, CHK1 inhibitors, WEE1 inhibitors, docetaxol, carboplatin, vincristine, vinblastine, methotrexate, cyclophosphamide, CPT-11, 5 -fluorouracil (5- FU), gemcitabine, estramustine, carmustine, adriamycin (doxorubicin), rsenic trioxide, irinotecan, and epothilone derivatives.

21. The method of any one of claims 14 or 19, wherein the at least one additional anti-cancer therapy is an immune checkpoint blockade (ICB) therapy.

22. The method of claim 21, wherein the ICB therapy is selected from at least one PD-1 inhibitor, PD-L1 inhibitor, CTLA-4 inhibitor, and / or LAG-3 inhibitor.

23. The method of claim 22, wherein the at least one PD-1 inhibitor, PD-L1 inhibitor, CTLA- 4 inhibitor, and / or LAG-3 inhibitor is selected from Pembrolizumab (Keytruda), Nivolumab (Opdivo), Cemiplimab (Libtayo), Atezolizumab (Tecentriq), Avelumab (Bavencio), Durvalumab (Imfinzi), Ipilimumab (Yervoy) tremelimumab (Imjuno), and Relatlimab.

24. The method of any one of the preceding claims, wherein the tumor is a recalcitrant tumor or a COLD tumor.

25. The method of any one of the preceding claims, wherein the tumor is selected from a lung cancer, a brain cancer, an ovarian cancer, a kidney cancer, a esophogeal cancer, a sarcoma,a liver cancer, a bone cancer, a stomach cancer, a bladder cancer, head and neck cancer and thyroid cancer.

26. The method of any one of the preceding claims, wherein the tumor is Small Cell Lung Cancer (SCLC).

27. The method of any one of the preceding claims, wherein said VMIA is administered in a pharmaceutically acceptable carrier via route selected from systemic, oral, intraperitoneal, intravenous, intracerebral, intratumoral and topical administration.

28. A pharmaceutical composition comprising at least one viral-mimicry-inducing agent (VMIA) and a pharmaceutically acceptable carrier.

29. The pharmaceutical composition of claim 28, wherein said at least one VMIA is a DHX9 modulator.

30. The pharmaceutical composition of claim 29, wherein the DHX9 modulator is an inhibitor of EWS-FLI1 binding to DHX9.

31. The pharmaceutical composition of claim 29, wherein the DHX9 modulator is TK216 or YK-4-279.

32. The pharmaceutical composition of claim 29, wherein said VMIA is an inhibitory nucleic acid which represses DHX9 expression and function in a cancer cell, selected from an siRNA, an antisense oligonucleotide, an shRNA, and a ribozyme having sufficient sequence homology to a DHX9 encoding nucleic acid to reduce expression of thereof in a target cell.

33. The pharmaceutical composition of claim 32, wherein said inhibitory nucleic acid comprises one or more modified nucleotides or nucleosides.

34. The pharmaceutical composition of claim 29, further comprising at least one additional anti-canccr therapy.

35. The pharmaceutical composition of claim 34, wherein the anti-cancer therapy is selected from Cisplatin, Etoposide, PARP inhibitors, CHK1 inhibitors, WEE1 inhibitors, docetaxol, carboplatin, vincristine, vinblastine, methotrexate, cyclophosphamide, CPT-11, 5-fluoro uracil (5- FU), gemcitabine, estramustine, carmustine, adriamycin (doxorubicin), rsenic trioxide, irinotecan, and epothilone derivatives.

36. The pharmaceutical composition of claim 34, wherein the at least one additional anticancer therapy is an immune checkpoint blockade (ICB) therapy.

37. The pharmaceutical composition of claim 36, wherein the ICB therapy is selected from at least one PD-1 inhibitor, PD-L1 inhibitor, CTLA-4 inhibitor, and / or LAG-3 inhibitor.

38. The pharmaceutical composition of claim 37, wherein the at least one PD-1 inhibitor, PD-L1 inhibitor, CTLA-4 inhibitor, and / or LAG-3 inhibitor is selected from Pembrolizumab (Keytruda), Nivolumab (Opdivo), Cemiplimab (Libtayo), Atezolizumab (Tecentriq), Avelumab (Bavencio), Durvalumab (Imfinzi), Ipilimumab (Yervoy) tremelimumab (Imjuno), and Relatlimab.

39. The pharmaceutical composition of any one of claims 28-38, wherein said composition is formulated for administration via a route selected from systemic, oral, intraperitoneal, intravenous, intracerebral, intratumoral and topical administration.

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