METHODS OF DIAGNOSING, PROGNOSING, AND TREATING CANCERS OVEREXPRESSING miR-155

Abstract

Disclosed herein are methods of diagnosing severity, aggressiveness, or prognosis of a solid cancer by determining if miR-155 is overexpressed. Also disclosed herein are methods of treating, preventing, decreasing, eliminating, and / or ameliorating a cancer by synthetic nucleic acids encoding administering inducible T-cell costimulatory ligand (ICOSL) and / or RELA, thereby upregulating ICOSL and MHC-I on cancerous cells to improve the efficacy of immune cell therapies and checkpoint inhibitors. Any of the disclosed methods can further be paired with administration of an miR-155 inhibitor (e.g., cobomarsen).

Description

[0001] METHODS OF DIAGNOSING, PROGNOSING, AND TREATING CANCERS OVEREXPRESSING miR-155

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS

[0003] This application claims the benefit of priority to U.S. Provisional Application No. 63 / 658,199, filed June 10, 2024, U.S. Provisional Application No. 63 / 709,026, filed October 18, 2024, U.S. Provisional Application No. 63 / 781,667, filed April 1, 2025, and U.S. Provisional Application No. 63 / 811,100, filed May 23, 2025, which are each incorporated by reference herein in their entireties.

[0004] REFERENCE TO SEQUENCE LISTING

[0005] The sequence listing submitted on June 10, 2025, as an .XML file entitled “103362- 007W01_ST26.xml” created on June 10, 2025, and having a file size of 34,157 bytes is hereby incorporated by reference pursuant to 37 C.F.R. § 1.52(e)(5).

[0006] BACKGROUND miR-155 is a well-characterized microRNA that plays a critical role in regulating immune responses, inflammation, and various cellular processes such as proliferation and differentiation. Encoded by the MIR155HG (also known as BIC) gene, miR-155 is processed into a mature 22-nucleotide RNA that can post-transcriptionally silence gene expression by binding to complementary sequences in the 3' untranslated regions (UTRs) of target mRNAs. It is normally expressed in several immune cell types, including B cells, T cells, and macrophages, where it modulates responses to stimuli such as cytokines and microbial signals. Dysregulation of miR-155 has been linked to pathological conditions, including autoimmune diseases and cancer, highlighting its role as a crucial regulatory node in human physiology (O’Connell et al., 2010; Vigorito et al., 2007; Thai et al., 2007).

[0007] In cancer, miR-155 is frequently upregulated and acts primarily as an oncomiR — an oncogenic microRNA — by repressing tumor suppressor genes and promoting pathways that drive cell survival, proliferation, and metastasis. Overexpression of miR-155 has been observed in a variety of malignancies, including diffuse large B-cell lymphoma (DLBCL), breast cancer, pancreatic cancer, and lung cancer (Eis et al., 2005; Kong et al., 2008; Jiang et al., 2010). Functionally, miR-155 contributes to oncogenesis by targeting genes such as SHIP1, SOCS1, and PTEN, which are involved in negative regulation of signaling pathways like PI3K / AKT and JAK / STAT (Costinean et al., 2006; Rai et al., 2008; Wang et al., 2010). Its aberrant expression is often associated with poor prognosis and resistance to therapy. Consequently, miR-155 has garnered significant attention as a potential biomarker for cancer diagnosis and prognosis, as well as a therapeutic target in strategies aimed at restoring normal gene expression profiles in tumor cells (Kaur et al., 2012).

[0008] Transgenic mice constitutively overexpressing miR-155 in B cells (Ep-miR-155) develop lymphoblastic leukemia and high-grade lymphomas. Controlled overexpression of miR-155 in mouse lymphoid tissues similarly causes the development of disseminated B cell lymphomas, confirming that sustained expression of miR-155 is causative for the development of this type of malignancies. Lymphomas regress once miR-155 has been switched back OFF. Furthermore, it was found that the speed of tumor regression in mice was increased once miR- 155 was turned OFF.

[0009] Thus, there is a need to identify miR-155 targets, and develop anti-cancer therapies and methods thereof to improve anti-tumor responses. The methods disclosed herein address these and other needs.

[0010] SUMMARY

[0011] Inducible T-cell costimulatory ligand (ICOSL) is a ligand which interacts with inducible T-cell costimulator (ICOS, also referred to as CD278) expressed by T cells to induce a T cell-mediated immune response. Similarly, major histocompatibility complex I (MHC-I) presents peptide fragments from within the cell to various immune cells, including T cells and natural killer (NK) cells, which can serve to indicate infection or abnormal (i.e., cancerous) growth and trigger an immune response. However, as disclosed herein, it has been discovered that expression of ICOSL and MHC-I on cancerous cells are down-regulated by miR-155, which is overexpressed in certain - and typically aggressive - cancers. In the absence of ICOSL and MHC-I, immune cells cannot recognize, monitor, or kill cancerous cells, making immune cell therapy and checkpoint inhibitors ineffective against the cancer.

[0012] Accordingly, the methods disclosed herein describe administering synthetic nucleic acid sequences including modified ICOSL genes and / or modified RELA genes (which are a key component for MHC-I synthesis) which lack the miR-155 target sequence (AGCATTA, SEQ ID NO: 9) present in the 3’-UTR region of these wildtype genes. These methods can further be paired with administration of miR-155 inhibitors, such as cobomarsen, or followed by immune cell therapies and / or administration of checkpoint inhibitors after the cancerous cells have begun re-expressing ICOSL and / or MHC-I. Determination and / or monitoring of miR-155 levels can further allow for prognosis and differential treatment of cancers which are overexpressing miR-155, including with more aggressive conventional treatments (e.g., chemotherapy and / or radiation therapy), miR-155 inhibitors, and / or modified ICOSL and / or RELA genes. Importantly, miR-155 overexpression can indicate that immune cell therapy and checkpoint inhibitors will be ineffective until miR-155 inhibitors and / or modified ICOSL and / or RELA genes are administered to restore ICOSL and / or MHC-I expression, allowing for more targeted and specialized treatment. Further, it has also been discovered that miR-155 overexpression is tied to current or prior infection by the Epstein-Barr virus (EB V), suggesting a potential class of patients who may particularly need to have miR-155 levels determined and / or monitored.

[0013] In some aspects, disclosed herein is a method of diagnosing severity, aggressiveness, or prognosis of a solid cancer in a subject in need thereof, the method including: a) obtaining a sample from the subject; b) determining if miR-155 is overexpressed compared to miR-155 in a control sample; c) calculating a risk score for the subject based on results of step b); and d) treating the subject with appropriate medication based on the risk score. As described herein, “severe” or “aggressive” cancers can be defined as cancers that require treatment to prevent, halt or reduce disease progression and potential further complications (such as metastases or metastatic progression).

[0014] In some aspects, also disclosed herein is a method of treating, preventing, decreasing, eliminating, and / or ameliorating a cancer in a subject in need thereof, the method including administering to the subject a synthetic nucleic acid sequence encoding inducible T-cell costimulatory ligand (ICOSL) or a functional fragment thereof, wherein expression and presentation of ICOSL or the functional fragment thereof by a cancerous cell induces and / or enhances an immune response against the cancer.

[0015] In some aspects, also disclosed herein is a method of treating, preventing, decreasing, eliminating, and / or ameliorating a cancer in a subject in need thereof, the method including administering to the subject a synthetic nucleic acid sequence encoding RELA or a functional fragment thereof, wherein expression of RELA or the functional fragment thereof by a cancerous cell induces and / or enhances expression of major histocompatibility complex class I (MHC-I), and wherein presentation of MHC-I by the cancerous cell induces and / or enhances an immune response against the cancer.

[0016] In some aspects, also disclosed herein is a method of treating a cancer in a subject in need In some aspects, also disclosed herein is a method of treating, preventing, decreasing, eliminating, and / or ameliorating a cancer in a subject in need thereof, the method including: a) administering a first synthetic nucleic acid sequence encoding ICOSL or a functional fragment thereof according to any of the disclosed methods of the same; and b) administering a second synthetic nucleic acid sequence encoding RELA or a functional fragment thereof according to any of the disclosed methods of the same; wherein expression of RELA or the functional fragment thereof by a cancerous cell induces and / or enhances expression of major histocompatibility complex class I (MHC-I); and wherein expression and presentation of ICOSL or the functional fragment thereof and MHC-I by the cancerous cell induces and / or enhances an immune response against the cancer.

[0017] BRIEF DESCRIPTION OF FIGURES

[0018] The accompanying figures, which are incorporated in and constitute a part of this specification, illustrate several aspects described below.

[0019] Figures 1A, IB, 1C, ID, IE, IF, and 1G show that miR-155 impairs T cell anti-tumor response at least in part by targeting ICOSL transcripts. Figure 1A shows the detection of miR- 155 (dark blue) in mice with miR-155 ON and OFF using ISH. Left, miR-155 detection (dark blue) when this microRNA is turned ON in Nestin-Cre x miR- l 55 / A / , / '1offsprings; Right, Lack of miR-155 expression when this microRNA has been turned OFF in the same mice. Figure IB shows the tumor infiltrates in mice with miR-155 ON for 3 months (top panels) then back OFF for one week (bottom panels). Top panels: Left: H&E staining of the kidney showing infiltrates (dark blue color); Middle: B220 (red signal) and CD4 (brown signal) staining of the infiltrate from the kidney indicated by the square); Right: B220 (red signal) and CD4 (brown signal) staining of a skeletal muscle. Bottom panels, tumor infiltrates in kidneys. From left to right: H&E staining at low (first panel) and high magnification (second panel); B220 staining (red, third panel, notice lack of B220 positive cells); CD4 staining (brown, fourth panel). Figure 1C shows the top panels: Left: IHC for CD4 (brown) showing T cell infiltration surrounding a lymph node. Right: magnification of a part of the image on the left. Bottom panels: IHC for Icos (brown) on lymphomas with miR-155 ON (left) and then turned back OFF (right). Figure ID shows the dual -luciferase reporter assays performed in Raw264.7 macrophages co-transfected with a Renilla luciferase reporter vector containing the human ICOSL-3’-UTR with either the WT miR-155 binding site or its mutated version, along with either a miR-Control RNA, a human miR-155 RNA, or a human miR-155-Inhibitor RNA, as indicated. n=6 replicates / each experimental setting; p= 5.09182E-07. Figure IE shows the raw264.7 cells transfected with either a miR-Control RNA, a mouse miR-155 RNA, or a mouse miR-155-Inhibitor RNA, as indicated, were treated with either LPS (100 ng / ml) or the vehicle 24 hours after transfection. Two days post-transfection, cell lysates were analyzed by western blot for ICOSL expression. GAPDH was used as a loading control. Figure IF shows two days after transfection with either a miR-Control RNA or human-miR-155 RNA, BL cell lines B JAB and NAMALWA were analyzed for ICOSL expression by western blotting, a-tubulin and GAPDH were used as loading control. Figure 1G shows the inverse correlation of miR-155 and ICOSL transcripts as measured by qRT-PCR in CLL (MEC1 and MEC2) and BL (NAMALWA, BJAB, RAJI, PH3R1, DAUDI) cell lines.

[0020] Figures 2A, 2B, 2C, and 2D show the turning ON the expression of miR-155 in lymphocytes of Nestin-Cre x miR-155LSLtTAmice leads to development of B cell lymphomas lacking ICOSL expression, while turning miR-155 back OFF allows the formation of B (ICOSL) - T (Icos) cells synapses mostly located around blood vessels. Figure 2A shows the flow cytometry analyses for B220 and ICOSL expression in lymph nodes of littermates with miR-155 OFF (left) or ON (right) for two months. Representative images. Figure 2B shows the 1strow: ICOSL (green; left) and CD20 (red; middle) staining in a WT spleen. Colocalization (yellow; right). 2ndrow: Lack of ICOSL (green; left) expression by CD20-positive cells (red; middle) in lymphomas with miR-155 ON (no yellow signal, right). ICOSL-positive cells in the left are distinct from malignant B cells. 3rdraw: ICOSL-positive cells (green; left) in lymphomas with miR-155 ON are CD31-positive (red, middle) with colocalization (yellow; right). 4thraw: A few benign B cells present in the mice with miR-155 ON then OFF for one- week had started to re-express ICOSL. CD20 (green; left); ICOSL (red; middle); colocalization (yellow; right). Panels are representative of staining of 3 mice / group. Figure 2C shows the IHC for B220 (red; top left), CD3 (green; top right) in infiltrates from mice with miR-155 ON for 10 weeks then back OFF for 4 days. Scattered B220-CD3 synapses are seen as yellow fluorescence in the pictures at the bottom left after merging B220-CD3 signals; Bottom right is a magnification of the square in the left picture. Figure 2D shows the IHC for Icos (red; top) and ICOSL (green; middle) and co-expression (yellow; bottom) in infiltrates from mice with miR-155 ON for 2-3 months then OFF for 4 days. The immunological synapses between B cells (ICOSL) and T cells (Icos) are located mostly around blood vessels (ovals). Each scale bar is 100 pm.

[0021] Figures 3A, 3B, and 3C show the loss of ICOSL expression as B cells progress toward malignancy in Ep-miR-155 mice. Figure 3A shows the representative flow cytometry images of splenocytes from WT and Ep-miR-155 transgenic mice (TG) stained for CD 19, B220, ICOSL, and PDL1. Gated CD19+B220+ in WT and TG mice and CD19+B220- splenocytes in TG mice were analyzed for ICOSL and PDL1 (two bottom panels). w=3 / genotype. Figure 3B shows the graphical presentation of the percentage of B cell subpopulations positive for ICOSL or PDL1 analyzed in Figure 3A. n=3 / group. *, p < 0.0285. Figure 3C shows the top panels: Left, ICOSL staining (brown) of a spleen section from a WT mouse. The circle indicates the T cell zone of the spleen; Middle, CD20 (brown) staining of a WT spleen; Right, lymphoma cells of Ep-miR-155 mice are B220 negative based on IHC. The circle indicates very few B220+ cells (brown) in this lymphoma. Bottom panels: Left, H&E staining of the chest organs of a terminally ill Ep-miR-155 mouse. Middle, the expression of ICOSL (brown) is completely lost in lymphoma cells of TGEp-miR-155 mouse; Right, Lymphoma cells from another terminally ill Ep-miR-155 mouse stained for ICOSL (brown) were also negative for ICOSL expression.

[0022] Figures 4A, 4B, 4C, 4D, and 4E show the expression of ICOSL in MC38 colon tumor cells injected subcutaneously causes tumor regression. Figure 4A shows the flow cytometry analysis of ICOSL expression in parental MC38 cells, and MC38 cells transfected with the CMV-ICOSL vector and selected on G418-containing medium for the expression of the neomycin gene present in the CMV-ICOSL vector. MC38-A4 cells do not express ICOSL and were further used as control, while MC38-A5 express high levels of ICOSL. Figure 4B shows the tumors originating from mice injected with MC38-A4 cells are larger than those injected with MC38-A5 cells and show skin ulceration indicated by arrowheads. Figure 4C shows the size in grams of tumors originating from MC38-A4 and MC38-A5 transformants was significantly different. / ? =0.0286. n=5 for MC38- A4 tumors, as one female was found dead and was not included in the experiment: n=6 for MC38-A5 tumors. Figure 4D shows the histopathological and cellular characterization of tumors derived from MC38-A4 and MC38- A5 cells. Extensive necrosis is indicative of rapid growth in tumors derived from MC38-A4 cells (arrowheads in top left panel). Less necrosis and presence of inflammatory infiltrates at the invading front of the tumor in tumors derived from MC38-A5 cells (arrowhead in lower left panel). The two middle panels in each row show staining for ICOSL, with minimal expression found in MC38-A4 tumors and many ICOSL positive (red) present in MC38-A5 tumors (p 0.00 1). The two right panels in each row show staining for CD8 with significantly higher infiltration of CD8 positive cells (brown) in MC38-A5 and minimal CD8 positive cell infiltration in control MC38-A4 tumors (p=0.0015). The right bottom panel also shows massive CD8-positive T cell infiltration at the border of tumor growth (rectangle). Figure 4E shows the co-expression analysis based on the immunofluorescence signals of ICOSL and CD8 in the heavy CD8-infiltrated areas of MC38-A5 tumors. Top left panel: The expression of ICOSL (red) and CD8 (brown) under visible light; Top middle panel: CD8 positive cells (green); Bottom left panel: ICOSL expression (red); Middle bottom panel: Co-expression of CD8 and ICOSL. Scale bars for these images are at 100 microns. El and E2 are enlargements of the red squares in the bottom middle panel where due to the close vicinity of ICOSL-positive and CD8 cells the overlay of the two signals becomes yellow.

[0023] Figure 5 shows the tumor infiltrate in in a lymph node of a miR-155LSLtTAmouse with miR-155 ON. Immunohistochemistry (IHC) showing that B220 + (brown) B cells dominate, and the variable intensity of the signal from cell to cell, typical of lymphomas.

[0024] Figure 6 shows a mi-155 inhibitor increases the levels of ICOSL on the cell surface of a CLL-derived cell line. ICOSL expression in MEC2-CLL cells transfected as indicated was determined by flow cytometry analyses 48hours post-transfection.

[0025] Figure 7 shows the CD 163 macrophages positive for ICOSL in a tumor from a mi- 155LSLtTAmouse with mi-155 ON. IHC shows cells positive for CD163 (ie. macrophages) colocalize with cells expressing ICOSL.

[0026] Figure 8 shows the turning OFF mi- 155 expression in mi-155LSLtTAmice increases Socsl expression in CD20-positive B cells. IHC for Socsl and CD20. Top panels: When miR- 155 is turned ON, the expression of Socsl, an established target of miR-155, in the spleen of miR-155LSLtTAmice is barely noticeable. Socsl (left panel, red); CD20 (middle panel, green); Rare co-localization (right panel, no yellow signal). Bottom panels: Socsl is re-expressed by B cells when miR-155 is turned back OFF: Socsl (left panel, green); CD20 (middle panel, red); Right panel: Colocalization (yellow signal).

[0027] Figure 9 shows the turning miR-155 expression back OFF in miR-155LSLtTAmice increases Icos expression in CD4 but not CD8 positive cells. Representative flow cytometry analysis of the spleens of miR-155LSLtTAmice showing the expression of Icos on gated CD4- positive (top panels) or CD8-positive (bottom) cells. Note the gradual increase of the percentage of Icos-positive CD4-positive cells (top panels) after miR-155 has been turned back OFF, without noticeable change of the percentage of Icos-positive CD8-positive cells (bottom panels).

[0028] Figure 10 shows the turning miR-155 expression back OFF in miR-155LSLtTAmice increases ICOSL in B cells. Representative flow cytometry analysis from the spleen of miR- 155LSLtTAmice showing the reduction of the percentage of B220-positive cells, but also the concomitant increase of the percentages of B cells B220-positive cells expressing ICOSL (arrow in the right panel). Figure 11 shows the CD19+cells from Emu-miR-155 transgenic mice are positive for MHC-II. The expression of MHC-II in splenocytes from WT and Ep-miR-155 mice was analyzed by flow cytometry in at least three male (M) and three female (F) mice per genotype. Representative images are shown.

[0029] Figure 12 shows the mouse-ICOSL expression analysis in HEK-293 cells transiently transfected with plasmids engineered to express the mouse ICOSL protein. Western blot analysis of a parental mouse cell lines (Em-miR-155 B2), and of human HEK-293 cells (293) transiently transfected with either the Tet-ON-CMV-mouse-ICOSL vector, the CMV-mouse- Socsl vector, or the CMV-mouse-ICOSL vector, as indicated. The inducibility of ICOSL expression in HEK-293 cells transiently transfected with the Tet-on CMV-ICOSL vector was checked by doxycycline treatment.

[0030] Figure 13 shows the wide-spread necrosis and overgrowth in tumors derived by MC38- A4 cells but not in those derived from MC38-A5 based on H&E staining. Tumors derived from MC38-A4 and MC38-A5 cells were partly fixed for histopathology analysis and partly processed for flow cytometry (See Figure 14).

[0031] Figure 14 shows the characterization of MC38-A4 and MC38-A5 tumors by flow cytometry. Tumors derived from MC38-A4 and MC38-A5 cells (a representative tumor from each cell line transformant is shown on the left) were analyzed by flow cytometry as indicated (MC38-A4: 3 top right panels; MC38-A5: 3 bottom right panels). The rulers on the images on the left show the millimeter scale. Notice that only a small fraction of cells from MC38-A5 tumors stained positive for ICOSL at the time of tumor collection compared to -65% of cells being positive for ICOSL at the day of injection.

[0032] Figure 15 shows the tumor regions that lack ICOSL-expressing cells also lack CD8+T cells. IHC for ICOSL (red) and CD8 (green) in tumors derived from MC38-A5 cells. These images were taken from areas of the tumor with few CD8 positive cell infiltration. They show the lack of CD8 infiltration in the absence of ICOSL-expressing cells. Scale bars for these two images is at 100 microns.

[0033] Figures 16A, 16B, and 16C show the inverse correlation between ICOSL and miR-155 expression in samples from DLBCL patients. Serial section of 29 CLBCL samples laid on a TMA microarray were analyzed for the expression of ICOSL and miR-155 by IHC and ISH, respectively. Figure 16A shows the relative expression of miR-155 and ICOSL in TMA- DLBCL samples. Letters followed by numbers indicate the ID of the samples in the TMA. Figure 16B shows the inverse correlation between the miR-155 and ICOSL levels in TMA- DLBCL samples. A3, B4, B5, C2, C3, C5, C6, E3, and E5 correspond to the IDs of the 9 tumors showing miR-155 dose-dependent effects. Figure 16C shows the representative images of the DLBCL samples showing strong miR-155 expression (dark blue) and no ICOSL (brown). A5, Bl, C2, and C6 indicate the ID / location of the corresponding samples on the TMA. The counterstain was nuclear fast red which yields a pink color.

[0034] Figures 17A, 17B, 17C, 17D, 17E, and 17F show the expression of miR-155, ICOSL, and MHC-I in ABD and GCB primary tumors (PTs) and cell lines (CLs). Quantitative RT-PCR analyses of miR-155 (Figure 17A), ICOSL (Figure 17B), and MHC-I (Figure 17E) expression in the indicated DLBCL primary tumors and DLBCL-derived cell lines. Normalization was done using U48 for miR-155 and GAPDHio ICOSL and MHC-I samples. Each sample was run in triplicates, n.d. : non-determines. Figure 17C shows the flow cytometry analysis of ICOSL expression on TOLEDO and SUDHL2 DLBCL-derived cell lines. Figures 17D and 17F show the MFI for ICOSL (Figure 17D) and MHC-I (Figure 17F) is given for 4 ABC- and 6 GCB-DLBCL cell lines.

[0035] Figures 18A, 18B, 18C, 18D, 18E, 18F, and 18G show the high miR-155 levels are associated with reduced levels of MHC-I in both mouse and human B cells. Figure 18A shows CD19+B220+B cells from Ep-miR-155 transgenic mice have lower MHC-I H2-Kblevels than their wild-type littermates. Figure 18B shows the MFI of MHC-I H2-Kbexpression in CD19+B220+B cells from wild-type (n=3) and Ep-miR-155 transgenic mice (n=5). *, P<0.008. Figure 18C shows the flow cytometry analyses of MHC-I expression in SUDHL5 (GCB- derived) and LY3 (ABC-derived) cell lines. SUDHL5 cells were transfected with either a miR- 155 mimic or a control RNA (left panel), while LY3 cells were transfected with either a miR- 155 antisense inhibitor RNA or a control inhibitor RNA (right panel). Figures 18D and 18E show the inverse correlation between miR-155 and MHC-I levels in TMA-DLBCLs samples (n=29), with evidence of miR-155 dose-dependent effects. In Figure 18E, the dots corresponding to the scores of the 9 tumors pointed to in Figure 16B are indicated. Figure 18F shows the representative images of MHC-I staining of DLBCL samples form the TMA. The signal is red with a hematoxylin counterstain. A5 samples are negative for MHC-I, while C2 and C6 show a strong signal. The staining for miR-155 of the same tumors is presented in Figure 16C. Figure 18G shows the positive correlation between MHC-I and ICOSL levels in the TMA-DLBCL samples. The dots corresponding to the scores of the 9 tumors pointed to in Figure 18E here above and in Figure 16B are indicated.

[0036] Figures 19A, 19B, 19C, 19D, 19E, and 19F depict histologic, viral, and mononuclear cell correlates of NPC. Figures 19A and 19B show the carcinoma nests strongly positive for EBV-EBER-1 / 2 RNA (star) amongst the many large aggregates of lymphoid cells (arrow) at low and high magnifications. Figure 19C shows a representative field where the EBV+ carcinoma nests (brown staining) and lymphoid aggregates (blue counterstain) occupy equivalent volumes of the tumor. Figure 19D shows the carcinoma cells (star) adjacent to the normal nasopharynx (arrow) (EBER-1 / 2 signal brown due to DAB with hematoxylin counterstain). Figure 19E shows that the T cells, as marked by CD3 (red), dominate the lymphoid aggregates in NPC and surround a nest of carcinoma cells (arrowheads). Figure 19F shows that the CD3 positive cells also infiltrate the carcinoma nests directly (CD3 signal from FAST RED with hematoxylin counterstain).

[0037] Figures 20A, 20B, 20C, and 20D depict that the EBV+ carcinoma cells in NPC show high expression of miR-155. Figures 20A and 20B show the individual channels for EBER- 1 / 2 and miR-155, respectively after co-expression of the targets in an NPC. The merged image (Figure 20C) shows that the EBER- 1 / 2+ cells strongly co-localize with miR-155. There is no co-localization when using the scrambled probe and EBER 1-2 (Figure 20D).

[0038] Figures 21A, 21B, 21C, 21D, 21E, 21F, 21G, 21H, and 211 depict that the EBV+ carcinoma cells in NPC lack expression of ICOSL, MHC-I, and RELA. Figures 21A and 21B show the normal nasopharynx adjacent to an NPC. Note that the lining epithelia (arrow) strongly expresses both MHC-I (red) and RELA (DAB, green) seen as fluorescent yellow on the co-localization image (Figure 2 IB). The NPC cancer cells directly adjacent to the normal nasopharynx do not demonstrate MHC-I expression (fluorescent red) but do contain EBER-1 / 2 (fluorescent green, Figure 21C). Figure 21D shows that the stroma of the adjacent cancer negative nasopharynx has ICOSL expression in mononuclear cells which is lacking in the cancer cells of the NPC (Figure 21E; signal from FAST RED). Co-localization of the T cell marker CD3 (fluorescent red) and ICOS (fluorescent green) in the tumor infiltrate of the NPC shows strong co-expression (fluorescent yellow). Figures 21G and 21H show the isolated channels for EBER-1 / 2 (Figure 21G) and PD-L1 (Figure 21H) after co-expression analyses. Merging the two channels (Figure 211) confirms the strong co-expression of the EBV RNA and PD-L1 in the tumor cells.

[0039] Figure 22 depicts a model of EBV-related NPC progression and malignant transformation unrecognized by massive T cell infiltrates. In the healthy nasopharyngeal epithelial tissue, the expression of miR-155 is low to undetectable, while these cells express ICOSL, MHC-I and RELA. Following EBV infection, there is a significant upregulation of miR-155 expression. This initial increase in miR-155, along with the EBV infection, is expected to activate T cells that infiltrate the tissue to fight the infection. These T cells start expressing ICOS and can successfully eliminate the initial benign EBV+ NPC cells. However, the sustained high levels of miR-155 lead to a reduction and eventual complete loss of ICOSL, as well as reduction of MHC-I expression. This decline impairs the cytotoxic T cells to recognize and eliminate tumor cells. At this stage, while the T cells express ICOS, they are unable to perform their function effectively, as they cannot bind to the NPC cells, resulting in “frustrated” T cells. Overtime time, these T cells may evolve into “exhausted” T cells. This exhaustion can help explain why, despite the presence of PD-1 expression on T cells and PD- L1 expression on tumor cells, immune checkpoint inhibitors often fail to elicit a robust immune response. The complex interplay of miR-155 and tumor evasion strategies underscores the challenges in developing effective therapies for NPC and highlights the need for the use of miR-155-inhibitors combined with strategies aimed at expressing ICOSL in tumor cells.

[0040] Figures 23A, 23B, 23C, and 23D depict examination of an EBV+ gastric cancer. Figure 23A shows hematoxylin and eosin examination of an EBV+ gastric cancer shows the endophytic growth pattern (mucosa and muscularis externa labeled with the arrow marking the muscularis mucosa). Note the many germinal centers (rectangle) among the tumor; the area of the oval is included in the insert after in situ analysis for EBER-1 / 2 (DAB signal, brown); note the poorly differentiated growth pattern. Figure 23B shows that EBER-1 / 2 (fluorescent green) strongly co-localizes with miR-155 (fluorescent turquoise) in the cancer cells with coexpression seen as fluorescent yellow. PDL1 (fluorescent red), however, is seen primarily in the CD8+ cytotoxic T cells infiltrating the tumor (arrow, Figure 23B); fluorescent blue (hematoxylin) is the counterstain. Figure 23C depicts that the intertumor CD8 cells (fluorescent green) do show extensive co-localization with ICOS (fluorescent red, coexpression seen as fluorescent yellow). Figure 23D shows that ICOSL (DAB, brown) is not expressed in the tumor cells (circle) but is seen in the adjacent benign gastric epithelia (arrow). Scale bars in Figures 23B and 23C equal 100 microns.

[0041] DETAILED DESCRIPTION

[0042] The following description of the disclosure is provided as an enabling teaching of the disclosure in its best, currently known embodiment s). To this end, those skilled in the relevant art will recognize and appreciate that many changes can be made to the various embodiments of the invention described herein, while still obtaining the beneficial results of the present disclosure. It will also be apparent that some of the desired benefits of the present disclosure can be obtained by selecting some of the features of the present disclosure without utilizing other features. Accordingly, those who work in the art will recognize that many modifications and adaptations to the present disclosure are possible and can even be desirable in certain circumstances and are a part of the present disclosure. Thus, the following description is provided as illustrative of the principles of the present disclosure and not in limitation thereof.

[0043] Reference will now be made in detail to the embodiments of the invention, examples of which are illustrated in the drawings and the examples. This invention may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein.

[0044] TERMINOLOGY

[0045] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood to one of ordinary skill in the art to which this disclosure belongs. The term “comprising” and variations thereof as used herein is used synonymously with the term “including” and variations thereof and are open, non-limiting terms. Although the terms “comprising” and “including” have been used herein to describe various embodiments, the terms “consisting essentially of’ and “consisting of’ can be used in place of “comprising” and “including” to provide for more specific embodiments and are also disclosed. As used in this disclosure and in the appended claims, the singular forms “a”, “an”, “the”, include plural referents unless the context clearly dictates otherwise.

[0046] The following definitions are provided for the full understanding of terms used in this specification.

[0047] Ranges can be expressed herein as from “about” one particular value, and / or to “about” another particular value. When such a range is expressed, another embodiment includes from the one particular value and / or to the other particular value. Similarly, when values are expressed as approximations, by use of the antecedent “about,” it will be understood that the particular value forms another embodiment. It will be further understood that the endpoints of each of the ranges are significant both in relation to the other endpoint, and independently of the other endpoint. It is also understood that there are a number of values disclosed herein, and that each value is also herein disclosed as “about” that particular value in addition to the value itself. For example, if the value “10” is disclosed, then “about 10” is also disclosed. It is also understood that when a value is disclosed that “less than or equal to” the value, “greater than or equal to the value” and possible ranges between values are also disclosed, as appropriately understood by the skilled artisan. For example, if the value “10” is disclosed the “less than or equal to 10”as well as “greater than or equal to 10” is also disclosed. It is also understood that the throughout the application, data is provided in a number of different formats, and that this data, represents endpoints and starting points, and ranges for any combination of the data points. For example, if a particular data point “10” and a particular data point 15 are disclosed, it is understood that greater than, greater than or equal to, less than, less than or equal to, and equal to 10 and 15 are considered disclosed as well as between 10 and 15. It is also understood that each unit between two particular units are also disclosed. For example, if 10 and 15 are disclosed, then 11, 12, 13, and 14 are also disclosed.

[0048] “Optional” or “optionally” means that the subsequently described event or circumstance may or may not occur, and that the description includes instances where said event or circumstance occurs and instances where it does not.

[0049] An "increase" can refer to any change that results in a greater amount of a symptom, disease, composition, condition, or activity. An increase can be any individual, median, or average increase in a condition, symptom, activity, composition in a statistically significant amount. Thus, the increase can be a 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100% or more increase so long as the increase is statistically significant.

[0050] A "decrease" can refer to any change that results in a smaller amount of a symptom, disease, composition, condition, or activity. A substance is also understood to decrease the genetic output of a gene when the genetic output of the gene product with the substance is less relative to the output of the gene product without the substance. Also, for example, a decrease can be a change in the symptoms of a disorder such that the symptoms are less than previously observed. A decrease can be any individual, median, or average decrease in a condition, symptom, activity, composition in a statistically significant amount. Thus, the decrease can be a 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100%, or more decrease so long as the decrease is statistically significant.

[0051] "Inhibit," "inhibiting," and "inhibition" mean to decrease an activity, response, condition, disease, or other biological parameter. This can include but is not limited to the complete ablation of the activity, response, condition, or disease. This may also include, for example, a 10% reduction in the activity, response, condition, or disease as compared to the native or control level. Thus, the reduction can be a 10, 20, 30, 40, 50, 60, 70, 80, 90, 100%, or any amount of reduction below, above, or in between the given ranges as compared to native or control levels.

[0052] By “reduce” or other forms of the word, such as “reducing” or “reduction,” means lowering of an event or characteristic (e.g., tumor growth). It is understood that this is typically in relation to some standard or expected value, in other words it is relative, but that it is not always necessary for the standard or relative value to be referred to. For example, “reduces tumor growth” means reducing the rate of growth of a tumor relative to a standard or a control.

[0053] By “prevent” or other forms of the word, such as “preventing” or “prevention,” is meant to stop a particular event or characteristic, to stabilize or delay the development or progression of a particular event or characteristic, or to minimize the chances that a particular event or characteristic will occur. Prevent does not require comparison to a control as it is typically more absolute than, for example, reduce. As used herein, something could be reduced but not prevented, but something that is reduced could also be prevented. Likewise, something could be prevented but not reduced, but something that is prevented could also be reduced. It is understood that where reduce or prevent are used, unless specifically indicated otherwise, the use of the other word is also expressly disclosed.

[0054] The term “subject” refers to any individual who is the target of administration or treatment. The subject can be a vertebrate, for example, a mammal. In one aspect, the subject can be human, non-human primate, bovine, equine, porcine, canine, or feline. The subject can also be a guinea pig, rat, hamster, rabbit, mouse, or mole. Thus, the subject can be a human or veterinary patient. The term “patient” refers to a subject under the treatment of a clinician, e.g., physician.

[0055] The term “therapeutically effective amount” refers to the amount of the composition used is of sufficient quantity to ameliorate one or more causes or symptoms of a disease or disorder. Such amelioration only requires a reduction or alteration, not necessarily elimination.

[0056] The terms “treat,” “treating,” and grammatical variations thereof as used herein, include partially or completely delaying, alleviating, mitigating or reducing the intensity of one or more attendant symptoms of a disorder or condition and / or alleviating, mitigating or impeding one or more causes of a disorder or condition. Treatments according to the disclosure may be applied preventively, prophylactically, palliatively or remedially. Treatments are administered to a subject prior to onset (e.g., before obvious signs of a cancer), during early onset (e.g., upon initial signs and symptoms of a cancer), or after an established development of a cancer.

[0057] The term “treatment” refers to the medical management of a patient with the intent to cure, ameliorate, stabilize, or prevent a disease, 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; preventative treatment, that is, treatment directed to minimizing or partially or completely inhibiting the development of the associated 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.

[0058] “Composition” refers to any agent that has a beneficial biological effect. Beneficial biological effects include both therapeutic effects, e.g., treatment of a disorder or other undesirable physiological condition, and prophylactic effects, e.g., prevention of a disorder or other undesirable physiological condition (e.g., a cancer). The terms also encompass pharmaceutically acceptable, pharmacologically active derivatives of beneficial agents specifically mentioned herein, including, but not limited to, a vector, polynucleotide, cells, salts, esters, amides, proagents, active metabolites, isomers, fragments, analogs, and the like. When the term “composition” is used, then, or when a particular composition is specifically identified, it is to be understood that the term includes the composition per se as well as pharmaceutically acceptable, pharmacologically active vector, polynucleotide, salts, esters, amides, proagents, conjugates, active metabolites, isomers, fragments, analogs, etc.

[0059] "Comprising" is intended to mean that the compositions, methods, etc. include the recited elements, but do not exclude others. "Consisting essentially of' when used to define compositions and methods, shall mean including the recited elements, but excluding other elements of any essential significance to the combination. Thus, a composition consisting essentially of the elements as defined herein would not exclude trace contaminants from the isolation and purification method and pharmaceutically acceptable carriers, such as phosphate buffered saline, preservatives, and the like. "Consisting of' shall mean excluding more than trace elements of other ingredients and substantial method steps for administering the compositions provided and / or claimed in this disclosure. Embodiments defined by each of these transition terms are within the scope of this disclosure.

[0060] “Expression” as used herein refers to the process by which information from a gene is used in the synthesis of a functional gene product that enables it to produce a peptide / protein end product, and ultimately affect a phenotype, as the final effect.

[0061] A “nucleotide” is a compound consisting of a nucleoside, which consists of a nitrogenous base and a 5-carbon sugar, linked to a phosphate group forming the basic structural unit of nucleic acids, such as DNA or RNA. The four types of nucleotides are adenine (A), cytosine (C), guanine (G), and thymine (T), each of which are bound together by a phosphodiester bond to form a nucleic acid molecule. A “nucleic acid” is a chemical compound that serves as the primary informationcarrying molecules in cells and make up the cellular genetic material. Nucleic acids comprise nucleotides, which are the monomers made of a 5-carbon sugar (usually ribose or deoxyribose), a phosphate group, and a nitrogenous base. A nucleic acid can also be a deoxyribonucleic acid (DNA) or a ribonucleic acid (RNA). A chimeric nucleic acid comprises two or more of the same kind of nucleic acid fused together to form one compound comprising genetic material.

[0062] The terms “percent identity” and “% identity,” as applied to polynucleotide sequences, refer to the percentage of residue matches between at least two polynucleotide sequences aligned using a standardized algorithm. Such an algorithm may insert, in a standardized and reproducible way, gaps in the sequences being compared in order to optimize alignment between two sequences, and therefore achieve a more meaningful comparison of the two sequences. Percent identity for a nucleic acid sequence may be determined as understood in the art. (See, e.g., U.S. Pat. No. 7,396,664, which is incorporated herein by reference in its entirety). A suite of commonly used and freely available sequence comparison algorithms is provided by the National Center for Biotechnology Information (NCBI) Basic Local Alignment Search Tool (BLAST) (Altschul, S. F. et al. (1990) J. Mol. Biol. 215:403 410), which is available from several sources, including the NCBI, Bethesda, Md., at its website. The BLAST software suite includes various sequence analysis programs including “blastn,” that is used to align a known polynucleotide sequence with other polynucleotide sequences from a variety of databases. Also available is a tool called “BLAST 2 Sequences” that is used for direct pairwise comparison of two nucleotide sequences. “BLAST 2 Sequences” can be accessed and used interactively at the NCBI website. The “BLAST 2 Sequences” tool can be used for both blastn and blastp (discussed above).

[0063] Percent identity may be measured over the length of an entire defined polynucleotide sequence or may be measured over a shorter length, for example, over the length of a fragment taken from a larger, defined sequence, for instance, a fragment of at least 20, at least 30, at least 40, at least 50, at least 70, at least 100, or at least 200 contiguous nucleotides. Such lengths are exemplary only, and it is understood that any fragment length may be used to describe a length over which percentage identity may be measured.

[0064] A “full length” polynucleotide sequence is one containing at least a translation initiation codon (e.g., methionine) followed by an open reading frame and a translation termination codon. A “full length” polynucleotide sequence encodes a “full length” polypeptide sequence.

[0065] A “variant,” “mutant,” “fragment”, or “derivative” of a particular nucleic acid sequence may be defined as a nucleic acid sequence having at least 50% sequence identity to the particular nucleic acid sequence over a certain length of one of the nucleic acid sequences using blastn with the “BLAST 2 Sequences” tool available at the National Center for Biotechnology Information's website. (See Tatiana A. Tatusova, Thomas L. Madden (1999), “Blast 2 sequences — a new tool for comparing protein and nucleotide sequences”, FEMS Microbiol Lett. 174:247-250). In some embodiments a variant polynucleotide may show, for example, at least 60%, at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% or greater sequence identity over a certain defined length relative to a reference polynucleotide.

[0066] For example, a fragment may comprise from 5 to 1000 contiguous nucleotides or contiguous amino acid residues of a reference polynucleotide or reference polypeptide, respectively. In some embodiments, a fragment may comprise at least 5, 10, 15, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 40, 50, 60, 70, 80, 90, 100, 150, 250, or 500 contiguous nucleotides or contiguous amino acid residues of a reference polynucleotide or reference polypeptide, respectively. Fragments may be preferentially selected from certain regions of a molecule, for example the N-terminal region and / or the C-terminal region of a polypeptide or the 5 '-terminal region and / or the 3' terminal region of a polynucleotide. The term “at least a fragment” encompasses the full length polynucleotide or full length polypeptide.

[0067] A “functional fragment” as referred to herein comprises a portion of a polypeptide which retains its functional ability.

[0068] As used herein, an “expression vector” includes, but is not limited to a plasmid or a virus or viral vector. A plasmid or a viral vector can be capable of extrachromosomal replication or, optionally, can integrate into the host genome. As used herein, the term "integrated" used in reference to an expression vector (e.g., a plasmid or viral vector) means the expression vector, or a portion thereof, is incorporated (physically inserted or ligated) into the chromosomal DNA of a host cell. As used herein, a “viral vector” refers to a virus-like particle containing genetic material which can be introduced into a eukaryotic cell without causing substantial pathogenic effects to the eukaryotic cell. A wide range of viruses or viral vectors can be used for transduction but should be compatible with the cell type the virus or viral vector are transduced into (e.g., low toxicity, capability to enter cells). Suitable viruses and viral vectors include adenovirus, lentivirus, retrovirus, among others. In some embodiments, the expression vector encoding a chimeric polypeptide is a naked DNA or is comprised in a nanoparticle (e.g., liposomal vesicle, porous silicon nanoparticle, gold-DNA conjugate particle, polyethyleneimine polymer particle, cationic peptides, etc.). As used herein, the term “integrated” refers to the act or process of fusing one composition into another composition, cell, or organism to form a new entity.

[0069] The term “administer,” “administering”, or derivatives thereof refer to delivering a composition, substance, inhibitor, or medication to a subject or object by one or more the following routes: oral, topical, intravenous, subcutaneous, transcutaneous, transdermal, intramuscular, intra-joint, parenteral, intra-arteriole, intradermal, intraventricular, intracranial, intraperitoneal, intralesional, intranasal, rectal, vaginal, by inhalation or via an implanted reservoir. The term “parenteral” includes subcutaneous, intravenous, intramuscular, intraarticular, intra-synovial, intrastemal, intrathecal, intrahepatic, intralesional, and intracranial injections or infusion techniques.

[0070] The terms “immunotherapy” and “immunotherapeutic” refers to the treatment of disease by activating or suppressing the immune system. In cancer treatment, the most effective immunotherapies are cell-based immunotherapies that utilize lymphocytes, macrophages, dendritic cells, natural killer cells, cytotoxic T lymphocytes, etc. to defend the body against cancer by targeting abnormal antigens expressed on the surface of tumor cells.

[0071] The terms “anticancer agent” and “anticarcinogen” refers to a substance, composition, or formula that counteracts the effects or inhibits the development of a cancerous cells and tissues.

[0072] The term “cancer” is used to address any neoplastic disease and is not limited to epithelial neoplasms (surface and glandular cancers; such a squamous cancers or adenomas)). It is used here to describe both solid tumors and hematologic malignancies, including epithelial (surface and glandular) cancers, soft tissue and bone sarcomas, angiomas, mesothelioma, melanoma, lymphomas, leukemias and myeloma.

[0073] A “T cell” refers to a type of lymphocyte that is one of the most important white blood cells of the immune system. T cells can be distinguished from other lymphocytes by the presence of a T-cell receptor (TCR) on their cell surface. The immune-mediated cell death function of T cells is carried by two major subtypes: CD8+“killer” T cells and CD4+“helper T cells.

[0074] A “pharmaceutically effective amount” or a “therapeutically effective amount” of a drug necessary to achieve a therapeutic effect may vary according to factors such as the age, sex, and weight of the subject. Dosage regimens can be adjusted to provide the optimum therapeutic response. For example, several divided doses may be administered daily, or the dose may be proportionally reduced as indicated by the exigencies of the therapeutic situation. "Pharmaceutically acceptable carrier" (sometimes referred to as a “carrier”) means a carrier or excipient that is useful in preparing a pharmaceutical or therapeutic composition that is generally safe and non-toxic and includes a carrier that is acceptable for veterinary and / or human pharmaceutical or therapeutic use. The terms "carrier" or "pharmaceutically acceptable carrier" can include, but are not limited to, phosphate buffered saline solution, water, emulsions (such as an oil / water or water / oil emulsion) and / or various types of wetting agents.

[0075] As used herein, the term “carrier” encompasses any excipient, diluent, filler, salt, buffer, stabilizer, solubilizer, lipid, stabilizer, or other material well known in the art for use in pharmaceutical formulations. The choice of a carrier for use in a composition will depend upon the intended route of administration for the composition. The preparation of pharmaceutically acceptable carriers and formulations containing these materials is described in, e.g., Remington's Pharmaceutical Sciences, 21st Edition, ed. University of the Sciences in Philadelphia, Lippincott, Williams & Wilkins, Philadelphia, PA, 2005. Examples of physiologically acceptable carriers include saline, glycerol, DMSO, buffers such as phosphate buffers, citrate buffer, and buffers with other organic acids; antioxidants including ascorbic acid; low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, arginine or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugar alcohols such as mannitol or sorbitol; salt-forming counterions such as sodium; and / or nonionic surfactants such as TWEEN™ (ICI, Inc.; Bridgewater, New Jersey), polyethylene glycol (PEG), and PLURONICS™ (BASF; Florham Park, NJ). To provide for the administration of such dosages for the desired therapeutic treatment, compositions disclosed herein can advantageously comprise between about 0.1% and 99% by weight of the total of one or more of the subject compounds based on the weight of the total composition including carrier or diluent.

[0076] METHODS OF DIAGNOSING AND / OR PROGNOSING CANCER

[0077] In some aspects, disclosed herein is a method of diagnosing severity, aggressiveness, or prognosis of a solid cancer in a subject in need thereof, the method including: a) obtaining a sample from the subject; b) determining if miR-155 is overexpressed compared to miR-155 in a control sample; c) calculating a risk score for the subject based on results of step b); and d) treating the subject with appropriate medication based on the risk score. As described herein, “severe” or “aggressive” cancers can be defined as cancers that require treatment to prevent, halt or reduce disease progression and potential further complications (such as metastases or metastatic progression).

[0078] According to the present invention, a “baseline” or “control” can include a normal or negative control and / or a disease or positive control, against which a test level of miR-155 expression can be compared. Therefore, it can be determined, based on the control or baseline level of miR-155 expression, whether a sample to be evaluated for miR-155 overexpression has a measurable difference or substantially no difference in miR-155 expression, as compared to the baseline level. In one aspect, the baseline control is indicative of the level of miR-155 expression as expected in a normal (e.g., healthy, negative control, non-depressive) patient. Therefore, the term “negative control” used in reference to a baseline level of miR-155 expression typically refers to a baseline level of expression from a population of individuals which is believed to be normal (i.e., not having or developing a solid cancer). In some aspects, it may also be useful to compare miR-155 expression in a test sample to a baseline that has previously been established from a patient or population of patients with a solid cancer. Such a baseline level, also referred to herein as a “positive control”, refers to a level of miR-155 expression established in a sample from one or preferably a population of individuals who had been positively diagnosed with a solid cancer.

[0079] By “overexpressed” means that miR-155 is expressed 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%,

[0080] 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%,

[0081] 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%,

[0082] 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%,

[0083] 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, or 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.25, 3.5, 3.75, 4, 5, 6, 7, 8, 9, or 10 times or more compared the control sample.

[0084] In some aspects, the control sample can include a sample from a healthy subject or a sample from a subject with a less aggressive cancer.

[0085] In some aspects, after a risk score is calculated, the subject can be placed into a risk category based on their risk score. In some aspects, a subject with a low risk level can be treated differently than a subject with a moderate or high risk level. For example, in some aspects, regular or only slightly elevated expression of miR-155 can indicate a low risk level, and overexpression of miR-155 can indicate a moderate or high risk level . In some aspects, treatment for a low risk level can include administration of immune cell therapy (such as, for example, T cell therapy, macrophage therapy, NK cell therapy, NK T cell therapy, CAR T cell therapy, and / or CARNK cell therapy) and / or one or more checkpoint inhibitors. For example, a low risk level can indicate regular or only slightly elevated expression of miR-155, thereby indicating that the cancerous cells are likely to express, to at least some extent, ICOSL and MHC-I. As discussed previously, ICOSL and MHC-I are necessary for successful treatment with immune cell therapy and / or checkpoint inhibitors. Accordingly, in some aspects, a low risk level can indicate that immune cell therapy and / or administration of one or more checkpoint inhibitors is likely to be successful or, at minimum, more successful than in a subject having a moderate or high risk level.

[0086] Examples of checkpoint inhibitors include, but are not limited to, a PD-L1 inhibitor, a PD-1 inhibitor, and a CTLA-4 inhibitor. In some embodiments, the PD-L1 inhibitor includes, but is not limited to Atezolizumab, Avelumab, Durvalumab, LY3300054 (Eli Lilly and Company), and monoclonal antibodies or monoclonal antibody conjugates that act as a PD-L1 inhibitor. In some embodiments, the PD-1 inhibitor includes, but is not limited to pembrolizumab, Nivolumab, Cemiplimab and monoclonal antibodies or monoclonal antibody conjugates that act as a PD-1 inhibitors. In some embodiments, the CTLA-4 inhibitor includes, but is not limited to Ipilimumab, AGEN1884 and monoclonal antibodies or monoclonal antibody conjugates that act as a CTLA-4 inhibitor.

[0087] In some aspects, treatment for a moderate or high risk level can include a higher dosage or more frequent administration of chemotherapy or radiation therapy than treatment for a low risk level.

[0088] Example chemotherapeutic agents include, but are not limited to, anti -estrogens (e.g. tamoxifen, raloxifene, and megestrol), LHRH agonists (e.g. goscrclin and leuprolide), antiandrogens (e.g. flutamide and bicalutamide), photodynamic therapies (e.g. vertoporfin (BPD- MA), phthalocyanine, photosensitizer Pc4, and demethoxy-hypocrellin A (2BA-2-DMHA)), nitrogen mustards (e.g. cyclophosphamide, ifosfamide, trofosfamide, chlorambucil, estramustine, and melphalan), nitrosoureas (e.g. carmustine (BCNU) and lomustine (CCNU)), alkyl sulphonates (e.g. busulfan and treosulfan), triazenes (e.g. dacarbazine, temozolomide), platinum containing compounds (e.g. cisplatin, carboplatin, oxaliplatin), vinca alkaloids (e.g. vincristine, vinblastine, vindesine, and vinorelbine), taxoids (e.g. paclitaxel or a paclitaxel equivalent such as nanoparticle albumin-bound paclitaxel (ABRAXANE), docosahexaenoic acid bound-paclitaxel (DHA-paclitaxel, Taxoprexin), polyglutamate bound-paclitaxel (PG- paclitaxel, paclitaxel poliglumex, CT-2103, XYOTAX), the tumor-activated prodrug (TAP) ANG1005 (Angiopep-2 bound to three molecules of paclitaxel), paclitaxel -EC- 1 (paclitaxel bound to the erbB2-recognizing peptide EC-1), and glucose-conjugated paclitaxel, e.g., 2'- paclitaxel methyl 2-glucopyranosyl succinate; docetaxel, taxol), epipodophyllins (e.g. etoposide, etoposide phosphate, teniposide, topotecan, 9-aminocamptothecin, camptoirinotecan, irinotecan, crisnatol, mytomycin C), anti -metabolites, DHFR inhibitors (e.g. methotrexate, dichloromethotrexate, trimetrexate, edatrexate), IMP dehydrogenase inhibitors (e.g. mycophenolic acid, tiazofurin, ribavirin, and EICAR), ribonucleotide reductase inhibitors (e.g. hydroxyurea and deferoxamine), uracil analogs (e.g. 5 -fluorouracil (5-FU), floxuridine, doxifluridine, ratitrexed, tegafur-uracil, capecitabine), cytosine analogs (e.g. cytarabine (ara C), cytosine arabinoside, and fludarabine), purine analogs (e.g. mercaptopurine and Thioguanine), Vitamin D3 analogs (e.g. EB 1089, CB 1093, and KH 1060), isoprenylation inhibitors (e.g. lovastatin), dopaminergic neurotoxins (e.g. l-methyl-4-phenylpyridinium ion), cell cycle inhibitors (e.g. staurosporine), actinomycin (e.g. actinomycin D, dactinomycin), bleomycin (e.g. bleomycin A2, bleomycin B2, peplomycin), anthracycline (e.g. daunorubicin, doxorubicin, pegylated liposomal doxorubicin, idarubicin, epirubicin, pirarubicin, zorubicin, mitoxantrone), MDR inhibitors (e.g. verapamil), Ca2+ ATPase inhibitors (e.g. thapsigargin), imatinib, thalidomide, lenalidomide, tyrosine kinase inhibitors (e.g., axitinib (AGO 13736), bosutinib (SKI-606), cediranib (RECENTIN™, AZD2171), dasatinib (SPRYCEL®, BMS- 354825), erlotinib (TARCEVA®), gefitinib (IRESSA®), imatinib (Gleevec®, CGP57148B, STI-571), lapatinib (TYKERB®, TYVERB®), lestaurtinib (CEP-701), neratinib (HKI-272), nilotinib (TASIGNA®), semaxanib (semaxinib, SU5416), sunitinib (SUTENT®, SU11248), toceranib (PALLADIA®), vandetanib (ZACTIMA®, ZD6474), vatalanib (PTK787, PTK / ZK), trastuzumab (HERCEPTIN®), bevacizumab (AVASTIN®), rituximab (RITUXAN®), cetuximab (ERBITUX®), panitumumab (VECTIBIX®), ranibizumab (Lucentis®), nilotinib (TASIGNA®), sorafenib (NEXAVAR®), everolimus (AFINITOR®), alemtuzumab (CAMPATH®), gemtuzumab ozogamicin (MYLOTARG®), temsirolimus (TORISEL®), ENMD-2076, PCI-32765, AC220, dovitinib lactate (TKI258, CHIR-258), BIBW 2992 (TOVOK™), SGX523, PF-04217903, PF-02341066, PF-299804, BMS-777607, ABT-869, MP470, BIBF 1120 (VARGATEF®), AP24534, JNJ-26483327, MGCD265, DCC- 2036, BMS-690154, CEP-11981, tivozanib (AV-951), OSI-930, MM-121, XL-184, XL-647, and / or XL228), proteasome inhibitors (e.g., bortezomib (VELCADE)), mTOR inhibitors (e.g., rapamycin, temsirolimus (CCI-779), everolimus (RAD-001), ridaforolimus, AP23573 (Ariad), AZD8055 (AstraZeneca), BEZ235 (Novartis), BGT226 (Norvartis), XL765 (Sanofi Aventis), PF-4691502 (Pfizer), GDC0980 (Genetech), SF1126 (Semafoe) and OSI-027 (OSI)), oblimersen, gemcitabine, caminomycin, leucovorin, pemetrexed, cyclophosphamide, dacarbazine, procarbizine, prednisolone, dexamethasone, campathecin, plicamycin, asparaginase, aminopterin, methopterin, porfiromycin, melphalan, leurosidine, leurosine, chlorambucil, trabectedin, procarbazine, discodermolide, caminomycin, aminopterin, and hexamethyl melamine.

[0089] In some aspects, treatment for a moderate or high risk level can include administering an miR-155 inhibitor to the subject. Examples of miR-155 inhibitors and treatment of cancers using the same is provided in more detail below.

[0090] In some aspects, treatment for a moderate or high risk level can include administering to the subject a first synthetic nucleic acid sequence encoding inducible T-cell costimulatory ligand (ICOSL) or a functional fragment thereof and / or a second synthetic nucleic acid sequence encoding RELA or a functional fragment thereof. Examples of synthetic nucleic acid sequences encoding ICOSL or a functional fragment thereof and / or RELA or a functional fragment thereof, as well as treatment of cancers using the same, is provided in more detail below.

[0091] In some aspects, particularly when the first synthetic nucleic acid sequence is administered, treatment for a moderate or high risk level can further include administering one or more chemokines or cytokines to the subject. In some aspects, the one or more chemokines or cytokines can increase expression of ICOSL or the functional fragment thereof. In some aspects, the one or more chemokines or cytokines can be administered at the same time as administration of the first synthetic nucleic acid sequence. In other aspects, the one or more chemokines or cytokines can be administered after administration of the first synthetic nucleic acid sequence. In some aspects, the one or more chemokines or cytokines can include INF-y, TNF-a, and / or LPS.

[0092] In some aspects, particularly when the second synthetic nucleic acid sequence is administered, treatment for a moderate or high risk level can further include administering one or more enzymes or nucleic acid sequences encoding enzymes for post-translational phosphorylation and / or acetylation of RELA or the functional fragment thereof. In some aspects, the one or more enzymes or nucleic acid sequences encoding enzymes can increase activation of NFKB. In some aspects, the one or more enzymes or nucleic acid sequences encoding enzymes can be administered at the same time as administration of the second synthetic nucleic acid sequence. In some aspects, the one or more enzymes or nucleic acid sequences encoding enzymes can be administered after administration of the second synthetic nucleic acid sequence. In some aspects, treatment for a moderate or high risk level can further include, only after administration of any combination of an miR-155 inhibitor, the first synthetic nucleic acid sequence, and / or the second synthetic nucleic acid sequence, administering an immune cell therapy (such as, for example, T cell therapy, macrophage therapy, NK cell therapy, NK T cell therapy, CAR T cell therapy, and / or CAR NK cell therapy) and / or one or more checkpoint inhibitors to the subject. Examples of checkpoint inhibitors are described above. In some aspects, the subject may not receive immune cell therapy and / or one or more checkpoint inhibitors prior to administration of any combination of an miR-155 inhibitor, the first synthetic nucleic acid sequence, and / or the second synthetic nucleic acid sequence. This is a key advantage of this method, as immune cell therapy and checkpoint inhibitors will not be effective prior to blocking or circumventing miR-155 expression. This method can allow the subject to receive a more appropriate and effective treatment while also, optionally, being treated to restore ICOSL, RELA, and / or MHC-I function to enable successful treatment with immune cell therapy and / or checkpoint inhibitors.

[0093] In some aspects, the method can further include, before step a), screening the subject for Epstein-Barr virus (EBV). In some aspects, this may be achieved by diagnostic testing or sequencing, or by obtaining a medical history of the subject, either formal or informal (i.e., as recalled by the patient).

[0094] Epstein-Barr virus (EBV), a double-stranded DNA virus belonging to the Herpesviridae family, is a ubiquitous human pathogen with a seroprevalence exceeding 90% globally. Primary infection typically occurs in childhood and is often asymptomatic; however, when acquired during adolescence or adulthood, it commonly presents as infectious mononucleosis, characterized by lymphocytosis, pharyngitis, fever, and cervical lymphadenopathy. EBV exhibits tropism for B lymphocytes, where it establishes a lifelong latent infection by integrating its genome into host cells and expressing a limited set of latency- associated proteins, including EBNA (Epstein-Barr nuclear antigens) and LMP (latent membrane proteins). The virus can periodically reactivate, particularly in immunocompromised individuals, contributing to a range of pathologies. It has been discovered herein that EBV infection is correlated to and may induce miR-155 overexpression. In some aspects, the method may be preferentially carried out on subjects who are currently or have previously been infected with EBV due to the increased likelihood of miR-155 overexpression in these subjects. In some aspects, current or previous infection with EBV can be further used to calculate the risk score in step c). In some aspects, step b) can further include determining that ICOSL, RELA, and / or MHC-I are underexpressed in cancerous cells derived from the subject compared to ICOSL, RELA, and / or MHC-I expression on control cells. In some aspects, underexpression of ICOSL, RELA, and / or MHC-I can be further used to calculate the risk score in step c). For example, in some aspects, regular or only slightly decreased expression of ICOSL, RELA, and / or MHC- I can indicate a low risk level, and underexpression of ICOSL, RELA, and / or MHC-I can indicate a moderate or high risk level.

[0095] By “underexpressed” means that ICOSL, RELA, and / or MHC-I are expressed 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%,

[0096] 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%,

[0097] 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%,

[0098] 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%,

[0099] 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, or 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 3, 4, 5, 6, 7, 8, 9, or 10 times or less compared to the control sample.

[0100] In some aspects, the sample can include a cell sample, a tissue sample, a bodily fluid sample (e.g., blood, urine, sweat, spit, etc.), or a cell lysate sample. In some aspects, the sample can be a tumor sample (i.e., a cell, tissue, blood, or cell lysate sample taken directly from the tumor or tumor environment).

[0101] In some aspects, the solid cancer can be breast cancer, lung cancer, pancreatic cancer, gastric cancer, or colon cancer. Other solid cancers include, but are not limited to acoustic neuroma, adenocarcinoma, adrenal gland cancer, anal cancer, angiosarcoma (e.g., lymphangiosarcoma, lymphangioendotheliosarcoma, hemangiosarcoma), appendix cancer, benign monoclonal gammopathy, biliary cancer (e.g., cholangiocarcinoma), bladder cancer, breast cancer (e.g., adenocarcinoma of the breast, papillary carcinoma of the breast, mammary cancer, medullary carcinoma of the breast), brain cancer (e.g., meningioma; glioma, e.g., astrocytoma, oligodendroglioma; medulloblastoma), bronchus cancer, carcinoid tumor, cervical cancer (e.g., cervical adenocarcinoma), choriocarcinoma, chordoma, craniopharyngioma, colorectal cancer (e.g., colon cancer, rectal cancer, colorectal adenocarcinoma), epithelial carcinoma, ependymoma, endotheliosarcoma (e.g., Kaposi's sarcoma, multiple idiopathic hemorrhagic sarcoma), endometrial cancer (e.g., uterine cancer, uterine sarcoma), esophageal cancer (e.g., adenocarcinoma of the esophagus, Barrett's adenocarinoma), Ewing's sarcoma, eye cancer (e.g., intraocular melanoma, retinoblastoma), familiar hypereosinophilia, gall bladder cancer, gastric cancer (e.g., stomach adenocarcinoma), gastrointestinal stromal tumor (GIST), head and neck cancer (e.g., head and neck squamous cell carcinoma, oral cancer (e.g., oral squamous cell carcinoma (OSCC), throat cancer (e.g., laryngeal cancer, pharyngeal cancer, nasopharyngeal cancer, oropharyngeal cancer)); and multiple myeloma (MM)), heavy chain disease (e.g., alpha chain disease, gamma chain disease, mu chain disease), hemangioblastoma, inflammatory myofibroblastic tumors, immunocytic amyloidosis, kidney cancer (e.g., nephroblastoma a.k.a. Wilms' tumor, renal cell carcinoma), liver cancer (e.g., hepatocellular cancer (HCC), malignant hepatoma), lung cancer (e.g., bronchogenic carcinoma, small cell lung cancer (SCLC), non-small cell lung cancer (NSCLC), adenocarcinoma of the lung), leiomyosarcoma (LMS), mastocytosis (e.g., systemic mastocytosis), myelodysplastic syndrome (MDS), mesothelioma, myeloproliferative disorder (MPD) (e.g., polycythemia Vera (PV), essential thrombocytosis (ET), agnogenic myeloid metaplasia (AMM) a.k.a. myelofibrosis (MF), chronic idiopathic myelofibrosis, hypereosinophilic syndrome (HES)), neuroblastoma, neurofibroma (e.g., neurofibromatosis (NF) type 1 or type 2, schwannomatosis), neuroendocrine cancer (e.g., gastroenteropancreatic neuroendoctrine tumor (GEP-NET), carcinoid tumor), osteosarcoma, ovarian cancer (e.g., cystadenocarcinoma, ovarian embryonal carcinoma, ovarian adenocarcinoma), papillary adenocarcinoma, pancreatic cancer (e.g., pancreatic adenocarcinoma, intraductal papillary mucinous neoplasm (IPMN), Islet cell tumors), penile cancer (e.g., Paget's disease of the penis and scrotum), pinealoma, primitive neuroectodermal tumor (PNT), prostate cancer (e.g., prostate adenocarcinoma), rectal cancer, rhabdomyosarcoma, salivary gland cancer, skin cancer (e.g., squamous cell carcinoma (SCC), keratoacanthoma (KA), melanoma, basal cell carcinoma (BCC)), small bowel cancer (e.g., appendix cancer), soft tissue sarcoma (e.g., malignant fibrous histiocytoma (MFH), liposarcoma, malignant peripheral nerve sheath tumor (MPNST), chondrosarcoma, fibrosarcoma, myxosarcoma), sebaceous gland carcinoma, sweat gland carcinoma, synovioma, testicular cancer (e.g., seminoma, testicular embryonal carcinoma), thyroid cancer (e.g., papillary carcinoma of the thyroid, papillary thyroid carcinoma (PTC), medullary thyroid cancer), urethral cancer, vaginal cancer and vulvar cancer (e.g., Paget's disease of the vulva).

[0102] METHODS OF TREATING AND / OR PREVENTING CANCER

[0103] In some aspects, disclosed herein is a method of treating, preventing, decreasing, eliminating, and / or ameliorating a cancer in a subject in need thereof, the method including administering to the subject a synthetic nucleic acid sequence encoding inducible T-cell costimulatory ligand (ICOSL) or a functional fragment thereof, wherein expression and presentation of ICOSL or the functional fragment thereof by a cancerous cell induces and / or enhances an immune response against the cancer (e.g., induces and / or enhances killing of cancerous cells by cytotoxic T cells).

[0104] Inducible T-cell costimulatory ligand (ICOSL) is a protein encoded by the ICOSL gene located at chromosome 21. ICOSL is a glycosylated transmembrane protein that is also a member of the B7 family of costimulatory ligands. ICOS is an essential protein from upregulating and promoting T cell immune responses. After stimulation by means including, but not limited to IFN-y, TNF-a, and LPS stimulation, ICOSL expression is upregulated in B cells, dendritic cells, monocytes, macrophages, and T cells. ICOSL expression is also shown to promote antitumor T cell responses, thus making ICOSL an effective candidate for cancer immunotherapi e s .

[0105] In some aspects, the synthetic nucleic acid sequence can have a mutated or at least partially deleted 3’-UTR region. A wildtype ICOSL gene includes a 3’-UTR region, which includes an miR-155 target site of SEQ ID NO: 9 (AGCATTA). In some aspects, the 3’-UTR sequence and / or the miR-155 target site may be mutated, partially deleted, or fully deleted in the synthetic nucleic acid sequence such that the synthetic nucleic acid sequence does not include a wildtype 3’-UTR sequence and, in some aspects where the synthetic nucleic acid sequence still includes at least a portion of a 3’-UTR sequence, also does not include SEQ ID NO: 9.

[0106] In some aspects, the miR-155 target site in the synthetic nucleic acid sequence can be partially or completely replaced with a restriction site for a restriction enzyme. For example, in some aspects, the miR-155 target site in the synthetic nucleic acid sequence can be partially or completely replaced by a restriction site for EcoRl, for example, SEQ ID NO: 10 (GAATTC). In some such aspects, the synthetic nucleic acid sequence can be prepared by: a) using SEQ ID NO: 1 and SEQ ID NO: 4 to obtain a 5’ fragment of the synthetic nucleic acid sequence (i.e., any portion of the synthetic nucleic acid sequence including the 5’ end); b) using SEQ ID NO: 2 and SEQ ID NO: 3 to obtain a 3’ fragment of the synthetic nucleic acid sequence i.e., any portion of the synthetic nucleic acid sequence including the 3’ end); and c) digesting the 5’ fragment and the 3’ fragment with EcoRl, thereby forming the synthetic nucleic acid sequence.

[0107] In some aspect, at least a portion of the 3’-UTR region of the synthetic nucleic acid sequence can be deleted. In some aspects, the entire 3’-UTR region may be deleted such that the synthetic nucleic acid sequence may not include a 3’-UTR region. In some aspects, the synthetic nucleic acid sequence can include about 80% similarity or more (e.g., about 81% similarity or more, about 82% similarity or more, about 83% similarity or more, about 84% similarity or more, about 85% similarity or more, about 86% similarity or more, about 87% similarity or more, about 88% similarity or more, about 89% similarity or more, about 90% similarity or more, about 91% similarity or more, about 92% similarity or more, about 93% similarity or more, about 94% similarity or more, about 95% similarity or more, about 96% similarity or more, about 97% similarity or more, about 98% similarity or more, about 99% similarity or more, about 100% similarity) to SEQ ID NO: 5.

[0108] In some aspects, the method can further include administering one or more chemokines or cytokines to the subject, for example, to increase expression of ICOSL after the synthetic nucleic acid sequence has been taken up by cells. In some aspects, the one or more chemokines or cytokines can be administered at the same time as administration of the synthetic nucleic acid sequence. In other aspects, the one or more chemokines or cytokines can be administered after administration of the synthetic nucleic acid sequence. In some aspects, the one or more chemokines or cytokines can include INF-y, TNF-a, and / or LPS.

[0109] In some aspects, also disclosed herein is a method of treating, preventing, decreasing, eliminating, and / or ameliorating a cancer in a subject in need thereof, the method including administering to the subject a synthetic nucleic acid sequence encoding RELA or a functional fragment thereof, wherein expression of RELA or the functional fragment thereof by a cancerous cell induces and / or enhances expression of major histocompatibility complex class I (MHC-I), and wherein presentation of MHC-I by the cancerous cell induces and / or enhances an immune response against the cancer (e.g., induces and / or enhances killing of cancerous cells by cytotoxic T cells).

[0110] RELA (p65) is a key transcription factor within the NFKB signaling pathway, which plays a pivotal role in regulating immune responses, inflammation, and cell survival. Upon activation by stimuli such as pathogen-associated molecular patterns (PAMPs) or damage- associated molecular patterns (DAMPs), IKB proteins are phosphorylated and degraded, leading to the dimerization of p65 / p50 complexes that translocate to the nucleus. In the nucleus, RELA binds to KB elements in the promoter regions of target genes, promoting the transcription of pro-inflammatory cytokines, adhesion molecules, and immune response genes. Notably, RELA also upregulates the expression of major histocompatibility complex class I (MHC-I) molecules on the surface of cells. MHC-I is critical for the presentation of endogenous antigens to cytotoxic T cells and natural killer (NK) cells, facilitating the immune system’s ability to detect and eliminate infected or transformed cells. In some aspects, the synthetic nucleic acid sequence can have a mutated or at least partially deleted 3’-UTR region. A wildtype RELA gene includes a 3’-UTR region, which includes an miR-155 target site of SEQ ID NO: 9 (AGCATTA). In some aspects, the 3’-UTR sequence and / or the miR-155 target site may be mutated, partially deleted, or fully deleted in the synthetic nucleic acid sequence such that the synthetic nucleic acid sequence does not include a wildtype 3’-UTR sequence and, in some aspects where the synthetic nucleic acid sequence still includes at least a portion of a 3’-UTR sequence, also does not include SEQ ID NO: 9.

[0111] In some aspect, at least a portion of the 3’-UTR region of the synthetic nucleic acid sequence can be deleted. In some aspects, the entire 3’-UTR region may be deleted such that the synthetic nucleic acid sequence may not include a 3’-UTR region.

[0112] In some aspects, the synthetic nucleic acid sequence can include about 80% similarity or more (e.g., about 81% similarity or more, about 82% similarity or more, about 83% similarity or more, about 84% similarity or more, about 85% similarity or more, about 86% similarity or more, about 87% similarity or more, about 88% similarity or more, about 89% similarity or more, about 90% similarity or more, about 91% similarity or more, about 92% similarity or more, about 93% similarity or more, about 94% similarity or more, about 95% similarity or more, about 96% similarity or more, about 97% similarity or more, about 98% similarity or more, about 99% similarity or more, about 100% similarity) to SEQ ID NO: 11.

[0113] In some aspects, the method can further include administering one or more enzymes or nucleic acid sequences encoding enzymes for post-translational phosphorylation and / or acetylation of RELA. In some aspects, the one or more enzymes or nucleic acid sequences encoding enzymes can increase activation of NFKB. In some aspects, the one or more enzymes or nucleic acid sequences encoding enzymes can be administered at the same time as administration of the synthetic nucleic acid sequence. In other aspects, the one or more enzymes or nucleic acid sequences encoding enzymes can be administered after administration of the synthetic nucleic acid sequence.

[0114] In some aspects, also disclosed herein is a method of treating, preventing, decreasing, eliminating, and / or ameliorating a cancer in a subject in need thereof, the method including: a) administering a first synthetic nucleic acid sequence encoding ICOSL or a functional fragment thereof according to any of the disclosed methods of the same; and b) administering a second synthetic nucleic acid sequence encoding RELA or a functional fragment thereof according to any of the disclosed methods of the same; wherein expression of RELA or the functional fragment thereof by a cancerous cell induces and / or enhances expression of major histocompatibility complex class I (MHC-I); and wherein expression and presentation of ICOSL or the functional fragment thereof and MHC-I by the cancerous cell induces and / or enhances an immune response against the cancer (e.g., induces and / or enhances killing of cancerous cells by cytotoxic T cells).

[0115] In some aspects, steps a) and b) can be performed simultaneously. In other aspects, step a) can be performed before step b). In yet other aspects, step a) can be performed after step b).

[0116] In some aspects, any of the disclosed synthetic nucleic acid sequences can be provided as a vector. In some aspects, the vector can include an expression vector including a viral vector, a plasmid, a nanoparticle, or any variation thereof. Non-limiting examples of viral vectors used to treat, prevent, decrease, eliminate, and / or ameliorate cancer are retroviral vectors, adenoviral vectors, adeno-associated viral vectors, and large payload viral vectors.

[0117] Retroviral Vectors: A retrovirus is an animal virus belonging to the virus family of Retroviridae, including any types, subfamilies, genus, or tropisms. Retroviral vectors, in general, are described by Verma, I.M., Retroviral vectors for gene transfer.

[0118] A retrovirus is essentially a package which has packed into it nucleic acid cargo. The nucleic acid cargo carries with it a packaging signal, which ensures that the replicated daughter molecules will be efficiently packaged within the package coat. In addition to the package signal, there are a number of molecules which are needed in cis, for the replication, and packaging of the replicated virus. Typically a retroviral genome, contains the gag, pol, and env genes which are involved in the making of the protein coat. It is the gag, pol, and env genes which are typically replaced by the foreign DNA that it is to be transferred to the target cell. Retrovirus vectors typically contain a packaging signal for incorporation into the package coat, a sequence which signals the start of the gag transcription unit, elements necessary for reverse transcription, including a primer binding site to bind the tRNA primer of reverse transcription, terminal repeat sequences that guide the switch of RNA strands during DNA synthesis, a purine rich sequence 5' to the 3' LTR that serve as the priming site for the synthesis of the second strand of DNA synthesis, and specific sequences near the ends of the LTRs that enable the insertion of the DNA state of the retrovirus to insert into the host genome. The removal of the gag, pol, and env genes allows for about 8 kb of foreign sequence to be inserted into the viral genome, become reverse transcribed, and upon replication be packaged into a new retroviral particle. This amount of nucleic acid is sufficient for the delivery of a one to many genes depending on the size of each transcript. It is preferable to include either positive or negative selectable markers along with other genes in the insert. Since the replication machinery and packaging proteins in most retroviral vectors have been removed (gag, pol, and env), the vectors are typically generated by placing them into a packaging cell line. A packaging cell line is a cell line which has been transfected or transformed with a retrovirus that contains the replication and packaging machinery, but lacks any packaging signal. When the vector carrying the DNA of choice is transfected into these cell lines, the vector containing the gene of interest is replicated and packaged into new retroviral particles, by the machinery provided in cis by the helper cell. The genomes for the machinery are not packaged because they lack the necessary signals.

[0119] Adenoviral Vectors: The construction of replication-defective adenoviruses has been described (Berkner et al., J. Virology 61 : 1213-1220 (1987); Massie et al., Mol. Cell. Biol. 6:2872-2883 (1986); Haj-Ahmad Davidson et al., J.

[0120] Virology 61 : 1226-1239 (1987); Zhang "Generation and identification of recombinant adenovirus by liposome-mediated transfection and PCR analysis" BioTechniques 15:868-872 (1993)). The benefit of the use of these viruses as vectors is that they are limited in the extent to which they can spread to other cell types, since they can replicate within an initial infected cell, but are unable to form new infectious viral particles. Recombinant adenoviruses have been shown to achieve high efficiency gene transfer after direct, in vivo delivery to airway epithelium, hepatocytes, vascular endothelium, CNS parenchyma and a number of other tissue sites (Morsy, J. Clin. Invest. 92: 1580-1586 (1993); Kirshenbaum, J. Clin. Invest. 92:381-387 (1993); Roessler, J. Clin. Invest. 92: 1085-1092 (1993); Moullier, Nature Genetics 4: 154-159 (1993); La Salle, Science 259:988-990 (1993); Gomez-Foix, J. Biol. Chem. 267:25129-25134 (1992); Rich, Human Gene Therapy 4:461-476 (1993); Zabner, Nature Genetics 6:75-83 (1994); Guzman, Circulation Research 73: 1201-1207 (1993); Bout, Human Gene Therapy 5:3- 10 (1994); Zabner, Cell 75:207-216 (1993); Caillaud, Eur. J. Neuroscience 5: 1287-1291 (1993); and Ragot, J. Gen. Virology 74:501-507 (1993)). Recombinant adenoviruses achieve gene transduction by binding to specific cell surface receptors, after which the virus is internalized by receptor-mediated endocytosis, in the same manner as wild type or replicationdefective adenovirus (Chardonnet and Dales, Virology (1970); Brown and

[0121] Burlingham, J. Virology 12:386-396 (1973); Svensson and Persson, J. Virology 55:442-449 (1985); Seth, et al., J. Virol. 51 :650-655 (1984); Seth, et al., Mol. Cell. Biol. 4: 1528-1533 (1984); Varga et al., J. Virology 65:6061-6070 (1991); Wickham et al., Cell 73:309-319 (1993)). A viral vector can be one based on an adenovirus which has had the El gene removed and these virons are generated in a cell line such as the human 293 cell line. In another preferred embodiment both the El and E3 genes are removed from the adenovirus genome.

[0122] Adeno-Associated Viral Vectors: Another type of viral vector is based on an adeno- associated virus (AAV). This defective parvovirus is a preferred vector because it can infect many cell types and is nonpathogenic to humans. AAV type vectors can transport about 4 to 5 kb and wild type AAV is known to stably insert into chromosome 19. Vectors which contain this site specific integration property are preferred. An especially preferred embodiment of this type of vector is the P4.1 C vector produced by Avigen, San Francisco, CA, which can contain the herpes simplex virus thymidine kinase gene, HSV-tk, and / or a marker gene, such as the gene encoding the green fluorescent protein, GFP.

[0123] In another type of AAV virus, the AAV contains a pair of inverted terminal repeats (ITRs) which flank at least one cassette containing a promoter which directs cell-specific expression operably linked to a heterologous gene. Heterologous in this context refers to any nucleotide sequence or gene which is not native to the AAV or B19 parvovirus.

[0124] Typically the AAV and B19 coding regions have been deleted, resulting in a safe, noncytotoxic vector. The AAV ITRs, or modifications thereof, confer infectivity and sitespecific integration, but not cytotoxicity, and the promoter directs cell-specific expression. United states Patent No. 6,261,834 is herein incorporated by reference for material related to the AAV vector.

[0125] Large Payload Viral Vectors: Molecular genetic experiments with large human herpesviruses have provided a means whereby large heterologous DNA fragments can be cloned, propagated and established in cells permissive for infection with herpesviruses (Sun et al., Nature genetics 8: 33-41, 1994; Cotter and Robertson,. Curr Opin Mol Ther 5: 633-644, 1999). These large DNA viruses (herpes simplex virus (HSV) and Epstein-Barr virus (EBV), have the potential to deliver fragments of human heterologous DNA > 150 kb to specific cells. EBV recombinants can maintain large pieces of DNA in the infected B-cells as episomal DNA. Individual clones carried human genomic inserts up to 330 kb appeared genetically stable The maintenance of these episomes requires a specific EBV nuclear protein, EBNA1, constitutively expressed during infection with EBV. Additionally, these vectors can be used for transfection, where large amounts of protein can be generated transiently in vitro. Herpesvirus amplicon systems are also being used to package pieces of DNA > 220 kb and to infect cells that can stably maintain DNA as episomes. Other useful systems include, for example, replicating and host-restricted nonreplicating vaccinia virus vectors.

[0126] In some aspects, any of the disclosed synthetic nucleic acid sequences can be under control of a tissue specific promoter or an inducible promoter. Exemplary tissue specific promoters include, but are not limited to surfactant protein B promoter (SP-B in lung), B29 promoter (B cells), CD14 promotor (monocytic cells), CD43 promoter (leukocytes and platelets), CD68 promoter (macrophages), Desmin promoter (muscle), Elastase- 1 promoter (pancreatic acinar cells), endoglin promoter (endothelial cells), Fibronectin promoter (differentiating cells and healing tissues), Flt-1 promoter (endothelial cells), GFAP promoter (astrocytes), Mb promoter (muscle), SYN1 promoter (neurons), SV40 / bAlb promoter (Liver)) and cancer specific promoters (including, but not limited to carcinoembryonic antigen (CEA) promoter, hTERT promoter, epidermal growth factor receptor (EGFR) promoter, human epidermal growth factor receptor / neu (HER2 / NEU) promoter, vascular endothelial growth factor receptor (VEGFR) promoter, folate receptor (FR) promoter, transferrin receptor (CD71) promoter, mucines promoters, tumor resistance antigen 1-60 (TRA-1-60) promoter, cyclooxygenase (COX) promoter, cytokeratin 18 promoter, cytokeratin 19 promoter, surviving promoter, and chimeric antigen receptor (CAR) promoters, alpha-fetoprotein (AFP) promoter, thyroid transcription factor 1 (TTF-1) promoter, glypican-3 protein (GPC3) promoter, human secretory leukocyte protease inhibitor (hSLPI) promoter, ERBB2 promoter, Mucin 1 (MUC1) promoter, L-plastin promoter, alpha-lactalbumin (LALBA) promoter, cyclooxygenase 2 (COX2) promoter, epithelial glycoprotein (EPG2) promoter, A33 promoter, uPAR promoter, breast cancer 1 (BRCA1) and BRCA2 promoters). Exemplary inducible promoters include, but are not limited to chemically inducible promoters (such as, for example the inducible tetracycline ON (Tet-On) system, the inducible pLac promoter, and the inducible pBad promoter), temperature inducible promoters (such as, for example heat shock protein 70(Hsp70) promoter, Hsp90 promoter, and derivatives thereof), and light inducible promoters (such as, for example the red flame plasmid pDawn two-component system). In some aspects, any of the discloed synthetic nucleic acids can be administered directly into a tumor of the subject.

[0127] In some aspects, any of the disclosed synthetic nucleic acid sequences may be delivered as naked nucleic acid (unpackaged) or via delivery vehicles. As used herein, the terms “delivery vehicle,” “transfer vehicle,” “nanoparticle” or grammatical equivalent, are used interchangeably. In some aspects, any of the disclosed synthetic nucleic acid sequences may be delivered via a single delivery vehicle. In some aspects, any of the disclosed synthetic nucleic acid sequences may be delivered via one or more delivery vehicles each of a different composition. According to various aspects, suitable delivery vehicles include, but are not limited to polymer based carriers, such as polyethyleneimine (PEI), lipid nanoparticles and liposomes, nanoliposomes, ceramide-containing nanoliposomes, proteoliposomes, both natural and synthetically-derived exosomes, natural, synthetic and semi-synthetic lamellar bodies, nanoparticulates, calcium phosphor-silicate nanoparticulates, calcium phosphate nanoparticulates, silicon dioxide nanoparticulates, nanocrystalline particulates, semiconductor nanoparticulates, poly(D-arginine), sol-gels, nanodendrimers, starch-based delivery systems, micelles, emulsions, niosomes, multi-domain-block polymers (vinyl polymers, polypropyl acrylic acid polymers, dynamic poly conjugates), cell- or platelet-derived exosomes, ethosomes, or transfersomes.

[0128] In some aspects, a suitable delivery vehicle can be a lipid nanoparticle. As used herein, “lipid nanoparticles” refer to particles having at least one dimension on the order of nanometers (e.g., 1-1000 nm) and including one or more lipids. In the context of the present invention, a lipid nanoparticle typically serves to transport a desired nucleic acid sequence to a target cell or tissue. The process of incorporation of a desired nucleic acid sequence into a lipid nanoparticle is often referred to as “loading”. The lipids and the nucleic acid sequence can create a self-assembled structure via counterion interactions. The purpose of incorporating a nucleic acid sequence into a transfer vehicle, such as a lipid nanoparticle, is often to protect the nucleic acid from an environment which may contain enzymes or chemicals that degrade nucleic acids and / or systems or receptors that cause the rapid excretion of the nucleic acids. Accordingly, in some aspects, a suitable delivery vehicle may be capable of enhancing the stability of any of the disclosed synthetic nucleic acid sequences contained therein and / or facilitate the delivery of any of the disclosed synthetic nucleic acid sequences to the target cell or tissue. In some aspects, the delivery vehicle is a viral particle which infects cancerous cells.

[0129] In some aspects, any of the disclosed methods can further include providing an miR- 155 inhibitor to the subject. In some aspects, the miR-155 inhibitor can include an antibody, an antibody fragment, a small molecule inhibitor, a polypeptide, an oligonucleotide, or any combination thereof. In some aspects, the miR-155 inhibitor can include cobomarsen or an anti-miR-155 oligonucleotide.

[0130] In some aspects, any of the disclosed methods can further include, after administering the synthetic nucleic acid sequence(s), administering an immune cell therapy (such as, for example, T cell therapy, macrophage therapy, NK cell therapy, NK T cell therapy, CAR T cell therapy, and / or CARNK cell therapy) and / or one or more checkpoint inhibitors to the subject. Examples of checkpoint inhibitors are described above. In some aspects, the subject may not have received immune cell therapy and / or one or more checkpoint inhibitors before administration of the synthetic nucleic acid sequence(s). This may be due either to the stage of treatment, or as an intentional decision to withhold immune cell therapy and / or checkpoint inhibitors until the cancer has begun to re-express ICOSL and / or MHC-I.

[0131] In some aspects, the method can further include, before administering the synthetic nucleic acid sequence(s), screening the subject for Epstein-Barr virus (EBV). In some aspects, this may be achieved by diagnostic testing or sequencing, or by obtaining a medical history of the subject, either formal or informal (i.e., as recalled by the patient). In some aspects, the subject may currently or may have previously been infected with Epstein-Barr virus (EBV).

[0132] In some aspects, any of the disclosed synthetic nucleic acid sequences may be administered in such amounts, time, and route deemed necessary in order to achieve the desired result. The exact amount of any of the disclosed synthetic nucleic acid sequences will vary from subject to subject, depending on the species, age, and general condition of the subject, the severity of the cancer, the particular composition, its mode of administration, its mode of activity, and the like. Any of the disclosed synthetic nucleic acid sequences are preferably formulated in dosage unit form for ease of administration and uniformity of dosage. It will be understood, however, that the total daily usage of any of the disclosed synthetic nucleic acid sequences will be decided by the attending physician within the scope of sound medical judgment. The specific therapeutically effective dose level for any particular subject will depend upon a variety of factors including the cancer being treated and the severity of the cancer / tumor; the activity of any of the disclosed synthetic nucleic acid sequences being employed; any of the disclosed synthetic nucleic acid sequences; the age, body weight, general health, sex and diet of the patient; the time of administration, route of administration, and rate of excretion of any of the disclosed synthetic nucleic acid sequences being employed; the duration of the treatment; drugs used in combination or coincidental with any of the disclosed synthetic nucleic acid sequences being employed; and like factors well known in the medical arts.

[0133] Any of the disclosed synthetic nucleic acid sequences may be administered by any route. In some embodiments, any of the disclosed synthetic nucleic acid sequences may be administered via a variety of routes, including intravenous, intramuscular, intra-arterial, intramedullary, intrathecal, subcutaneous, intraventricular, transdermal, interdermal, rectal, intravaginal, intraperitoneal, mucosal, nasal, buccal, enteral, sublingual; by intratracheal instillation, bronchial instillation, and / or inhalation; and / or as an oral spray, nasal spray, and / or aerosol. In general, the most appropriate route of administration will depend upon a variety of factors including the nature of any of the disclosed synthetic nucleic acid sequences (e.g., its stability in the environment of the subject’s body), the condition of the subject (e.g., whether the subject is able to tolerate the chosen route of administration), etc.

[0134] The exact amount of any of the disclosed synthetic nucleic acid sequences required to achieve a therapeutically effective amount will vary from subject to subject, depending on species, age, and general condition of a subject, severity of the side effects, identity of the particular compound(s), mode of administration, and the like. The amount to be administered to, for example, a child or an adolescent can be determined by a medical practitioner or person skilled in the art and can be lower or the same as that administered to an adult.

[0135] In some aspects, any of the disclosed synthetic nucleic acid sequences can be administered 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24,

[0136] 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,

[0137] 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74,

[0138] 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99,

[0139] 100, or more times. In some aspects, any of the disclosed synthetic nucleic acid sequences can be administered daily. In some aspects, any of the disclosed synthetic nucleic acid sequences can be administered every day, every 2 days, every 3 days, every 4 days, every 5 days, every 6 days, every 7 days, or more. In some aspects, any of the disclosed synthetic nucleic acid sequences can be administered every week, every 2 weeks, every 3 weeks, every 4 weeks, or more. In some aspects, any of the disclosed synthetic nucleic acid sequences can be administered every month, every 2 months, every 3 months, every 4 months, every 5 months, every 6 months, every 7 months, every 8 months, every 9 months, every 10 months, every 11 months, every 12 months, or more. In some aspects, any of the disclosed synthetic nucleic acid sequences can be administered every year, every 2 years, every 3 years, every 4 years, every 5 years, or more.

[0140] In some aspects, the cancer targeted in any of the disclosed methods can be lymphoma, breast cancer, lung cancer, pancreatic cancer, gastric cancer, or colon cancer. Other cancers include, but are not limited to acoustic neuroma, adenocarcinoma, adrenal gland cancer, anal cancer, angiosarcoma (e.g., lymphangiosarcoma, lymphangioendotheliosarcoma, hemangiosarcoma), appendix cancer, benign monoclonal gammopathy, biliary cancer (e.g., cholangiocarcinoma), bladder cancer, breast cancer (e.g., adenocarcinoma of the breast, papillary carcinoma of the breast, mammary cancer, medullary carcinoma of the breast), brain cancer (e.g., meningioma; glioma, e.g., astrocytoma, oligodendroglioma; medulloblastoma), bronchus cancer, carcinoid tumor, cervical cancer (e.g., cervical adenocarcinoma), choriocarcinoma, chordoma, craniopharyngioma, colorectal cancer (e.g., colon cancer, rectal cancer, colorectal adenocarcinoma), epithelial carcinoma, ependymoma, endotheliosarcoma (e.g., Kaposi's sarcoma, multiple idiopathic hemorrhagic sarcoma), endometrial cancer (e.g., uterine cancer, uterine sarcoma), esophageal cancer (e.g., adenocarcinoma of the esophagus, Barrett's adenocarinoma), Ewing's sarcoma, eye cancer (e.g., intraocular melanoma, retinoblastoma), familiar hypereosinophilia, gall bladder cancer, gastric cancer (e.g., stomach adenocarcinoma), gastrointestinal stromal tumor (GIST), head and neck cancer (e.g., head and neck squamous cell carcinoma, oral cancer (e.g., oral squamous cell carcinoma (OSCC), throat cancer (e.g., laryngeal cancer, pharyngeal cancer, nasopharyngeal cancer, oropharyngeal cancer)), hematopoietic cancers (e.g., leukemia such as acute lymphocytic leukemia (ALL) (e.g., B-cell ALL, T-cell ALL), acute myelocytic leukemia (AML) (e.g., B-cell AML, T-cell AML), chronic myelocytic leukemia (CML) (e.g., B-cell CML, T-cell CML), and chronic lymphocytic leukemia (CLL) (e.g., B-cell CLL, T-cell CLL); lymphoma such as Hodgkin lymphoma (HL) (e.g., B-cell HL, T-cell HL) and non-Hodgkin lymphoma (NHL) (e.g., B-cell NHL such as diffuse large cell lymphoma (DLCL) (e.g., diffuse large B-cell lymphoma (DLBCL)), follicular lymphoma, chronic lymphocytic leukemia / small lymphocytic lymphoma (CLL / SLL), mantle cell lymphoma (MCL), marginal zone B-cell lymphomas (e.g., mucosa- associated lymphoid tissue (MALT) lymphomas, nodal marginal zone B-cell lymphoma, splenic marginal zone B-cell lymphoma), primary mediastinal B-cell lymphoma, Burkitt lymphoma, lymphoplasmacytic lymphoma (i.e., “Waldenstrom's macroglobulinemia”), hairy cell leukemia (HCL), immunoblastic large cell lymphoma, precursor B-lymphoblastic lymphoma and primary central nervous system (CNS) lymphoma; and T-cell NHL such as precursor T-lymphoblastic lymphoma / leukemia, peripheral T-cell lymphoma (PTCL) (e.g., cutaneous T-cell lymphoma (CTCL) (e.g., mycosis fungiodes, Sezary syndrome), angioimmunoblastic T-cell lymphoma, extranodal natural killer T-cell lymphoma, enteropathy type T-cell lymphoma, subcutaneous panniculitis-like T-cell lymphoma, anaplastic large cell lymphoma); a mixture of one or more leukemia / lymphoma as described above; and multiple myeloma (MM)), heavy chain disease (e.g., alpha chain disease, gamma chain disease, mu chain disease), hemangioblastoma, inflammatory myofibroblastic tumors, immunocytic amyloidosis, kidney cancer (e.g., nephroblastoma a.k.a. Wilms' tumor, renal cell carcinoma), liver cancer (e.g., hepatocellular cancer (HCC), malignant hepatoma), lung cancer (e.g., bronchogenic carcinoma, small cell lung cancer (SCLC), non-small cell lung cancer (NSCLC), adenocarcinoma of the lung), leiomyosarcoma (LMS), mastocytosis (e.g., systemic mastocytosis), myelodysplastic syndrome (MDS), mesothelioma, myeloproliferative disorder (MPD) (e.g., polycythemia Vera (PV), essential thrombocytosis (ET), agnogenic myeloid metaplasia (AMM) a.k.a. myelofibrosis (MF), chronic idiopathic myelofibrosis, chronic myelocytic leukemia (CML), chronic neutrophilic leukemia (CNL), hypereosinophilic syndrome (HES)), neuroblastoma, neurofibroma (e.g., neurofibromatosis (NF) type 1 or type 2, schwannomatosis), neuroendocrine cancer (e.g., gastroenteropancreatic neuroendoctrine tumor (GEP-NET), carcinoid tumor), osteosarcoma, ovarian cancer (e.g., cystadenocarcinoma, ovarian embryonal carcinoma, ovarian adenocarcinoma), papillary adenocarcinoma, pancreatic cancer (e.g., pancreatic adenocarcinoma, intraductal papillary mucinous neoplasm (IPMN), Islet cell tumors), penile cancer (e.g., Paget's disease of the penis and scrotum), pinealoma, primitive neuroectodermal tumor (PNT), prostate cancer (e.g., prostate adenocarcinoma), rectal cancer, rhabdomyosarcoma, salivary gland cancer, skin cancer (e.g., squamous cell carcinoma (SCC), keratoacanthoma (KA), melanoma, basal cell carcinoma (BCC)), small bowel cancer (e.g., appendix cancer), soft tissue sarcoma (e.g., malignant fibrous histiocytoma (MFH), liposarcoma, malignant peripheral nerve sheath tumor (MPNST), chondrosarcoma, fibrosarcoma, myxosarcoma), sebaceous gland carcinoma, sweat gland carcinoma, synovioma, testicular cancer (e.g., seminoma, testicular embryonal carcinoma), thyroid cancer (e.g., papillary carcinoma of the thyroid, papillary thyroid carcinoma (PTC), medullary thyroid cancer), urethral cancer, vaginal cancer and vulvar cancer (e.g., Paget's disease of the vulva).

[0141] In some aspects, any of the disclosed methods can decrease cancer by 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, 100%, by less than, by more than, or a percentage in-between any previously percentage relative to a control.

[0142] A number of embodiments of the disclosure have been described. Nevertheless, it will be understood that various modifications may be made without departing from the spirit and scope of the invention. Accordingly, other embodiments are within the scope of the following claims.

[0143] By way of non-limiting illustration, examples of certain embodiments of the present disclosure are given below. EXAMPLES

[0144] The following examples are set forth below to illustrate the compositions, devices, methods, and results according to the disclosed subject matter. These examples are not intended to be inclusive of all aspects of the subject matter disclosed herein, but rather to illustrate representative methods and results. These examples are not intended to exclude equivalents and variations of the present invention which are apparent to one skilled in the art.

[0145] EXAMPLE 1: miR-155-targeted ICOSL controls tumor rejection

[0146] Elevated levels of miR-155 in solid and liquid malignancies correlate with aggressiveness of the disease. Herein, it is shown that miR-155 targets transcripts encoding ICOSL, the ligand for Inducible T-cell co-stimulator (Icos), thus impairing the ability of T cells to recognize and eliminate malignant cells. Specifically, overexpression of miR-155 in B cells of Ep-miR-155 mice was found to cause loss of ICOSL expression as they progressed towards malignancy. Similarly, in mice where miR-155 expression is controlled by a Cre-Tet-OFF system, miR-155 induction led to malignant infiltrates lacking ICOSL expression. Conversely, turning miR-155 OFF led to tumor regression and emergence of infiltrates including ICOSL- positive B cells and Icos-positive T cells forming immunological synapses. Therefore, malignant cells were engineered to express ICOSL, in order to determine whether ICOSL expression would increase tumor infiltration by cytotoxic T cells and reduce tumor progression. Indeed, overexpressing an ICOSL-encoding cDNA in MC38 murine colon cancer cells before injection into syngeneic C57BL6 mice reduced tumor size and increased intra-tumor CD8+ T cell infiltration, that formed synapses with ICOSL-expressing MC38 cells. These results underscore the fact that by targeting ICOSL transcripts, miR-155 impairs the infiltration of cancer cells by cytotoxic T cells, as well as the importance of ICOSL on enhancing the immune response against malignant cells. These findings lead to the development of more effective anti-cancer treatments based on maintaining, increasing, or restoring ICOSL expression by malignant cells, along with impairing miR-155 activity.

[0147] Herein, one crucial aspect of microRNA miR-155 oncogenic activity is the targeting of ICOSL, that is needed for the recognition and elimination of malignant cells by CD8 positive T cells. The data indicate that ICOS-ICOSL immune checkpoint is critical for enhancing the effectiveness of immune therapies, not only for B cell lymphomas, but also for breast, colon, pancreatic, and lung solid tumors. This is supported by the findings herein that these solid tumors likewise exhibit elevated levels of miR-155, showing that they may evade the antitumor immune response through similar molecular mechanisms. Coercing cancer cells to express ICOSL, along with reducing miR-155 levels, could enhance their elimination by immune cells leading to tumor eradication and cure.

[0148] It was found that miR-155 is highly expressed not only in B cell malignancies, but also in colon, breast, pancreatic, and lung cancers. High levels of miR-155 expression in these solid malignancies, as in B-cell malignancies is associated with increased aggressiveness, showing that miR-155 may hinder the recognition of malignant cells by the immune system. Given that microRNAs can target multiple transcripts simultaneously, the key question regarding the oncogenic effects of miR-155 in these malignancies is to identify specific target transcripts that encode factors critical for the recognition and elimination of malignant cells by T cells. To address this important question, immune checkpoints factors expressed by B cells that could be targeted by miR-155 were the focus. Attention was particularly drawn to Inducible T-cell co-stimulator Ligand (ICOSL), as bioinformatic analysis revealed a conserved miR-155 site in the 3’-UTR of ICOSL gene that is particularly expressed in B cells. ICOSL acts as the ligand for ICOS / CD278, a T cell-specific surface costimulatory molecule structurally related to CD28 and CTLA-4. ICOS expression is induced upon T cell activation and is necessary for mounting effective T cell-dependent immune responses. ICOS-expressing T cells in the germinal centers (GC) are implicated in T-B cell communication and T-cell dependent B cell responses through ICOS- ICOSL interactions. ICOSL binding to ICOS induces the formation of immune synapses, T cell activation, and T cell effector functions.

[0149] Herein, it is shown that ICOSL is a direct target of miR-155, and that high levels of miR-155 result in reduction or loss of ICOSL expression in two different mouse models of B cell lymphoma. Turning miR-155 expression OFF after being active, induced a strong lymphocytic infiltration with the formation of immunological synapses between T-B cells through Icos-ICOSL interactions. To demonstrate that ICOSL is indeed the critical target of miR-155 responsible for the elimination of cancer cells and tumor rejection by the immune system, the coding sequence of ICOSL was overexpressed, without its 3’-UTR, in a murine MC38 colon cancer cell line derived from C57BL6 mice. Herein, the aim was to test the ability of ICOSL to induce an immune response targeting tumors induced in mice by the subcutaneous injection of MC38 tumor cells. These cells are highly mutated, aggressive, and lead rapidly to terminal illness when subcutaneously injected into syngeneic mice. Mice injected with ICOSL- overexpressing MC38 cells showed tumors smaller than those found in mice injected with MC38 cells that did not express ICOSL. In addition, the tumors derived from ICOSL- expressing MC38 cells showed extensive CD8 cell infiltration that aggregated in the areas where ICOSL-expressing cells were present as demonstrated by co-expression analyses. Furthermore, it was found that CD8 positive cells and ICOSL-expressing cells were forming immunological synapses within the tumors.

[0150] These results present new opportunities for the development of future T-cell-based immune therapies, not only for B cell malignancies but also for other very common tumors that express high levels of miR-155, such as breast, lung, pancreatic, or colon cancers. These therapies focus on enhancing the activity of the ICOS-ICOSL immune checkpoint by either blocking miR-155 activity or by re-expressing the ICOSL gene.

[0151] Results

[0152] MiR-155 activity in malignant B cells impairs T cell anti-tumor activity at least in part by targeting ICOSL transcripts: To identify miR-155 targets involved in anti -tumor immune response, the effects of miR-155 were first analyzed on B cell malignancy and T cell infiltration by turning the expression of miR-155 ON or OFF in lymphocytes of the Nestin-Cre x miR-155LSLtTAoffspring. The expression of miR-155 was confirmed in these mice is induced or repressed by withdrawal or addition of doxycycline in their food, respectively, using in situ hybridization (ISH) (Figure 1A). Turning ON the expression of miR-155 for three months led to extensive malignant B cell infiltrates in different organs such as the spleen, kidneys, and muscles (Figure IB, top panel and Figure 5). When miR-155 was subsequently turned back OFF for one week, infiltrates remained visible in different organs (Figure IB, bottom panel), however, this time the infiltrates were predominantly of CD4-positive T cells (brown signal on the bottom right) and only a few B cells could still be found (Figure IB, bottom panel). As B cell tumors regressed, focal infiltrations of T cells expressing Icos (Figure 1C) was observed. It is worth noting that the presence of T cells expressing Icos in contrast was rare when miR- 155 was ON (Figure 1C, bottom panel). These results showed that high levels of miR-155 inhibited T cell ingression and that when this microRNA was turned OFF, B cells were eliminated by infiltrating T cells. Thus, it was reasoned that, beside intrinsic mechanisms inducing the apoptosis of malignant B cells, in particular the de-repression of Shipl, that it was previously established as a direct target of miR-155 there should be additional, extrinsic mechanisms also involved in the elimination of malignant B cells, likely through their recognition and elimination by T cells. Among miR-155 putative targets able to interfere with T cell activity, Inducible T-cell co-stimulator Ligand (ICOSL) became the focus because: 1) The bioinformatic analysis showed a conserved miR-155 site in the 3’-UTR of ICOSL transcripts; 2) ICOSL in B cells serves as the ligand for ICOS / CD278, a T cell-specific surface costimulatory molecule structurally related to CD28 and CTLA-4; 3) ICOS expression is induced upon T cell activation and is necessary for mounting effective T cell-dependent immune responses; and 4) T cells in the tumor infiltrates were actually expressing Icos once miR-155 had been turned OFF (Figure 1C). It was checked whether miR-155 could directly target ICOSL transcripts. It was found that: (i) In Raw264.7 macrophages, miR-155 significantly reduced the activity of a Luciferase reporter construct containing the WT 3’-UTR of human ICOSL (Figure ID). This effect was abolished by mutating the consensus miR-155 binding site present in ICOSL 3’-UTR (Figure ID). Similar results were obtained with HEK- 293 cells (not shown); (ii) Treating Raw264.7 cells with lipopolysaccharide (LPS), thatit was previously shown to increase miR-155 expression, or transfecting these cells with miR-155, both reduced the expression of ICOSL (Figure IE); (iii) Transfecting miR-155 into BJAB and NAMALWA Burkitt’s lymphoma (BL) cell lines, that do not express miR-155, reduced the expression of ICOSL (Figure IF); (iv) It has also been previously shown that miR-155 is highly expressed by CLL cells but not in BL cells. A comparative analysis showed an inverse correlation between miR-155 and ICOSL levels of expression, with BL cells expressing high levels of ICOSL and virtually no miR-155 versus CLL cells showing high levels of miR-155 and less ICOSL (Figure 1G); and finally (v) Transfecting MEC2-CLL cells with a miR-155- inhibitor increased the levels of ICOSL on the cell surface as measured by flow cytometry (Figure 6). Altogether, the above results indicated that miR-155 activity in malignant B cells may potentially impair T cell infiltration, activation, and anti-tumor response at least in part through the targeting of ICOSL transcripts.

[0153] MiR-155 activity leads to accumulation of malignant B cells lacking ICOSL expression: Given the above results, it was investigated the impact of miR-155 activity on ICOSL expression and tumor progression / regression by turning the expression of miR-155 ON or OFF in CvQ-Neslin x miR-155LSLtTAmice. With miR-155 ON, the percentage of B cells positive for ICOSL in the lymph nodes of CvQ-Neslin x miR-155LSLtTAmice with miR-155 ON was reduced compared with lymph nodes of wild-type (WT) littermates, based on both flow cytometry (Figure 2A) and IHC analyses. Specifically, in contrast to B splenocytes from WT mice that stained positive for ICOSL (Figure 2B, 1strow), B cell infiltrates in miR-155LSLtTAmice with miR-155 ON for three months completely lacked ICOSL expression, and they exhibited a malignant phenotype characterized by their round shape and typical variable intensity of labeling with the B cell marker CD20 (Figure 2B, 2ndrow). Although certain cells within these infiltrates stained positive (green) for ICOSL, they were not B cells, as morphologically they resembled either endothelial cells or macrophages. Indeed, this was confirmed by co-staining for ICOSL and CD31 endothelial marker (Figure 2B, 3rdrow) or CD 163, a macrophage marker (Figure 7). Turning miR-155 back OFF for one week after it had been ON for three months led to the disappearance of malignant B cells in Nestin-Cre x miR- 155LSLtTAmice (Figure 2B, 4throw), and as shown in Figure IB and consistent with previous reports in this mouse model. At this timepoint, a few CD20 positive cells were found that presented with the morphology of benign B cells. These B cells expressed ICOSL based on the colocalization of CD20 and ICOSL signals (Figure 2B, 4throw). Socs an established target of miR-155, was also reduced and then re-expressed along ICOSL in B cell infiltrates with miR-155 turned ON and then back OFF again, respectively (Figure 8).

[0154] Loss of ICOSL expression in malignant B cells impairs the formation of B-T cell immune synapses and tumor immune surveillance: As shown in Figure 1C, once miR-155 was turned back OFF and ICOSL was de-repressed, the number of Icos-positive T cells increased along with tumor regression. These T cells showed a focal perivascular accumulation pattern with areas containing a high number of Icos-positive cells (Figure 1C). Thus, the average number of Icos-positive T cells counted automatically in tumors from mice with miR- 155 ON shown in Figure 1C was 5.3 / 200x microscopic field but increased dramatically to 131 / 200x microscopic field after miR-155 was turned back OFF again for one week. Similar results were also obtained in the spleen; however, the difference did not reach statistical significance most probably due to the existence of variable patterns as compared to diffuse pattern of T cell infiltrates (Figure 9). Nevertheless, the induction of ICOSL once miR-155 was turned back OFF strongly showed that, in addition to undergoing apoptosis due the derepression of Socsl (Figure 8) or Shipl malignant B cells were also being eliminated through extrinsic mechanisms, likely through their recognition and elimination by T cells. This elimination of malignant B cells was likely facilitated by Icos-ICOSL engagement. Namely, when miR-155 was induced for 10-12 weeks and then turned OFF for a period as short as four days, Icos-positive T cells were found to form immune synapses with ICOSL-positive B cells in remaining infiltrates (Figure 2C). Interestingly, the T (Icos) - B (ICOSL) interactions were primarily observed in the vicinity of blood vessels (Figure 2D). The localization of synapses next to blood vessels likely explains the presence of focal areas containing Icos-positive T cells seen on Figure 1C and may also reflect the ability of ICOSL-expressing B cells to recruit and activate T cells at the interface between lymphoma and normal tissue. The formation of immune synapses due to the de-repression of ICOSL gene was also observed in the spleen of miR-155LSLtTAmice, where a small, however increasing, percentage of B cells positive for ICOSL was recorded, i.e., from 3.94% when miR-155 was turned back OFF up to 13.94% four days later (Figure 10). The loss of ICOSL expression is also associated with B cell malignant transformation in Ep-miR-155 mice: Next, it was examined whether the targeting of ICOSL by miR-155 is part of a broader phenomenon. To this end, the expression of ICOSL was examined in B cells from Ep-miR-155 transgenic mice. Splenocytes from WT and Ep-miR- 155 transgenic littermates were stained for CD19 and B220 B-cell surface markers and were then analyzed for ICOSL expression using flow cytometry. Of note, CD 19+ splenocytes from Ep-miR-155 transgenic express MHC-II based on flow cytometry analyses (Figure 11). In addition, flow cytometry analysis of splenocytes from Ep-miR-155 transgenic mice showed the existence of both CD19+B220- and CD19+B220+ cell subpopulations. The expression of ICOSL in CD19+B220+ B cells was slightly reduced in Ep-miR-155 mice. However, the expression of ICOSL was completely lost in CD19+B220- cell subpopulation that was only found in Ep-miR-155 transgenic spleens (Figure 3A and 3B). Lymphomas from Ep-miR-155 mice were found to be negative for both, B220 and ICOSL expression based on IHC analyses (Figure 3C). A highly malignant B220low7CD19+B cell immunophenotype, when Bcor frameshift mutations were introduced in NP23 HSPCs. Interestingly, both, CD19+B220+ and CD19+B220- subpopulations in Ep-miR-155 transgenic mice, expressed elevated levels of PDL1, another immune checkpoint (Figure 3A and 3B). Without excluding the possibility that PDL1 is also involved in immune escape, B cell malignancy in mice, nevertheless, is likely to be due to the complete loss of ICOSL in addition to the gain of PDL1 expression. Whether high levels of PDL1 are also involved in the loss of ICOSL expression remains to be shown. Altogether, the data from flow cytometry and IHC showed that lymphoma cells in Ep-miR- 155 transgenic mice do not express ICOSL. In addition, the above data showed that while B220-positive and B220-negative (lymphoma cells) inEp-miR-155 mice express similar levels of PDL1, the two subpopulations could be distinguished by the loss of ICOSL expression in malignant cells.

[0155] The expression of ICOSL in malignant cells disrupts tumor development in syngeneic C57BL6 mice: The above data showed that dysfunction of the ICOS-ICOSL immune checkpoint may play a critical role in B cell malignancies. A strategy was devised to assess the ability of ICOSL to induce tumor rejection when expressed in malignant B cells. Initially, a cell line was created from an Ep-miR-155 lymphoma, which as anticipated, did not express ICOSL. Subsequently, these cells were transfected with either a construct containing an inducible tetracycline-responsive element upstream of a CMV-ICOSL (Tet-on-CMV- ICOSL), or a CMV-ICOSL construct that constitutively expressed the mouse ICOSL gene. The expression of ICOSL by the Tet-on-CMV-ICOSL or the CMV-ICOSL constructs was tested by western blot analysis on lysates from transiently transfected HEK-293 cells, as indicated in Figure 12. It is important to note that the constructs used herein only contained the coding region of the mouse ICOSL gene, making the derived transcripts insensitive to miR-155 activity. However, despite multiple attempts, it was repeatedly observed that ICOSL expression was progressively lost in lymphoma cells during their selection on G418-containing medium.

[0156] On the other hand, it was established that miR-155 expression is significantly elevated in several solid tumors, including colon, breast, pancreatic, and lung cancer. The C57BL6- derived MC38 colon cancer cell line expressed extremely low levels of ICOSL based on flow cytometry (Figure 4A) and was therefore used in ensuing experiments. Ultimately, two transformants of the MC38 cell line that were transfected and selected on G418 to contain a CMV-ICOSL vector. The A4 transformant served as a control, as it did not express ICOSL, while the A5 transformant expressed high levels of ICOSL based on flow cytometry analysis (Figure 4A).

[0157] Subcutaneous injection of one million cells / per mouse, either MC38-A4 (control) or MC38-A5 (ICOSL) was performed on the same day on 6 mice / experimental group (3 males and 3 females). MC38-A4-derived tumors showed focal surface ulceration (arrowheads in Figure 4B) indicative of rapid growth, a feature that was not found in mice injected with ICOSL-expressing MC38-A5 cells. In addition, the tumors derived from MC38-A4 cells were significantly larger than tumors derived from MC38-A5 cells (Figure 4C). Histopathological analysis using H&E staining showed extensive necrosis in control group, but much less in the ICOSL-expressing tumors (Figure 4D arrowheads in top left panel). Additional images showing rapid overgrowth and necrosis in MC38-A4-derived tumors vs. a relative lack of necrosis in MC38-A5 tumors are shown in Figure 13. Unlike the tumors derived from MC38- A4 cells, MC38-A5-derived tumors that showed minimal necrosis, marked T cell infiltration, and presented with inflammatory infiltrates at the invading front of the tumor (Figure 4D arrowhead in bottom left panel). The flow cytometry analysis of tumors showed no difference in the percentage of CD4 positive cells in control and ICOSL-expressing tumors, but there was a highly significant increase in the percentage of CD8 positive T cells in ICOSL-expressing tumors (Figure 13). Therefore, it was contemplated that the inflammatory infiltrates in MC38- A5 cells-derived tumors were likely CD8 positive cytotoxic T cells. Therefore, the MC38-A4 and MC38-A5 tumors were analyzed for both, ICOSL and CD8 expression using IHC. Many cells in MC38-A5 tumors stained positive for ICOSL (Figure 4D, bottom middle panel; red signal). Importantly, the presence of ICOSL-positive cells (red signal) in these tumors was paralleled by a concomitant heavy infiltration of cytotoxic CD8 T cells (Figure 4£>, bottom right panel; brown signal). On the other hand, the CD8 infiltration was minimal in MC38-A4 control tumors, as was the expression of ICOSL (Figure 4D, top middle, and right panels). Detailed co-expression analyses of CD8 and ICOSL in the MC38-A5-derived tumors showed that in fact the CD8 T cells strongly concentrated in regions rich in ICOSL-expressing cells (Figure 4E), showing for a critical crosstalk taking place between the two cellular subsets. In addition, colocalization analyses showed that CD8-positive and ICOSL-positive cells were indeed forming immunological synapses as evidenced by the yellow signals on the enlargements (El and E2) of the two red squares on the lower middle panel (Figure 4E, third column). Interestingly, flow cytometry analysis for MC38-A5-tumors showed that a large part of injected cells had lost ICOSL expression (Figure 14), indicating a selective pressure against the expression of the ICOSL gene, that enabled these cells to escape the immune surveillance. This argument is further supported by the fact that although it was a rare event, a areas with few CD8-positive and ICOSL-positive cells in MC38- A5 -derived tumors (Figure 15) were found. Of note, H4C staining, and analyses were done blinded of the tumor origin and performed by different investigators that performed the flow cytometry analysis. Overall, the reduced size of the tumors, the enhanced presence of CD8 positive T cells in the vicinity of ICOSL-expressing cells, and the formation of synapse between these two cell types in MC38- A5-derived tumors showed a genuine immune response toward MC38 ICOSL-expressing cells in C57BL6 mice, in contrast to MC38 cells that did not express ICOSL.

[0158] Discussion

[0159] It has previously been shown that overexpressing miR-155 in B cells under the Eu- enhancer results in the development of aggressive B cell malignancies. Similarly, inducing miR-155 expression in lymphoid tissue using a tetracycline-inducible system led to the formation of aggressive B cell lymphomas. However, lymphoma infiltrates rapidly disappeared when miR-155 expression was turned back OFF in 755LSLtTAmice. The disappearance of malignant infiltrates coincided with an increased infiltration of T cells, leading to the consideration the possibility that high levels of miR-155 in B cells may impair T cell recruitment to the tumor site as well as B-T cell interactions. This prompted for the search for surface antigens that are both expressed by B cells and predicted targets of miR-155. The ICOSL checkpoint was the focus, and it was found that in two mouse models of miR-155- induced lymphomas, the malignant cells were negative for ICOSL. In addition, turning miR- 155 back OFF in miR- l 55 / A / ' / ;Imice led to reappearance of benign B cells re-expressing ICOSL and forming immunological synapses with T cells through ICOSL-Icos interactions. It is also worth emphasizing that as B cells from Ep-miR-155 mice progressed toward a more aggressive phenotype, both the pre-malignant and malignant B cells kept expressing PDL1 at similar levels and thus could not be differentiated based on PDL1 expression alone. However, these two kinds of cells could be discriminated by the level of ICOSL expression, with the malignant B cells showing complete loss of ICOSL. Finally, this disclosure provides strong evidence that ICOSL is a genuine target of miR-155 based on Luciferase assays, miR-155 overexpression, inhibition of ICOSL expression, and by assessing both, ICOSL and miR-155 expression in two different types of B cell malignancies, CLLs and BLs.

[0160] CLLs originate from mature naive B cells that have not yet encountered antigens, while BL cells arise from GC B cells that have already encountered T cell-dependent antigens. Activated B cells in GC express high levels of ICOSL that facilitates their interactions with T follicular helper cells through Icos. Such B-T cell interactions are vital for the development of high affinity antibody responses, class switching, and the generation of long-lived plasma cells and memory B cells. MiR-155 also is a critical player in immune responses within GC B cells. Namely, in addition to promoting B cell survival and proliferation, miR-155 controls somatic hypermutation and class switch recombination in GC B cells by targeting AICD (Activation- Induced Cytidine Deaminase). Dynamic changes in miR-155 expression are thus predicted to occur during B cell maturation, in particular the transient decrease of miR-155 levels needed to allow the expression of AICD and ICOSL upon B cell activation. Thus, single cell sequencing of B cells from the GCs showed that miR-155J / G (the host gene from which miR- 155 primary transcript is produced) is expressed at different levels in the different B cell clusters. Therefore, genomic alterations or other molecular malfunctions that disrupt the tight control of the expression of miR-155, ICOSL, or other critical factors controlling B cell maturation may potentially contribute to the development of distinct tumors, such as CLL and BL.

[0161] These findings indicate not only that ICOSL acts as a costimulatory factor for T cells during the late stages of the immune response after T cells have reached the inflamed tissues, but that ICOSL also plays a crucial role in promoting T cell recruitment at the tumor site. Thus, in two different cancer models found with re-expression of ICOSL by B cells or the overexpression of ICOSL in MC38 tumor cells significantly increased both, T cell infiltration, the formation of immunological synapses between T cells and cancer cells, and the elimination of malignant cells. Injecting mice with MC38 cells that expressed ICOSL led to substantial infiltration of tumors by CD8-positive cells and delayed the onset of terminal illness. These results indicate that ICOSL activity in tumor cells can make them more responsive to anti- cancer immunity, pointing to a critical role of ICOSL in the effectiveness of anti -tumor immune responses. This is in accordance with previous studies showing that: 1- Patients with higher levels of ICOSL transcripts have a better prognosis for nasopharyngeal carcinomas; 2- The activation of Icos-ICOSL signaling pathway in colon cancer is associated with improved survival; and 3- Chemotherapies enhance ICOSL expression in the B cells of breast cancer patients, thereby improving survival outcomes. This better outcome was attributed to the greater recruitment of cytotoxic T cells by ICOSL-expressing B cells.

[0162] T cell responses elicited by Icos-ICOSL costimulatory pathway have been shown to be critical for both, CD4+ T cell regulatory activities, and CD8+ cytotoxic T cell responses. These results critically establish that the effects of ICOSL on T cells vary depending on the type of malignant cells being recognized and eliminated. Namely, in B cell malignancies, ICOSL effects primarily increased the capability of CD4+ T cells to form immune synapses with B cells through Icos-ICOSL interactions. Furthermore, flow cytometry analysis of B cells from Ep-miR-155 mice showed that CD19+ cells actually express MHC-II, which can explain a CD4-T cell-based response. In contrast, the immune response to MC38 solid tumor cells that were expressing ICOSL was mostly mounted by CD8+ T cells, with no detectable effects on the size of CD4+ T cell population within the tumor. Different groups have shown that MC38 cells express H-2Kbclass I MHC (44) as well as MHC-I-restricted antigens. In addition, similar to this disclosure, a CD8-response toward MC38 cells has been reported. On the other hand, research on possible effects of ICOSL or miR-155 on the expression of MHC -I and MHC-II genes should be the subject of interesting studies in the future. Nevertheless, in two different mouse models, solid tumor, and B-cell lymphoma, an enhanced T cell infiltration in response to ICOSL expression by malignant cells was observed followed by synapse formation between T (Icos)-cancer (ICOSL) cells and the elimination of these malignant cells.

[0163] Overall, in the present disclosure, the manipulation of miR-155 expression levels was essential to understanding of the deleterious consequences of the targeting of ICOSL transcripts by miR-155. These findings thus establish ICOSL as a critical factor in immune rejection of tumors by boosting host antitumor immunity and show that the targeting of ICOSL transcripts by miR-155 is instrumental in tumor development. This discovery has important implications not only for T-cell-based immune therapies of B-cell malignancies but also of common solid tumors that exhibit high levels of miR-155, such as lung, breast, pancreatic, and colon cancer. By harnessing the Icos-ICOSL immune checkpoint to enhance the body’s immune response, either by blocking miR-155 activity or by re-expressing ICOSL gene, it presents the opportunity to markedly enhance the efficacy of cancer treatments. These findings show that ICOS-ICOSL checkpoint is a critical target for future immune therapies.

[0164] Materials and Methods

[0165] Mice. Ep-miR-155 mice were used herein. miR-155 Cre-loxP tetracycline-controlled knock-in mouse model, miR-155LSLfZ4, in which miR-155 is targeted to the ROSA26 locus, were donated by Dr. Slack. They were mated to Nestin-Cxe. mice (Jax. Org; Catalog number 003771). Doxycycline withdrawal was done at day Pl. Littermates from parallel mating fed with Doxycycline were used as control. Doxycycline Grain-Based Rodent Diet, Sterile, was purchased from Bio-Serv (Catalog number S3888).

[0166] DNA Constructs. Preparation of ICOSL-luciferase reporter clones. A 492 bp-long fragment of human ICOSL 3'-UTR (corresponding to nucleotides 2321-2812 of NCBI Reference Sequence: NM_015259.6) that contains the putative target site for miR-155-5p was cloned downstream of the Renilla luciferase gene in the psiCHECK2 vector (Promega). The construct containing the mutated / deleted miR-155 target site (AGCATTA) (psiCHECK2- ICOSL Mut) was built based on this construct. Specifically, the putative miR-155-5p site was replaced by 6 nucleotides that constitute the EcoRl restriction site (GAATTC). The sequences of the DNA oligonucleotides used to create these two constructs are:

[0167] ICOSLG Dir 5’-GGCTCGCTCGAGCCCTCCTTCTTACTTCCCAG-3’ (SEQ ID NO: 1) ICOSLG Rev 5’-AGAGTCGCGGCCGCCCCAGGGCACCTCCCGGGAC-3’ (SEQ ID NO: 2)

[0168] ICOSLG MUT DIR 5’-GAAGAATTCGAAGATGTGTGGTGTTTATAAAAG-3’ (SEQ ID NO: 3)

[0169] ICOSLG MUT REV 5’-TTCGAATTCTTCATGTTAAAAGCGGGAGTG-3’ (SEQ ID NO: 4)

[0170] For preparing the psiCHECK2-ICOSL_Mut / deletion, two fragments of the WT 3 ’ -UTR of ICOSL that correspond to the upstream and downstream regions of the miR-155 binding site were first PCR amplified and an EcoRl restriction enzyme cutting site (GAATTC) was introduced. Specifically, the ICOSLG Dir and ICOSLG MUT REV oligonucleotides were used to obtain the 5 ’-part of ICOSLG sequence and ICOSLG MUT DIR and ICOSLG Rev oligonucleotides were used to obtain 3 ’-part of ICOSLG sequence. The two fragments were then digested with the EcoRl enzyme and ligated, replacing the miR-155 binding site with the GAATTC sequence. This strategy was designed due to the difficulty of directly mutating the miR-155 binding-site in ICOSL 3’-UTR due to the great richness in adenine and thymine of sequences surrounding the site. The deletion of the miR-155 binding site or the introduction of the SNP were confirmed by sequencing.

[0171] Flow cytometry. Flow cytometry analyses were done on single cell suspension of either spleen, lymph nodes, peritoneal lymphomas, MC38 cell grown in culture, and MC38- A4- or MC38-A5-derived tumors. Splenocytes were treated with red blood cell lysis buffer (Sigma) for 5 minutes on ice prior to staining. After tissue were smashed using the plungers of a 1ml syringes, they were passed through cell strainers, to prepare single cell suspensions. Staining was always done in parallel for transgenic or WT littermate mice, or for MC38-A4, MC38-A5 tumors. Antibodies were as follow: anti-CD3-APC: 17-0031-81, eBioscience; anti- CD3-PeCy7: 25-0031-81, eBioscience; anti-CD4-APC: 561091, BD-Bioscience; anti-CD4- PE: 12-0041-82, eBioscience; anti-CD4-PeCy7: 563933, BD-Bioscience; anti-CD8-PeCy7: 25-0081-81, eBioscience; anti-CD8-FITC: 11-0081-82, eBioscience; anti-CD19: 17-0193-82, eBioscience; anti-B220-APC: 17-0452-81, eBioscience; anti-B220-PeCy7: 552772, BD- Bioscience, anti-B220-FITC: 11-0452-82, eBioscience; anti-ICOSL-PE: 12-5985-82, eBioscience; anti-Icos-APC: 17-9949-82, eBioscience; anti-Icos-FITC: 11-9942-82, eBioscience; anti-PDl-FITC: 11-9985-81, eBioscience; anti-PDLl-PeCy7: 25-5982-80, eBioscience. Samples were analyzed on a Calibur (BD-Biosciences) machine. Data were analyzed using FloJow software (Ashland, OR, USA).

[0172] Immunohistochemistry (IHC) and in situ hybridization (ISH). Tissues were fixed in 4% formalin for at least 48h before further processing. Fixed tissues were embedded into paraffin blocks, sectioned, and placed into microscopic slides at the Comparative Pathology and Digital Imaging Shared Resource, Department of Veterinary Biosciences, College of Veterinary Medicine, The Ohio State University, as pay per service. Staining for CD4, CD8, B220, and H&E were done in this facility. Reagent used were: Primary antibodies: CD4 (Abeam; abl83685; 0.25pg / mL); CD8 (Cell Signaling; CST98941; 2pg / mL); CD45R (BD Biosciences; 550286; 0.125pg / mL). Secondary antibody: Horse Anti-Rabbit (Vector Laboratories; MP-7401; ready to use); Goat Anti -Rat (Vector Laboratories; MP-5444; ready to use); Substrate DAB Dako (K3468, as per manufacturer recommendation); ImmPACT (Vector Red Vector Laboratories; SK-5105; as per manufacturer recommendation); endogenous enzyme block BLOXALL (Vector Laboratories; SP-6000; ready to use); Protein block: Normal Horse Serum (Vector Laboratories; MP-7401; ready to use); Normal Goat Serum (Vector Laboratories; MP-5444, ready to use); Antigen retrieval buffer: Target Retrieval Solution Dako; S2367; IX.

[0173] IHC for specific antibodies such as ICOS, ICOSL, CD163, CD3, and Cd20, and the ISH for miR-155 were done by Dr. Nuovo, as previously reported (47-49). The specific antibodies used (source and catalog numbers) were as follows: ICOS (ProSci; 8685), ICOSL (ProSci; 8687), T cell (CD3) (Abeam; abl6669), CD163 (Abeam; abl82422), CD31 (Abeam; ab28364); and CD20 B cells (Biogenex; AM537GP). All required pretreatments (antigen retrieval) were done for 30 minutes at 95°C using an EDTA solution. The IHC protocol used the Leica Bond Max (Buffalo Grove, IL) automated platform; the Fast Red (DS 9820) and the DAB (DS 9800) detection kits gave equivalent results. The protocol for ISH for microRNAs. All sections were dealt with blindly using an automated Leica Bond Max platform. Coexpression analyses were done using the Nuance system (CRI). Tissues / slides were first stained for two different targets of interest using Fast Red, NBT / BCIP or DAB as the chromogens. Coexpression experiments were performed by analyzing a given tissue section for one protein using the DAB (brown) chromogen and analyzing the other protein with Fast Red chromogen. Co-localization of the signals were then analyzed by the Nuance and InForm advanced image analyses software systems (PerkinElmer) with the Zeiss Axioskop microscope. Quantification for the signal with either single immunohistochemistry' or multi -lab eled immunohistochemistry’ was done using either the InForm software or manual counting which yielded equivalent results. IHC scoring for Figures 4A, 4B, 4C, 4D, and 4E was done blinded to the treatment regimens. The optimal conditions for ICOSLG (ProSci, #8687) and CD8 (ABCAM, #ab316778) were each pretreatment for 30 minutes with an EDTA antigen retrieval solution (pH 9.0). Three 200X fields were analyzed for a given tumor with each field scored for the number of cells with the target of interest. This allowed the analysis of over 75% of each tumor.

[0174] Quantitative RT-PCR. RNAs were extracted either with TRIzol (Life Technologies) or the RNA purification kit from Norgen (Thorold, ON, Canada). MicroRNA and gene qRT- PCRs were respectively performed using the corresponding Assays from Life Technologies as follow: Actin-P (mouse) Taqman gene expression assay (Mm00607939_sl); Actin-P Taqman (human) gene expression assay (HS03023943_gl); Hsa-miR-155 Taqman miRNA assay (002623); Mmu-miR-155 Taqman miRNA assay (002571); Control miRNA Assay U6 snRNA (001973); ICOSL: HS01055793_ml; ICOSL: Mm00497237_ml; Icos: Mm00497600_ml; Values were normalized using U6 for microRNAs assays, and P-Actin or GAPDH for gene expression assays. The cDNA synthesis for gene expression assays was set up on 1 pg RNA. Ten nanogram RNAs were used as starting material for microRNA qRT-PCR assays. The high- capacity cDNA reverse transcriptase kit with RNA inhibitor was used to prepare the cDNAs, Catalog number: 4368814. Taqman Fast Universal PCR Master Mix catalog number 4367846.

[0175] Western blot. Protein lysates were run on ready to use 4-20% gradient agarose gels from Biorad. Antibodies were as follow: ICOSL: Abeam (ab209262, 1 :500 dilution); Icos: Abeam (ab224644, 1 :500 dilution); a-Tubulin: Cell Signaling Technologies (CST9099, 1 : 1000 dilution); P-actin: Cell Signaling Technologies (CST3700, 1 : 1000 dilution); GAPDH: GeneTex, (Gtx627408, 1 : 1000 dilution); SOCS1 : Abclonal (A7754, 1 :500 dilution).

[0176] Luciferase assays. Cells were plated a night before transfection in 24 well plates. Transfection was done using Lipofectamine following standard protocols as showed by the manufacturer. Whenever needed, cells were treated 24 hours post-transfection. Luciferase assays were run 48 hours after transfection. The luciferase activity was read on a Luminometer (Promega), using The Dual -Luciferase® Reporter (DLR™) Assay System.

[0177] Cell lines and DNA / microRNA transfection. THP-1 and Raw264.7 cells were purchased from ATCC. Cells were grown following standard procedures in RPMI1640 medium supplemented with heat-inactivated bovine fetal serum at concentration 10% and penicillin / streptomycin. The MC38 cell line was established in the laboratory of Dr. Jeffrey Schlom at NCI. This cell line has a high mutation burden. All reagents for tissue culture were purchased from Sigma. LPS and phorbol 12-myristate 13-acetate (PMA) were purchased from Sigma, diluted in PBS as stock, and used. Raw264.7 and HEK-293 cells were transfected using Lipofectamine 2000 (Invitrogen). MC38 cells were transfected with JetPei Polyplus using the manufacturer’s instructions with some modifications. One million cells were seeded in a 10 cm dish and the next day transfected with two micrograms of CMV-mouse ICOSL plasmid and six microliters of JetPei transfection reagent. Two days later the cells were seeded in a 15 cm dish with medium containing 800 pg / ml of G418. Single colonies were isolated and expanded with continued selection (final concentration of G418 400 pg / ml) and tested for ICOSL expression by flow cytometry. Clone A4 (non-expressing) and clone A5 were used for mouse tumor studies. THP-1, BL, and CLL cell lines were electroporated using Amaxa® Cell Line Nucleofector® Kit V from Lonza. Mouse miR-155 RNA was used to transfect Raw264.7, while the rest of the cells (BLs, CLLs, THP-1, and HEK-293 were transfected with human miR-155-RNA. MicroRNAs were transfected at lOOnM final concentration. MicroRNAs, purchased from Ambion / Life technologies, are as follows: PM13058 Pre-miR miRNA Precursors mmu-miR-155; AM13058 Anti-miR miRNA Inhibitors mmu-miR-155-5p; AM17110 Pre-miR™ miRNA Precursor Negative Control #1; AM17010 Anti-miR™ miRNA Inhibitor Negative Control #1; AM12601 Pre-miR miRNA Precursors Hsa-miR-155; PM12601 Anti-miR miRNA Inhibitors Hsa-miR-155-5p. HEK-293 cells were plated on 6-well plates and transfected with either CMV-mICOSL or pENTRY-rtTA-mICOSL clone using Lipofectamine 2000 (Invitrogen). The expression of mICOSL in cells transfected with the pENTRY-rtTA-m ICOSL clone was induced 48h later using doxycycline Ipg / ml. The expression of mICOSL was checked 48 hours-post doxycycline treatment using western blot.

[0178] Statistical analyses were done using the Student t test, and P values are provided in the Figure legends. The the multispectral and co-expression statistical analyses were done using the InStat Statistical Analysis Software (version 3.36) and a paired t-test (also referred to as a “repeated measure t-test”). The null hypothesis was rejected if the significance level was below 5%.

[0179] MicroRNA predicted site analyses. In silico analysis to identify the microRNAs that can potentially target ICOSL transcripts was done using Targetscan.org software.

[0180] EXAMPLE 2: miR-155 impairs ICOSL and MHC-I expression in DLBCL lymphomas

[0181] Elevated miR-155 levels in B-cell malignancies, such as CLL and DLBCL, correlate with increased aggressiveness of the disease. It was recently reported that, in two different mouse models of miR-155-driven B cell malignancy, miR-155 targets and down- regulates transcripts encoding ICOSL, the ligand for the Inducible T-cell co-stimulator (ICOS), thereby impairing the capacity of T lymphocytes to recognize and eliminate malignant cells. In this new report, the findings in Example lare extended to human by showing that miR-155 levels negatively correlate with those of both ICOSL and MHC-I in samples from DLBCL patients. The present disclosure presents evidence of miR-155 reducing the levels of ICOSL transcripts in ABC, but not in GCB primary tumors and cell lines. In contrast, there was no evidence of miR-155 targeting MHC-I transcript levels in both types of DLBCLs. Nevertheless, miR-155 and MHC-I levels inversely correlated in DLBCLs samples, showing the existence of indirect regulatory effects of miR-155. There was also evidence of dose-dependent effects at low miR-155 levels. Altogether, the findings indicate that the deficiency of both ICOSL and MHC-I activity, driven by high levels of miR-155, may be causative in the failure of the host immune system to recognize and eliminate malignant B cells.

[0182] The present disclosure shows that miR-155, a gene highly expressed in different hematological malignancies and solid tumors, modifies the expression of two important genes, namely ICOSL and MHC-I in diffuse large B cell lymphoma. ICOSL and MHC-I play a critical role in immune response toward malignant cells. These findings pave the way for the development of innovative immune therapies aimed at reaching complete tumor cell rejection in B cell malignancies as well as in solid tumors. Additionally, since elevated levels of miR-155 have been associated with poorer survival outcomes for cancer patients, the present disclosure aims to improve both the survival and quality of life of individuals affected by these malignancies.

[0183] It has been established that miR-155 is expressed at high levels in several B-cell malignancies, including Chronic Lymphocytic Leukemia (CLL) and Diffuse Large B-Cell Lymphoma (DLB CL). Elevated miR-155 levels in CLL or in the plasma of DLBC patient are associated with increased tumor aggressiveness. Similarly, high miR-155 levels were also found in Hodgkin lymphoma patients with unfavorable responses to first-line therapy and those who had shorter survival times. Several targets of miR-155 have been validated in B cell malignancies, primarily corresponding to genes involved in either B cell maturation, such as Pu.l, or in the control of cell proliferation and / or cell death, such as Socsl, Shipl, and Weel. In mouse, the present disclosure shows that miR-155 targets transcripts encoding the ligand for Inducible T-cell co-stimulator (ICOSL), that is essential for T helper and B cell functions during T cell-dependent B cell immune response. Major histocompatibility complex class I (MHC-I) genes A, B and C are expressed in nearly all nucleated cells. They encode cell surface proteins that play a crucial role in the anti-tumor immune response by presenting tumor and viral antigens to T lymphocytes. Tumor immune evasion is often associated with a reduced or a lack of expression of MHC-I genes, and loss of MHC-I expression has been reported in different cancers including DLBCLs. In DLBCL, the loss of functional P2-Microglobulin (P2M), an obligate associate of MHC-I proteins, impairs antigen presentation and causes tumor cells to escape from CD8+ cytotoxic T cell recognition and killing activity. P2M mutations have been found in both activated B cell- like (ABC)-DLBCL and germinal center B-cell (GCB)-DLBCL subtypes but are rare in other B cell lymphomas. MHC-I expression is under the control of RELA / p65 NFKB transcription factor, that is an established target of miR-155. Herein, it is shown that an inverse correlation between miR-155 and ICOSL transcripts in ABC-DLBCL tumors and cell lines, miR-155 decreases the level of MHC-I at the cell surface of both ABC and GCB cell lines as well as in mice that constitutively over-express miR-155. In addition, the data show dosedependent effects of miR-155 on both ICOSL and MHC-I expression in DLBCL tumors expressing low to moderate levels of this microRNA. The present disclosure demonstrates that miR-155 dose-dependent reduction of both ICOSL and MHC-I activities is instrumental in decreasing anti-tumor immune response in DLBCLs.

[0184] Results

[0185] The expression of ICOL and miR-155 inversely correlate in DLBCL samples except for those with lowest miR-155 levels: As miR-155 expression is elevated in DLBCLs and ICOSL transcripts are targets of miR-155, the levels of ICOSL were first assessed by immunohistochemistry (IHC) and those of miR-155 by in situ hybridization (ISH) on serial sections of a set of 29 DLBCL samples laid out in a DLBCL tissue microarray (TMAs). The scoring of ICOSL and miR-155 expression in each tumor was done with the computer- based Nuance system in which a score from 0 to 100 is given for each target based on both the percentage of tumor cells expressing the target and the intensity of the signal in cells positive for the target, with a value of 100 assigned to the tumor with 100% cells expressing the target gene signal at a level > 3. The miR-155 data was read blinded to the ICOSL data. An inverse correlation (R2=-0.4585) w a s fo u n d between miR-155 and ICOSL levels in these samples (Figures 16A and 16B). Figure 16C shows representative images of miR-155 and ICOSL staining on serial sections of 4 DLBCL samples on TMA, in which the same tumor cells were scored for each target. A close consideration of the regression line in Figure IB for miR-155 scores < 40 brings up an interesting feature: tumors C2, C5 and C6 show very high ICOSL scores, while tumors A3, B4 and C3 in contrast show very low ICOSL scores. In contrast, the ICOSL scores for the 23 other tumors are roughly proportional to the miR-155 scores. All things being otherwise equal, this shows that the capacity of miR- 155 to impair ICOSL expression varies greatly between tumors presenting the lower range of miR-155 scores. Overall, these data nevertheless clearly demonstrate that DLBCLs that express high levels of miR-155 have decreased levels of ICOSL, further supporting that miR-155 plays a critical role in the regulation of ICOSL expression in this type of tumor.

[0186] High miR-155 levels are associated with low levels of ICOSL transcripts in ABC- DLBCLs but not in GCB-DLBCLs primary tumor: Two main subtypes of DLBCLs have been described: GCB-DLBCLs, that are derived from germinal center light zone and ABC- DLBCLs, that present with characteristics of plasmablasts, i.e., of late germinal center cells already committed to plasma cell differentiation. As the status (ABC, GCB or other) of the 29 DLBCL sample of the TMA from Figure 1A is not provided by the manufacturer, qRT-PCR was used to determine the levels of miR-155 and ICOSL transcripts in 12 other primary DLBCLs and 21 DLBCL cell lines of known status. As anticipated, miR-155 was up-regulated in both ABC and GCB primary tumors (Figure 17A). Among the 7 ABC cell lines, 4 (LY3, TMD8, SUDHL2 and HBL1) have levels of miR-155 comparable to those of primary ABC tumors, however miR-155 levels were lower in the three other ABC cell lines (RIVA, U2932 and HLY1) and rather comparable to those in the 14 GCB cell lines (Figure 17A). Of note, the measures of miR-155 relative levels in LY3 and U2932 were similar to those previously measured by Anastasiadou et al (E. Anastasiadou, et al, Cobomarsen, an oligonucleotide inhibitor of miR-155, slows DLBCL tumor cell growth in vitro and in vivo. Clin Cancer Res. 27, 1139-1149 (2021). In addition, Anastasiadou et al. showed that miR-155 levels in U2932 cells are significantly higher than those in B cells from healthy donors. Thus, the results herein confirm that miR-155 expression is greatly increased in both ABC- and GCB-DLBCL primary tumors.

[0187] It was previously shown that miR-155 can target ICOSL transcripts directly and found that the levels of ICOSL transcripts were reduced in MEC1 and MEC2 CLL cell lines that have high levels of miR-155 expression. In addition, transfecting MEC2 cells with a miR-155- inhibitor RNA showed an increased ICOSL levels at the cell surface. Therefore, qRT-PCR was then used to assess the levels of ICOSL transcripts in the DLBCLs primary tumors and cell lines used in Figure 17A. The levels of ICOSL transcripts were very low in the five ABC primary tumors as well as in ABC cell lines, with the exception of RIVA cells (Figure 17B). Unexpectedly, however, ICOSL transcript levels were higher in all GCB primary tumors but 2026 as well as in 9 out of the 14 GCB cell lines (Figure 17B). This result shows that miR-155 targeting of ICOSL is impaired in GCB tumors or may even possibly concur to increase ICOSL expression in GCB-DLBCLs. To explore further how ICOSL expression is regulated in these two types of DLBCLs, flow cytometry was first used to compare the expression of ICOSL at the cell surface of SUDHL2 (ABC) and TOLEDO (GCB) cell lines. The difference in mean fluorescence intensities (Figure 17C) was roughly proportional to the respective levels of ICOSL transcripts in these two cell lines (Figure 17B). In a new set of flow cytometry experiments, the mean fluorescence intensities for 3 ABC cell lines were similar to those of isotope control (Figure 17D), with LY3 showing a greater MFI despite its low ICOSL transcript levels (Figure 17B). In contrast, among the 6 GCB cell lines tested, only the two with highest levels of ICOSL transcripts (TOLEDO and LY1) showed expression of ICOSL at the cell surface (Figure 17B). Together, these results show that miR- 155 regulates the levels of ICOSL transcripts in ABC but not in GCB cell lines, and also that the levels of ICOSL protein at the cell surface depend on additional factors whose activity may or may not depend on miR-155. miR-155 affects MHC-I expression indirectly: Since (1) The NFKB transcription factor controls the expression of MHC-I; (2) The p65 / RELA subunit of NFKB, is a known target of miR-155 (20); (3) TNF activates MHC-I expression through CREB1, SMARCA4 and HIFla factors; and (4) SMARCA4 and HIFla are validated targets of miR-155 (24-26), and CREB1 3’-UTR contains a miR-155 consensus target site, flow cytometry was used to determine the density of MHC-I (H2-Kb) at the surface of B cells from both Ep-miR-155 mice and their non-transgenic littermates. Ep-miR-155 are transgenic mice whose B cells over-express miR-155 under the control of the Ep enhancer. A significant reduction of the density of the MHC-I H2-Kbsurface protein was found in CD19+B220+-gated cells from Ep- miR-155 transgenic mice as compared with those from littermate controls (Figures 18A and 18B). This result showed that in addition of ICOSL (see here above), miR-155 may possibly reduce the expression of MHC-I, which would further impair the ability of T cells to recognize tumor antigens.

[0188] In human, neither HI.A-A. HLA-B or HLA-C gene contains a miR-155 target site in its 3’-UTR. Nevertheless, to determine whether miR-155 may reduce MHC-I expression indirectly, SUDHL5 cells were transfected, that were found to express miR-155 at low levels (Figure 16A), with a miR-155 mimic: this reduced the density of MHC-I at the surface of B cells, with the MFI decreasing from 2714 to 1548 (Figure 18C, left panel). In contrast, a miR-155 inhibitory RNA proved unable to significantly increase the density of MHC-I at the surface of LY3 cells (Figure 18C, right panel) that previously expressed the highest levels of miR-155 (Figure 17A). This shows that miR-155 can reduce the expression of MHC-I at the cell surface at least when miR-155 is expressed at moderate levels. The DLBCL samples of the TMA presented in Figures 17A, 17B, 17C, 17D, 17E, and 17F were then screened for the expression of MHC-I and found a moderate inverse correlation between MHC-I and miR-155 scores (R2= -0.2858) (Figures 18D and 18E). Figure 18F shows representative staining of MHC-I positive and negative tumors on the TMA. Interestingly, as previously found with ICOSL (Figure 16B), MHC-I scores for DLBCLs with a miR-155 score < 40 showed a greater dispersion than the scores of the other tumors (Figure 18E), with tumors C2, E3 and C6 showing high MHC-I scores, and tumors B4, B5 C3, C5 and E5 in contrast showing low or very low MHC-I scores. These are again indicative of miR-155 dose-dependent effects. Of note, on the previous experiments with ICOSL (Figure 16B), tumor E3 did not have a high score, tumors B5 and E5 did not have a low score, however tumor A3 had a low score and C5 a high score. These in-between tumor score differences for ICOSL and MHC-I only found in tumors with the lower range of miR-155 scores exclude the possibility of sample processing biases. qRT-PCR was further used to determine whether miR-155 levels would correlate with the levels of MHC-I transcripts in ABC and GCB DLBCLs and cell lines. The levels of MHC-I transcripts were elevated in both types of tumors and were very low in both types of cell lines, irrespective of miR-155 levels (Figure 17E). Thus, in contrast to ICOSL transcripts (Figure 17B), MHC-I transcripts were not sensible to miR-155 activity in DLBCL tumors, indicating that miR-155 effects on MHC-I density on cell membrane (Figures 17D and 17E) were indirect. This was later confirmed by flow cytometry analyses showing that the density of MHC-I at the cell surface, as deduced from the MFI, was high in LY3, TMD8, HBL1 and SUDHL5 and low in SUDHL2, SUDHL4, LY7, SUDHL10 and LY1 (Figure 17F) although they did not show dramatic differences in MHC-I transcript levels (Figure 17E). Of note, the measures of MHC-I relative levels in LY3, LY1 and SUDHL2 were similar to those previously determined by Challa-Malladi et al. (M. Challa-Malladi et al. Combined genetic inactivation of P2-microglobulin and CD58 reveals frequent escape from immune recognition in diffuse large B cell lymphoma. Cancer Cell. 20, 728-740 (2011). The above TMA arrays were further used to check whether miR-155 effects on MHC-I may be due to its negative effects on RELA expression. This was not the case, as no appreciable correlation was found between miR-155 and RELA scores within the tumors, and, in contrast to ICOSL (Figure 16B) and MHC-I (Figure 18E) scores, those for RELA were greatly variable along the whole range of miR-155 scores (not shown).

[0189] Finally, the correlation observed between the scores of ICOSL and MHC-I in DLBCLs from the same TMAs (Figure 18G) show that the expression of these two factors critical for the anti-tumor immune response is under the control of common regulators that include miR-155. Altogether, these results that miR-155 regulates the levels of ICOSL and MHC-I at the cell surface through distinct mechanisms in both ABC and GCB DLBCLs, in addition to its direct targeting of ICOSL transcripts in ABC DLBCLs.

[0190] Discussion

[0191] It was previously reported that the expression of ICOSL is reduced in two mouse models of miR-155-induced B cell malignancy and that ICOSL is a direct target of miR-155. Herein, the present example expands on both ICOSL and MHC-I expression in human DLBCL primary tumors and cell lines. The main results are: (1) ICOSL expression in DLBCLs is generally inversely correlated with miR-155 levels, except for tumors with low miR-155 expression; (2) The levels of ICOSL transcripts are low in ABC, but not GCB, tumors and cell lines; and (3) miR-155 activity affects MHC-I expression without decreasing MHC-I transcript levels significantly. As for ICOSL, tumors with low levels of miR-155 expression show a great dispersion of MHC-I levels. According to published results, the present disclosure found high levels of miR-155 expression in ABC tumors (2173, 2197, 2127, 2152 and 2154) and four out of 6 ABC cell lines (LY3, TMD8, SUDHL2 and HBL1), as well as in three out of six GCB primary tumors (2113, 2079 and 2109) (23, 28). This shows that miR-155 is instrumental in the development of DLBCLs. Indeed, the miR-155 inhibitor cobomarsen has been found to reduce the proliferation and induce the apoptosis of ABC- DLBCL cell lines both in vitro and in vivo.

[0192] It was further found that the levels of ICOSL transcripts were low in the tested ABC primary tumors and cell lines, in accordance with previous results showing that miR-155 targets and decreases the levels of ICOSL transcripts. It was thus not surprising to find a higher level of ICOSL transcripts in RIVA cells, that had a lower level of miR-155 than other ABC cell lines. While high or very high levels of ICOSL transcripts were also found in most of the GCB cell lines that all had low levels of miR-155 expression, the very high levels of ICOSL transcripts in 2113, 2079 and 2107 GCB primary tumors, that expressed miR-155 at levels comparable to those of ABC primary tumors, show that miR-155 does not reduce the levels of ICOSL transcripts in GCB- DLBCLs. In these primary tumors, ICOSL and other transcripts may possibly be protected against miR-155 inhibition by circular RNAs such as CircRNA_0075723 that has been shown to inhibit macrophage pyroptosis by sponging miR- 155 and thus regulating the expression of the miR-155 target SHIP1. Given its many target transcripts, however, it is also likely that miR-155 may simultaneously target a number of transcripts encoding factors needed for GCB-DLBCLs development and cell survival. In this respect, it is worth noting that miR-155 has been shown to impair Caspase 3 and PTEN pro-apoptotic effects, thus increasing PI3K / AKT activity and consequently the proliferation of nasopharyngeal carcinoma cells. GCB-DLBCs originate in a tissue (the dark zone of the GC) that, in order to allow somatic mutations and isoform switches to take place, reduces the activity of DNA repair enzymes needed for maintaining the integrity of the genome, down-regulates genes encoding tumor suppressor factors such as CDKN1A, CDKN1B and TP53, and provides GC B cells with an enhanced proliferation capability and metabolic adaptations supporting cell proliferation. Thus, it is not surprising that GCB-DLBCs carry an extremely high load of mutations and show multiple abnormalities in gene expression, all likely to impact the expression of a number of other genes, which does not facilitate the identification of significant molecular abnormalities in GCB tumors. Of note, miR-155 targets transcripts encoding anti -proliferative factors such as CDKN1B, and TP53INP1, a factor implicated in autophagic cell death. BCL6, that is needed for the GC formation, represses the expression of the MIR155HG, from which mature miR-155 is produced. By reducing miR-155 levels in Ip-HA-BCL6 transgenic mice, BCL6 increases the levels of AICDA / AID, SPI1, IRF8 and MYB proteins that are required for the GC reaction. Of note, this increase was not associated with higher transcript levels, except for IRF8, which is in agreement with the results herein showing post-transcriptional effects of miR-155 on the expression of ICOSL and MHC-I. In addition, FBX011 is needed for downregulating BCL6 at the end of the GC reaction. Reduced FBX011 activity increases the number of GC B cells with higher BCL6 expression, and FBX011 inactivation causes lymphoproliferative disorder in mice. As the 3’-UTR of FBX011 contains a miR-155 target sequence, it is likely that miR-155 may favor GC lymphoma development in many different ways through dose-dependent effects on multiple target genes.

[0193] The observation of higher ICOSL transcript levels in the tested GCB primary tumors and cell lines may at least in part explain why patients with GCB-DLBCLs usually have a better prognosis than ABC-DLBCLs. However, the levels of ICOSL transcripts may not always reflect the actual ICOSL density at the cell surface, as shown by the fact that the MFI of LY3 and TOLEDO cells were nearly identical despite considerable difference in ICOSL transcripts levels in the two cell lines. Also, the results herein strongly demonstrate that miR- 155 effects may be dose-dependent in DLBCLs. Although the status of DLBCLs from the TMA arrays (GCB, ABC or other) is not known, there was a clear correlation between the levels of ICOSL and miR-155 levels. However, for miR-155 scores lower than 40, the ICOSL scores of DLBCLs C2, C5 and C6 showed high levels of ICOSL expression, while ICOSL scores of tumors A3, C3 and B4 were very low. A similar observation was done for MHC-I scores, with high scores for C2, E3 and C6 and low scores for B4, B5 and C3 again for miR-155 scores lower than 40. This effect was not observed for RELA scores, and accordingly no correlation was found between miR-155 and RELA scores (not shown).

[0194] Interestingly, ICOSL unusual highest scores were limited to the below 40 region of MHC-I scores, suggesting that miR-155 effects on ICOSL and MHC-I expression were not similar. Of note, a dose-dependent miR-155 targeting of Quaking transcripts was previously found, and it was further found that miR-155 targets different groups of genes in acute myeloid leukemia (AML) cells at intermediate and high levels. These dose-dependent effects of miR-155 effects may also possibly explain why, while high levels of miR-155 are usually associated with increased tumor aggressiveness and favor chemo-resistance in lung, breast, colon and other cancers, R-CHOP treatment increased overall survival and progression-free survival as compared with CHOP treatment for patients with high levels, but not those with low levels of miR-155. In addition, the data herein show that B cells from Ep-miR-155 transgenic mice that over-express miR-155 have reduced expression of H2- Kbat their surface, and that there is an inverse correlation between miR-155 and MHC-I levels in DLBCL patients. The RELA / p65 subunit of NFKB, that has been previously shown to control MHC-I expression, is a validated target of miR-155. Thus, the indirect effects of miR-155 on expression of MHC-I could be at different levels along the NFKB pathway. However, there was no correlation between RELA and miR-155 scores in DLBCL samples, contrary to the significant reduction of RelA expression in Ep-miR-155 splenocytes (not shown). Similarly, the levels of RELA expression did not show significant variation among different DLBCL-derived cell lines, as determined by western blot analysis (not shown).

[0195] The functional antigen-presenting complex is a heterodimer including one MHC-I molecule (HLA-A, B or C) and one P2M molecule. It has been shown that, among mature B cell malignancies, DLBCLs predominantly display a loss of HLA-I expression, and that this loss can be due to a lack of MHC-I, P2M, or both, as a result of somatic mutations. MHC-I loss in DLBCLs was found in roughly 60% of GBC- and 40% of ABC-DLBCLs, in relation with their high burden of mutations, especially those affecting fl2M or MHC-I genes. However, the precise cause of MHC- I malfunctions in the remaining MHC-I negative DLBCLs that still possess one (43%) or both (26%) intact P2M and HLA-I alleles remains to be found. Nevertheless, the existence of an inverse correlation between miR-155 and MHC- I score in the TMA strongly supports that miR-155 also is implicated in MHC-I downregulation in DLBCLs, either alone or in combination with gene inactivation. With 3297 human transcripts containing at least one miR-155 target sites, there are many possibilities for miR-155 to influence, directly or indirectly, the anti -tumor immune response. For example, in glioma cells, TNF induces MHC-I expression by increasing CREB1 phosphorylation through HIFla activity, which leads to the replacement of SMARCA2 / Brahma by SMARCA4 / BRG1 on the MHC-I promoter, leading to its transcriptional activation. Interestingly, the transcripts encoding CREB1, SMARCA4 and HIFla contain a miR- 155 consensus target site, with SMARCA4 and HIFla transcripts being validated targets of this microRNA. Therefore, miR-155 is predicted to somewhat impair MHC-I expression and function. This also shows that the activity of several components of the complex regulatory network associated with the B cell selection and function may be malfunctioning in DLBCL patients, where miR-155 potentially could influence multiple pathways involved in immune response or immune evasion. As MHC-I is expressed in nearly all nucleated cells and miR-155 levels are elevated in different solid tumors, the data herein strongly support the idea that abnormal miR-155 expression can decrease the antitumor activity of CD8 T cells through reducing both antigen presentation by tumor cells and CD8 T cell activation by simultaneously reducing the expression of ICOSL and MHC- I through dose-dependent effects.

[0196] Overall, an inverse correlation between the levels of miR-155 and those of ICOSL and MHC-I, with DLBCLs with higher miR-155 levels expressing less ICOSL and less MHC- I than the DLBCLs with lower miR-155 levels was observed.

[0197] Materials and Methods

[0198] DLBCL-TMAs and DLBCL-cell lines: LY301 Lymph node diffuse large B cell lymphoma (DLBCL) tissue arrays (30 cases / 30 cores / histological slide) were purchased from Tissue Array. DLBCL cell lines were grown in culture. Their classification is explained in the results section.

[0199] Flow cytometry: Flow cytometry analyses were done on single cell suspension of either spleens or lymph nodes. Splenocytes were treated with red blood cell lysis buffer (Sigma) for 5 minutes on ice prior to staining. After tissue were smashed using the plungers of a 1ml syringes, they were passed through cell strainers, to prepare single cell suspensions. Staining was always done in parallel for transgenic or WT littermate mice. Antibodies were as follow: anti-MHC Class I H2-Kb(APC-labelled) antibody was purchased from Invitrogen / eBioscience (catalog number: 17-5958-80); anti-mouse B220 (PE-labelled) antibody was from BD Pharmingen (catalog number: 561878); anti -mouse CD 19 (PE-Cy5- labelled) antibody was from Invitrogen (catalog number: 45-0193-80). APC-labelled antihuman HLA-A, B and C antibody (Cat no: 311409) as well as the corresponding APC- labelled isotype control (Cat no: 400219) were from BD Pharmingen. PE-labelled anti- human-ICOSL antibody (Cat no: 12-5889-42; BD Pharmingen) and corresponding PE- labelled isotype control (Cat no: 553930) were purchased from Samples were analyzed on a Calibur (BD-Biosciences) machine. Data were analyzed using FloJo software (Ashland, OR, USA).

[0200] DLBCL cell line electroporation: DLBCL cell lines were electroporated using Kit V and program T16 on a Lonza / Amaxa equipment, following manufacturer’s protocol. SUDHL5 cell line was electroporated either with hsa-miR-155-5p-Pre-miR™ miRNA Precursor (PM12601) or Pre-miR™ miRNA Precursor Negative Control #1 (PM1711) while LY3 was electroporated with hsa-miR-155-5p-Anti-miR™ miRNA Inhibitor (AM12601) or Anti- miR™ miRNA Inhibitor Negative Control #1 (AM17010). MicroRNAs used for electroporation were purchased from Ambion / Life Technologies. Cells were analyzed for HLA-expression using flow cytometry 2 days later.

[0201] IHC: IHC for proteins of interest and the ISH for miR-155 were performed. The specific antibodies used (source and catalog numbers) were as follows: anti-ICOSL (ProSci; 8687), Anti-HLA Class I antibody [W6 / 32] was purchased from Abeam (ab22432). MHC Class I (H2-D1) Polyclonal Antibody Invitrogen: PA5144130 was also used and gave similar results as ab22432. The shown data come from ab22432 antibody. Anti-NF-kB p65 antibody was from Abeam (ab7970). As previously reported (9, 41, 42), all required pretreatments (antigen retrieval) were done for 30 minutes at 95°C using an EDTA solution. The IHC protocol used the Leica Bond Max (Buffalo Grove, IL) automated platform; the Fast Red (DS 9820) and the DAB (DS 9800) detection kits gave equivalent results. The protocol for ISH for microRNAs has been previously reported. Quantification for the signal with either single immunohistochemistry or multi-labeled immunohistochemistry was done using either the InForm software or manual counting which yielded equivalent results. The optimal conditions for ICOSL and MHC-I were each pretreatment for 30 minutes with an EDTA antigen retrieval solution (pH 9.0).

[0202] Scoring of miR-155, ICOSL, and MHC-I expression: The scoring of the ICOSL, MHC-I, and miR-155 data was done with the computer-based Nuance system in which a score is given from 0 to 100 based on the percentage of tumor cells expressing the target as well as the intensity of the signal in the positive cells with 100 representing each tumor cell expressing high levels of the protein / miRNA. The miR-155, MHC-I, and the ICOSL data were obtained blinded to the other results.

[0203] Quantitative RT-PCR: RNAs were extracted either with TRIzol (Life Technologies) or the RNA purification kit from Norgen (Thorold, ON, Canada). MicroRNA and gene qRT- PCRs were respectively performed as indicated by the manufacturer, using the corresponding TaqMan Assays from Life Technologies as follows: Hsa-miR-155 (assay ID: 002623); RNU48 (assay ID: 001006); ICOSL (assay ID: HS01055793_ml); HLA-A (assay ID: HS01058806 G1), and GAPDH (assay ID: Hs02758991). Values were normalized using RNU48 for microRNAs assays, and GAPDH for gene expression assays. The cDNA synthesis for gene expression assays was set up on 1 pg RNA. Ten nanogram RNAs were used as starting material for microRNA qRT-PCR assays, as indicated by the manufacturer and previously described. The high-capacity cDNA reverse transcriptase kit with RNA inhibitor was used to prepare the cDNAs, Catalog number: 4368814. Taqman Fast Universal PCR Master Mix catalog number 4367846. Samples were run in technical triplicates.

[0204] Mice: Ep-miR-155 mice were used herein.

[0205] EXAMPLE 3: Nasopharyngeal Carcinoma

[0206] Nasopharyngeal carcinomas (NPC) causes about 65,000 deaths / year and shows a striking variability in its geographic distribution where it is rare in the USA and common in southeastern Asia where it can comprise up to 20% of all cancers.1-4Most NPCs are WHO type 2 and 3 in which the carcinoma cells are poorly differentiated, and these tumors are strongly associated with infection by EBV.5The five-year survival rate for patients in stages I, II, III and IV are 66.7%, 55.6%, 41.8% and 25.9%, respectively.

[0207] Although NPC is sensitive to radiotherapy and chemotherapy, around 15% to 58% of patients experience recurrent disease.6Combination immunotherapy for recurrent / metastatic cancers using anti-PD-1, PD-L1, and / or CTLA-4 has shown relatively poor responses, with the overall response and best overall response rates ranging from 20.5 to 38.0% with the median progression-free survival rate and overall survival at 5.3 and 19.5 months, respectively.4,7’8

[0208] EBV associated NPC are unique among human cancers for showing an intense inflammatory response as a major feature of the tumor histopathology. The cytotoxic CD8+ T cell is the most abundant lymphocyte in these mononuclear infiltrates that include CD4+ T cells, dendritic cells, NK cells and, to a much less extent CD20+ B cells.9

[0209] This data raised the question: how are the EBV+ tumor cells able to avoid immune elimination being surrounded by activated cytotoxic T cells which are the backbone of current successful cancer immunotherapies.10,11Although intensive inter-cell communication between the CD8 T cells and the malignant cells in NPC have been recorded,12the mechanism whereby the CD8 T cells are unable to eliminate the tumor cells needs explanation. Here a study was conducted which demonstrates that the intense up-regulation of miR-155 by the EBV genome in the NPC tumor cells is associated with the loss of two of its targets on these cells, ICOSL and MHC-I which would help render the tumor invisible to the intense surrounding CD8 T cell infiltrates.

[0210] Methods

[0211] Clinical Samples and Controls: The patient samples (formalin fixed paraffin embedded biopsy material) originated from the files of Folio Biosciences. Clinical information included the diagnosis and age / sex of patient. There was biopsy material from 9 patients (mean age 55.2 years). Five of the cases had normal adjacent nasopharynx tissue which, with 5 unremarkable tonsils, served as the negative controls. Four-micron tissue samples were placed on sequentially labeled slides, baked at 60°C for 30 min, and stored at RT with an hematoxylin and eosin stain performed to verify the diagnosis.

[0212] Immunohistochemistry: Immunohistochemistry was done as previously reported.13,14The specific antibodies used (source and catalogue numbers; Ab = ABCAM, Prot = Proteintech) were as follows: CD3 (Abl6669), CD4 (Roche 790-4423), CD20 (Roche 760- 2531), ICOSL (Prot 8687), ICOS (Prot 8685), RELA (Ab7970), for MHC-I, two antibodies were tested, Ab22432 and InvitrogenPAS-14413 that gave similar results), PD-L1 (Ab205921) and PD1 (Ab237728). The protocols for these antibodies have been published.13,14In brief, all required antigen retrieval that was done for 30 minutes at 950C using an EDTA solution. The immunohistochemistry protocol used the Leica Bond Max (Buffalo Grove, IL) automated platform; the Fast red (DS9390) and the DAB (DS9800) detection kits were used and gave equivalent results.

[0213] In situ Hybridization: In situ hybridization for miR-155 was performed as previously described.15In brief, the tissue was pretreated for 30 minutes at 95°C using the EDTA solution and then hybridized overnight at 37°C with an LNA anti -miR-155 probe digoxigenin tagged at its 5’ end and then detected with an anti-digoxigenin-AP conjugate with NBT / BCIP as the chromogen. In situ hybridization for the EBER-1 / 2 RNA was done with the RNAscope assay as previously described.13'16In brief, the target sequence was the human herpes 4 isolate SDTW400 EBER-1 and -2 genes, complete sequence from ACD (catalogue # 310271). The assay was done per the manufacturer’s protocol that used DAB as the chromogen.

[0214] Multispectral and Co-Expression Testing And Statistical Analyses: As previously described13,14co-expression experiments were performed by analyzing a given tissue section for one protein using the DAB (brown) chromogen and analyzing the other protein with Fast Red chromogen or, with tri-localization, using methylene blue (Enzo Life Sciences). Coexpression analyses were done using the Nuance software. Quantification for the signal with either single immunohistochemistry or multi-labeled immunohistochemistry was done using either the InForm software or manual counting which yielded equivalent results. Statistical analysis was done using the InStat Statistical Analysis Software (version 3.36) and a paired t-test (also referred to as a “repeated measure t-test”). The null hypothesis was rejected if the significance level was below 5%.

[0215] Immunohistochemistry / / ^ situ Scoring: Immunohistochemistry and in situ hybridization scoring was done blinded to the target tested. The standard pathology system of 0, 1+, 2+, and 3+ was used as defined in this study by 1-24% target cells positive (1+), 25-49% target cells positive (2+) and 50% or more target cells positive (3+). Six 200X fields are scored for a given target that yields an average value for each data point.

[0216] Results

[0217] Clinical Pathologic Data: Nasopharyngeal biopsies were available from 9 people that ranged in age from 49 to 64 years (mean 55.2); 6 were men. Race was listed for only two people (African- American and Oriental) and regional lymph node metastases were documented in one case. The histologic findings in each case were equivalent: there were nests of undifferentiated epithelial cells dispersed with varying sized groups of mononuclear cells (Figures 19A, 19B, 19C, 19D, 19E, and 19F). Although there was much variation in a given case and between cases, overall a near equivalent volume of the tumor included the malignant epithelial cells and the intense lymphoid infiltrates; in 5 cases the adjacent non -involved nasopharynx epithelia were identified (Figures 19A, 19B, 19C, 19D, 19E, and 19F). In 9 / 9 cases, a 3+ signal was seen in the carcinoma cells for EBV-EBER-1 / 2 RNAs by in situ hybridization. As seen in Figures 19A, 19B, 19C, 19D, 19E, and 19F, theEBV-RNA was evident in basically all of the carcinoma cells and in none of the surrounding mononuclear cells. Immunohistochemistry stains for CD3 demonstrated that over 95% of the mononuclear cells in the lymphoid infiltrates were T cells (3+ signal); the CD8 / CD3 co-expression data showed that over 67% of the CD3 cells were cytotoxic T cells (Figures 19A, 19B, 19C, 19D, 19E, and 19F). CD20 positive cells were rarely evident (1+ signal) and made up about 1% of the mononuclear cells.

[0218] The EBER-1 / 2 Positive Cells Strongly Express miR-155: TheNPC and normal tonsil tissues were tested for miR-155 by in situ hybridization. As evident in Figures 20A, 20B, 20C, and 20D, there was a 1 : 1 relationship between the EBV-RNAs and miR-155 as demonstrated by co-expression analyses. No signal was seen when a scrambled LNA probe was used in place of the miR-155 probe (Figures 20 A, 20B, 20C, and 20D). As evident, miR-155 was not detected in the mononuclear cell infiltrate nor was it present in the adjacent normal epithelia.

[0219] Expression at Protein Level of miR-155 Targets: MiR-155 targets at least two proteins that can modulate the immune response against a tumor, including ICOSL14and RELA17, both of which are established targets of miR-155. Furthermore, since RELA controls the expression of MHC-I18'20miR-155 is predicted to indirectly reduce MHC-I expression. MHC-I molecules play a key role in presenting tumor antigens to CD8+ T cells,21and reduced MHC-I expression is associated with tumor immune evasion.22,23Therefore, the elevated levels of miR-155, are predicted to lead to decreased MHC-I expression and further facilitate tumor evasion, in addition to the direct targeting of ICOSL transcripts by miR-155. Therefore, the NPC tissues were tested for each of these proteins by immunohistochemistry. The normal adjacent nasopharynx showed strong expression of MHC- I and RELA as well as ICOSL (Figures 21A, 21B, 21C, 21D, 21E, 21F, 21E, 21G, 21H, and 211). Figures 21A, 21B, 21C, 21D, 21E, 21F, 21E, 21G, 21H, and 211 also demonstrate the near total loss of MHC-I, ICOSL, and RELA expression in the cancer cells of NPC, and coexpression of MHC-I and RELA in the adjacent benign tissue (Figure 21C). In Figure 21D, normal epithelial cells are ICOSL positive while NPC cells are ICOSL negative (Figure 2 IE). However, the T cells strongly expressed ICOS as seen by co-expression of ICOS and CD3 in the tumor-associated lymphoid infiltrates (Figure 2 IF), indicating that the T cells were nevertheless expressing this protein whose coupling with ICOSL is essential for tumor destruction.

[0220] Discussion

[0221] NPCs are an excellent model to study the topic which has dominated cancer therapy over the last decade: immunotherapy. The reason why NPC is an excellent model is that it is one of the two human solid cancers where massive T cell infiltration is a pathognomonic feature used by pathologists to render the diagnosis, the other being seminoma, and explains why it is sometimes referred to as a lymphoepithelial carcinoma. The ability of checkpoint inhibitors to allow the resident cytotoxic T cells in a given tumor to actuate clinically documented reduction in the tumor mass has been well documented but is effective in only about 20-30% of cases. Importantly, it is well documented, that the degree of PD1 and / or PD-L1 expression as well as CD8 infiltration are histologic factors associated with a clinical response to checkpoint inhibitors.24,25NPC have been well documented to show strong expression of PD-L1 on the tumor cells in nearly 100% of cases, as was corroborated in this study, yet combined checkpoint inhibitors of NPC using either anti-PD-Ll, anti-PD-1, and / or anti-CTLA-4 is effective in only about 20-30% of cases.26'29

[0222] MiR-155 is upregulated in various cancers,30,31including NPCs32'36although the latter studies did not focus on the specific cellular source of this particular microRNA. It has recently been documented that miR-155 targets ICOSL in two distinct mouse models of B-cell driven malignancies.14The study also showed that ICOSL induces a strong co-stimulatory signal for cytotoxic T cells upon binding to ICOS, leading to T cell activation.14This study shows that the expression of miR-155 inversely correlates with that of ICOSL in NPC. Furthermore, the study shows that in NPC, there is an inverse correlation between the levels of miR-155 and the expression of MHC-I and RELA. RELA is a published target of miR-15517and serves as the main transcription factor for the MHC-I locus, suggesting that elevated levels of miR- 155 could indirectly reduce MHC-I expression by downregulating RELA. In addition to RELA, other potential transcription factors that control MHC-I expression may also be modulated by miR- 155 activity. It is proposed that by concurrently co-inhibiting ICOSL and MHC-I, high levels of miR-155 found in NPC are the cause of tumor evasion.

[0223] The key finding of this study is that the EBV+ carcinoma cells in NPC are the primary source of the markedly elevated levels of miR-155. This upregulation is associated with a loss of expression of ICOSL, MHC-I-HLA, and RELA in the tumor cells. Additionally, it was observed that each of these three proteins is strongly expressed in in the normal epithelial cells of the nasopharynx, which are believed to be the progenitor cells of NPC. This suggests that the dysregulation of miR-155 and its targets play a critical role in the pathogenesis of NPC, contributing to the immune evasion mechanisms employed by the tumor.

[0224] The main clinical correlate of this study is that it provides insights into why the failure rate of checkpoint inhibitors is often as high as 70-80% in cancers in general and NPC in particular, even though the latter tumor shows a 100% high expression of PD-L1 on the tumor cells. The cytotoxic CD8 T cells in NPC are activated in the sense of intense proliferation and their abundant expression of ICOS which primes them for tumor eradication. However, the miR-155 induced loss of ICOSL in the tumor cells renders ICOS ineffective and the loss of MHC-I, also documented by others in NPC, 37 will also assist the tumor cells in avoiding T cell obliteration. In this sense the cytotoxic T cells are not exhausted, but simply rendered incapable of recognizing and binding to the tumor cells. It can be argued that the EBV infection that is at the epicenter of NPCs has evolved to render the immune system incapable of destroying the tumor at multiple pathways, including the loss of ICOSL / MHC-I and the gain of PD-L1, as otherwise the cancer cells would have been eradicated by the massive CD8 infiltration.

[0225] The interplay between EBV-induced changes in infected cells, the immune response to viral infection, the accumulation of genomic alterations (regardless of whether these changes are related to the infection) and the initiation of cellular transformation, which in turn leads to the infiltration of immune cells is inherently complex. This dynamic interaction is predicted to evolve over time. Recent studies have shown that NPCs express the TRIM21 protein, which plays a role in modulating the cGAS / STING cytosolic DNA sensing pathway. This modulation can lead to a reduction in CD8 T cell activity against carcinoma cells.38Furthermore, research by Jin et al39has documented that NPC tumor cells exhibit characteristics of both carcinoma and immune cells, effectively repressing IFN-y production in CD8 T cells. This repression diminishes the T cells’ capacity to mount an effective response against the cancer cells. These findings underscore the intricate relationship between tumor biology and immune evasion mechanisms, suggesting that NPCs exploit both viral infection and immune modulation to create an environment that supports tumor growth and evasion. Understanding these interactions is crucial for developing targeted therapies that can enhance anti-tumor immunity and improve patient outcomes.

[0226] Overall, the data shows that miR-155 shuts off both ICOSL and MHC-I and thus ICOS positive cytotoxic T cells cannot bind ICOSL negative tumor cells failing to kill them. NPCs are unique among human cancers in allowing the cytotoxic tumor cells to invariably accumulate in massive numbers which, in turn may relate to why the tumor has evolved to shut down several key pathways needed by the CD8+ cells to eliminate the cancer. As mentioned, NPC are 100% positive for PD / PD-L1 but only 20% seem to be somewhat responsive to immunotherapy.

[0227] EXAMPLE 4: EBV-positive gastric cancers and the loss of tumor immune surveillance

[0228] Cytotoxic T cells are able to eradicate cancer cells and, thus, cancers had to evolve mechanisms to avoid immune mediated destruction. EBV positive cancers demonstrate a key way cancer cells can do this: the miR-155 mediated elimination of ICOSL expression by the tumor cells, which makes them invisible to cytotoxic T cells.

[0229] As described above, EBV positive carcinoma cells in nasopharyngeal cancers (NPC) are the primary source of the markedly elevated levels of miR-155 and that the upregulation of miR-155 correlated with the loss of both ICOSL and MHC-I expressions in these cells.40By reducing the expression of both ICOSL and MHC-I, high levels of miR-155 found in nasopharyngeal cancer may contribute to the immune evasion mechanisms employed by the tumor. Here a study was conducted which shows that a similar mechanism occurs in a different EBV-induced solid tumor.

[0230] Gastric carcinomas can be divided into two groups on histological grounds: exophytic adenocarcinomas and endophytic poorly differentiated carcinomas. The latter include EBV positive gastric cancers, comprising 10% of gastric cancers, and signet ring cancer, which comprises about 30% of such tumors. The mean survival of signet ring gastric carcinoma is about 10 months. Despite their undifferentiated and deeply invasive pattern, EBV positive gastric cancers had a median survival in one study of 8.5 years.41The study explored a tissue microarray (TMA) from Folio of 80 gastric cancers that included eight cases of EBV positive cancers and 20 cases of signet ring / poorly differentiated cancer for miR-155, PDL1, CD8, ICOSL, MHC1, Granzyme, and ICOS. The study added five other EBV positive gastric cancer cases. Each of the EBV-positive gastric cancers showed a deeply infiltrative, poorly differentiated, endophytic pattern on hematoxylin and eosin stain (Figure 23A). The EBV positive tumors showed an intense lymphocytic infiltrate that included many germinal centers which is exceedingly rare even in CD8 positive rich carcinomas (Figure 23 A) with an average CD8 density of more than 500 cytotoxic T cells / 500 microns. Germinal centers are the epicenter of coordinated B and T cell activation and, thus, symbolize the intense immune reaction directed against these cancers. The presence of numerous germinal centers in the tumor might be the reason of the longer survival of EBV positive gastric cancer patients. The signet ring cancers showed no CD8 positive T cell infiltrate in 14 / 20 (70%) of cases and the 6 cases positive for T cells had a maximum density of 93.4 CD8 positive cells / 500 microns. PDL1 was strongly expressed in each of the EBV positive cancers (Figure 23B, arrow) whereas only 4 / 20 (20%) of the signet ring cancers showed PDL1 expression. Importantly, miR-155 was markedly up-regulated in 13 / 13 EBV positive gastric cancers but was not expressed in any of the 20 signet ring cancers (Figure 23B; note the co-localization of miR-155 and EBER-1 / 2). The CD8 cells in the EBV positive stomach cancers were primed for tumor elimination as seen by the strong co-expression with ICOS (Figure 23 C). This study has demonstrated that miR- 155 directly reduces ICOSL expression, which is an important ligand for the CD8 positive / ICOS positive cytotoxic T cell mediated elimination of tumor cells.42As noted, the miR- 155-positive gastric cancer cells expressed EBER-1 / 2 (Figure 23B) but not ICOSL, though the latter was strongly expressed by the adjacent normal gastric mucosa (Figure 23D). Indeed, ICOSL expression in the tumor cells was 0 / 13 for the EBV positive gastric cancers and 13 / 20 (65%) for the signet ring cancers.

[0231] The balance between immune-mediated elimination of cancer cells versus their evasion in the tumor microenvironment is a key factor in determining the tumor’s clinical presentation. Cancer associated neoantigens primes the immune response and in EBV positive cancers viral associated neoantigens are present a priori. From an immune microenvironment perspective, it is not surprising that signet ring gastric cancer has a much worse prognosis than EBV positive gastric cancers given the near absence of a T cell response in the former and an intense CD8 positive T cell presence in the latter. In both EBV positive gastric cancers and NPC, despite the intense cytotoxic T cell presence with strong co-expression with PDL1, checkpoint inhibitors only show clinical efficacy in about 10-20% of cases.40

[0232] An important part of the why and what other factor(s) is / are preventing the massive numbers of CD8 positive T cells from eliminating these tumors even when the PDL1 is inactivated is the finding that miR-155 in the tumor cells directly targets ICOSL.42MiR-155 indirectly also reduces MHC-I expression. The end result is that despite the release of the PDL1 inhibition, the tumor cells still are able to avoid immune-mediated elimination by the massive numbers of CD8 positive / ICOS positive cytotoxic T cells due to their loss of ICOSL as well as MHC-I. The study refers to these T cells as “frustrated” rather than “exhausted”. The ability of EBV to up-regulate miR-155 is well documented via its LMP1 protein. In addition, it was recently shown that EBV strains encoding for the specific LMP-1 peptide variants GGDPHLPTL or GGDPPLPTL, presented by HLA-E, elicit strong inhibitory NKG2A positive NK and CD8 positive T-cell responses.43With regards to PDL1, two microRNAs of EBV, BART 11 and 17, target two negative regulators of PDL1, F0XP1 and PBRM1, and thus up- regulate PDL1.44

[0233] This inverse correlation between miR-155 and ICOSL is not just a feature of EBV positive tumors. Specifically, in mice where miR-155 expression is controlled by tetracycline, it was documented that miR-155 induction led to B cell malignancies lacking ICOSL expression. Conversely, turning miR-155 off led to lymphoma regression with concomitant synapsing of ICOSL positive B lymphoma cells and ICOS positive T cells. The study found an equivalent inverse correlation between ICOSL and miR-155 in human DLBCL.

[0234] In sum: (1) the EBV positive carcinoma cells in gastric cancers are the primary source of the markedly elevated levels of miR-155; and (2) this upregulation correlates with the loss of both ICOSL and MHC-I expression, which would help render the tumor cells invisible to the intense surrounding T cell infiltrates, even with checkpoint inhibition. These data highlight the need for therapeutic strategies combining the use of checkpoint inhibitors with miR-155- inhibitors in order to drive re-expression of ICOSL in tumor cells.

[0235] Clinical samples and controls: The patient samples (formalin fixed paraffin embedded biopsy material) originated from the files of Folio Biosciences. Clinical information included the diagnosis and age / sex of patient. This study, based on de-identified formalin fixed, paraffin embedded tissues, was exempt from IRB review.40A TMA of 80 gastric cancer cores (each 1mm) was studied. Five additional EBV+ gastric cancers were studied. Five normal gastric biopsies served as the negative controls. Four-micron tissue samples were placed on sequentially labeled slides, baked at 600C for 30 min, and stored at RT with an hematoxylin and eosin stain performed to verify the diagnosis.

[0236] Immunohistochemistry (IHC): The specific antibodies used (source and catalogue numbers; Ab = ABCAM, Prot = Proteintech) were as follows: CD3 (Ab 16669), CD8 (Roche 790-4460), ICOSL (Prot 8687), ICOS (Prot 8685), MHC-I (Ab22432), PDL1 (Ab205921) and PD1 (Ab237728). All required antigen retrieval was done for 30 minutes at 950C using an EDTA solution. The IHC protocol used the Leica Bond Max (Buffalo Grove, IL) automated platform; the Fast red (DS9390) and the DAB (DS9800) detection kits were used and gave equivalent results.

[0237] In situ hybridization (ISH): In brief, the tissue was pretreated for 4 minutes in proteinase K (0.1 mg / ml) then hybridized overnight at 370C with an LNA anti-miR-155 probe digoxigenin tagged at its 5’ end and then detected with an anti-digoxigenin-AP conjugate with NBT / BCIP as the chromogen. ISH for the EBER-1 / 2 RNA was done with the RNAscope assay. In brief, the target sequence was the human herpes 4 isolate SDTW400 EBER-1 and -2 genes, complete sequence from ACD (catalogue # 310271). The assay was done per the manufacturer’s protocol that used DAB as the chromogen.

[0238] Multispectral and co-expression testing and statistical analyses: Co-expression experiments were performed by analyzing a given tissue section for one protein using the DAB (brown) chromogen and analyzing the other protein with Fast Red chromogen or, with tri- localization, using methylene blue (Enzo Life Sciences). Co-expression analyses were done using the Nuance software. Quantification for the signal with either single IHC or multi-labeled IHC was done using either the InForm software or manual counting which yielded equivalent results. Statistical analysis was done using the InStat Statistical Analysis Software (version 3.36) and a paired t-test (also referred to as a “repeated measure t-test”). The null hypothesis was rejected if the significance level was below 5%.

[0239] IHC-zn situ scoring: IHC and ISH scoring was done blinded to the target tested. The standard pathology system of 0, 1+, 2+, and 3+ was used as defined in this study by 1-24% target cells positive (1+), 25-49% target cells positive (2+) and 50% or more target cells positive (3+). Six 200X fields are scored for a given target that yields an average value for each data point.

[0240] EXAMPLE ASPECTS

[0241] Example 1 : A method of diagnosing severity, aggressiveness, or prognosis of a solid cancer in a subject in need thereof, the method comprising: a) obtaining a sample from the subject; b) determining if miR-155 is overexpressed compared to miR-155 in a control sample; c) calculating a risk score for the subject based on results of step b); and d) treating the subject with appropriate medication based on the risk score. Example 2: The method of any examples herein, particularly Example 1, wherein the control sample comprises a sample from a healthy subject or a sample from a subject with a less aggressive solid cancer.

[0242] Example 3: The method of any examples herein, particularly Examples 1-2, wherein after a risk score is calculated, the subject is placed into a risk category based on their risk score.

[0243] Example 4: The method of any examples herein, particularly Example 3, wherein a subject with a low risk level is treated differently than a subject with a moderate or high risk level.

[0244] Example 5: The method of any examples herein, particularly Example 4, wherein treatment for a low risk level comprises administration of immune cell therapy and / or one or more checkpoint inhibitors.

[0245] Example 6: The method of any examples herein, particularly Examples 4-5, wherein treatment for a moderate or high risk level comprises a higher dosage or more frequent administration of chemotherapy or radiation therapy than treatment for a low risk level.

[0246] Example 7: The method of any examples herein, particularly Examples 4-6, wherein treatment for a moderate or high risk level comprises administering an miR-155 inhibitor to the subject.

[0247] Example 8: The method of any examples herein, particularly Example 7, wherein the miR-155 inhibitor comprises an antibody, an antibody fragment, a small molecule inhibitor, a polypeptide, an oligonucleotide, or any combination thereof.

[0248] Example 9: The method of any examples herein, particularly Examples 7-8, wherein the miR-155 inhibitor comprises cobomarsen or an anti-miR-155 oligonucleotide.

[0249] Example 10: The method of any examples herein, particularly Examples 7-9, wherein treatment for a moderate or high risk level further comprises, only after administration of the miR-155 inhibitor, administering an immune cell therapy and / or one or more checkpoint inhibitors to the subject.

[0250] Example 11 : The method of any examples herein, particularly Example 10, wherein the subject does not receive immune cell therapy and / or one or more checkpoint inhibitors prior to administration of the miR-155 inhibitor.

[0251] Example 12: The method of any examples herein, particularly Examples 4-11, wherein treatment for a moderate or high risk level comprises administering to the subject a first synthetic nucleic acid sequence encoding inducible T-cell costimulatory ligand (ICOSL) or a functional fragment thereof and / or a second synthetic nucleic acid sequence encoding RELA or a functional fragment thereof.

[0252] Example 13: The method of any examples herein, particularly Example 12, wherein the first synthetic nucleic acid sequence has a mutated, partially deleted, or fully deleted 3’-UTR region compared to a wildtype nucleic acid sequence encoding ICOSL.

[0253] Example 14: The method of any examples herein, particularly Example 13, wherein the 3’-UTR region of the wildtype nucleic acid sequence comprises an miR-155 target site of SEQ ID NO: 9 (AGCATTA), and wherein the 3’-UTR region of the first synthetic nucleic acid sequence, if present, does not include SEQ ID NO: 9.

[0254] Example 15: The method of any examples herein, particularly Example 14, wherein the miR-155 target site in the 3’-UTR region of the first synthetic nucleic acid sequence is partially or completely replaced with a restriction site for a restriction enzyme.

[0255] Example 16: The method of any examples herein, particularly Example 15, wherein the miR-155 target site in the 3’-UTR region of the first synthetic nucleic acid sequence is partially or completely replaced by a restriction site for EcoRl

[0256] Example 17: The method of any examples herein, particularly Example 16, wherein the miR-155 target site in the 3’-UTR region of the first synthetic nucleic acid sequence is partially or completely replaced by SEQ ID NO: 10 (GAATTC).

[0257] Example 18: The method of any examples herein, particularly Examples 12-17, wherein the first synthetic nucleic acid sequence comprises about 80% similarity or more to SEQ ID NO: 5.

[0258] Example 19: The method of any examples herein, particularly Examples 12-18, wherein treatment for a moderate or high risk level further comprises administering one or more chemokines or cytokines to the subject.

[0259] Example 20: The method of any examples herein, particularly Example 19, wherein the one or more chemokines or cytokines increase expression of ICOSL or the functional fragment thereof.

[0260] Example 21 : The method of any examples herein, particularly Examples 19-20, wherein the one or more chemokines or cytokines are administered at the same time as or after administration of the first synthetic nucleic acid sequence.

[0261] Example 22: The method of any examples herein, particularly Examples 19-21, wherein the one or more chemokines or cytokines comprise INF-y, TNF-a, and / or LPS. Example 23 : The method of any examples herein, particularly Examples 12-22, wherein the second synthetic nucleic acid sequence has a mutated or at least partially deleted 3’-UTR region compared to a wildtype nucleic acid sequence encoding RELA.

[0262] Example 24: The method of any examples herein, particularly Example 23, wherein the 3’-UTR region of the wildtype nucleic acid sequence comprises an miR-155 target site of SEQ ID NO: 9 (AGCATTA), and wherein the 3’-UTR region of the second synthetic nucleic acid sequence, if present, does not include SEQ ID NO: 9.

[0263] Example 25: The method of any examples herein, particularly Examples 12-24, wherein the second synthetic nucleic acid sequence comprises about 80% similarity or more to SEQ ID NO: 11.

[0264] Example 26: The method of any examples herein, particularly Examples 12-25, wherein treatment for a moderate or high risk level further comprises administering one or more enzymes or nucleic acid sequences encoding enzymes for post-translational phosphorylation and / or acetylation of RELA or the functional fragment thereof.

[0265] Example 27: The method of any examples herein, particularly Example 26, wherein the one or more enzymes or nucleic acid sequences encoding enzymes increase activation of NFKB.

[0266] Example 28: The method of any examples herein, particularly Examples 26-27, wherein the one or more enzymes or nucleic acid sequences encoding enzymes are administered at the same time as or after administration of the second synthetic nucleic acid sequence.

[0267] Example 29: The method of any examples herein, particularly Examples 12-28, wherein the first synthetic nucleic acid sequence and / or second synthetic nucleic acid sequence is provided as a vector.

[0268] Example 30: The method of any examples herein, particularly Examples 12-29, wherein the first synthetic nucleic acid sequence and / or second synthetic nucleic acid sequence is delivered via a lipid nanoparticle, viral particle, or any other suitable carrier.

[0269] Example 31 : The method of any examples herein, particularly Examples 12-30, wherein treatment for a moderate or high risk level further comprises, only after administration of the first synthetic nucleic acid sequence and / or second synthetic nucleic acid sequence, administering an immune cell therapy and / or one or more checkpoint inhibitors to the subject.

[0270] Example 32: The method of any examples herein, particularly Example 31, wherein the subject does not receive immune cell therapy and / or one or more checkpoint inhibitors prior to administration of the first synthetic nucleic acid sequence and / or second synthetic nucleic acid sequence. Example 33: The method of any examples herein, particularly Examples 1-32, further comprising, before step a), screening the subject for Epstein-Barr virus (EBV).

[0271] Example 34: The method of any examples herein, particularly Example 33, wherein current or previous infection with EBV is further used to calculate the risk score in step c).

[0272] Example 35: The method of any examples herein, particularly Examples 1-34, wherein step b) further comprises determining if ICOSL, RELA, and / or MHC-I are underexpressed in cancerous cells derived from the subject compared to ICOSL, RELA, and / or MHC-I expression on control cells.

[0273] Example 36: The method of any examples herein, particularly Example 35, wherein underexpression of ICOSL, RELA, and / or MHC-I is further used to calculate the risk score in step c).

[0274] Example 37: The method of any examples herein, particularly Examples 1-36, wherein the sample comprises a cell sample, a tissue sample, a bodily fluid sample, or a cell lysate sample.

[0275] Example 38: The method of any examples herein, particularly Example 37, wherein the sample is a tumor sample.

[0276] Example 39: The method of any examples herein, particularly Examples 1-38, wherein the solid cancer is breast cancer, lung cancer, pancreatic cancer, gastric cancer, or colon cancer.

[0277] Example 40: A method of treating a cancer in a subject in need thereof, the method comprising administering to the subject a synthetic nucleic acid sequence encoding inducible T-cell costimulatory ligand (ICOSL) or a functional fragment thereof, wherein expression and presentation of ICOSL or the functional fragment thereof by a cancerous cell induces and / or enhances an immune response against the cancer.

[0278] Example 41 : The method of any examples herein, particularly Example 40, wherein expression and presentation of ICOSL or the functional fragment thereof by the cancerous cell induces and / or enhances killing of the cancerous cell by cytotoxic T cells.

[0279] Example 42: The method of any examples herein, particularly Examples 40-41, wherein the synthetic nucleic acid sequence has a mutated, partially deleted, or fully deleted 3’-UTR region compared to a wildtype nucleic acid sequence encoding ICOSL.

[0280] Example 43 : The method of any examples herein, particularly Example 42, wherein the 3’-UTR region of the wildtype nucleic acid sequence comprises an miR-155 target site of SEQ ID NO: 9 (AGCATTA), and wherein the 3’-UTR region of the synthetic nucleic acid sequence, if present, does not include SEQ ID NO: 9. Example 44: The method of any examples herein, particularly Example 43, wherein the miR-155 target site in the 3’-UTR region of the synthetic nucleic acid sequence is partially or completely replaced with a restriction site for a restriction enzyme.

[0281] Example 45: The method of any examples herein, particularly Example 44, wherein the miR-155 target site in the 3’-UTR region of the synthetic nucleic acid sequence is partially or completely replaced by a restriction site for EcoRl

[0282] Example 46: The method of any examples herein, particularly Example 45, wherein the miR-155 target site in the 3’-UTR region of the synthetic nucleic acid sequence is partially or completely replaced by SEQ ID NO: 10 (GAATTC).

[0283] Example 47: The method of any examples herein, particularly Examples 40-46, wherein the synthetic nucleic acid sequence comprises about 80% similarity or more to SEQ ID NO: 5.

[0284] Example 48: The method of any examples herein, particularly Examples 40-47, wherein the synthetic nucleic acid sequence is provided as a vector.

[0285] Example 49: The method of any examples herein, particularly Examples 40-48, wherein the synthetic nucleic acid sequence is delivered via a lipid nanoparticle, viral particle, or any other suitable carrier.

[0286] Example 50: The method of any examples herein, particularly Examples 40-49, further comprising providing an miR-155 inhibitor to the subject.

[0287] Example 51 : The method of any examples herein, particularly Example 50, wherein the miR-155 inhibitor comprises an antibody, an antibody fragment, a small molecule inhibitor, a polypeptide, an oligonucleotide, or any combination thereof.

[0288] Example 52: The method of any examples herein, particularly Examples 50-51, wherein the miR-155 inhibitor comprises cobomarsen or an anti-miR-155 oligonucleotide.

[0289] Example 53: The method of any examples herein, particularly Examples 40-52, further comprising administering one or more chemokines or cytokines to the subject.

[0290] Example 54: The method of any examples herein, particularly Example 53, wherein the one or more chemokines or cytokines increase expression of ICOSL or the functional fragment thereof.

[0291] Example 55: The method of any examples herein, particularly Examples 53-54, wherein the one or more chemokines or cytokines are administered at the same time as or after administration of the synthetic nucleic acid sequence.

[0292] Example 56: The method of any examples herein, particularly Examples 53-55, wherein the one or more chemokines or cytokines comprise INF-y, TNF-a, and / or LPS. Example 57: The method of any examples herein, particularly Examples 40-56, further comprising, after administering the synthetic nucleic acid sequence, administering an immune cell therapy and / or one or more checkpoint inhibitors to the subject.

[0293] Example 58: The method of any examples herein, particularly Example 57, wherein the subject has not received immune cell therapy and / or one or more checkpoint inhibitors before administration of the synthetic nucleic acid sequence.

[0294] Example 59: The method of any examples herein, particularly Examples 40-58, wherein the cancer is lymphoma, breast cancer, lung cancer, pancreatic cancer, gastric cancer, or colon cancer.

[0295] Example 60: The method of any examples herein, particularly Examples 40-59, further comprising, before administration of the synthetic nucleic acid sequence, screening the subject for Epstein-Barr virus (EBV).

[0296] Example 61 : The method of any examples herein, particularly Example 60, wherein the subject is currently or has previously been infected with EBV.

[0297] Example 62: A method of treating a cancer in a subject in need thereof, the method comprising administering to the subject a synthetic nucleic acid sequence encoding RELA or a functional fragment thereof, wherein expression of RELA or the functional fragment thereof by a cancerous cell induces and / or enhances expression of major histocompatibility complex class I (MHC-I), and wherein presentation of MHC-I by the cancerous cell induces and / or enhances an immune response against the cancer.

[0298] Example 63: The method of any examples herein, particularly Example 62, wherein presentation of MHC-I by the cancerous cell induces and / or enhances killing of the cancerous cell by cytotoxic T cells.

[0299] Example 64: The method of any examples herein, particularly Examples 62-63, wherein the synthetic nucleic acid sequence has a mutated or at least partially deleted 3’-UTR region compared to a wildtype nucleic acid sequence encoding RELA.

[0300] Example 65: The method of any examples herein, particularly Example 64, wherein the 3’-UTR region of the wildtype nucleic acid sequence comprises an miR-155 target site of SEQ ID NO: 9 (AGCATTA), and wherein the 3’-UTR region of the synthetic nucleic acid sequence, if present, does not include SEQ ID NO: 9.

[0301] Example 66: The method of any examples herein, particularly Examples 62-65, wherein the synthetic nucleic acid sequence comprises about 80% similarity or more to SEQ ID NO: 11. Example 67: The method of any examples herein, particularly Examples 62-66, wherein the synthetic nucleic acid sequence is provided as a vector.

[0302] Example 68: The method of any examples herein, particularly Examples 62-67, wherein the synthetic nucleic acid sequence is delivered via a lipid nanoparticle, viral particle, or any other suitable carrier.

[0303] Example 69: The method of any examples herein, particularly Examples 62-68, further comprising providing an miR-155 inhibitor to the subject.

[0304] Example 70: The method of any examples herein, particularly Example 69, wherein the miR-155 inhibitor comprises an antibody, an antibody fragment, a small molecule inhibitor, a polypeptide, an oligonucleotide, or any combination thereof.

[0305] Example 71 : The method of any examples herein, particularly Examples 69-70, wherein the miR-155 inhibitor comprises cobomarsen or an anti-miR-155 oligonucleotide.

[0306] Example 72: The method of any examples herein, particularly Examples 62-71, further comprising administering one or more enzymes or nucleic acid sequences encoding enzymes for post-translational phosphorylation and / or acetylation of RELA or the functional fragment thereof.

[0307] Example 73 : The method of any examples herein, particularly Example 72, wherein the one or more enzymes or nucleic acid sequences encoding enzymes increase activation of NFKB.

[0308] Example 74: The method of any examples herein, particularly Examples 72-73, wherein the one or more enzymes or nucleic acid sequences encoding enzymes are administered at the same time as or after administration of the synthetic nucleic acid sequence.

[0309] Example 75: The method of any examples herein, particularly Examples 62-74, further comprising, after administering the synthetic nucleic acid sequence, administering an immune cell therapy and / or one or more checkpoint inhibitors to the subject.

[0310] Example 76: The method of any examples herein, particularly Example 75, wherein the subject has not received immune cell therapy and / or one or more checkpoint inhibitors before administration of the synthetic nucleic acid sequence.

[0311] Example 77: The method of any examples herein, particularly Examples 62-76, wherein the cancer is lymphoma, breast cancer, lung cancer, pancreatic cancer, gastric cancer, or colon cancer.

[0312] Example 78: The method of any examples herein, particularly Examples 62-77, further comprising, before administration of the synthetic nucleic acid sequence, screening the subject for Epstein-Barr virus (EBV). Example 79: The method of any examples herein, particularly Example 78, wherein the subject is currently or has previously been infected with EBV.

[0313] Example 80: A method of treating a cancer in a subject in need thereof, the method comprising: a) administering a first synthetic nucleic acid sequence encoding ICOSL or a functional fragment thereof according to the method of any examples herein, particularly Examples 40-61; and b) administering a second synthetic nucleic acid sequence encoding RELA or a functional fragment thereof according to the method of any examples herein, particularly Examples 62-79; wherein expression of RELA or the functional fragment thereof by a cancerous cell induces and / or enhances expression of major histocompatibility complex class I (MHC-I); and wherein expression and presentation of ICOSL or the functional fragment thereof and MHC-I by the cancerous cell induces and / or enhances an immune response against the cancer.

[0314] Example 81 : The method of any examples herein, particularly Example 80, wherein expression and presentation of ICOSL or the functional fragment thereof and MHC-I by the cancerous cell induces and / or enhances killing of cancerous cells by cytotoxic T cells.

[0315] Example 82: The method of any examples herein, particularly Examples 80-81, wherein steps a) and b) are performed simultaneously.

[0316] Example 83 : The method of any examples herein, particularly Examples 80-81, wherein step a) is performed before or after step b).

[0317] Example 84: The method of any examples herein, particularly Examples 80-83, further comprising providing an miR-155 inhibitor to the subject.

[0318] Example 85: The method of any examples herein, particularly Example 84, wherein the miR-155 inhibitor comprises an antibody, an antibody fragment, a small molecule inhibitor, a polypeptide, an oligonucleotide, or any combination thereof.

[0319] Example 86: The method of any examples herein, particularly Examples 84-85, wherein the miR-155 inhibitor comprises cobomarsen or an anti-miR-155 oligonucleotide.

[0320] Example 87: The method of any examples herein, particularly Examples 80-86, further comprising, after administering the first and second synthetic nucleic acid sequences, administering an immune cell therapy and / or one or more checkpoint inhibitors to the subject.

[0321] Example 88: The method of any examples herein, particularly Example 87, wherein the subject has not received immune cell therapy and / or one or more checkpoint inhibitors before administration of the synthetic nucleic acid sequence. Example 89: The method of any examples herein, particularly Examples 80-88, wherein the cancer is lymphoma, breast cancer, lung cancer, pancreatic cancer, gastric cancer, or colon cancer.

[0322] Example 90: The method of any examples herein, particularly Examples 80-89, further comprising, before administration of the synthetic nucleic acid sequence, screening the subject for Epstein-Barr virus (EBV).

[0323] Example 91 : The method of any examples herein, particularly Example 90, wherein the subject is currently or has previously been infected with EBV.

[0324] It will be apparent to those skilled in the art that various modifications and variations can be made in the present disclosure without departing from the scope or spirit of the invention. Other embodiments of the disclosure will be apparent to those skilled in the art from consideration of the specification and practice of the methods disclosed herein. It is intended that the specification and examples be considered as exemplary only, with a true scope and spirit of the invention being indicated by the following claims.

[0325] The following patents, applications and publications as listed below and throughout this document are hereby incorporated by reference in their entirety herein.

[0326] Reference List

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[0376] SEQUENCES

[0377] 1. SEQ ID NO: 1

[0378] GGCTCGCTCGAGCCCTCCTTCTTACTTCCCAG

[0379] 2. SEQ ID NO: 2

[0380] AGAGTCGCGGCCGCCCCAGGGCACCTCCCGGGAC

[0381] 3. SEQ ID NO: 3

[0382] GAAGAATTCGAAGATGTGTGGTGTTTATAAAAG

[0383] 4. SEQ ID NO: 4

[0384] TTCGAATTCTTCATGTTAAAAGCGGGAGTG

[0385] 5. SEQ ID NO: 5 - Human ICOSL nucleotide sequence gcagttagagccgatctcccgcgccccgaggttgctcctctccgaggtctcccgcggcccaagttctccgcgccccgaggtctccgc gccccgaggtctccgcggcccgaggtctccgcccgcaccatgcggctgggcagtcctggactgctcttcctgctcttcagcagccttc gagctgatactcaggagaaggaagtcagagcgatggtaggcagcgacgtggagctcagctgcgcttgccctgaaggaagccgtttt gatttaaatgatgtttacgtatattggcaaaccagtgagtcgaaaaccgtggtgacctaccacatcccacagaacagctccttggaaaac gtggacagccgctaccggaaccgagccctgatgtcaccggccggcatgctgcggggcgacttctccctgcgcttgttcaacgtcacc ccccaggacgagcagaagtttcactgcctggtgttgagccaatccctgggattccaggaggttttgagcgttgaggttacactgcatgt ggcagcaaacttcagcgtgcccgtcgtcagcgccccccacagcccctcccaggatgagctcaccttcacgtgtacatccataaacgg ctaccccaggcccaacgtgtactggatcaataagacggacaacagcctgctggaccaggctctgcagaatgacaccgtcttcttgaac atgcggggcttgtatgacgtggtcagcgtgctgaggatcgcacggacccccagcgtgaacattggctgctgcatagagaacgtgcttc tgcagcagaacctgactgtcggcagccagacaggaaatgacatcggagagagagacaagatcacagagaatccagtcagtaccgg cgagaaaaacgcggccacgtggagcatcctggctgtcctgtgcctgcttgtggtcgtggcggtggccataggctgggtgtgcaggga ccgatgcctccaacacagctatgcaggtgcctgggctgtgagtccggagacagagctcactggccacgtttgaccggagctcaccgc ccagagcgtggacagggcttccgtgagacgccaccgtgagaggccaggtggcagcttgagcatggactcccagactgcagggga gcacttggggcagcccccagaaggaccactgctggatcccagggagaacctgctggcgttggctgtgatcctggaatgaggcccttt caaaagcgtcatccacaccaaaggcaaatgtccccaagtgagtgggctccccgctgtcactgccagtcacccacaggaagggactg gtgatgggctgtctctacccggagcgtgcgggattcagcaccaggctcttcccagtaccccagacccactgtgggtcttcccgtggga tgcgggatcctgagaccgaagggtgtttggtttaaaaagaagactgggcgtccgctcttccaggacggcctctgtgctgctggggtca cgcgaggctgtttgcaggggacacggtcacaggagctcttctgccctgaacgctcccaacctgcctcccgcccggaagccacagga cccactcatgtgtgtgcccacaagtgtagttagccgtccacaccgaggagcccccggaagtccccactgggcttcagtgtcctctgcc acattccctgggaggaacaatgtccctcggctgttccggtgaaaagttgagccacctttggaagacgcacgggtggagtttgccagaa gaaaggctgtgccagggccgtgtttggctacaggggctgccggggctcttggctctgcagcgagaaagacacagcccagcagggc tggagacgcccatgtccagcaggcgcaggcctggcaacacggtccccagagtcctgagcagcagttaggtgcatggagagggtat cacctggtggccacagtcccccttctcacctcagcaatgatccccaaagtgagaggtggctcccccggcccccaccaccctcagcag ccccaccccactcaaccctgagggtccccagggtcctgatgaagacctccgaccccagcgccaggctcctcggagcccaacagtcc caagggggcaggagacggggtggtccagtgctgaggggtacagccctgggccctgaccagccccggcacctgccatgctggttcc cggaatgaatcagctgctgactgtctccagaagggctggaaaggatgctgccaggtgacccgaggtgcactcgccccagggagatg gagtagacagcctggcctggccctcgggacacattgtctgccccggggctatgggcaaatgcccctccttcttacttcccagaatcccc tgacattcccagggtcagccaggacctgttacagccctggtcacttggaactgacagctgtgtgaggcctgcacttctcagacccaga cttagaacaaaaggaggagtgaggactcaaggctacaatgaggttccagtacttgttacaagaaattggttttctgcaaaaaaagtccct acctgagcctttaggtgaatgtgggatccactcccgcttttaacatgaaagcattagaagatgtgtggtgtttataaaagaacagttgtcat caccgggcattgattggcagggacaaggagctgcttgggtgtggaaagttggggcgttggaaagtgggctgtggtgcccatttgcag tgactgtgaagtgactccaggacggacctgcgggggcacccagaggtcctaagccccaggactgagggtcgtgcatcaccactcg ggtgtcccgggaggtgccctgggcccggggacctcacaggcaggacggcgacactaatgcagggagagggagtctggccccag cttttcctatcagaggcgattttccttcaccaggggatgggcaggaaagaggcaggggccccagaagcttctgtccctcatgcctgag ggcacgggggacacttggaggctgctgtcaccactgtgcgtccaaggccatgctctctgcgggtcagtgcctgagtctcgcctccctg ctggtccctgaagccccctcagaagccctgcctgtcacgtcggcatttgtgagacctaccctgtaacgcctgcccctctcagcccaaca tcagcttcctctttctcccttgctgtagacaggctggattccagtgttgggacagccatctccagaaacctgacttaagagagtaagatgc aaatcgtgcctgtatccagtggctttggtgggtgcagggagtcttgggcacagccagctcagctgtctgtggtatgagcaggaacagg tgccactcctgctcaggggaccctgccctacaccaggctgttccgtccccctggaggacatggggccaggtctggaggcattttgggt tgtcacagctgggggctgttcctcggcttcagcgggtggaagcctcagatgctgttcaacatcttctggacacgggaggccccgacag agagaagcgtccacccgcaagtccacagtctgaggtctcccctcagagaccctgccctgcacacccacctccagccaaaggtcctg cctgccccagggctcaggggaaccttgccggtctgtggaacaggagaggggactctcgccagctgcaccaccctgcacgtagtag gtgtgcggtaaacatccaccagggaggctccagtcaaggctggcagatggggcggtccatccctagggcaggtgacagaagggaa aaggctgcctgctggcccccgagccaggtagcacatgcttgtgcctcagtttcccctcctgtaaagtgaggcgctggatccaggttctg tctactgggctctgcagcttggacgctcctaagaccaagcgacccaccctggggagggcagctatggctttggaatagctgtccaggc ccgggtgcctccaagacggccaccacaccctgcctgtgctgcaggggtgcaggggtaaggggcaagactccagaggcctcctctct gcatctccttgtcttcagtggccggaggtgaggcctgagctcaggggaggggcttctgccacgaaccctatggcggggcacagcac acttttcccagggaggacccctgggccccctgcattatccccagcggagtgtggggtcaccttccaagagcgacattgagaagctcca gctctaggagtgtgcagactcttaaccaggcaggcccaggccctggggcacacaaaggcggggcctgctctccccagctgcccctg ccaatgggggctggactgtcctaccctcctcccttctacctccccactgtcttccctctccactgtcaccactgcctccctcttccactgtc ctccatgcactgccctccctccaccttcccccacccccaccactccccatgctgtccccaggctccccccgctctcccccctccccact gtccccctccccatgctgtacccagctcaccccgctctcccctctccccactgtcccccctcccactccccatgctgtccccagctcacc ctacatggacttggcgatgtccttccatggctcaccggtctgaatttccatgatgagccgggcctgcagctttgctcccctatccctgccc aggctgcagctgtccatgcagggagcgagctccagcacctgcggagtccttccgtgggggcctctccgtgccacagcagccaggg acctcaggtgcctgtgcatgacaccaccgcccatcctcatcctgagccagcctctcaggatcaggacttggtttggcggcgttaacctt agagcctgcaaggggcttcctcctggtgggtctggccgtagcctggggaggccacagctccaggccactccagacctcccttcctct gggccttccatgtggtggcaaccaccgcagctgtaagggagggaaaatggagcgtttgttctcgggctgggctggggtctggggga agccatgggcgtgaagactggagtattatttgatggagaagcggccactcctggagaccggcggcaaacacagaagcacagcgtg gaaggtgctggtgtcagcccacacgggtgatggggtcagactcaggagtcacactcaggagtcaccaggctcaaagggcccaggc accgcaagtcctgctcagccccagacacaatgcattcctgttgccctcgccctcagccaggccccacgcaggccagggagcactgg caaagcttggcaaccctctgggggccagccttcatccaggccgaaggtggtcagtggcccaccatggcccaggtagaaaactcacg gattaagatttcatgcccgactccaaaggcaagagactttattattttattttttttgagccagagtatcgctctgtcacctaggctggagtgc aatctctgctcattgcaacatctgcctcccgaactcaagcaattctgcctcagcctcccaagtagctgggattacaggtgtgcgccacca tgcccaggtaattgtatttttagtagagacagggtttcaccatgttggtcaggctggtttcaaactcctgacctcaaatgatctgcccacctc gacctcccaaagtgctgggattacaggtgcgagccaccgcacctggctaccagacacttcagagttacaggttagtttttctttttcttttat ttttttttttttggcggaggtgcagggggagttaaacaaacaaacaaaataaacaggccgggtgcggtggctcatgcctgtaatcccagc actttaggaggcctaggtgggtggatcacgagatcaggggttcaagaccagcctggccgagatggtaaaaccccgtctccactaaaa atacaaaaattggccaggcacggtggctcacacctgtaatcccagtactttgggaggctgaggtgggcagatcacctgaggtcagga gttcaagaccaacctgaccaacatggagaaaccccatctctactaaaaatacaaaattagccaggtgtggtggtgcatgcctgtaattcc agctactcgggaggctgaggcaggagaattgcttgaacccaggaggcagaggttgcagtgggccaagatggcgccattgcactcca gcctgggaacaagagcgaaactctgactaaaaaagaaagaaagaaagaaaaaaattagttgggcacggtggcaggcgcctgtaatc ccaggtactcaggaggctgaggcaggagaattgcttgaacccgggaggcagaggtcgcagtgagccgagattgcaccactgccct ccagcctgggtgacagagcaagactccgtctcaaaaaaaaaaaaaaaaaaaattggatacattgtaatacctcaaatacttgtaagtga agcaccccagttcccatagagctgccgcactcagaggcttctgtaacctgcctgctcccagcattctatttagggtctggtatgtccaga atttgcagacacagcaattcctgcagcagcagtgcaccatgtggaaggggccccatgaccagcccactgtgagctcacacgtgatga ctgaggcttcttcacacagcagggctctgggtgtgatacccagggcacacgcgtttgcacaggcacaggccacacaagttctcacatg ctcagccccataagccgtgctggacaggcatggccatttacacccaggatcctgctgagaacagcaaccaactcaccaccctcgcat catgatccttgccacacaggggctctggtggctttggtggcctgggctgtggctctgctgccagccaccttgagtgaagatccgggttc tctgggtgctactcagctgctatgtggggagctggcccctggggtgatgagggcccttcccaacccgccctcagcccttggacagcc aggatcacccggggctgtctgcatacagacttctcaggggagttctcagcttggacccttatctccccagaatcctggaacctgctcctt ctgctctcgtgactgactgtgttctctatgcaacttccaataaaacctcttcatttgaaaggaaaaaa

[0386] 6. SEQ ID NO: 6 - Human ICOSL polypeptide sequence

[0387] MRLGSPGLLFLLFSSLRADTQEKEVRAMVGSDVELSCACPEGSRFDLNDVYVYWQT SESKTVVTYHIPQNSSLENVDSRYRNRALMSPAGMLRGDFSLRLFNVTPQDEQKFHC LVLSQSLGFQEVLSVEVTLHVAANFSVPVVSAPHSPSQDELTFTCTSINGYPRPNVYW INKTDNSLLDQALQNDTVFLNMRGLYDVVSVLRIARTPSVNIGCCIENVLLQQNLTV GSQTGNDIGERDKITENPVSTGEKNAATWSILAVLCLLVVVAVAIGWVCRDRCLQHS

[0388] YAGAWAVSPETELTGHV

[0389] 7. SEQ ID NO: 7 - Mouse ICOSL nucleotide sequence agcgggtctcctgccgccaaagcctcaagaaccccagatttcagcgccccaagcctggaagctccccagttcttcgtggcccccaac agctccggaaccccagccgctgcaactctccgcgtccgaaatccagcaccccgcagtctgcgctcgcaccatgcagctaaagtgtcc ctgttttgtgtccttgggaaccaggcagcctgtttggaagaagctccatgtttctagcgggttcttttctggtcttggtctgttcttgctgctgt tgagcagcctctgtgctgcctctgcagagactgaagtcggtgcaatggtgggcagcaatgtggtgctcagctgcattgacccccacag acgccatttcaacttgagtggtctgtatgtctattggcaaatcgaaaacccagaagtttcggtgacttactacctgccttacaagtctccag ggatcaatgtggacagttcctacaagaacaggggccatctgtccctggactccatgaagcagggtaacttctctctgtacctgaagaat gtcacccctcaggatacccaggagttcacatgccgggtatttatgaatacagccacagagttagtcaagatcttggaagaggtggtcag gctgcgtgtggcagcaaacttcagtacacctgtcatcagcacctctgatagctccaacccgggccaggaacgtacctacacctgcatg tccaagaatggctacccagagcccaacctgtattggatcaacacaacggacaatagcctaatagacacggctctgcagaataacactg tctacttgaacaagttgggcctgtatgatgtaatcagcacattaaggctcccttggacatctcgtggggatgttctgtgctgcgtagagaat gtggctctccaccagaacatcactagcattagccaggcagaaagtttcactggaaataacacaaagaacccacaggaaacccacaat aatgagttaaaagtccttgtccccgtccttgctgtactggcggcagcggcattcgtttccttcatcatatacagacgcacgcgtccccacc gaagctatacaggacccaagactgtacagcttgaacttacagaccacgcctgacaggactctgcccaggatatggacagggtttctgt gagttgccaccaggtggatgtcagacacaacttcagagtggacccccacaggcctggtgacagaggacaacgagctgtctgcttatg ggctgtgatggaggccaggaatccctggctttacgaggcacagagacttcatcccagaaaccccgagggagatctctccagtgggc agcagcaacatcatcggaatatggagcctccggtgagctgtcggcacagagagcagcagcttgtgagaagatccttccttggcacgtt actactcaggcctaggagctttataaaagagcgtttgagccactctgaaagccctacagagtctactggagactttccctgcaggacctt cagttggggaggaagcctgactttatttaggtctcaggctacttgggcctcttcgaggatatgtgggattttgtctactgcaaacctgtttct ggctgacaatggttgggctcagaggcactcagcttcacaacatcaatgggacacgcctcatccttgacttcctgtggctacagaagcttt ccgaaagccttgagctctttcagactgaacagctctgcccagtctcagcagcccatgaagatctcaactccagcttcctgggtctccgtg ttgctggccagaatagagctagctcttttgtttcaagatggttctgcaaagttggctgcttggaaacctagggatgtatgtacaagctccag gctgatgcagtagggggcacggactccccgatggaacacagtatctgaccctaggtgagggcaagctccttcccacgcagaggact ggaaattctggaccgtcaaggcctgtctgctatgtggctggggctcagtgctgatggatgtgtgagatctcaggaatgaggagtgaga accctgggctcaggactaggaagacctgtccatttttttttttttttaatgcccacatggactttttattcttcacaccgatgtattcaatgagtg tagagagaactacttaagtccttcccgagtacaaagcattacctacctgcagaatagcaactgttgttatgggtcttgagttggcagctac agcaaacaagcacaaggagcagttggggtgcaagaagatggggtgcagcgcccccaaggacagacatttgggaattagtggtctc cctgatgcccatagttccccaggaactcaggtgggtctgcggcagcacagtaggagtattcctcctactttaacttttcttgtcagacgta gtttaggttcagaaagaggtcaactcagcaagccagctagccgccttggggcaccagacacactgccccccaccccctgcttatgtag gcattgggaacccttcacagaccactggctgtacagtcaccatcacctgctgattccagcaggcccccaccttcttgtggaatcctggg agcactcccctcttacccctcactgccccccaccccctgcacatcagcattcattagatttgccctgtaacgtctgattcctcctttatctgg gttgtagatggggcatagtgacttctagaaacctaacaagggaataaatgtaagatgtgctttc

[0390] 8. SEQ ID NO: 8 - Mouse ICOSL polypeptide sequence

[0391] MQLKCPCF VSLGTRQP VWKKLHVS SGFF SGLGLFLLLLS SLC AAS AETEVGAMVGSN VVLSCIDPHRRHFNLSGLYVYWQIENPEVSVTYYLPYKSPGINVDSSYKNRGHLSLDS MKQGNF SLYLKNVTPQDTQEFTCRVFMNTATELVKILEEVVRLRVAANF STP VISTS DSSNPGQERTYTCMSKNGYPEPNLYWINTTDNSLIDTALQNNTVYLNKLGLYDVIST LRLPWTSRGDVLCCVENVALHQNITSISQAESFTGNNTKNPQETHNNELKVLVPVLA VLAAAAFVSFIIYRRTRPHRSYTGPKTVQLELTDHA

[0392] 9. SEQ ID NO: 9 - miR- 155 target site AGCATTA

[0393] 10. SEQ ID NO: 10 - EcoRl restriction site GAATTC

[0394] 11. SEQ ID NO: 11 - RELA nucleotide sequence atttccgcctctggcgaatggctcgtctgtagtgcacgccgcgggcccagctgcgaccccggccccgcccccgggaccccggccat ggacggtgaggtcgccctctgaccccgcggggtggcatcgccggggccgccgcggctgtggcgggagcccccgcgccgctttcc gccccttcctgcgccgacgggcgctgcgcagggagcggtcggcctgccggatggtggcgcggggcggggcaggcggggaggg gtctgactcagtttcccctctgggtggagggggtggcccttgactcagcatcctccctgggtgggggagcagagggaaccttgactca gtttccctccacaccgtccatgggggcagctctggcttcgtttccctattaggggagaacgcatctgattcagtttcctctctggagggag gtggggcgtgccctccctcaattttccttaagaaagtacaacaggccctgattcagcttccctctggagaagaaggattccttaccccgtt ttccctcaagaagcaggtgtttctggttcctttcctccaccccttagtttcaccgcaggttctagggagcaggtcctgactcagttcccctg ggagggagcctggcttctcctctccgtggagagcagattcctccttgtgagaatgggcattgggtgtgtgctgaccctgatctccctccc ctttcagaactgttccccctcatcttcccggcaggtaagtggcccccgcggcggtcggggaggtgctccctggggtagggcagtggg ggcagccgcctaatggggctgcggtgtcccctggcagagccagcccaggcctctggcccctatgtggagatcattgagcagcccaa gcagcggggcatgcgcttccgctacaagtgcgaggggcgctccgcgggcagcatcccaggcgagaggagcacagataccacca agacccaccccaccatcaaggtcagcactgcgccagggtgtgcggggaagggactgtggaaccccagccctctgtgtttgggagtc aggtttggatgtctgggcctctgtactttggacagatcaatggctacacaggaccagggacagtgcgcatctccctggtcaccaagga ccctcctcaccggcctcacccccacgagcttgtaggaaaggactgccgggatggcttctatgaggctgagctctgcccggaccgctg catccacaggtgaggtccctgcaggctaggaggcccagcatgaggggacaggaggcgggctatgaagtagccgctacagtgttact cctttctggacccagggcagaattcctcactccttgccctgactcagctctgcaagtaaactgcctttgtcttgaaattatctatttgtacgttt actctagcagatgagttccaggggatacggatgggtccctaatcacttctgaattcctgacccctgtagctggaaggacaagtccctca gagtcacgtctcttgtttgaggaagaaaaccattgaaccagcactctcccctggagttgacattgcagttgacagagacaggctgtaaa aaaatatacaggccgggcatagtggctcatgcttataatcctagcacttcgggaggccaaggcaggaagatctcttgagcctaggaatt caagactagccaaggcaacatagtgagaccccatgtctacaaaaaataagaaattagctgggtatggtggcatgcgcctatagtccca gggaggctgaggtgggaggatcgcctgagcccaggaggtcactgctgcggcaagctatgattgcaccactgcacttgggtggcag ggcgataccttgtctttttttttttttttttttttttaagaaagagtcttgctctgtccccagactggagtgcagtggcgcgatctcagctcactgc aacctccaccttccgggttcatgccattctcctgcctcagcttcccgaatagctgggactacaggtgcctgccaccacgcctggctaattt tttgtattttttagtagagacagggtttcaccgtgttagccaggatggtctcaatctcctgaccttgtgatctgcctgtcttggcctcccaaag tgctgggattacaggcttgagccaccgcacccggcccgacactgtgtctcttaaaaataaataaatacacagataaataaacaaggtgc tgtcagagtaacagtttcagtcctgaaaataaaacgaggtcataggatagaaggataacctggggtgggggagttaggggagctggt gtgtgatgactttagattgggaggtcagggaaggcctccctcagctaagacttgaatggtgagaaggaggagagaagtaggaaaaga acattccaagccgaccaaacaagtgcaaaggccttgaggtgggaatgagtgtcctgtgtttagagagtggaaaaggagccccggtgg ctgtaagcagggagcccacagggaggctggcaggggcaggggcacggaggtgcttgtcagctgagtgaagggacagggttcgtt gcagcacagtaggaagccactggagagggttaggcatggagcgatgatctaggggtgggcagagtaggaaggagcaggctgggc aaggcaggaaggtctgggctctgtgagagacagtgggacagacgactgggggcgctcagtttccagaacctgggaatccagtgtgt gaagaagcgggacctggagcaggctatcagtcagcgcatccagaccaacaacaaccccttccaaggtgagggcaaacctgcctgc cacacccgcaccctctgtcaccctccccagcccgcctttcctgcatctccctcactggccaagacacagtaaagctggaatcttggtgc ccattcagtactctcccactttaccactgggaaactgagtcccaggaggggacatgacattcaggccacagtggagtcagtgctgggtt ctgcctcctctaggacttgtgtttcacacacgcaccagcggccctcccagcgcagagctccctgcccccactgatagcacctatatccc cacttcccctgctctcctgatcgcctgttttcttggcagttcctatagaagagcagcgtggggactacgacctgaatgctgtgcggctctg cttccaggtgacagtgcgggacccatcaggcaggcccctccgcctgccgcctgtcctttctcatcccatctttgacaatcgtgagtagc gagggagacgcaggaaggggagagggtgggcctgaataggaggaagggggttggggtgagtgtggcctgcaagaggcgctctg gcttcattcaatcatcttgcgtatttgtctactcttctctgcccagctctttgttggctggggagagagggcaaagaagacccagagctgtc cttgaggtgtagctctggcctggcaggaagtctagcagagagtctgatgggagatgggcattttcagtattgtgtgatgagaaggtggt gtgcccacaactgcgataatgactctaggaaatgaactcccaggaacaagatagtgttcgagaatgatgattcagtgatggaagccca cagatgaattgggctgactgatcagctgcagtctttagttaagtaaccagcaggatgaatggaaataaaacgaatctggagccccaag gatgggaaaccaggataaactgggggtcaggagggaagggctcattgccaaggtgggtatggactggccttaaagattgctttggtc aggtggagaaagagtgtgatcagattggcacccactggactataagctccgggagagcagagaggtttgggttttgtgtgtggtcgtat tcttgcttagatcagtgttgagtgcacagtaggcaaatacttgttgaatcggttggggtgaggagctaataagaaggtctgactttggcta caaaggtgtaggtggcaagctctgggcaaccttgactctgggctgggcagggaagttgcatctcctgcaggccaccctggggacctc tcgtggctcaggtcatcctcccttgttttcactgcctcaggtgcccccaacactgccgagctcaagatctgccgagtgaaccgaaactct ggcagctgcctcggtggggatgagatcttcctactgtgtgacaaggtgcagaaaggtatacatcaggaggcaggggtgggctcttgg gagcaaggggtgaagctgagcagagaagtggagtgtcaggtaactggctttggatacttcctcctgtgcctcgggggcctcaggtgg gccgaagggtgatgtatcagaagacaagcatcagaagttgagggggagaccatggagggttagacacactgacccacactgccac cgcatctcctgcagaggacattgaggtgtatttcacgggaccaggctgggaggcccgaggctccttttcgcaagctgatgtgcaccga caagtggccattgtgttccggacccctccctacgcagaccccagcctgcaggctcctgtgcgtgtctccatgcagctgcggcggcctt ccgaccgggagctcagtgagcccatggaattccagtacctgccagatacaggtacacagctggggtggcagcatcagggcagctg ggctctgtgccgtgagagagaggccaagactgcatgtctggggctgtggttgagggccagaagctggagtcagagcttgggatcca agaaactggaatccatttcttgcctcatttaataataatgatacctcagacattgagctagtactacacatctgcccctattccaggcacttc actggattacctgattttaatctcatgacccttttgaagtaggagatagtgtccaacttgcctgggttcacgtggctagtaagtagcagagt ctagcagagtcagcgttggaacccaggccatctggcttcaggatccttttgccacactggttgtcttgtcactaccatacagcccagcca cattttggtcgaggtttgagtgtccacttggtaccagggcctgtgccaggtattgtgaatacaaaaaatgagtaagacatgctccctgccc ttcagagtctcagtcacttgaggaagacaaatgtgcaaaacagatccttaagtcacggaatgacatgtgaaatggaggtgtgcgcaga gaagcacgggggcacagtggcctccggggagctggagacagctgcatggagaaggtggcattgaagctggagaaagacacaga agtgtgttctccaggcaggtggcccagcacctggaagggaaggggtgttggcaactgcacggcctattcagggccaccttcgtggttt gagctgatgttgggcagtgagcaggctcagagattggagggcctggttaagctgaaggcctctaagcagagaggtgacaggagca gatttggaagttgactcaggaggccacgtgtagaagatcagagacagacagactcaaagcagggaggccattctggaaatgttgctg tagtccaggaagagaagatgcggcacaaaccttacagaaggaatagcagagcaagatcacatctccaccactgtcctcagttaccctt cagccctgcagccgttttgcgtcttacagaccaagctcagtctcacctcaggacctgtgtctagctgttgcctctgcttggaatgcccaag gttttaatgcaagtggctcatgccagtccttccctgacctctaaattagcccttccctctacatttagactgtcctgacctcacttccagtatt gttacatcaccatatcgtccctggaccaccgtcacttgagattttctggtttggttttgtttgtttgtttgagacagagtctcactcttgtcgccc aggctggagtgcagtggtgcaatctcggctcactgcaacctccacctcccgggttcaagtgattctccctgcctcagcctcccgagtag ctgggactacaggcgcccgccaccacatccggctaattttttgtatttttagtagagacggggttttcaccatgttggccagggtggtctc gatctcttgacctcgtgatccacccgcctcgacctcccaaagtgctgggattacaggcgtgagccaccacgcctggccgagattttctg gtttgtatatatgattgtgtccctctcactttttcactaggatgccaactttttgggagcagacaccttgtgtgcatactcacggccacatccc cagtgtctagaagggaggctggcacatagtaggtgcttgataaatatatgtcaagttactggcggggagtgagagaaaagaagataat cattccaaggtttctagctttggtgacagggcagatggcggatggtcagggaatgcaggagaaagaacctagtttttgagggatgatga ggttgtctttgaattgatggaattgaagtgctttggagctactgaaataccactgtaattagaaagacagttctgaaagctctgactaaaat atgaaaccattagagataagaataagagtctctgctgaagtcttaaggcagttgtgcctcagctgccccagagtgccaagaaattctcg ccaggcctgggtggaggctgacaatgagccaggatgtgagcagtgcaggaaggtgtgagctggaggctggggccccccaggtctc ctctttgcccagagcatggcccctgtactcggcctcaccatgtggtcttagaggggcagattcagccaacagaggcctccaaaagctg tgggattctcagaactgctgagtgtcacacatcccttctcattggcagagaaggttgggagaatgggtcttgggaaagcctggctcctc cccttcctcctgagggacctttgagagcgtcggggacccacctgaggagagcaagtcccacttctcccccttactttcccagacgatcg tcaccggattgaggagaaacgtaaaaggacatatgagaccttcaagagcatcatgaagaagagtcctttcagcggtgagatggggac tgggaaagccagagaggaaggcagcgggcagggaggggacaccctggggctgagaactgacctagcccttgccctacaggacc caccgacccccggcctccacctcgacgcattgctgtgccttcccgcagctcagcttctgtccccaagccaggtaaggatttccttttgtc ccactggaacgacaggttcagctctgttcagctgaggagcagtggagatgaagactcctgggccccattacagacctactgaatcaga ggctttgaaaggatggcgcaggaaccagcgacttcctgggatgctaatgcacactgaagcctgggaaccaccgtgccacttaaattct atattgggtatcttgggaatcagaagttaaaaatgcagaatctctgctctcatctctcacttgaattgggggaacaaaaatcacctctaaatt agcaagatgtgtgtgcctgtgcgaggctcaaaaactgacaagctctgcacttcaggccgctgctcacttggtgctccatgggctcattg cctctcaggacttagtttttcagctgtaaactgaggagtccagcttatctctccaagtgtcccttcctctaaaaggctggaagagtttcgtg ggcttgagtagtcagggaggactccaaggtggaggtgtggctagaactggaccctacaggctgggtcagatggggtaagaggaag gagatagggtgttggcagtgactccctagagagggattgagcctgagcaccatgaagaggggctggggtacagaggacggggtgg atctctagggctttctctgacccctgcctctctgtctctctcctccagcaccccagccctatccctttacgtcatccctgagcaccatcaact atgatgagtttcccaccatggtgtttccttctgggcagatcagccaggcctcggccttggccccggcccctccccaagtcctgccccag gctccagcccctgcccctgctccagccatggtatcagctctggcccaggccccagcccctgtcccagtcctagccccaggccctcctc aggctgtggccccacctgcccccaagcccacccaggctggggaaggaacgctgtcagaggccctgctgcagctgcagtttgatgat gaagacctgggggccttgcttggcaacagcacagacccagctgtgttcacagacctggcatccgtcgacaactccgagtttcagcag ctgctgaaccagggcatacctgtggccccccacacaactgagcccatgctgatggagtaccctgaggctataactcgcctagtgaca ggggcccagaggccccccgacccagctcctgctccactgggggccccggggctccccaatggcctcctttcaggagatgaagactt ctcctccattgcggacatggacttctcagccctgctgagtcagatcagctcctaagggggtgacgcctgccctccccagagcactggg ttgcaggggattgaagccctccaaaagcacttacggattctggtggggtgtgttccaactgcccccaactttgtggatgtcttccttggag gggggagccatattttattcttttattgtcagtatctgtatctctctctctttttggaggtgcttaagcagaagcattaacttctctggaaaggg gggagctggggaaactcaaacttttcccctgtcctgatggtcagctcccttctctgtagggaactctggggtcccccatccccatcctcc agcttctggtactctcctagagacagaagcaggctggaggtaaggcctttgagcccacaaagccttatcaagtgtcttccatcatggatt cattacagcttaatcaaaataacgccccagataccagcccctgtatggcactggcattgtccctgtgcctaacaccagcgtttgagggg ctggccttcctgccctacagaggtctctgccggctctttccttgctcaaccatggctgaaggaaaccagtgcaacagcactggctctctc caggatccagaaggggtttggtctgggacttccttgctctccctcttctcaagtgccttaatagtagggtaagttgttaagagtgggggag agcaggctggcagctctccagtcaggaggcatagtttttactgaacaatcaaagcacttggactcttgctctttctactctgaactaataaa tctgttgccaagctgg

[0395] 12. SEQ ID NO: 12 - RELA polypeptide sequence

[0396] MDELFPLIFPAEPAQASGPYVEIIEQPKQRGMRFRYKCEGRSAGSIPGERSTDTTKTHP TIKINGYTGPGTVRISLVTKDPPHRPHPHELVGKDCRDGFYEAELCPDRCIHSFQNLGI QCVKKRDLEQAISQRIQTNNNPFQVPIEEQRGDYDLNAVRLCFQVTVRDPSGRPLRLP PVLSHPIFDNRAPNTAELKICRVNRNSGSCLGGDEIFLLCDKVQKEDIEVYFTGPGWE ARGSFSQADVHRQVAIVFRTPPYADPSLQAPVRVSMQLRRPSDRELSEPMEFQYLPD TDDRHRIEEKRKRTYETFKSIMKKSPFSGPTDPRPPPRRIAVPSRSSASVPKPAPQPYPF TSSLSTINYDEFPTMVFPSGQISQASALAPAPPQVLPQAPAPAPAPAMVSALAQAPAP VPVLAPGPPQAVAPPAPKPTQAGEGTLSEALLQLQFDDEDLGALLGNSTDPAVFTDL ASVDNSEFQQLLNQGIPVAPHTTEPMLMEYPEAITRLVTGAQRPPDPAPAPLGAPGLP NGLLSGDEDFSSIADMDFSALLSQISS

Claims

CLAIMS1. A method of diagnosing severity, aggressiveness, or prognosis of a solid cancer in a subject in need thereof, the method comprising: a) obtaining a sample from the subject; b) determining if miR-155 is overexpressed compared to miR-155 in a control sample; c) calculating a risk score for the subject based on results of step b); and d) treating the subject with appropriate medication based on the risk score.

2. The method of claim 1, wherein the control sample comprises a sample from a healthy subject or a sample from a subject with a less aggressive solid cancer.

3. The method of any one of claims 1-2, wherein after a risk score is calculated, the subject is placed into a risk category based on their risk score.

4. The method of claim 3, wherein a subject with a low risk level is treated differently than a subject with a moderate or high risk level.

5. The method of claim 4, wherein treatment for a low risk level comprises administration of immune cell therapy and / or one or more checkpoint inhibitors.

6. The method of any one of claims 4-5, wherein treatment for a moderate or high risk level comprises a higher dosage or more frequent administration of chemotherapy or radiation therapy than treatment for a low risk level.

7. The method of any one of claims 4-6, wherein treatment for a moderate or high risk level comprises administering an miR-155 inhibitor to the subject.

8. The method of claim 7, wherein the miR-155 inhibitor comprises an antibody, an antibody fragment, a small molecule inhibitor, a polypeptide, an oligonucleotide, or any combination thereof.

9. The method of any one of claims 7-8, wherein the miR-155 inhibitor comprises cobomarsen or an anti-miR-155 oligonucleotide.

10. The method of any one of claims 7-9, wherein treatment for a moderate or high risk level further comprises, only after administration of the miR-155 inhibitor, administering an immune cell therapy and / or one or more checkpoint inhibitors to the subject.

11. The method of claim 10, wherein the subject does not receive immune cell therapy and / or one or more checkpoint inhibitors prior to administration of the miR-155 inhibitor.

12. The method of any one of claims 4-11, wherein treatment for a moderate or high risk level comprises administering to the subject a first synthetic nucleic acid sequence encoding inducible T-cell costimulatory ligand (ICOSL) or a functional fragment thereof and / or a second synthetic nucleic acid sequence encoding RELA or a functional fragment thereof.

13. The method of claim 12, wherein the first synthetic nucleic acid sequence has a mutated, partially deleted, or fully deleted 3’-UTR region compared to a wildtype nucleic acid sequence encoding ICOSL.

14. The method of claim 13, wherein the 3’-UTR region of the wildtype nucleic acid sequence comprises an miR-155 target site of SEQ ID NO: 9 (AGCATTA), and wherein the 3’-UTR region of the first synthetic nucleic acid sequence, if present, does not include SEQ ID NO: 9.

15. The method of claim 14, wherein the miR-155 target site in the 3’-UTR region of the first synthetic nucleic acid sequence is partially or completely replaced with a restriction site for a restriction enzyme.

16. The method of claim 15, wherein the miR-155 target site in the 3’-UTR region of the first synthetic nucleic acid sequence is partially or completely replaced by a restriction site for EcoRl17. The method of claim 16, wherein the miR-155 target site in the 3’-UTR region of the first synthetic nucleic acid sequence is partially or completely replaced by SEQ ID NO: 10 (GAATTC).

18. The method of any one of claims 12-17, wherein the first synthetic nucleic acid sequence comprises about 80% similarity or more to SEQ ID NO: 5.

19. The method of any one of claims 12-18, wherein treatment for a moderate or high risk level further comprises administering one or more chemokines or cytokines to the subject.

20. The method of claim 19, wherein the one or more chemokines or cytokines increase expression of ICOSL or the functional fragment thereof.

21. The method of any one of claims 19-20, wherein the one or more chemokines or cytokines are administered at the same time as or after administration of the first synthetic nucleic acid sequence.

22. The method of any one of claims 19-21, wherein the one or more chemokines or cytokines comprise INF-y, TNF-a, and / or LPS.

23. The method of any one of claims 12-22, wherein the second synthetic nucleic acid sequence has a mutated or at least partially deleted 3’-UTR region compared to a wildtype nucleic acid sequence encoding RELA.

24. The method of claim 23, wherein the 3’-UTR region of the wildtype nucleic acid sequence comprises an miR-155 target site of SEQ ID NO: 9 (AGCATTA), and wherein the 3’-UTR region of the second synthetic nucleic acid sequence, if present, does not include SEQ ID NO: 9.

25. The method of any one of claims 12-24, wherein the second synthetic nucleic acid sequence comprises about 80% similarity or more to SEQ ID NO: 11.

26. The method of any one of claims 12-25, wherein treatment for a moderate or high risk level further comprises administering one or more enzymes or nucleic acid sequencesencoding enzymes for post-translational phosphorylation and / or acetylation of RELA or the functional fragment thereof.

27. The method of claim 26, wherein the one or more enzymes or nucleic acid sequences encoding enzymes increase activation of NFKB.

28. The method of any one of claims 26-27, wherein the one or more enzymes or nucleic acid sequences encoding enzymes are administered at the same time as or after administration of the second synthetic nucleic acid sequence.

29. The method of any one of claims 12-28, wherein the first synthetic nucleic acid sequence and / or second synthetic nucleic acid sequence is provided as a vector.

30. The method of any one of claims 12-29, wherein the first synthetic nucleic acid sequence and / or second synthetic nucleic acid sequence is delivered via a lipid nanoparticle, viral particle, or any other suitable carrier.

31. The method of any one of claims 12-30, wherein treatment for a moderate or high risk level further comprises, only after administration of the first synthetic nucleic acid sequence and / or second synthetic nucleic acid sequence, administering an immune cell therapy and / or one or more checkpoint inhibitors to the subject.

32. The method of claim 31, wherein the subject does not receive immune cell therapy and / or one or more checkpoint inhibitors prior to administration of the first synthetic nucleic acid sequence and / or second synthetic nucleic acid sequence.

33. The method of any one of claims 1-32, further comprising, before step a), screening the subject for Epstein-Barr virus (EBV).

34. The method of claim 33, wherein current or previous infection with EBV is further used to calculate the risk score in step c).

35. The method of any one of claims 1-34, wherein step b) further comprises determining if ICOSL, RELA, and / or MHC-I are underexpressed in cancerous cells derived from the subject compared to ICOSL, RELA, and / or MHC-I expression on control cells.

36. The method of claim 35, wherein underexpression of ICOSL, RELA, and / or MHC-I is further used to calculate the risk score in step c).

37. The method of any one of claims 1-36, wherein the sample comprises a cell sample, a tissue sample, a bodily fluid sample, or a cell lysate sample.

38. The method of claim 37, wherein the sample is a tumor sample.

39. The method of any one of claims 1-38, wherein the solid cancer is breast cancer, lung cancer, pancreatic cancer, gastric cancer, or colon cancer.

40. A method of treating a cancer in a subject in need thereof, the method comprising administering to the subject a synthetic nucleic acid sequence encoding inducible T-cell costimulatory ligand (ICOSL) or a functional fragment thereof, wherein expression and presentation of ICOSL or the functional fragment thereof by a cancerous cell induces and / or enhances an immune response against the cancer.

41. The method of claim 40, wherein expression and presentation of ICOSL or the functional fragment thereof by the cancerous cell induces and / or enhances killing of the cancerous cell by cytotoxic T cells.

42. The method of any one of claims 40-41, wherein the synthetic nucleic acid sequence has a mutated, partially deleted, or fully deleted 3’-UTR region compared to a wildtype nucleic acid sequence encoding ICOSL.

43. The method of claim 42, wherein the 3’-UTR region of the wildtype nucleic acid sequence comprises an miR-155 target site of SEQ ID NO: 9 (AGCATTA), and wherein the 3’-UTR region of the synthetic nucleic acid sequence, if present, does not include SEQ ID NO: 9.

44. The method of claim 43, wherein the miR-155 target site in the 3’-UTR region of the synthetic nucleic acid sequence is partially or completely replaced with a restriction site for a restriction enzyme.

45. The method of claim 44, wherein the miR-155 target site in the 3’-UTR region of the synthetic nucleic acid sequence is partially or completely replaced by a restriction site for EcoRl46. The method of claim 45, wherein the miR-155 target site in the 3’-UTR region of the synthetic nucleic acid sequence is partially or completely replaced by SEQ ID NO: 10 (GAATTC).

47. The method of any one of claims 40-46, wherein the synthetic nucleic acid sequence comprises about 80% similarity or more to SEQ ID NO: 5.

48. The method of any one of claims 40-47, wherein the synthetic nucleic acid sequence is provided as a vector.

49. The method of any one of claims 40-48, wherein the synthetic nucleic acid sequence is delivered via a lipid nanoparticle, viral particle, or any other suitable carrier.

50. The method of any one of claims 40-49, further comprising providing an miR-155 inhibitor to the subject.

51. The method of claim 50, wherein the miR-155 inhibitor comprises an antibody, an antibody fragment, a small molecule inhibitor, a polypeptide, an oligonucleotide, or any combination thereof.

52. The method of any one of claims 50-51, wherein the miR-155 inhibitor comprises cobomarsen or an anti-miR-155 oligonucleotide.

53. The method of any one of claims 40-52, further comprising administering one or more chemokines or cytokines to the subject.

54. The method of claim 53, wherein the one or more chemokines or cytokines increase expression of ICOSL or the functional fragment thereof.

55. The method of any one of claims 53-54, wherein the one or more chemokines or cytokines are administered at the same time as or after administration of the synthetic nucleic acid sequence.

56. The method of any one of claims 53-55, wherein the one or more chemokines or cytokines comprise INF-y, TNF-a, and / or LPS.

57. The method of any one of claims 40-56, further comprising, after administering the synthetic nucleic acid sequence, administering an immune cell therapy and / or one or more checkpoint inhibitors to the subject.

58. The method of claim 57, wherein the subject has not received immune cell therapy and / or one or more checkpoint inhibitors before administration of the synthetic nucleic acid sequence.

59. The method of any one of claims 40-58, wherein the cancer is lymphoma, breast cancer, lung cancer, pancreatic cancer, gastric cancer, or colon cancer.

60. The method of any one of claims 40-59, further comprising, before administration of the synthetic nucleic acid sequence, screening the subject for Epstein-Barr virus (EBV).

61. The method of claim 60, wherein the subject is currently or has previously been infected with EBV.

62. A method of treating a cancer in a subject in need thereof, the method comprising administering to the subject a synthetic nucleic acid sequence encoding RELA or a functional fragment thereof, wherein expression of RELA or the functional fragment thereof by a cancerous cell induces and / or enhances expression of major histocompatibility complex class I (MHC-I), and wherein presentation of MHC-I by the cancerous cell induces and / or enhances an immune response against the cancer.

63. The method of claim 62, wherein presentation of MHC-I by the cancerous cell induces and / or enhances killing of the cancerous cell by cytotoxic T cells.

64. The method of any one of claims 62-63, wherein the synthetic nucleic acid sequence has a mutated or at least partially deleted 3’-UTR region compared to a wildtype nucleic acid sequence encoding RELA.

65. The method of claim 64, wherein the 3’-UTR region of the wildtype nucleic acid sequence comprises an miR-155 target site of SEQ ID NO: 9 (AGCATTA), and wherein the 3’-UTR region of the synthetic nucleic acid sequence, if present, does not include SEQ ID NO: 9.

66. The method of any one of claims 62-65, wherein the synthetic nucleic acid sequence comprises about 80% similarity or more to SEQ ID NO: 11.

67. The method of any one of claims 62-66, wherein the synthetic nucleic acid sequence is provided as a vector.

68. The method of any one of claims 62-67, wherein the synthetic nucleic acid sequence is delivered via a lipid nanoparticle, viral particle, or any other suitable carrier.

69. The method of any one of claims 62-68, further comprising providing an miR-155 inhibitor to the subject.

70. The method of claim 69, wherein the miR-155 inhibitor comprises an antibody, an antibody fragment, a small molecule inhibitor, a polypeptide, an oligonucleotide, or any combination thereof.

71. The method of any one of claims 69-70, wherein the miR-155 inhibitor comprises cobomarsen or an anti-miR-155 oligonucleotide.

72. The method of any one of claims 62-71, further comprising administering one or more enzymes or nucleic acid sequences encoding enzymes for post-translational phosphorylation and / or acetylation of RELA or the functional fragment thereof.

73. The method of claim 72, wherein the one or more enzymes or nucleic acid sequences encoding enzymes increase activation of NFKB.

74. The method of any one of claims 72-73, wherein the one or more enzymes or nucleic acid sequences encoding enzymes are administered at the same time as or after administration of the synthetic nucleic acid sequence.

75. The method of any one of claims 62-74, further comprising, after administering the synthetic nucleic acid sequence, administering an immune cell therapy and / or one or more checkpoint inhibitors to the subject.

76. The method of claim 75, wherein the subject has not received immune cell therapy and / or one or more checkpoint inhibitors before administration of the synthetic nucleic acid sequence.

77. The method of any one of claims 62-76, wherein the cancer is lymphoma, breast cancer, lung cancer, pancreatic cancer, gastric cancer, or colon cancer.

78. The method of any one of claims 62-77, further comprising, before administration of the synthetic nucleic acid sequence, screening the subject for Epstein-Barr virus (EBV).

79. The method of claim 78, wherein the subject is currently or has previously been infected with EBV.

80. A method of treating a cancer in a subject in need thereof, the method comprising: a) administering a first synthetic nucleic acid sequence encoding ICOSL or a functional fragment thereof according to the method of any one of claims 40-61; and b) administering a second synthetic nucleic acid sequence encoding RELA or a functional fragment thereof according to the method of any one of claims 62-79; wherein expression of RELA or the functional fragment thereof by a cancerous cell induces and / or enhances expression of major histocompatibility complex class I (MHC-I); andwherein expression and presentation of ICOSL or the functional fragment thereof and MHC-I by the cancerous cell induces and / or enhances an immune response against the cancer.

81. The method of claim 80, wherein expression and presentation of ICOSL or the functional fragment thereof and MHC-I by the cancerous cell induces and / or enhances killing of cancerous cells by cytotoxic T cells.

82. The method of any one of claims 80-81, wherein steps a) and b) are performed simultaneously.

83. The method of any one of claims 80-81, wherein step a) is performed before or after step b).

84. The method of any one of claims 80-83, further comprising providing an miR-155 inhibitor to the subject.

85. The method of claim 84, wherein the miR-155 inhibitor comprises an antibody, an antibody fragment, a small molecule inhibitor, a polypeptide, an oligonucleotide, or any combination thereof.

86. The method of any one of claims 84-85, wherein the miR-155 inhibitor comprises cobomarsen or an anti-miR-155 oligonucleotide.

87. The method of any one of claims 80-86, further comprising, after administering the first and second synthetic nucleic acid sequences, administering an immune cell therapy and / or one or more checkpoint inhibitors to the subject.

88. The method of claim 87, wherein the subject has not received immune cell therapy and / or one or more checkpoint inhibitors before administration of the synthetic nucleic acid sequence.

89. The method of any one of claims 80-88, wherein the cancer is lymphoma, breast cancer, lung cancer, pancreatic cancer, gastric cancer, or colon cancer.

90. The method of any one of claims 80-89, further comprising, before administration of the synthetic nucleic acid sequence, screening the subject for Epstein-Barr virus (EBV).

91. The method of claim 90, wherein the subject is currently or has previously been infected with EBV.

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