Oncolytic herpes simplex virus (OHSV) prognostic biomarkers and combination therapy

Abstract

Disclosed herein is a method for predicting prognosis of a subject being with a cancer, such as a malignant glioma, being treated with an oncolytic herpes simplex virus (oHSV) that involves assaying a sample from the subject for Acyl-CoA-binding protein (ACBP) levels, wherein an elevated level of ACBP compared to a control is an indication of a poor prognosis. Also disclosed herein is a method for treating a cancer, such as a malignant glioma, in a subject that involves administering to the subject an effective amount of an oncolytic herpes simplex virus (oHSV) in combination with a glioma Bmi1 inhibitor or a fatty acid oxidation (FAO) inhibitor optionally followed by one or more immune checkpoint inhibitors.

Description

TH Docket No. 222120-2150ONCOLYTIC HERPES SIMPLEX VIRUS (OHSV) PROGNOSTIC BIOMARKERS AND COMBINATION THERAPY CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of U. S. Provisional Application No.63 / 738,617 filed on December 24, 2024 and U. S. Provisional Application No. 63 / 915,203 filed on November 11, 2025, each of which is incorporated herein by reference in its entirety.STATEMENT OF GOVERNMENT INTEREST

[0002] This invention was made with Government Support under Grant No. HT9425-23-1-0792 awarded by the Department of Defense. The Government has certain rights in the invention.BACKGROUND OF THE INVENTION

[0003] Malignant glioma (MG) remains an intractable problem with uniformly fatal outcomes in patients. With standard therapy, prognosis is still unfavorable with a median survival of less than 2 years. Clearly, there is an unmet need for the development of novel agents that can suppress MG growth and recurrence.SUMMARY OF THE INVENTION

[0004] Disclosed herein is a method for predicting prognosis of a subject being with a cancer, such as a malignant glioma, being treated with an oncolytic herpes simplex virus (oHSV) that involves assaying sample from the subject, including but not limited to a blood sample, a serum sample, or a tumor sample following biopsy or resection, for Acyl-CoA-binding protein (ACBP) levels, wherein an elevated level of ACBP compared to a control is an indication of a poor prognosis.

[0005] Also disclosed herein is a method for treating a cancer, such as a malignant glioma, in a subject that involves administering to the subject an effective amount of an oncolytic herpes simplex virus (oHSV) in combination with a glioma Bmi1 inhibitor or a fatty acid oxidation (FAO) inhibitor, optionally followed by one or more immune checkpoint inhibitors (ICI).

[0006] Also disclosed herein is a method for treating a cancer, such as a malignant glioma, in a subject being treated with an oHSV that involves detecting in a blood, serum, or tumor sample from the subject elevated levels of ACBP compared to a control, and administering to the subject an effective amount of a glioma Bmi1 inhibitor or an FAO inhibitor optionally followed by one or more immune checkpoint inhibitors (ICI).

[0007] In some embodiments, the oHSV comprises a nucleic acid sequence that encodes for mouse or human interleukin 12 (IL-12). In some embodiments, the glioma Bmi1 inhibitor is PTC596 (PTC). In some embodiments, the glioma FAO inhibitor is Etomoxir (Eto). In some embodiments, the immune checkpoint inhibitors can be anti-PD-1, or anti-CTLA-4,TH Docket No. 222120-2150or anti-PD-L1, or anti-Lag3, or anti-Tim3, or anti-TIG IT, other agents that lead to immune cell activation, or a combination thereof. In some embodiments, the malignant glioma is glioblastoma multiforme (GBM). In some embodiments, the cancer is a breast cancer, or a melanoma (skin cancer), or any solid tumor. In some embodiments, the cancer is a metastasized tumor.

[0008] This combined strategy optimizes the administering schedule of clinically relevant agents to maximize the therapeutic efficacy, which is distinct from most of current oncolytic virus-based combined therapies entailing concurrently administered agents, thereby transforming cancer treatment in the field of oncolytic virus therapy of MG toward a new direction.

[0009] The details of one or more embodiments of the invention are set forth in the accompanying drawings and the description below. Other features, objects, and advantages of the invention will be apparent from the description and drawings, and from the claims. BRIEF DESCRIPTION OF FIGURES

[0010] FIG. 1 shows M002 treatment prolonged mice survival. C57BL / 6J (B6) mice (n=10) were intracranially injected with GSC005 (5 x 104) at day 0, and then injected with saline (S) or M002 (M) at day 11. Median survival (S=53d; M=70d). Log-rank test, **P = 0.0035.

[0011] FIG. 2 shows a proposed model where oHSV therapy combined with glioma Bmi1 inhibition restricts refractory tumor cells and promotes the TME remodeling by modulating the glioma secretome, leading to improved therapeutic efficacy.

[0012] FIG. 3A shows a comparison of each marker in GSC005 and PN20 cells cultured under neurosphere condition. FIG. 3B shows confirmation of ACBP knockout (KO) in GSC005 cells. The mean fluorescence intensity (MFI) of ACBP is indicated. Iso Ctrl: isotype control. FIG. 3C shows 1:1 mixed culture of EGFP-GSC005 and EBFP-PN cells. FIG. 3D shows cell counts and percent viable cells of GSC005 and PN20 cells at day 2 after infected with or without (-) M002 (M) at MOI 1.5. ***P < 0.001 and ****P < 0.0001 (3D, unpaired 2-tailed t-test). Bars, mean ± SEM.

[0013] FIGs. 4A and 4B show expression of CD44, Bmi1 and ICOSL in GSC005 cells (FIG. 4A) or expression of CD44 and CD24 in PN20 cells (FIG. 4B) after 2 days of culture with Control (C) or MOORedtreated GSC-CM (M). Percent CD44+Bmi1+ and CD44+ICOSL+ cells in (4A) and CD44 MFI in (4B). **P < 0.01 (unpaired 2-tailed t-test). Bars, mean ± SEM.

[0014] FIGs. 5A and 5B show ACBP was highly expressed in conditioned medium (CM) collected from various cells 2 days post-oHSV treatment. FIG. 5A shows mass spectrometry (MS) results with numbers indicating fold changes in GSC005. FIG. 5B shows ELISA analysis of ACBP in CM (C, Ctrl; M, M002-treated) from indicated cells inTH Docket No. 222120-2150duplicates. *P < 0.05, **P < 0.01 and ***P < 0.001 (FIG. 5B, unpaired 2-tailed t-test). Bars, mean ± SEM.

[0015] FIG. 6A shows REMBRANDT dataset: High expression of ACBPwas correlated with poor survival of patients with brain tumors. FIG. 6B shows IvyGAP database: Core tumors had higher levels of ACBP compared to those at the edge.

[0016] FIGs. 7A and 7B show B6 mice implanted with GSC005 and treated with saline or M002, as in FIG. 1. FIG. 7A shows tumors harvested at day 58 post-implantation (day 47 posttreatment). Sections were stained with ACBP antibody and visualized with Alexa Fluor 488 secondary antibody. Nuclei were counterstained with DAPI. Normal brain section as controls. FIG. 7B left, shows serum ACBP levels (Saline: n=3-4; M002: n=3-6). FIG. 7B right, shows frequency of CD44+CD45~CD11 b“ tumor cells. *P < 0.05 and **P < 0.01 (Two-way ANOVA with Tukey’s multiple comparisons test). Bars, mean ± SEM.

[0017] FIG. 8A shows analysis of neurosphere formation of GSC005 treated with Control (C) or M002 GSC-CM (M) in the presence of anti-ACBP or its isotype (Iso) control antibody (Ab) (0.5 pg / ml) under extreme limiting dilution assay conditions in complete stem cell media for 7 days. The corresponding dash lines indicate the ranges of each condition. ***P < 0.001 (b vs a), ****p < 0.0001 (c vs a; c vs b; c vs d), *P < 0.05 (d vs a) and NS (no significance) (b vs d) (Chisq test). FIG. 8B shows B6 mice implanted with 105mixed (1:1) PN20 and GSC005 cells that express doxycycline (DOX)-inducible ACBP knockdown (shiACBP ) or control (shCtrl), and were treated with saline and M002 at day 11. DOX was given to mice at days 5, 7 and 9 post-implantation. Kaplan Meier survival curve (n=8-10) with log-rank test. *P< 0.05, shCtrl+Saline vs shCtrl+M002; NS, shCtrl+Saline vs shiACBP+Saline; *P< 0.05, shCtrl+M002 vs shiACBP+M002; **P< 0.01, shiACBP+saline vs shiACBP+M002. FIG. 8C show B6 mice implanted with 1.5x105mixed (1:2) GSC005 (ACBP WT or KO) and PN20 cells followed by treatment with saline or M002 at day 11. Kaplan Meier survival curve (n=8-9) with log-rank test. **P < 0.01, ACBP WT+Saline vs ACBP WT+M002; *P < 0.05, ACBP KO+Saline vs ACBP KO+Saline; *P < 0.05, ACBP WT+M002 vs ACBP KO+M002; *P < 0.05, ACBP KO+Saline vs ACBP KO+M002. M, median survival.

[0018] FIG. 9 shows live CD45_CD11 c_CD11 b" F4 / 80“ tumor cells were concatenated after downsampling to 100,000 events for subsequent high-dimensional data analysis by FlowJo t-SNE to normalize the contribution among brain tumors from mice treated with saline or M002 (day 47 post-treatment).

[0019] FIG. 10A shows live CD45+cells were concatenated after downsampling to 100,000 events for subsequent high-dimensional data analysis to normalize the contribution among brain tumor samples, as in FIG. 9. Manually gated Treg (circles) and microglial (irregular shapes) populations were overlaid onto the total t-SNE map. FIG. 10B showsTH Docket No. 222120-2150expression of intracellular iNOS in CD11 b+F4 / 80+(left) and surface PD-L1 on CD11 b+iNOS+(right) microglial cells at day 2 after treatment with GSC-CM (C, control; M, M002-treated). U, unstained. FIG. 10C shows naive CD4+T-cells cultured with TGFp and anti-CD3 / CD28 antibodies for 5 days to differentiate into Tregs. Tregs were then treated with Control (C) or M002 GSC-CM (M) for 24 hours with or without adding 50 pM Etoxomir (Eto) for the last 12 hours prior to mitochondrial stress test. OCR was performed after immobilizing cells on the assay plate using Cell-Tak cell attachment solution. Results are expressed as mean ± SEM.

[0020] FIG. 11 A shows B6 mice intracranially injected with 1,5X105GSC005 cells with lentiviral shRNA control (shCtrl) or shRNA knockdown of Bmi1 (shBmil) and survival was monitored. Kaplan Meier survival curve (n=10) with log-rank test (**P < 0.01). M, median survival. Experiments end at day 65. FIGs. 11B-11C show live CD45“CD11c_CD11b“F4 / 80“ cells (FIG. 11 B) or CD45+cells (FIG. 11 C) were concatenated after downsampling to ~15,000 events for subsequent high-dimensional data analysis to normalize the contribution among brain tumors from mice implanted with shCtrl or shBmil GSC005 cells (at day 86 post-implantation) in an experiment different from FIG. 11 A.Manually gated Treg and microglial populations were overlaid onto the total t-SNE map in FIG. 11C.

[0021] FIGs. 12A-12C show analysis of glioma Tregs and CD8+T-cells at the endpoint of mice as in FIG. 11B. Representative flow plots of Foxp3+CD4+Tregs (FIG. 12A), frequencies of Foxp3+Tregs, MFI of Foxp3 and CD25 (n=4) (FIG. 12B), and representative flow plots of IFNy, TNFa expression in Tregs and IFNy, granzyme B expression in CD8+T-cells (FIG. 12C). *P < 0.05 and ***p< 0.001 (12B, unpaired two-tailed Student’s t-test). Bars, mean ± SEM.

[0022] FIG. 13A shows OCR under mitochondrial stress test conditions and ECAR under glycolysis stress test conditions of cultured GSC005 cells (shCtrl vs shBmil). Results are normalized to the protein levels and expressed as mean ± SEM. FIG. 13B shows GSC005 cell media collected from 3-day culture and subject to MS analysis. Candidate proteins related to metabolism are listed. FC, fold change (shBmil vs shCtrl). FIG. 13C shows intracellular expression of FABP5 and ACBP in GSC005 cells. FIG. 13D shows GBM patient dataset analysis (GlioVis) revealed the correlations of expression of BMI1 and ACBP (TCGA) or FABP5 (REMBRANDT). Spearman’s rank correlation 2-sided.

[0023] FIG. 14A shows Treg cells were FACS-sorted from Foxp3-GFP mice and cultured with anti-CD3, anti-CD28 plus IL-2 in the presence or absence of recombinant FABP5 (500 ng / ml) for 3 days. Intracellular cytokines were analyzed. FIG. 14B shows TCGA analysis of GBM patients expressing FABP5hiversus FABP510. GO analysis revealed differentially expressed pathways. FIG. 14C shows combined knockdown of Bmi1 andTH Docket No. 222120-2150FABP5 in GSC005 cells (1.5*105injected) impaired mice survival. Kaplan Meier survival curve (n=9-10) with log-rank test. ***p< 0.001, shCtrl vs shBmil; NS, shCtrl vs shFABP5; **P < 0.01, shBmil vs shBmi1 / shFABP5; NS, shFABP5 vs shBmi1 / shFABP5. M, median survival.

[0024] FIGs. 15A-15B show ACBP from M002-treated conditioned medium upregulated MES markers and altered mitochondrial potential. FIG. 15A shows PN20 cells were cultured with control (Ctrl) CM or CM collected from GSC005 WT or ACBP KO cells after 2 days of infection with M002. At 48 hr of culture, markers associated with mesenchymal (MES) phenotype (CD44, ICOSL, Bmi-1), proliferation (Ki-67) and mitochondrial membrane potential state (TMRM) were analyzed via flow cytometry. The mean fluorescent intensity (MFI) of each marker was indicated. FIG. 15B shows ACBP levels in each CM in triplicates were analyzed by ELISA. **P< 0.01, ***p< 0.001 and ****P < 0.0001 (One-way ANOVA with Tukey’s multiple comparisons test). Bars, mean ± SEM.

[0025] FIG. 16 shows rACBP increased the expression of CD44 and the mesenchymal phenotype of human T98G glioma cells. T98G cells were starved in FBS free media (DMEM + 1mM sodium pyruvate) for 6 h. Human recombinant ACBP (rACBP) at indicated doses was added to the cells for 48 hr in DMEM+10%FBS in triplicates prior to flow cytometry analysis. Representative histogram overlays of CD44 with indicated MFI for each condition (left) and quantitation (right) are shown. *P< 0.05 and **P< 0.01 (One-way ANOVA with Tukey’s multiple comparisons test). Bars, mean ± SEM.

[0026] FIG. 17 shows ACBP deletion in GSC005 cells reduced the mesenchymal phenotype of glioma cells. B6 mice (n = 3-4) were implanted with 105GSC005 WT or ACBP KO cells followed by treatment with saline or M002 at day 11. Brain tumor was isolated at day 24 post-treatment for flow cytometry analysis. Representative plots (left) and percent of CD44+Bmi-1+tumor cells (live CD45-) (right) are shown. **P< 0.01 and ***p< 0.001 (Oneway ANOVA with Tukey’s multiple comparisons test). Bars, mean ± SEM.

[0027] FIGs. 18A-18D show single-cell RNA sequencing (scRNA-seq) analysis of the tumor and the TME. B6 mice were implanted with 1.5*105mixed (1:2) GSC005 (ACBP WT or KO) and PN20 cells at day 0 followed by treatment with saline or M002 at day 11. CD45+and CD45~ cells were sorted from tumors at day 45 post-treatment and subject to scRNA-seq analysis. FIG. 18A shows sub-clustering of all cells presented by the UMAP plot. FIG.18B heatmap shows each cell cluster identified based on mean expression of indicated marker genes, as revealed from A. FIG. 18C shows sub-clustering of all cell types after cluster grouping based on A, B. FIG. 18D stacked bar plots show relative proportions of all cell types across groups.

[0028] FIGs. 19A-19C show the absence of tumor-secreted ACBP impacts the GBM heterogeneity. FIG. 19A shows sub-clustering (left) and relative proportions (right) of tumorTH Docket No. 222120-2150cells as shown in FIG. 18C. FIGs. 19B-19C show four cellular states all groups combined or separately depicted on the UMAP plot.

[0029] FIGs. 20A-20C show the tumor-secreted ACBP promotes MES states and reduces NPC / OPC states of GBM. Neftel Classification of Glioma Cellular States for tumor clusters, as in FIGs. 19A-19C, for indicated treatment groups. FIG. 20A shows violin plots depicting the distribution of module scores for each cellular state-associated geneset. The bar plots show the means and SEM for each group. FIG. 20B shows two-dimensional representation (left) and relative proportions (right) of cellular states. Each quadrant corresponds to one cellular state for individual cells (dots). FIG. 20C shows sankey plot of cell states relative to the tumor population in each group.

[0030] FIGs. 21A-21B show pathway analysis of each cluster of tumor cells. Dot plots in FIG. 21A show the relatively enriched pathways in each cluster of tumor cells. FIG.21 B shows the relatively enriched pathways in clusters 0, 2 and 4.

[0031] FIGs. 22A-22B show analysis of microglia / macrophage clusters. FIG. 22A shows UMAP plots depicting myeloid and microglia clusters. FIG. 22B shows UMAP (left) and relative proportions (right) of myeloid and microglia populations.

[0032] FIGs. 23A-23C show scRNA-seq analysis of myeloid cells. FIG. 23A-C show myeloid clusters (FIG. 23A), proportion (FIG. 23B), and representative genes (FIG. 23C) in each cluster / population.

[0033] FIGs. 24A-24D show scRNA-seq analysis of microglia. FIG. 24A-24D show microglial clusters (FIG. 24A), proportion (FIG. 24B), representative genes (FIG. 24C) in each cluster / population, and UMAP plot at each condition (FIG. 24D).

[0034] FIG. 25 shows scRNA-seq analysis of T-cell clusters. FIG. 25 shows UMAP (left) and relative proportions (right) of T-cell populations.

[0035] FIGs. 26A-26C show scRNA-seq analysis of Treg cells. T-cells expressing Foxp3 > 1 were further subclustered from FIG. 25. FIGs. 26A-26C show clusters (FIG. 26A), proportion (FIG. 26B), and representative genes (FIG. 26C) in each cluster / population. FIG.26D shows B6 mice (n = 4) were implanted with 105GSC005 WT or ACBP KO cells followed by treatment with saline or M002 at day 11. Brain tumor was isolated at day 24 posttreatment for flow cytometry analysis of Treg cells and quantitation of Foxp3+Treg frequency is shown. *P < 0.05 and **P < 0.01 (One-way ANOVA with Tukey’s multiple comparisons test). Bars, mean ± SEM.

[0036] FIG. 27 shows combined M002 treatment with Bmi-1 knockdown in GSC005 significantly prolonged mice survival. B6 mice were implanted with 1.5xio5mixed (1:2) GSC005 and PN20 cells followed by treatment with saline or M002 at day 11. Kaplan Meier survival curve (n=10) with log-rank test. *P< 0.05, shCtrl +Saline vs shCtrl+M002; *P< 0.05,TH Docket No. 222120-2150shCtrl +Saline vs shBmil+Saline; *P< 0.05, shBmil+Saline vs shBmi1+M002; *P< 0.05, shCtrl+M002 vs shBmi1+M002. M, median survival.

[0037] FIG. 28 shows combined M002 and PTC596 treatment significantly prolonged mice survival. B6 mice were implanted with 5*104GSC005 cells followed by treatment with saline or M002 at day 11. 5 mg / kg PTC596 or vehicle (10% DMSO + 90% oil) was given twice a week via oral gavage starting day 25 post-tumor implantation for 15 days. Kaplan Meier survival curve (n=9-10) with log-rank test. **P< 0.01, Saline+Vehicle vs M002+Vehicle; **P < 0.01, M002+Vehicle vs M002+PTC; *P < 0.05, Saline+Vehicle vs Saline+PTC. M, median survival.

[0038] FIGs. 29A-29F show M002 treatment upregulates FAO and ACBP / Dbi in tumor cells. FIGs. 29A-29B show B6 mice were implanted with GSC005 WT and treated with saline or M002. scRNA-seq analysis of tumor cells and TME at day 45 post-treatment. Cell types (FIG. 29A) and proportion of each cell type (FIG. 28B). FIG. 29C shows GSEA analysis of top 6 enriched pathways in M002-treated versus saline-treated tumor cells. FIG.29D shows GSEA plot of FAO pathway in FIG. 29C. FIG. 29E shows expression levels of ACBP / Dbi in saline vs M002-treated tumor cells in FIG. 29A. FIG. 29F shows similar analysis was performed on days 3 and 10 post-treatment, and enriched FAO (M002 vs Saline) in tumor cells is shown.

[0039] FIG. 30 shows deletion of ACBP in GSC005 cells provides survival benefits upon M002 treatment, which is dependent of CD8+T-cells. B6 mice were implanted with 105GSC005 (ACBP WT or KO) cells and all were then treated with M002 at day 11. Anti-CD8 (aCD8) or its isotype (Iso) control antibody (300 pg) was administered on day -1 pretreatment and days 2 and 5 post-treatment. Kaplan Meier survival curve (n=8) with log-rank test. **P < 0.01, ACBP WT+lso vs ACBP KO+lso. NS, ACBP WT+lso vs ACBP WT+aCD8. *P < 0.05, ACBP KO+lso vs ACBP KO+aCD8. M, median survival.

[0040] FIGs. 31 A-31C show deletion of ACBP in tumor cells reshapes CD4+T-cells. FIGs. 31A-31C show B6 mice were implanted with GSC005 (ACBP WT or KO) cells and treated with saline or M002. scRNA-seq analysis of CD4+T-cells on day 45 post-treatment. Cell clusters (FIG. 31 A), proportion (FIG. 31 B) and representative genes (FIG. 31 C) in each cluster / population.

[0041] FIGs. 32A-32D show ablation of exhausted CD8+T-cells while expansion of Tpex in the TME of M002-treated ACBP KO tumor. scRNA-seq analysis of CD8+T-cells on day 45 post-treatment. Cell clusters (FIG. 32A), proportion (FIG. 32B), UMAP plot at each condition (FIG. 32C) and representative genes (FIG. 32D) in each cluster.

[0042] FIG. 33 shows reduced mesenchymal phenotype and proliferation of glioma cells treated with conditioned medium from etomoxir-treated cells. GSC005 cells were treated with Etomoxir (Eto, 5 pM) or vehicle (Ctrl) for 30 min followed by culture with GSCTH Docket No. 222120-2150medium for 2 days before collection of conditioned medium (CM). CM was then added into fresh GSC005 cells for 48 hr in triplicates before flow cytometry analysis of CD44 and Ki-67 (upper). Quantitation of MFI of each marker (bottom). **P< 0.01 and ***p< 0.001 (Unpaired Student’s t-test). Bars, mean ± SEM.

[0043] FIG. 34 shows condition medium collected from Etomoxir-treated GSC005 cells increased the expression of GzmB and Tcf1 but reduced PD-1 levels in CD8+T-cells. GSC005 cells were treated with Eto (5 pM) or vehicle (Veh) for 30 min followed by culture with GSC medium for 2 days before collection of conditioned medium (CM). CM was then added into CD8+T-cells during activation with anti-CD3 plus anti-CD28 for 3 days followed by flow cytometry analysis. The MFI of indicated markers is shown. * P < 0.05, and ** P < 0.01 (unpaired two-tailed Student’s t-test). Bars, mean ± SEM.

[0044] FIG. 35 shows a proposed model where oHSV therapy combined with glioma FAO inhibition followed by ICI administration restricts refractory tumor cells, remodels TME and boosts CD8+T-cell anti-GBM activity, leading to improved therapeutic efficacy. ACBP is closely linked to FAO. A second strategy by generating a new oncolytic virus carrying shRNA or gRNA / Cas9 to knockdown or knockout ACBP (marked by stars) may also provide survival benefit.DETAILED DESCRIPTION

[0045] Before the present disclosure is described in greater detail, it is to be understood that this disclosure is not limited to particular embodiments described, and as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present disclosure will be limited only by the appended claims.

[0046] Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limit of that range and any other stated or intervening value in that stated range, is encompassed within the disclosure. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges and are also encompassed within the disclosure, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the disclosure.

[0047] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present disclosure, the preferred methods and materials are now described.TH Docket No. 222120-2150

[0048] All publications and patents cited in this specification are herein incorporated by reference as if each individual publication or patent were specifically and individually indicated to be incorporated by reference and are incorporated herein by reference to disclose and describe the methods and / or materials in connection with which the publications are cited. The citation of any publication is for its disclosure prior to the filing date and should not be construed as an admission that the present disclosure is not entitled to antedate such publication by virtue of prior disclosure. Further, the dates of publication provided could be different from the actual publication dates that may need to be independently confirmed.

[0049] As will be apparent to those of skill in the art upon reading this disclosure, each of the individual embodiments described and illustrated herein has discrete components and features which may be readily separated from or combined with the features of any of the other several embodiments without departing from the scope or spirit of the present disclosure. Any recited method can be carried out in the order of events recited or in any other order that is logically possible.

[0050] Embodiments of the present disclosure will employ, unless otherwise indicated, techniques of chemistry, biology, and the like, which are within the skill of the art.

[0051] The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to perform the methods and use the probes disclosed and claimed herein. Efforts have been made to ensure accuracy with respect to numbers (e.g., amounts, temperature, etc.), but some errors and deviations should be accounted for. Unless indicated otherwise, parts are parts by weight, temperature is in °C, and pressure is at or near atmospheric. Standard temperature and pressure are defined as 20 °C and 1 atmosphere.

[0052] Before the embodiments of the present disclosure are described in detail, it is to be understood that, unless otherwise indicated, the present disclosure is not limited to particular materials, reagents, reaction materials, manufacturing processes, or the like, as such can vary. It is also to be understood that the terminology used herein is for purposes of describing particular embodiments only, and is not intended to be limiting. It is also possible in the present disclosure that steps can be executed in different sequence where this is logically possible.

[0053] It must be noted that, as used in the specification and the appended claims, the singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise.

[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. Thus, the subjectTH Docket No. 222120-2150can 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” 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 term “sample from a subject” refers to a tissue (e.g., tissue biopsy), organ, cell (including a cell maintained in culture), cell lysate (or lysate fraction), biomolecule derived from a cell or cellular material (e.g. a polypeptide or nucleic acid), or body fluid from a subject. Non-limiting examples of body fluids include blood, urine, plasma, serum, tears, lymph, bile, cerebrospinal fluid, interstitial fluid, aqueous or vitreous humor, colostrum, sputum, amniotic fluid, saliva, anal and vaginal secretions, perspiration, semen, transudate, exudate, and synovial fluid.

[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] As used herein, “cancer” or “tumor” as used interchangeably herein is meant to a group of diseases which can be treated according to the disclosure and involve abnormal cell growth with the potential to invade or spread to other parts of the body. Not all tumors are cancerous; benign tumors do not spread to other parts of the body. Possible signs and symptoms include: a new lump, abnormal bleeding, a prolonged cough, unexplained weight loss, and a change in bowel movements among others. There are over 100 different known cancers that affect humans. The present disclosure is preferably applicable to solid tumors. Non-limiting examples of cancer or tumor are bladder cancer, basal cell carcinoma, cholangiocarcinoma, colon cancer, endometrial cancer, esophageal cancer, Ewing's sarcoma, prostate cancer, gastric cancer, glioma, hepatocellular carcinoma, Hodgkin lymphoma, laryngeal carcinoma, liver cancer, lung cancer, melanoma, mesothelioma, pancreatic cancer, rectal cancer, renal cancer, thyroid cancer, malignantTH Docket No. 222120-2150peripheral nerve cell tumors, malignant peripheral nerve sheath tumors (MPNST), cutaneous and plexiform neurofibromas, leiomyoadenomatoid tumor, fibroids, uterine fibroids, leiomyosarcoma, papillary thyroid cancer, anaplastic thyroid cancer, medullary thyroid cancer, follicular thyroid cancer, hurthle cell carcinoma, thyroid cancer, ascites, malignant ascites, mesothelioma, salivary gland tumors, mucoepidermoid carcinoma of the salivary gland, acinic cell carcinoma of the salivary gland, gastrointestinal stromal tumors (GIST), tumors that cause effusions in potential spaces of the body, pleural effusions, pericardial effusions, peritoneal effusions aka ascites, giant cell tumors (GOT), GCT of bone, pigmented villonodular synovitis (PVNS), tenosynovial giant cell tumor (TGCT), TCGT of tendon sheath (TGCT-TS), and other sarcomas. In preferable embodiment, the present disclosure is used to treat esophageal cancer, lung cancer, prostate cancer, or bladder cancer.Oncolytic Herpes Simplex Virus

[0059] Several different oncolytic herpes simplex virus type 1 (HSV-1) strains have proven to be safe in phase I human clinical trials. See Aghi & Martuza, Oncogene (2005) 24:7802-7816. Viral genetic analysis has established that HSV-1 can be effectively neuroattenuated by deleting the y134.5 neuropathogenesis genes. Chou et, al., Science (1990) 250:1262-1266. The cellular interferon-induced elF2a kinase PKR, a major innate host defense component, phosphorylates the critical host cell translation initiation factor elF2a in response to viral infection. Phosphorylated elF2a blocks translation initiation thereby precluding the manufacturing of viral polypeptides and progeny. The y134.5 gene encodes a regulatory subunit of the cellular protein phosphatase 1 and directs dephosphorylation of elF2a which results in the production of viral proteins and progeny. Chou et al., Proc. Natl. Acad. Sci. USA (1995) 92:10516-10520; He et. al., Proc. Natl, Acad. Sci. USA (1997) 94:843-848.

[0060] The oHSV may be derived from a herpes simplex virus (HSV) strain. The HSV strain may be an HSV-1 or HSV-2 strain, or a derivative thereof. For example, a variant HSV of may be derived from a wild-type HSV-1, strain 17, having GenBank Accession No. X14112.

[0061] Derivatives of HSV include but are not limited to inter-type recombinants containing DNA from HSV-1 and HSV-2 strains. Such inter-type recombinants are described in the art, for example in Thompson et al., “DNA sequence and RNA transcription through a site of recombination in a non-neurovirulent herpes simplex virus intertypic recombinant,” Virus Genes, 1(3): 275-286, 1998; and Meignier et al., “In vivo behaviour of genetically engineered herpes simplex viruses R7017 and R7020: construction and evaluation in rodents,” J. Infect. Dis., 158(3): 602-614, 1988. Derivatives preferably have at least 70% sequence homology to either the HSV-1 or HSV-2 genome, more preferably at least 80%, even more preferably at least 90 or 95%. More preferably, a derivative has at least 70%TH Docket No. 222120-2150sequence identity to either the HSV-1 or HSV-2 genome, more preferably at least 80% identity, even more preferably at least 90%, 95% or 98% identity.

[0062] In some embodiments the oncolytic HSV is an HSV that is modified with respect to wild-type so as to inactivate at least one y134.5 gene copy, most preferably both copies of the y134.5 gene.

[0063] In some embodiments the oncolytic herpes simplex virus is a mutant of HSV-1 strain 17. In preferred embodiments the oncolytic herpes simplex virus is HSV1716 (ECACC Accession No. V92012803). HSV1716 is also called SEPREHVIR®. In some embodiments the herpes simplex virus is a mutant of HSV-1 strain 17 mutant 1716.

[0064] In some embodiments all copies of the ICP34.5 gene in the genome of the oncolytic herpes simplex virus are modified such that the ICP34.5 gene is incapable of expressing a functional ICP34.5 gene product. As such the oncolytic herpes simplex virus may be an ICP34.5 null mutant. In some embodiments one or both of the ICP34.5 genes in the genome of the oncolytic herpes simplex virus are modified such that the ICP34.5 gene is incapable of expressing a functional ICP34.5 gene product.

[0065] oHSV having an intact endogenous Us12 encoding gene and an intact endogenous Us11 encoding gene, lacking functional ICP34.5 encoding genes, wherein each ICP34.5 encoding gene is replaced by a polynucleotide cassette comprising: (a) a Us11 encoding gene operably associated with an immediate early (IE) promoter; and (b) at least one heterologous gene encoding a polypeptide capable of enhancing an anti-tumor response is provided in U. S. Patent No. 11,147,846, which is incorporated by reference in its entirety for the teaching of these oHSV and their uses.

[0066] A heterologous gene can encode an immunomodulatory polypeptide, such as one selected from the group consisting of a TAP 1 / 2 (“TAP”) inhibitor, granulocyte macrophage colony stimulating factor (GM-CSF), tumor necrosis factor (TNF)-alpha and CD40 ligand (CD40L). Other non-limiting examples of immunomodulatory polypeptides include for example, IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-b 11, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, IL-18, G-CSF, IFN-a, IFN-y, (MDA-7), and costimulator molecules such as B7-1 (CD80) and B7-2 (CD86).

[0067] The heterologous gene can also encode a prodrug converting enzyme. The heterologous gene can also encode an enzyme that degrades or modifies extra-cellular matrix components in order to facilitate viral spread through the tumor, for example, a matrix metalloproteinase. The heterologous gene can also encode the components that reduce or deplete certain factors of the tumor to inhibit tumor growth and / or progression.Bmi1 inhibitor

[0068] Bmi-1 inhibitors are described in U. S. Patent No. 10,370,371, which is incorporated by reference in its entirety for the teaching of these inhibitors.TH Docket No. 222120-2150

[0069] In some embodiments, the Bmi-1 inhibitor is a compound of Formula (I):R,or a form thereof, whereinX is N or N substituted with an oxygen atom substituent to form an N-oxide;Ri is bicyclic heteroaryl selected from the group consisting of 1 H-indolyl, 2H-indazolyl, 1H-benzimidazolyl, pyrazolo[1,5-a ]pyridinyl, pyrazolo[1,5-c]pyrimidinyl, 1H-imidazo[4,5-b ]pyridinyl, 1H-imidazo[4,5-c ]pyridinyl, imidazo[1,2-a]pyrazinyl, 7H-purinyl and quinolinyl, wherein the heteroatom is N, wherein the bicyclic heteroaryl is substituted on a carbon atom ring member with one, two, three or four R5substituents or on a nitrogen atom ring member with an oxygen atom substituent to form an N-oxide;R2is cyano, hydroxyl, nitro, C-|.8alkoxy, amino, Ci.3alkyl-amino, (Ci.3alkyl)2-amino, hydroxyl-amino, hydroxyl-Ci.8alkyl-amino, Ci-8alkoxy-Ci-8alkyl-amino, Ci8alkyl-thio, C-|_8alkyl-carbonyl, C-|.8alkyl-carbonyl-amino, amino-carbonyl, Ci3alkyl-amino-carbonyl, (Ci.8alkyl)2-amino-carbonyl, amino-carbonyl-amino, Ci.8alkyl-amino-carbonyl-amino, (Ci.8alkyl)2-amino-carbonyl-amino, C 1 _8a I koxy- carbonyl, Ci.8alkoxy-carbonyl-amino, amino-sulfonyl, C-|.8alkyl-amino-sulfonyl, (Ci.8alkyl)2-amino-sulfonyl, amino-sulfonyl-amino, C-i.8alkyl-amino-sulfonyl-amino, (Cq-salkyl^-amino-sulfonyl-amino, P(0)(R7)2-amino or heteroaryl, wherein heteroaryl is optionally substituted with one, two, three or four C-i.8alkyl substituents;R8is hydrogen, cyano, halo, C-i.8alkyl, amino, Ci.8alkyl-amino or (Ci.8alkyl)2-amino; R s Cs ^cycloalkyl, aryl, heteroaryl or heterocyclyl, each optionally substituted with one, two, three or four R8substituents;Rds independently selected from the group consisting of cyano, halo, hydroxyl, nitro, oxo, Ci-8alkyl, cyano-C-i.8alkyl, halo-Ci.8alkyl, hydroxyl-Ci.8alkyl, Ci.8alkoxy, Ci.8alkoxy-Ci.8alkyl, halo-Ci.8alkoxy, C2.8alkenyl, Ci.8alkoxy-C2.8alkenyl, C2.8alkynyl, Ci.8alkoxy-C2.8alkynyl, carboxyl, amino, C-|.8alkyl-amino, (Ci.8alkyl)2-amino, amino-Ci.8alkyl, Ci.8alkyl-amino-Ci.8alkyl, (Ci-8alkyl)2-amino-Ci-8alkyl, hydroxyl-Ci.8alkyl-amino, hydroxyl-Ci.8alkyl-amino-Ci.8alkyl, hydroxyl-Ci-8alkyl-amino-Ci-8alkyl-amino, C-i.8alkyl-thio, Ci.8alkyl-carbonyl, Ci.8alkyl-carbonyl-amino, Ci.8alkyl-carbonyl-oxy, Ci.8alkyl-carbonyl-oxy-Ci.8alkyl, Ci.8alkoxy-carbonyl, Ci-8alkoxy-carbonyl-Ci-8alkyl, Ci.8alkoxy-carbonyl-amino, Ci.8alkyl-sulfonyl, C3.i4cycloalkyl, aryl, aryl-Ci.8alkyl, aryl-amino, aryl-C-i.3alky-amino, heteroaryl, heteroaryl-Ci_3alkyl and heterocyclyl, wherein C3-i4cycloalkyl, aryl, heteroaryl or heterocyclyl and the aryl and heteroaryl portions of aryl-Ci_8alkyl, aryl-amino, aryl-Ci.8alky-amino and heteroaryl-C-i.8alkylTH Docket No. 222120-2150are each optionally substituted with one, two, three or four halo, Ci-8alkyl, halo-Ci.8alkyl, hydroxyl-Ci-8alkyl, Ci.8alkoxy, halo-C-i.8alkoxy, hydroxyl-Ci.8alkoxy or carboxyl substituents;R6is independently selected from the group consisting of cyano, halo, hydroxyl, nitro, Ci-8alkyl, halo-Ci-8alkyl, hydroxyl-C-i.8alkyl, C-i.8alkoxy, halo-C-i.8alkoxy, C2-8alkenyl, C-i.8alkoxy-C2-8alkenyl, C2-8alkynyl, Ci.8alkoxy-C2-8alkynyl, carboxyl, formyl, formyl-oxy, C-i.8alkyl-carbonyl, halo-Ci.8alkyl-carbonyl, C-i.8alkyl-thio, halo-C-i.8alkyl-thio, amino, C-i.8alkyl-amino, (Ci.8alkyl)2-amino, Ci.8alkyl-carbonyl, C-i.8alkyl-carbonyl-oxy, Ci.8alkyl-carbonyl-oxy-Ci.8alkyl, C-|.8alkoxy-carbonyl, halo-C-i.8alkoxy-carbonyl, Ci.8alkoxy-carbonyl-Ci.8alkyl, Ci.8alkoxy-carbonyl-amino, Ci.8alkoxy-carbonyl-amino-Ci.8alkyl, amino-carbonyl, Ci.8alkyl-amino-carbonyl, (Ci.8alkyl)2-amino-carbonyl, Ci.8alkyl-carbonyl-amino, Ci.8alkyl-carbonyl-amino-Ci.8alkyl, amino-Ci.8alkyl, Ci.8alkyl-amino-Ci.8alkyl, (Ci-8alkyl)2-amino-Ci-8alkyl, amino-Ci.8alkyl-amino, Ci.8alkyl-amino-Ci.8alkyl-amino, (Ci.8alkyl)2-amino-Ci.8alkyl-amino, hydroxyl-Ci.8alkyl-amino, hydroxyl-Ci.8alkyl-amino-Ci.8alkyl, hydroxyl-Ci.8alkyl-amino-Ci.8alkyl-amino, imino-C-i.8alkyl, hydroxyl-imino-Ci.8alkyl, Ci-8alkoxy-imino-Ci-8alkyl, Ci.8alkyl-sulfonyl, halo-C-i.8alkyl-sulfonyl, amino-sulfonyl, C-i.8alkyl-amino-sulfonyl, (Ci-8alkyl)2-amino-sulfonyl, B(OR8)2, C3. i4cycloalkyl, heterocyclyl, aryl and heteroaryl, wherein C3.i4cycloalkyl, heterocyclyl, aryl and heteroaryl are each optionally substituted with one, two, three or four halo or Ci.3alkyl substituents;R? is independently hydroxyl or (Ci.8alkoxy)n, wherein n represents an integer from 1 to 5; andR8is independently hydrogen or C-|.8alkyl;wherein the form of the compound is selected from the group consisting of a salt, hydrate, solvate, stereoisomer, racemate, enantiomer, diastereomer and tautomer thereof.

[0070] In some embodiments, the Bmi-1 inhibitor is selected from the group consisting of: N2-[4-(trifluoromethyl)phenyl]-6-(1,3, 5-tri methyl- 1 H-pyrazol-4-yl)pyrimidine-2,4-diamine, 6-(6-fluoro-2-methyl-1H-benzimidazol-1-yl)-N2-[4-(trifluoromethyl)phenyl]pyrimidine-2,4-diamine, N2-[4-(difluoromethoxy)phenyl]-6-(6-fluoro-2-methyl-1H-benzimidazol-1-yl)pyrimidine-2,4-diamine, 4-chloro-6-(2-methyl-1H-benzimidazol-1-yl)-N-[4-(trifluoromethyl)phenyl]pyrimidin-2-amine, 6-(2-methyl-1H-benzimidazol-1-yl)-N2-[4-(trifluoromethyl)phenyl]pyrimidine-2,4-diamine, 4-chloro-N-[3-fluoro-4-(trifluoromethyl)phenyl]-6-(2-methyl-1H-benzimidazol-1-yl)pyrimidin-2-amine, N2-[4-(difluoromethoxy)-3-fluorophenyl]-6-(6-fluoro-2-methyl-1H-benzimidazol-1-yl)pyrimidine-2,4-diamine, N2-[4-(difluoromethoxy)-3-fluorophenyl]-6-(2-ethyl-6-fluoro-1H-benzimidazol-1-yl)pyrimidine-2,4-diamine, 6-(2-cyclopropyl-6-fluoro-1H-benzimidazol-1-yl)-N2-[4-(difluoromethoxy)-3-fluorophenyl]pyrimidine-2,4-diamine, N2-[3-fluoro-4-(trifluoromethyl)phenyl]-6-(2-methyl-1H-benzimidazol-1-yl)pyrimidine-2,4-diamine, N2-[4-(difluoromethoxy)-3-fluorophenyl]-6-(2-methyl-1H-benzimidazol-1-yl)pyrimidine-2,4-diamine,TH Docket No. 222120-21502-{[6-(2-methyl-1 H-benzimidazol-1 -yl)-2-{[4-(trifluoromethyl)phenyl]amino}pyrimidin-4-yl]amino}ethanol, 2-{[2-{[3-fluoro-4-(trifluoromethyl)phenyl]amino}-6-(2-methyl-1H-benzimidazol-1-yl)pyrimidin-4-yl]amino}ethanol, 2-{[2-{[4-(difluoromethoxy)-3-fluorophenyl]amino}-6-(2-methyl-1H-benzimidazol-1-yl)pyrimidin-4-yl]amino}ethanol, 4-chloro-6-(5,6-difluoro-2-methyl-1 H-benzimidazol-1 -yl)-N-[4-(trifluoromethyl)phenyl]pyrimidin-2-amine, 4-chloro-6-(5,6-difluoro-2-methyl-1 H-benzimidazol-1 -yl)-N-[3-fluoro-4-(trifluoromethyl)phenyl]pyrimidin-2-amine, 6-(5,6-difluoro-2-methyl-1H-benzimidazol-1-yl)-N2-[4-(trifluoromethyl)phenyl]pyrimidine-2,4-diamine, 2-{[6-(5,6-difluoro-2-methyl-1H-benzimidazol-1-yl)-2-{[4-(trifluoromethyl)phenyl]amino}pyrimidin-4-yl]amino}ethanol, N4-hydroxy-6-(2-methyl-1H-benzimidazol-1-yl)-N2-[4-(trifluoromethyl)phenyl]pyrimidine-2,4-diamine, 6-(2-ethyl-1 H-benzimidazol-1 -yl)-N2-[4-(trifluoromethyl)phenyl]pyrimidine-2, 4-diamine, 6-(5,6-difluoro-2-methyl-1 H-benzimidazol-1 -yl)-N2-[3-fluoro-4-(trifluoromethyl)phenyl]pyrimidine-2,4-diamine, 6-(quinolin-4-yl)-N2-[4-(trifluoromethyl)phenyl]pyrimidine-2,4-diamine, 6-(2-ethyl-6-fluoro-1 H-benzimidazol-1 -yl)-N2-[4-(trifluoromethyl)phenyl]pyrimidine-2,4-diamine, 6-(2-cyclopropyl-6-fluoro-1H-benzimidazol- 1-yl)-N2-[4-(trifluoromethyl)phenyl]pyrimidine-2,4-diamine, 4-chloro-N-[4-(difluoromethoxy)-3-fluorophenyl]-6-(2-methyl-1 H-benzimidazol-1 -yl)pyrimidin-2-amine, 6-(6-chloro-2-methyl-1H-imidazo[4,5-b]pyridin-1-yl)-N2-[4-(trifluoromethyl)phenyl]pyrimidine-2,4-diamine, 6-(6-chloro- 2-ethyl-1H-imidazo [4,5-b]pyridin-1-yl)-N2-[4-(trifluoromethyl)phenyl]pyrimidine-2,4-diamine, 6-[2-(difluoromethyl)-6-fluoro-1 H-benzimidazol-1 -yl]-N2-[4-(trifluoromethyl)phenyl]pyrimidine-2,4-diamine, 6-(4,6-difluoro-2-methyl-1 H-benzimidazol-1 -yl)-N2-[4- (trifluoromethyl)phenyl]pyrimidine-2,4-diamine, 6-(4-fluoro-2-methyl-1 H-benzimidazol-1 -yl)-N2-[4-(trifluoromethyl)phenyl]pyrimidine-2,4-diamine, 4-chloro-6-(2-methyl-1H-benzimidazol- 1-yl)-N-[6-(trifluoromethyl)pyridin-3-yl]pyrimidin-2-amine, 6-(2-methyl-1 H-benzimidazol-1 -yl)-N2-[6-(trifluoromethyl)pyridin-3-yl]pyrimidine-2,4-diamine, 4-chloro-6-(6-fluoro-2-methyl-1H-benzimidazol-1-yl)-N-[6-(trifluoromethyl)pyridin-3-yl]pyrimidin-2-amine, 6-(5-fluoro-2-methyl-1H-benzimidazol-1-yl)-N2-[6-(trifluoromethyl)pyridin-3-yl]pyrimidine-2,4-diamine, 6-(6-fluoro- 2-methyl-1 H-benzimidazol-1 -yl)-N2-[6-(trifluoromethyl)pyridin-3-yl]pyrimidine-2,4-diamine, 6-(6-bromo-2-methyl-1H-benzimidazol-1-yl)-N2-[4-(trifluoromethyl)phenyl]pyrimidine-2,4-diamine, -6-(2,6-dimethyl-1 H-benzimidazol-1 -yl)-N2-[4-(trifluoromethyl)phenyl]pyrimidine-2, 4-diamine, 6-(2-ethyl-5-fluoro-1 H-benzimidazol-1 -yl)-N2-[6-(trifluoromethyl)pyridin-3-yl]pyrimidine-2,4-diamine, 6-(2-ethyl-6-fluoro-1 H-benzimidazol-1 -yl)-N2-[6-(trifluoromethyl)pyridin-3-yl]pyrimidine-2,4-diamine, 6-(6-chloro-2-methyl-1H-benzimidazol-1-yl)-N2-[4-(trifluoromethyl)phenyl]pyrimidine-2,4-diamine, 6-(6-chloro-2-methyl-1H-benzimidazol-1-yl)-2-{[4-(trifluoromethyl)phenyl]amino}pyrimidin-4-ol, 6-(5-chloro-2-methyl-1H-benzimidazol-1-yl)-N2-[4-(trifluoromethyl)phenyl]pyrimidine-2,4-diamine, N2-[4-(difluoromethoxy)phenyl]-6-(5,7-difluoro-2-methyl-1H-benzimidazol-1-yl)pyrimidine-2,4-TH Docket No. 222120-2150diamine, N2-[4-(difluoromethoxy)phenyl]-6-(4-fluoro-2-methyl-1H-benzimidazol-1-yl)pyrimidine-2,4-diamine, 6-(4-fluoro-2-methyl-1H-benzimidazol-1-yl)-N2-[3-methyl-4-(trifluoromethyl)phenyl]pyrimidine-2,4-diamine, 6-(2,6-dimethyl-1H-benzimidazol-1-yl)-N2-[3-methyl-4-(trifluoromethyl)phenyl]pyrimidine-2,4-diamine, N2-[4-(difluoromethoxy)phenyl]-6-(2,6-dimethyl-1H-benzimidazol-1-yl)pyrimidine-2,4-diamine, 5-fluoro-6-(2-methyl-1H-benzimidazol-1-yl)-N2-[4-(trifluoromethyl)phenyl]pyrimidine-2,4-diamine, 6-(2-ethyl-1H-benzimidazol-1-yl)-5-fluoro-N2-[4-(trifluoromethyl)phenyl]pyrimidine-2,4-diamine, 5-fluoro-6-(6-fluoro-2-methyl-1H-benzimidazol-1-yl)-N2-[4-(trifluoromethyl)phenyl]pyrimidine-2,4-diamine, 6-(2-ethyl-6-fluoro-1H-benzimidazol-1-yl)-5-fluoro-N2-[4- (trifluoromethyl)phenyl]pyrimidine-2,4-diamine, N2-[4-(difluoromethoxy)phenyl]-6-(4,6-difluoro-2-methyl-1H-benzimidazol-1-yl)pyrimidine-2,4-diamine, 6-(2-cyclopropyl-1H-benzimidazol-1-yl)-N2-[4-(trifluoromethyl)phenyl]pyrimidine-2,4-diamine, 5-fluoro-6-(2-methyl-1H-imidazo[4,5-b]pyridin-1-yl)-N2-[4-(trifluoromethyl)phenyl]pyrimidine-2,4-diamine, 6-(2-cyclopropyl-5,6-difluoro-1H-benzimidazol-1-yl)-N2-[4-(trifluoromethyl)phenyl]pyrimidine-2,4-diamine, 6-(2-ethyl-5,6-difluoro-1H-benzimidazol-1-yl)-N2-[4- (trifluoromethyl)phenyl]pyrimidine-2,4-diamine, 6-(2-cyclopropyl-5,6-difluoro-1H-benzimidazol-1-yl)-N2-[4-(difluoromethoxy)phenyl]pyrimidine-2,4-diamine, 6-(2-cyclopropyl- 5.6-difluoro-1H-benzimidazol-1-yl)-2-{[4-(trifluoromethyl)phenyl]amino}pyrimidine-4-carbonitrile, 6-(5,6-difluoro-2-methyl-1H-benzimidazol-1-yl)-2-{[4- (trifluoromethyl)phenyl]amino}pyrimidine-4-carbonitrile, 6-[2-(difluoromethyl)-1H-benzimidazol-1-yl]-N2-[4-(trifluoromethyl)phenyl]pyrimidine-2,4-diamine, 6-(2-cyclopropyl-6-fluoro-1H-benzimidazol-1-yl)-N2-[4-(difluoromethoxy)phenyl]pyrimidine-2,4-diamine, N2-[4-(difluoromethoxy)phenyl]-6-(2-ethyl-6-fluoro-1H-benzimidazol-1-yl)pyrimidine-2,4-diamine, N2-[4-(difluoromethoxy)phenyl]-6-[2-(difluoromethyl)-6-fluoro-1H-benzimidazol-1-yl]pyrimidine-2,4-diamine, 6-[2-(methoxymethyl)-1H-benzimidazol-1-yl]-N2-[4-(trifluoromethyl)phenyl]pyrimidine-2,4-diamine, 6-[2-(propan-2-yl)-1H-benzimidazol-1-yl]-N2-[4-(trifluoromethyl)phenyl]pyrimidine-2,4-diamine, N2-[4-(difluoromethoxy)phenyl]-6-(2-ethyl- 5.6-difluoro-1H-benzimidazol-1-yl) pyrimidine-2,4-diamine, N2-[4-(difluoromethoxy)phenyl]-6-(5,6-difluoro-2-methyl-1H-benzimidazol-1-yl)pyrimidine-2,4-diamine, N2-[4- (difluoromethoxy)phenyl]-6-[6-fluoro-2-(methoxymethyl)-1H-benzimidazol-1-yl]pyrimidine-2,4-diamine, N2-[4-(difluoromethoxy)phenyl]-6-[6-fluoro-2-(propan-2-yl)-1H-benzimidazol-1-yl]pyrimidine-2,4-diamine, 6-(2-cyclopropyl-5-fluoro-1 H-benzimidazol-1-yl)-N2-[4- (difluoromethoxy)phenyl]pyrimidine-2,4-diamine, N2-[4-(difluoromethoxy)phenyl]-6-[5-fluoro-2-(propan-2-yl)-1H-benzimidazol-1-yl]pyrimidine-2,4-diamine, 6-(2-methylpyrazolo[1,5-a]pyridin-3-yl)-N2-[4-(trifluoromethyl)phenyl]pyrimidine-2,4-diamine, N2-[4- (difluoromethoxy)phenyl]-6-(2-methyl-1H-benzimidazol-1-yl)pyrimidine-2,4-diamine, N2-[4-(difluoromethoxy)phenyl]-6-(2-ethyl-1H-benzimidazol-1-yl)pyrimidine-2,4-diamine, 6-(2-TH Docket No. 222120-2150cyclopropyl-1H-benzimidazol-1-yl)-N2-[4-(difluoromethoxy)phenyl]pyrimidine-2,4-diamine, 6-(5-chloro-2-cyclopropyl-1H-benzimidazol-1-yl)-N2-[4-(trifluoromethyl)phenyl]pyrimidine-2,4-diamine, 6-(6-chloro-2-cyclopropyl-1H-benzimidazol-1-yl)-N2-[4- (trifluoromethyl)phenyl]pyrimidine-2,4-diamine, N2-[4-(difluoromethoxy)phenyl]-6-[2-(difluoromethyl)-1H-benzimidazol-1-yl]pyrimidine-2,4-diamine, N2-[4-(difluoromethoxy)phenyl]-6-[2-(propan-2-yl)-1H-benzimidazol-1-yl]pyrimidine-2,4-diamine, 6-(2-ethylpyrazolo[1,5-a]pyridin-3-yl)-N2-[4-(trifluoromethyl)phenyl]pyrimidine-2,4-diamine, N2-[4-(difluoromethoxy)phenyl]-6-(2-ethylpyrazolo[1,5-a]pyridin-3-yl)pyrimidine-2,4-diamine, 6-(5-chloro-2-cyclopropyl-1H-benzimidazol-1-yl)-N2-[4-(difluoromethoxy)phenyl]pyrimidine-2,4-diamine, 6-(6-chloro-2-cyclopropyl-1H-benzimidazol-1-yl)-N2-[4- (difluoromethoxy)phenyl]pyrimidine-2,4-diamine, N2-[4-(difluoromethoxy)phenyl]-5-fluoro-6-(2-methyl-1H-benzimidazol-1-yl)pyrimidine-2,4-diamine, 6-(2-ethyl-1H-benzimidazol-1-yl)-N2-[6-(trifluoromethyl)pyridin-3-yl]pyrimidine-2,4-diamine, 6-(5-chloro-2-ethyl-1H-benzimidazol-1-yl)-N2-[4-(trifluoromethyl)phenyl]pyrimidine-2,4-diamine, 6-(6-chloro-2-ethyl-1H-benzimidazol-1-yl)-N2-[4-(trifluoromethyl)phenyl]pyrimidine-2,4-diamine, N2-[4-(trifluoromethyl)phenyl]-6-[2-(trifluoromethyl)pyrazolo[1,5-a]pyridin-3-yl]pyrimidine-2,4-diamine, [6-(6-fluoro-2-methyl-1 H-benzimidazol-1-yl)-2-{[4- (trifluoromethyl)phenyl]amino}pyrimidin-4-yl]phosphoramidic acid, 6-(6-fluoro-2-methyl-1H-imidazo[4,5-b]pyridin-1-yl)-N2-[4-(trifluoromethyl)phenyl]pyrimidine-2,4-diamine, N2-[4-(difluoromethoxy)phenyl]-6-(6-fluoro-2-methyl-1H-imidazo[4,5-b]pyridin-1-yl)pyrimidine-2,4-diamine, 6-(5-fluoro-2-methyl-1H-benzimidazol-1-yl)-N2-[4- (trifluoromethyl)phenyl]pyrimidine-2,4-diamine, 6-(2-cyclopropyl-5-fluoro-1H-benzimidazol-1-yl)-N2-[4-(trifluoromethyl)phenyl]pyrimidine-2,4-diamine, 6-(2-ethyl-5-fluoro-1H-benzimidazol-1-yl)-N2-[4-(trifluoromethyl)phenyl]pyrimidine-2,4-diamine, N2-[4-(difluoromethoxy)-3 -fluorophenyl]-6-(5-fluoro-2-methyl-1H -benzimidazol-1-yl)p yrimidine-2,4-diamine, 6-(2-cyclopropyl-5-methoxypyrazolo [1,5-a]p yridin-3-yl)-N2-[4-(trifluoromethyl)phenyl]pyrimidine-2,4-diamine, 6-(2-cyclopropyl-5-fluoropyrazolo[1,5-a]pyridin-3-yl)-N2-[4-(trifluoromethyl)phenyl]pyrimidine-2,4-diamine, 6-(6-fluoro-2-methyl-4-oxido-1H-imidazo[4,5-b]pyridin-1-yl-N2-[4-(trifluoromethyl)phenyl]pyrimidine-2,4-diamine, 6- (5-fluoro-2-methylpyrazolo[1,5-a]pyridin-3-yl)-N2-[4-(trifluoromethyl)phenyl]pyrimidine-2,4-diamine, 6-(2-methyl-1H-benzimidazol-1-yl)-N2-[4-(trifluoromethyl)phenyl]pyrimidine-2,4-diamine 3-oxide, 6-(2-ethyl-5-fluoropyrazolo[1,5-a]pyridin-3-yl)-N2-[4- (trifluoromethyl)phenyl]pyrimidine-2,4-diamine, 6-(6-fluoro-2-methyl-1H-benzimidazol-1-yl)-N2-[4-(trifluoromethyl)phenyl]pyrimidine-2,4-diamine 3-oxide, 4-chloro-6-(5-methoxy-2-methylpyrazolo[1,5-a]pyridin-3-yl)-N-[4-(trifluoromethyl)phenyl]pyrimidin-2-amine, 6-(5-methoxy-2-methylpyrazolo[1,5-a]pyridin-3-yl)-N2-[4-(trifluoromethyl)phenyl]pyrimidine-2,4-diamine, N2-[4-(difluoromethoxy)phenyl]-6-(5,6-difluoro-2-methyl-1H-benzimidazol-1-TH Docket No. 222120-2150yl)pyrimidine-2,4-diamine 3-oxide, 6-(2-cyclopropyl-1H-benzimidazol-1-yl)-N2-[4-(trifluoromethyl)phenyl]pyrimidine-2,4-diamine 3-oxide, 3-(6-amino-2-{[4-(trifluoromethyl)phenyl]amino}pyrimidin-4-yl)-2,5,6-trimethylpyrazolo[1,5-c]pyrimidin-7(6H)-one, 6-(2-ethyl-1H-benzimidazol-1-yl)-N2-[4-(trifluoromethyl)phenyl]pyrimidine-2,4-diamine 3-oxide, 6-(2-ethyl-6-fluoro-1H-benzimidazol-1-yl)-N2-[4-(trifluoromethyl)phenyl]pyrimidine- 2.4-diamine 3-oxide, 6-(5-amino-2-methylpyrazolo[1,5-a]pyridin-3-yl)-N2-[4-(trifluoromethyl)phenyl]pyrimidine-2,4-diamine, 6-(5-chloro-2-methylpyrazolo[1,5-a]pyridin-3-yl)-N2-[4-(difluoromethoxy)phenyl]pyrimidine-2,4-diamine, 6-(5-chloro-2-methylpyrazolo[1,5-a]pyridin-3-yl)-N2-[4-(trifluoromethyl)phenyl]pyrimidine-2,4-diamine, 6-[2-(methylsulfanyl)-1H-benzimidazol-1-yl]-N2-[4-(trifluoromethyl)phenyl]pyrimidine-2,4-diamine, 6-(5-chloro-2-ethylpyrazolo[1,5-a]pyridin-3-yl)-N2-[4-(trifluoromethyl)phenyl]pyrimidine-2,4-diamine, 6-(5-chloro-2-ethylpyrazolo[1,5-a]pyridin-3-yl)-N2-[4-(difluoromethoxy)phenyl]pyrimidine-2,4-diamine, 6-(2-cyclopropylpyrazolo[1,5-a]pyridin-3-yl)-N2-[4-(trifluoromethyl)phenyl]pyrimidine-2,4-diamine, 6-(2-methyl-1H-benzimidazol-1-yl)-N2-(3-methylphenyl)pyrimidine-2,4-diamine, 6-(2-methyl-1H-benzimidazol-1-yl)-N2-(4-methylphenyl)pyrimidine-2,4-diamine, N2-(4-methoxyphenyl)-6-(2-methyl-1H-benzimidazol-1-yl)pyrimidine-2,4-diamine, N2-(1,3-benzodioxol-5-yl)-6-(2-methyl-1H-benzimidazol-1-yl)pyrimidine-2,4-diamine, N2-(4-bromophenyl)-6-(2-methyl-1H-benzimidazol-1-yl)pyrimidine- 2.4-diamine, 6-(2-methyl-1H-benzimidazol-1-yl)-N2-(4-nitrophenyl)pyrimidine-2,4-diamine, 6-(6-fluoro-2-methyl-1H-benzimidazol-1-yl)-N2-(4-methoxyphenyl)pyrimidine-2,4-diamine, 6-(6-fluoro-2-methyl-1H-benzimidazol-1-yl)-N2-(4-methylphenyl)pyrimidine-2,4-diamine, N2-(4-chlorophenyl)-6-(6-fluoro-2-methyl-1H-benzimidazol-1-yl)pyrimidine-2,4-diamine, N2-[4-(dimethylamino)phenyl]-6-(6-fluoro-2-methyl-1 H-benzimidazol-1-yl)pyrimidine-2,4-diamine, 4-{[4-amino-6-(6-fluoro-2-methyl-1H-benzimidazol-1-yl)pyrimidin-2-yl]amino}benzonitrile, 6-(6-fluoro-2-methyl-1H-benzimidazol-1-yl)-N2-(3-fluorophenyl)pyrimidine-2,4-diamine, 6-(6-fluoro-2-methyl-1H-benzimidazol-1-yl)-N2-(3-methylphenyl)pyrimidine-2,4-diamine, 6-(6-fluoro-2-methyl-1H-benzimidazol-1-yl)-N2-(3-methoxyphenyl)pyrimidine-2,4-diamine, N2-(3-chlorophenyl)-6-(6-fluoro-2-methyl-1H-benzimidazol-1-yl)pyrimidine-2,4-diamine, N2-(3-fluoro-4-methoxyphenyl)-6-(6-fluoro-2-methyl-1H-benzimidazol-1-yl)pyrimidine-2,4-diamine, 6-(6-fluoro-2-methyl-1H-benzimidazol-1-yl)-N2-phenylpyrimidine-2,4-diamine, N2-[4-(dimethylamino)phenyl]-6-(2-methyl-1H-benzimidazol-1-yl)pyrimidine-2,4-diamine, 4-{[4-amino-6-(2-methyl-1 H-benzimidazol-1-yl)pyrimidin-2-yl]amino}benzonitrile, 6-(2-methyl-1 H-benzimidazol-1-yl)-N2-[4-(trifluoromethoxy)phenyl[pyrimidine-2,4-diamine, N2-(2,2-difluoro-1,3-benzodioxol-5-yl)-6-(2-methyl-1H-benzimidazol-1-yl)pyrimidine-2,4-diamine, N2-(3-fluoro-4-methoxyphenyl)-6-(2-methyl-1H-benzimidazol-1-yl)pyrimidine-2,4-diamine, N2-(3-chloro-4-methoxyphenyl)-6-(2-methyl-1H-benzimidazol-1-yl)pyrimidine-2,4-diamine, N2-(4-chlorophenyl)-6-(2-ethyl-1 H-benzimidazol-1-yl)pyrimidine-2,4-diamine, 6-(2-ethyl- 1 H-TH Docket No. 222120-2150benzimidazol-1-yl)-N2-(4-methoxyphenyl)pyrimidine-2,4-diamine, 6-(2-ethyl-1H-benzimidazol-1-yl)-N2-(2-methylphenyl)pyrimidine-2,4-diamine, 6-(2-ethyl-1H-benzimidazol-1-yl)-N2-(4-methylphenyl)pyrimidine-2,4-diamine, N2-(1,3-benzodioxol-5-yl)-6-(2-ethyl-1H-benzimidazol-1-yl)pyrimidine-2,4-diamine, 6-(2-ethyl-1H-benzimidazol-1-yl)-N2-(3-fluoro-4-methoxyphenyl)pyrimidine-2,4-diamine, N2-(3-chloro-4-methoxyphenyl)-6-(2-ethyl-1H-benzimidazol-1-yl)pyrimidine-2,4-diamine, N2-(6-methoxypyridin-3-yl)-6-(2-methyl-1H-benzimidazol-1-yl)pyrimidine-2,4-diamine, N2-(4-chlorophenyl)-6-(2-ethyl-6-fluoro-1H-benzimidazol-1-yl)pyrimidine-2,4-diamine, 6-(2-ethyl-6-fluoro-1H-benzimidazol-1-yl)-N2-phenylpyrimidine-2,4-diamine, 6-(2-ethyl-6-fluoro-1H-benzimidazol-1-yl)-N2-(4-methylphenyl)pyrimidine-2,4-diamine, 6-(2-ethyl-6-fluoro-1H-benzimidazol-1-yl)-N2-(4-methoxyphenyl)pyrimidine-2,4-diamine, N2-[4-(dimethylamino)phenyl]-6-(2-ethyl-6-fluoro-1H-benzimidazol-1-yl)pyrimidine-2,4-diamine, 6-(2-ethyl-6-fluoro-1H-benzimidazol-1-yl)-N2-(3-fluorophenyl)pyrimidine-2,4-diamine, N2-(3-chlorophenyl)-6-(2-ethyl-6-fluoro-1H-benzimidazol-1-yl)pyrimidine-2,4-diamine, 6-(2-ethyl-6-fluoro-1H-benzimidazol-1-yl)-N2-(3-methoxyphenyl)pyrimidine-2,4-diamine, 6-(2-ethyl-6-fluoro-1H-benzimidazol-1-yl)-N2-(3-methylphenyl)pyrimidine-2,4-diamine, 4-{[4-amino-6-(2-ethyl-6-fluoro-1H-benzimidazol-1-yl)pyrimidin-2-yl]amino}benzonitrile, 6-(2-ethyl-6-fluoro-1H-benzimidazol-1-yl)-N2-(3-fluoro-4-methoxyphenyl)pyrimidine-2,4-diamine, N2-(4-chloro-3-fluorophenyl)-6-(6-fluoro-2-methyl-1H-benzimidazol-1-yl)pyrimidine-2,4-diamine, N2-(4-chloro-3-fluorophenyl)-6-(2-ethyl-6-fluoro-1H-benzimidazol-1-yl)pyrimidine-2,4-diamine, 5-fluoro-N2-(4-methoxyphenyl)-6-(2-methyl-1H-benzimidazol-1-yl)pyrimidine-2,4-diamine, N2-(4-chlorophenyl)-6-(2-methyl-1H-benzimidazol-1-yl)pyrimidine-2,4-diamine, 5-fluoro-6-(2-methyl-1H-benzimidazol-1-yl)-N2-(4-methylphenyl)pyrimidine-2,4-diamine, N2-(4-chloro-3-fluorophenyl)-6-(2-methyl-1H-benzimidazol-1-yl)pyrimidine-2,4-diamine, 6-(5,6-difluoro-2-methyl-1H-benzimidazol-1-yl)-N2-(4-methoxyphenyl)pyrimidine-2,4-diamine, 6-(5,6-difluoro-2-methyl-1H-benzimidazol-1-yl)-N2-(4-methylphenyl)pyrimidine-2,4-diamine, N2-(4-chlorophenyl)-6-(5,6-difluoro-2-methyl-1H-benzimidazol-1-yl)pyrimidine-2,4-diamine, N2-(4-methylphenyl)-6-(2,5,6-trimethyl-1H-benzimidazol-1-yl)pyrimidine-2,4-diamine, N2-(4-chlorophenyl)-6-(2,5,6-trimethyl-1H-benzimidazol-1-yl)pyrimidine-2,4-diamine, N2-(4-methoxyphenyl)-6-(2,5,6-trimethyl-1H-benzimidazol-1-yl)pyrimidine-2,4-diamine, N2-(3-fluoro-4-methoxyphenyl)-6-(2,5,6-trimethyl-1H-benzimidazol-1-yl)pyrimidine-2,4-diamine, N2-(3-chloro-4-methoxyphenyl)-6-(2,5,6-trimethyl-1H-benzimidazol-1-yl)pyrimidine-2,4-diamine, N2-[4-(difluoromethoxy)phenyl]-6-(2,5,6-trimethyl-1H-benzimidazol-1-yl)pyrimidine-2,4-diamine, 6-(3,5-dimethyl-1,2-oxazol-4-yl)-N2-(4-methoxyphenyl)pyrimidine-2,4-diamine, 6-(3,5-dimethyl-1,2-oxazol-4-yl)-N2-(4-methylphenyl)pyrimidine-2,4-diamine, N2-(4-chlorophenyl)-6-(5-fluoro-2-methyl-1H-benzimidazol-1-yl)pyrimidine-2,4-diamine, N2-(4-chloro-3-fluorophenyl)-6-(5-fluoro-2-methyl-1H-benzimidazol-1-yl)pyrimidine-2,4-diamine, N2-(3-chlorophenyl)-6-(5,6-difluoro-2-methyl-TH Docket No. 222120-21501H-benzimidazol-1-yl)pyrimidine-2,4-diamine, 6-(5,6-difluoro-2-methyl-1H-benzimidazol-1-yl)-N2-(4-nitrophenyl)pyrimidine-2,4-diamine, 6-(5,6-difluoro-2-methyl-1H-benzimidazol-1-yl)-N2-(3-methylphenyl)pyrimidine-2,4-diamine, 6-(5,6-difluoro-2-methyl-1H-benzimidazol-1-yl)-N2-(3-methoxyphenyl)pyrimidine-2,4-diamine, 5-chloro-N2-(4-methoxyphenyl)-6-(2-methyl-1H-benzimidazol-1-yl)pyrimidine-2,4-diamine, N2-(4-chlorophenyl)-6-(3,5-dimethylisoxazol-4-yl)pyrimidine-2,4-diamine; and 6-(5-fluoro-2-methyl-1 H-benzimidazol-1-yl)-N2-[4-(trifluoromethyl)phenyl]pyrimidine-2,4-diamine3-oxide.

[0071] In some embodiments, the Bmi-1 inhibitor is Unesbulin (PTC596), having the structure:

[0072] In some embodiments, the FAO inhibitor is Etomoxir (Eto) having the structure:o

[0073] In other embodiments, the FAO inhibitor can be oxfenicine, perhexiline, mildronate, trimetazidine, or any combination thereof.

[0074] In some embodiments, the immune checkpoint inhibitor (ICI) can be anti-PD-1, or anti-CTLA-4, or anti-PD-L1, or anti-Lag3, or anti-Tim3, or anti-TIGIT, other agents that lead to immune cell activation, or a combination thereof, including, but not limited to, specific agents selected from pembrolizumab, nivolumab, atezolizumab, durvalumab, ipilimumab, or any combination thereof. However, other ICI are contemplated and should be considered disclosed.

[0075] A number of embodiments of the invention 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.EXAMPLESExample 1:TH Docket No. 222120-2150

[0076] Oncolytic herpes simplex virus (oHSV) was developed to treat both adult and pediatric brain tumor patients given its potential to selectively kill tumor cells while sparing normal brain cells. This includes an “unarmed” G207 oHSV for MG, and also an "armed" oHSV-expressing human interleukin 12 (IL-12) (M032, IND #14,946), which is recently completed in Phase I trial at UAB (NCT02062827). Both G207 and M032 are safe and have shown promise with disease regression or stabilization in approximately half of oHSV-treated recurrent MG patients, and exceptional responses in some. However, all patients eventually died of tumor relapse and growth. Treatment of mice bearing glioma with the oHSV expressing the murine IL-12 (M002) can effectively inhibit glioma growth and prolong mice survival. However, all mice eventually succumbed to death (FIG. 1).

[0077] It was reasoned that oHSV therapy-induced tumor cell lysis, a desired outcome may come with undesirable bystander effects. The dying cell-derived components post-oHSV not only represent potential factors facilitating the surviving fraction of tumor cells but also likely potentiate immunosuppression by affecting glial and immune cells in the glioma microenvironment. Thus, a precise definition of these confounding factors may aid in a better understanding of potential pitfalls of this very promising modality and developing improved therapeutic strategies.

[0078] Conditioned medium (CM) derived from glioma stem cells (GSCs) post-oHSV treatment (oHSV GSC-CM) induced pro-tumoral phenotypic and proliferative changes, which were associated with increased expression of the poor MG prognostic marker Bmi1, in the surviving cells along with their potent effects on immune compartments, including the upregulation of microglial iNOS and PD-L1, as well as elevated mitochondrial respiratory capacity of Treg cells (FIGs. 4A-4B and 10B-10C). Mass spectrometry analysis of the GSC-CM revealed the factor acyl-CoA-binding protein (ACBP), also known as diazepam-binding inhibitor (DBI), was the most highly upregulated factor related to lipid metabolism in oHSV GSC-CM (Table 1 and FIGs. 5A-5B). Most importantly, in vitro and in vivo findings strongly suggest that tumor-secreted ACBP (tsACBP) post-oHSV treatment promotes persisting GSCs toward a more pronounced aggressive phenotype, potentially leading to refractory to oHSV therapy, tumor recurrence and impaired mice survival (FIGs. 8A-8C).

[0079] It was therefore reasoned that preventing the tsACBP-induced secondary effects may improve the oHSV therapy while bypassing the hurdles from directly targeting soluble tsACBP. Interestingly, lentiviral-mediated shRNA knockdown (KD) of glioma Bmi1 (shBmil) prolonged mice survival and altered immune cells in the tumor microenvironment (TME), associated with changes in glioma cell metabolism, downregulation of tsACBP and increased secretion of fatty acid-binding protein 5 (FABP5) (FIGs. 11A-14B), a chaperone required to facilitate uptake and intracellular trafficking of free fatty acid. Moreover, KD of FABP5 (shFABP5) in shBmil glioma cells mitigated the beneficial effect from KD of Bmi1TH Docket No. 222120-2150alone on mice survival (FIG. 14C). These results strongly suggest that inhibiting glioma Bmi1 alters glioma secretome, resulting in the TME remodeling beyond its conventional role in glioma biology.

[0080] As disclosed herein, oHSV therapy combined with glioma Bmi1 inhibition restricts refractory tumor cells and promotes the TME remodeling by modulating the glioma secretome, leading to improved therapeutic efficacy (FIG. 2).

[0081] FIGs. 3A-3D show characterization of GSC005 and PN glioma stem cell lines. GSC005: Mesenchymal (MES) type; ACBP-secreting GSCs. PN cells: Proneural (PN) type; Responder cells; resistance to oHSV killing and the minimal source of tsACBP in the mixed model post-oHSV treatment.

[0082] Treatment of naive GSC005 or PN cells with oHSV GSC-CM upregulated the MES makers (FIGs. 4A-4B). Upregulation of MES markers (PN to MES subtype shifting) is associated with treatment resistance and enhanced tumorigenicity.

[0083] Mass Spectrometry (MS) analysis of GSC-CM revealed ACBP as the most highly upregulated factor related to lipid metabolism in oHSV GSC-CM (FIGs. 5A-5B, Table 1). ACBP is known to control the transit of fatty acyl-CoA into mitochondria to initiate fatty acid β-oxidation (FAO).Table 1: Significantly differentially regulated KEGG Pathway in GSC-CM (oHSV vs Ctrl) Enrichment FDR Functional Category0.008343424 Focal adhesion0.008343424 ECM-receptor interaction0.014443861 Carbon metabolism0.028312478 Glycolysis / Gluconeogenesis 0.028312478 Biosynthesis of amino acids 0.028312478 Peroxisome0.028312478 Gap junction0.04313273 Apoptosis0.04313273 Estrogen signaling pathway 0.043425083 PI3K-Akt signaling pathway 0.048359427 Protein processing in endoplasmic reticulum0.048359427 Phagosome0.049055875 Metabolic pathways

[0084] Glioblastoma multiforme (GBM) is the most commonly occurring MG in adults. High expression of ACBP was correlated with poor survival of GBM patients (FIGs.6A-6B).

[0085] There were higher levels of ACBP in glioma tissue and serum from oHSV-treated mice at a later disease stage (FIGs. 7A-7B). Serum ACBP level was significantly elevated in mice treated with M002 compared to mice given saline, even at day 47 post-TH Docket No. 222120-2150treatment, which mirrored the pattern of CD44 expression and potential subtype shifting in tumor cells.

[0086] Culture of GSC005 with oHSV GSC-CM increased, while the addition of an anti-ACBP antibody into the culture to neutralize ACBP, reduced their neurosphere formation capability (FIG. 8A). oHSV treatment combined with in vivo inhibition or knockout of ACBP expression extended mice survival further compared to mice treated with oHSV alone (FIGs.8B-8C).

[0087] M002 treatment not only induced substantial changes of live tumor cells compared to saline controls, but also resulted in the emergence of different cellular clusters (FIG. 9). The effect of exogenous ACBP on cellular metabolism remains unclear. tsACBP could alter the heterogeneity of tumor cells due to the subtype shifting.

[0088] M002 treatment also induced substantial changes of intratumoral immune cells (FIGs. 10A-10C). The role of tsACBP in immune regulation is poorly understood.Microglia and regulatory T cells (Tregs) displayed marked differences (M002 vs Saline) (FIG.10A). oHSV GSC-CM upregulated iNOS and PD-L1 in primary microglia (FIG. 10B). oHSV GSC-CM enhanced OCR of in vitro differentiated Tregs that was diminished by pretreatment of Tregs with etomoxir (metabolic changes) (FIG. 10C).

[0089] Kinetic analysis of serum ACBP and glioma CD44 expression (FIG. 7B) suggests that serum ACBP level could serve as a biomarker to predict treatment resistance and tumor recurrence, which can be used to direct a combined therapeutic approach to mitigate risk in MG patients post-oHSV therapy. A global inhibition of ACBP is not practicable, since it may cause systemic toxicity and the collapse of overall functioning of the organism. To bypass the hurdles from directly targeting soluble tsACBP alone, a multipronged approach can be used to improve oHSV therapeutic efficacy-Bmil inhibition, which can suppress glioma growth, reduce ACBP expression / secretion, and promote antitumor immunity (FIGs. 11A-14C). Although Bmi1 inhibition retards glioma growth, the effects of Bmi1 inhibition on immune cells within the TME remains largely unclear.

[0090] Mice bearing glioma with knockdown of Bmi1 had longer survival associated with reduced tumoral Tregs (FIGs. 11A-11C). Intratumoral Tregs in mice bearing glioma with knockdown of Bmi1 were converted into effector cells (FIGs. 12A-12C). The reprogrammed Tregs may constitute a new source of antitumor effector activity that synergizes with enhanced CD8+T-cell activity, contributing to better survival of mice bearing shBmil glioma. Reduced Bmi1 expression altered glioma cell metabolism, reduced ACBP but increased secretion of FABP5 (FIGs. 13A-13D). The correlations of Bmi1 and ACBP or FABP5 in GBM patients (FIG. 13D) and the changes of these factors in the shBmil CM (FIG. 13B) strongly justify that Bmi1 regulates glioma cells through the alterations of these lipid metabolism facilitators. Increased secretion of FABP5 promoted Treg conversion, and KD of FABP5 inTH Docket No. 222120-2150shBmil glioma cells compromised the beneficial effect from Bmi1 KD alone on mice survival (FIGs. 14A-14C). TOGA analysis revealed that the FABP5 level in GBM was associated with gene signatures related to immune or inflammatory response (FIG. 14B).

[0091] Kinetic analysis of serum ACBP and glioma CD44 expression (FIG. 7B) suggests that serum ACBP level could serve as a biomarker indicating the emergence of refractory tumor cells, which can be used to direct the administering timing of Bmi1 inhibitor.

[0092] Unless defined otherwise, all technical and scientific terms used herein have the same meanings as commonly understood by one of skill in the art to which the disclosed invention belongs. Publications cited herein and the materials for which they are cited are specifically incorporated by reference.

[0093] Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the following claims.

[0094] PN20 cells cultured with conditioned medium (CM) collected from M002-treated GSC005 WT cells, but not CM from M002-treated GSC005 ACBP KO cells, upregulated the mesenchymal (MES) phenotype and reduced TMRM signals, indicative of depolarized mitochondrial membrane (FIG. 15A). ELISA analysis of CM showed that ACBP levels were increased in M002-treated GSC005 WT cells and significantly reduced in GSC005 ACBP KO cells (FIG. 15B). To confirm the direct effect of ACBP on MES phenotype shifting, we treated human glioma cells, T98G, with recombinant human ACBP. The increases in the CD44 expression were evident in a dose-dependent manner (FIG. 16), which was also confirmed using recombinant murine ACBP to treat GSC005 cells (not shown). We also tested the effects of ACBP on GSCs in vivo. Analysis of tumor cells at day 24 post-treatment showed that M002 treatment increased the proportion of CD44+Bmi-1+MES subsets, which were substantially reduced in mice implanted with GSC005 ACBP KO cells (FIG. 17). Taken together, these results have further confirmed that ACBP from GSC tumor cells promoted proneural-to-mesenchymal (PMT) transition, potentially contributing to impaired oHSV therapeutic efficacy.

[0095] To define the mechanisms by which tsACBP post-oHSV alters GSCs. We have performed single-cell RNA sequencing (scRNA-seq) analysis of tumor cells and the cells in the TME isolated from mice implanted with GSC005 WT or GSC005 ACBP KO cells at day 45 post-treatment with saline or M002. M002 treatment of mice bearing GSC005 WT tumor substantially affected the tumor cells and other cells in the TME compared to saline-treated controls (FIGs. 18A-18D). Specifically, the proportion of T / NK cells, dendritic cells (DC) and B cells were increased, while the proportion of microglia and tumor cells were reduced (FIG. 18D). Interestingly, the relative cellular proportions in saline-treated miceTH Docket No. 222120-2150bearing GSC005 ACBP KO tumor did not differ significantly than saline-treated mice bearing GSC005 WT tumor (FIG. 18D). Analysis of cellular proportions in M002-treated GSC005 ACBP KO mice revealed that the fraction of tumor cells was similar to that in M002-treated GSC005 WT mice, while the microglia fraction was restored to the level as that in saline-treated GSC005 WT mice, and the proportion of myeloid cells was reduced to the smallest cell types among the four groups (FIG. 18D). Zooming in the tumor populations confirmed the GBM heterogeneity (FIGs. 19A-19C). Based on the Neftel classification of glioma cellular states (PMID: 31327527), including four different states, MES, AC, OPC, NPC, our analysis has shown that M002 treatment increased the MES and reduced NPC / OPC states of WT tumor (FIGs. 19B-19C). Notably, M002 treatment of GSC005 ACBP KO tumors reduced the MES states but increased NPC / OPC states compared to M002-treated GSC005 WT tumors (FIGs. 19B-19C). This finding was further supported by applying the metamodule scores derived from each cellular state-associated geneset (FIGs. 20A-20C). Although the tumor population was reduced in M002-treated WT tumor, which mainly comprises the MES states (FIGs. 20B-20C). The pathway analysis further revealed that the MTORC, EMT and OXPHOS-related pathways were enriched in clusters 0, 2 and 4 (FIGs. 21A-21B), among which cluster 0 was the predominant cluster in M002-treated WT tumor, but it was significantly reduced and cluster 4 was absent in M002-treated ACBP KO tumors (FIG. 19A). Taken together, these results support our hypothesis that ACBP produced by glioma cells after oHSV-mediated lysis promoted residual tumor cells toward a MES-like aggressive phenotype (a bystander effect resulting from the oHSV-mediated lysis), while reduced ACBP from tumor cells prevented this transition.

[0096] We also analyzed CD45+ tumor-infiltrating cells. Resident microglia and tumor-infiltrating myeloid cells in the TME are heterogenous (FIG. 22A). Interestingly, M002-treated WT tumor had increased tumor-infiltrating myeloid cells but reduced resident microglia, which was not the case in M002-treated ACBP KO tumors (FIG. 22B). The pathway analysis further revealed that cluster 0 was enriched with the EMT pathway, while cluster 3 was enriched with the OXPHOS-related pathway (not shown). Both clusters largely belonged to tumor-infiltrating myeloid cells (FIGs. 22A-22B), suggesting that ACBP from GSC tumor cells could impact resident and tumor-infiltrating myeloid cells with increased cell types contributing to the MES transition.

[0097] We further analyzed myeloid cells and microglia by sub-clustering (FIGs. 23A-23C). Combined cluster and pathway analysis of myeloid population revealed that M002 treatment of WT tumor increased the M1 -like population (cluster 1), which was enriched with the EMT pathway, and decreased the proliferative subset (cluster 4). In contrast, the proinflammatory subset (cluster 2) was substantially expanded in M002-treated ACBP KO tumor (FIGs. 23A-23C). In addition, the M2-like subset (cluster 0) and the population relatedTH Docket No. 222120-2150to tissue repair and tumor progression (cluster 3) were reduced in M002-treated ACBP KO tumor. Sub-clustering of microglial population coupled with pathway analysis also revealed that homeostatic (cluster 0) and IFN-responsive (cluster 2) populations were increased, but neuron-interacting (cluster 1) and disease-associated microglia (cluster 4) were reduced in M002-treated WT tumor (FIGs. 24A-24D). While IFN-responsive population (cluster 2) was reduced, the pro-inflammatory subset (cluster 3) was increased in M002-treated ACBP KO tumor (FIGs. 24A-24D). All of these findings have further supported that ACBP from GSC tumor cells can also impact resident and tumor-infiltrating myeloid cells with increased populations contributing to the MES transition.

[0098] Treg cells were reduced in M002-treated tumors irrespective of WT or ACBP KO tumors (FIG. 25). Notably, only Treg clusters were enriched with EMT-related pathways among all T-cell clusters. We further analyzed Treg cells by sub-clustering (FIGs. 26A-26C). Intratumoral Treg cells are heterogenous. M002 treatment of mice bearing WT tumor increased the naive Treg subset (cluster 2) enriched with OXPHOS-related pathways, which were further increased in ACBP KO tumor (FIGs. 26A-26C). The suppressive Treg populations (clusters 0, 3 and 4) were reduced after M002 treatment, which was more evident in ACBP KO tumors (FIGs. 26A-26C). Correspondingly, analysis of Treg cells isolated from brain tumors at day 24 post-treatment by flow cytometry revealed that Treg cells were significantly decreased after M002 treatment (FIG. 26D).

[0099] Bmi1 inhibition can suppress glioma growth, reduce ACBP expression / secretion, and promote anti-tumor immunity. oHSV treatment combined with inhibiting glioma Bmi1 could improve the oHSV therapeutic efficacy. M002 treatment in mice bearing shBmil GSC005 tumor extended mice survival compared to mice injected with shCtrl tumor cells or mice implanted with shBmil tumors without M002 treatment (FIG. 27), suggesting the synergistic protective effect of oHSV and reduced glioma Bmi1.

[0100] A constitutive inhibition of glioma Bmi1 may not be practicable in the clinic. The FDA-designated Bmi1 inhibitor PTC596 (PTC), which is reported to target cancer stem cells and efficiently induce GBM regression without obvious toxicity, is currently used in multiple clinical trials, including an ongoing Phase 1 b study for patients with newly diagnosed diffuse intrinsic pontine glioma and high grade glioma (NCT03605550). PTC can cross the blood-brain barrier and is distributed into the central nervous system. Treatment of mice with Bmi-1 inhibitor, PTC596, had some benefit in survival. Notably, combined PTC596 and M002 treatment significantly improved survival compared to mice treated with M002 or PTC596 alone (FIG. 28).

[0101] M002 treatment prolongs mouse survival and reduces the tumor size, which is further confirmed in our scRNA-seq analysis (FIGs. 29A-29B). Similar to the effect elicited by another oncolytic virus, multiple metabolic pathways, including FAO, were upregulated inTH Docket No. 222120-2150M002-treated tumors (FIGs. 29C-29D). Importantly, the gene encoding ACBP or Dbi, which is known to control the transit of fatty acyl-CoA into mitochondria to drive FAO and GBM progression, was highly increased in M002-treated tumors (FIG. 29E). Notably, the upregulation of FAO in residual tumor cells appeared to begin on day 10 post-M002 treatment (FIG. 29F). The increased FAO and ACBP / Dbi expression in tumor cells after M002 treatment indicative of emerging refractory glioma.

[0102] Depletion of CD8+T-cells largely abrogated the survival advantage conferred by M002 treatment in ACBP KO tumor-bearing mice (FIG. 30), suggesting the therapeutic effects of ACBP deletion rely on CD8+T-cell-mediated immunity.

[0103] Consistent with the essential role of CD4+T-cells in durable control of tumor in M002-treated mice (PMID: 39885128), Treg cells (clusters 2,5, Foxp3, Nrp1) were reduced and memory-like population (clusters, Tcf1, Slamf6) are increased in M002-treated CD4+T-cells (FIGs. 31A-31C). While the marked expansion of CD4+effector / memory subset (cluster 1, Gzmk, Cxcr3, Tbx21) likely accounts for the survival benefits of M002-treated ACBP KO mice (FIGs. 31A-31C), the most marked difference was observed in CD8+T-cells. M002-treated WT TME had increased terminally exhausted (cluster 0, Lag3, Havcr2, Cx3cr1) and intermediate exhausted (cluster 1, Nr4a1, Dusp5) populations, while these 2 populations are largely absent in M002-treated ACBP KO TME, which instead had increased Tpex (cluster 2, Tcf1, Slamf6) and effector populations (clusters, Gzmk, Cxcr3) (FIGs. 32A-32D).

[0104] ACBP is important to drive fatty acid oxidation (FAO) in GBM cells, contributing to tumor progression. Although we are establishing the strategy to directly target tumoral ACBP, we wondered if diminishing FAO of tumor cells could lead to similar consequence as depletion of tumoral ACBP. GSCs cultured with CM collected from etomoxir (Eto)-treated cells had reduced MES phenotype (i.e. CD44) and proliferation (i.e. Ki-67) (FIG. 33). Eto targets CPT-1 to reduce FAO. This finding has suggested that reduced FAO generated similar effects as reduced ACBP to influence the proliferation and MES phenotype of GSC tumor cells.

[0105] Interestingly, inhibition of FAO in GSC005 cells using Eto has revealed that conditioned medium (CM) collected from Eto-treated cells increased Tcf1 and GzmB expression but downregulated PD-1 levels in CD8+T-cells after co-culture (FIG. 34), suggesting that Eto-treated GSC005 CM, as a result of diminished FAO, increased both stem-like and effector signatures of CD8+T-cells, an effect similar to that observed in CD8+T-cells from M002-treated ACBP KO tumors (FIGs. 32A-32D). Given the increased FAO and ACBP expression in tumor cells after M002 treatment indicative of emerging refractory glioma cells, we reasoned that Eto may not only directly inhibit FAO and tumor growth butTH Docket No. 222120-2150also enhance CD8+T-cell-mediated targeting of residual tumor cells, potentially improving M002 treatment efficacy.

[0106] Eto has recently received the FDA-designated orphan drug status and has been the IND-enabled therapy currently advancing toward Phase 1 trials for GBM patients. Since diminishing FAO of tumor cells using genetic and pharmacological methods robustly blocked CD8+T-cell exhaustion and simultaneously expanded the progenitor exhausted (Tpex) and effector CD8+T-cell subsets (FIGs. 32A-32D and 34), and the presence of Tpex predicts the response to ICI therapy, this finding suggests the potential of combining metabolic rewiring and ICI with oHSV treatment to maximize the therapeutic efficacy for GBM patients (FIG. 35).

[0107] A combined treatment using oHSV, a drug that alters how tumor cells use energy (FAO inhibitor, Eto), and checkpoint blockade (ICI) may eliminate therapy-resistant glioma cells and activate CD8+T-cells for a stronger anti-tumor response. Unlike most current combination therapies, our approach repurposes the oHSV combination therapy by delivering the agents in a carefully timed sequence rather than all at once (guided by the sequential changes in tumor FAO and CD8+T-cells after M002 treatment), offering a new strategy for treating GBM and advancing the field of oHSV therapy. For example, treatment with oHSV is followed by Eto and finally ICI (e.g., anti-PD-1 + / - anti-CTLA-4, etc.) (FIG. 35)

[0108] We are also establishing the strategy to directly target tumoral ACBP by generating a new oncolytic virus carrying shRNA or gRNA / Cas9 to knockdown or knockout ACBP (FIG. 35).

Claims

TH Docket No. 222120-2150CLAIMS1. A method for treating a malignant glioma in a subject, comprising administering to the subject an effective amount of an oncolytic herpes simplex virus (oHSV) in combination with a glioma Bmi 1 inhibitor or a fatty acid oxidation (FAO) inhibitor.

2. The method of claim 1, wherein the oHSV comprises a nucleic acid sequence that encodes for human interleukin 12 (IL-12).

3. The method of claim 1, wherein the glioma Bmi1 inhibitor is PTC596 (PTC).

4. The method of claim 1, wherein the glioma FAO inhibitor is Etomoxir (Eto)5. The method of claim 1, wherein the malignant glioma is glioblastoma multiforme (GBM).

6. A method for treating a malignant glioma in a subject, comprising(a) detecting in a blood, serum, or tumor sample from the subject elevated levels of Acyl- CoA-binding protein (ACBP) compared to a control, and(b) administering to the subject an effective amount of an oncolytic herpes simplex virus (oHSV) in combination with a glioma Bmi1 inhibitor, a glioma fatty acid oxidation (FAO) inhibitor, or both a glioma Bmi1 inhibitor and a glioma FAO inhibitor.

7. The method of claim 6, comprising administering the oHSV in combination with the glioma FAO inhibitor, followed by administering one or more immune checkpoint inhibitors.

8. The method of claim 7, wherein the one or more immune checkpoint inhibitors comprise pembrolizumab, nivolumab, atezolizumab, durvalumab, ipilimumab, or any combination thereof.

9. The method of claim 6, wherein the oHSV comprises a nucleic acid sequence that encodes for human interleukin 12 (IL-12).

10. The method of claim 6, wherein the glioma Bmi1 inhibitor is PTC596 (PTC).

11. The method of claim 6, wherein the glioma FAO inhibitor is etomoxir (Eto).

12. The method of claim 6, wherein the malignant glioma is glioblastoma multiforme (GBM).

13. A method for treating a malignant glioma in a subject being treated with an oncolytic herpes simplex virus (oHSV), comprising(a) detecting in a blood, serum, or tumor sample from the subject elevated levels of Acyl- CoA-binding protein (ACBP) compared to a control, andTH Docket No. 222120-2150(b) administering to the subject an effective amount of a glioma Bmi1 inhibitor, an effective amount of a glioma fatty acid oxidation (FAO) inhibitor, or both a glioma Bmi1 inhibitor and a glioma FAO inhibitor.

14. The method of claim 13, comprising administering the oHSV in combination with the glioma FAO inhibitor, followed by administering one or more immune checkpoint inhibitors.

15. The method of claim 14, wherein the one or more immune checkpoint inhibitors comprise pembrolizumab, nivolumab, atezolizumab, durvalumab, ipilimumab, or any combination thereof.

16. The method of claim 13, wherein the glioma Bmi1 inhibitor is PTC596 (PTC),17. The method of claim 13, wherein the glioma FAO inhibitor is etomoxir (Eto).

18. The method of claim 13, wherein the malignant glioma is glioblastoma multiforme (GBM).

19. A method for predicting prognosis of a subject being with a malignant glioma being treated with an oncolytic herpes simplex virus (oHSV), comprising assaying a blood, serum, or tumor sample from the subject for Acyl-CoA-binding protein (ACBP) levels, wherein an elevated level of ACBP compared to a control is an indication of a poor prognosis.

20. The method of claim 19, wherein the malignant glioma is glioblastoma multiforme (GBM).

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