PTP1b inhibition improves the efficacy of radiotherapy in cancer treatment

Abstract

In one aspect, the disclosure relates to a method for treating or preventing cancer in a subject, the method including.at least the steps of administering radiotherapy to the subject and administering a PTP1b inhibitor to the subject; In an aspect, the PTP1b inhibitor can be BVT948, although other PTP1b inhibitors used in combination with radiotherapy are also disclosed. In some aspects, at least one immunotherapeutic agent, such as, for example, a PD-1 inhibitor, a PD-L1 inhibitor, or a CTLA4 inhibitor, can also be administered to the subject. In one aspect, the method reduces tumor growth or stops tumor progression. Also disclosed is a method for enhancing the efficacy of radiotherapy, the method including at least the step of administering a PTP1b inhibitor to the subject.

Description

ATTORNEY DOCKET NO. 222120-2100PTP1B INHIBITION IMPROVES THE EFFICACY OF RADIOTHERAPY IN CANCER TREATMENTCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. Provisional Application Ser. No. 63 / 689,890, filed September 3, 2024, which is incorporated herein by reference in its entirety.CROSS REFERENCE TO SEQUENCE LISTING

[0002] The genetic components described herein are referred to by sequence identifier numbers (SEQ ID NO). The sequence listing in written computer readable format (CRF) as a file named “222119-8871_Sequence_Listing.xml” created on August 19, 2024, and having a size of 10,829 bytes, is incorporated by reference in its entirety.BACKGROUND

[0003] Cancer patients often receive limited or transient benefit from traditional therapeutic strategies such as chemo- or radiotherapy. Failure to respond has been attributed, in part, to tumor cell intrinsic survival mechanisms, reprogramming to evade specific targeted therapies (e.g., pathway inhibition), and tumor-mediated manipulation of the immune response. Macrophages form frequently one of the most abundant intra-tumoral immune cell subsets and their activation state has been associated with patient outcomes, with predominantly pro-wound healing (M2) phenotypes characteristic of reduced survival, and with therapeutic resistance. Following chemo- or radiotherapy, increased numbers of M2 macrophages have been observed within the tumor microenvironment. This is paradoxical because cytotoxic therapies cause tumor cell death and the release of Damage Associated Molecular Patterns (DAMPs) which activate pro-inflammatory signaling cascades like Toll Like Receptors (TLRs) .

[0004] Previously tumor cells that secrete Prosl , a soluble Mer ligand, that inhibits the macrophage M1 response, have been identified. Genetic deletion of Prosl in tumor cells improved the in vivo efficacy of TLR agonism, leading to increased macrophage M1 activation and doubling of survival duration in a preclinical melanoma model. Based on these findings, it was hypothesized that in the context of chemo- or radiotherapy, tumor-secreted Prosl may reduce the ability of macrophages to respond to tumor DAMPs, many of which are TLR agonists, and potentially limit inflammation following therapy.ATTORNEY DOCKET NO. 222120-2100

[0005] Prosl is not an ideal pharmacologic target because it also acts as an anti-coagulant in the blood. In fact, genetic deficiency can lead to severe thrombotic events, which reduces the potential benefits of a direct depletion strategy. While Mer kinase inhibitors are currently in clinical trials, it is unclear whether they play a role in Pros'! :Mer signaling. PTP1b, however, was identified as a novel downstream intermediary of Prosl :Mer signaling in an in vitro model and it was hypothesized that it may improve macrophage activation in vivo as well. Clinically, PTP1b inhibitors have been well tolerated by patients in the treatment of Type 2 diabetes with limited toxicity. However, PTP inhibitors have yielded mixed results as direct anti-tumor agents.

[0006] Despite advances in anticancer research, there is still a scarcity of methods of cancer treatment that are effective at bypassing tumor cell survival mechanisms, pathway inhibition, and tumor-mediated manipulation of the immune response, while also avoiding off-target effects and improving macrophage activation. These needs and other needs are satisfied by the present disclosure.SUMMARY

[0007] In accordance with the purpose(s) of the present disclosure, as embodied and broadly described herein, the disclosure, in one aspect, relates to a method for treating or preventing cancer in a subject, the method including. at least the steps of administering radiotherapy to the subject and administering a PTP1 b inhibitor to the subject; In an aspect, the PTP1 b inhibitor can be BVT948, although other PTP1b inhibitors used in combination with radiotherapy are also disclosed. In some aspects, at least one immunotherapeutic agent, such as, for example, a PD- 1 inhibitor, a PD-L1 inhibitor, or a CTLA4 inhibitor, can also be administered to the subject. In one aspect, the method reduces tumor growth or stops tumor progression. Also disclosed is a method for enhancing the efficacy of radiotherapy, the method including at least the step of administering a PTP1b inhibitor to the subject.

[0008] Other systems, methods, features, and advantages of the present disclosure will be or become apparent to one with skill in the art upon examination of the following drawings and detailed description. It is intended that all such additional systems, methods, features, and advantages be included within this description, be within the scope of the present disclosure, and be protected by the accompanying claims. In addition, all optional and preferred features and modifications of the described embodiments are usable in all aspects of the disclosure taught herein. Furthermore, the individual features of the dependent claims, as well as all optional and preferred features and modifications of the described embodiments are combinable andATTORNEY DOCKET NO. 222120-2100 interchangeable with one another.BRIEF DESCRIPTION OF THE DRAWINGS

[0009] Many aspects of the present disclosure can be better understood with reference to the following drawings. The components in the drawings are not necessarily to scale, emphasis instead being placed upon clearly illustrating the principles of the present disclosure. Moreover, in the drawings, like reference numerals designate corresponding parts throughout the several views.

[0010] FIGs. 1A-1H show PTP inhibition improves the efficacy of radio- and chemotherapy in preclinical models. (FIG. 1A) Radiotherapy (6 Gy, local, once daily for 3 days), BVT948 (10 mg / kg, ip, once daily), or the combination were administered mice bearing subcutaneously implanted (FIG. 1B) B16F10 or (FIG. 1C) LLC tumors measurements taken to ascertain changes in tumor growth relative to vehicle (PBS, ip, once daily) treatment (n=8 per group). (FIG. 1 D) Cisplatin (5mg / kg, ip, once daily for 3 days), BVT948 (10 mg / kg, ip, once daily), or combination therapy were administered to (FIG. 1E) B16F10 or (FIG. 1 F) LLC tumor bearing mice and subcutaneous tumor volume measured over time compared to vehicle treated (PBS, ip, once daily) mice (n=9 per group). (FIG. 1G) GEMM6 tumor bearing mice were treated with vehicle (PBS, ip, once daily), vemurafenib (30 mg / kg, ip, once daily for 3 days), BVT948 (10 mg / kg, ip, once daily), or the combination once tumor volume reached 200 mm3and (FIG. 1 H) subcutaneous tumor volume measured (n=7 per group), p values were calculated by two-way ANOVA with Tukey post hoc test, p values are shown. Experiments were conducted twice.

[0011] FIGs. 2A-2H show tumor-secreted Prosl limits the anti-tumor immune response during chemotherapy and macrophages are essential for the efficacy of combination therapy. (FIG. 2A) Vehicle or cisplatin treatment were administered to parental B16F10 or Prosi-deficient (BdP) tumor bearing mice and tumor volumes measured over time (n=7). (FIG. 2B) Heatmap of differentially expressed genes from end-stage whole tumor mRNA. (n=4) (FIG. 2C) Volcano plot illustrating changes in immune related gene expression between cisplatin treated BdP and B16F10 tumors (n=4). Pathway upregulation in cisplatin treated BdP compared to B16F10 tumors from (FIG. 2D) KEGG and (FIG. 2E) Reactome databases. (FIG. 2F) Combination therapy was administered to B16F10 tumor bearing mice with full immune complement (encapsome, isotype antibody control), macrophage / phagocyte depletion (clodronate, isotype antibody control) or CD8+ T cell depletion (encapsome, anti-CD8+ antibody) and tumor volumes measured (n=6 mice per group). (FIG. 2G) H&E staining of B16F10 tumor sections from immune replete or respectiveATTORNEY DOCKET NO. 222120-2100 depletion mice (representative images of n=4). (FIG. 2H) Clodronate depletion reduces the number of intra-tumoral CD68+ macrophages as well as CD86 expression as quantitated with immunoflourescent staining (n=4, representative images from photos of the entire tumor section). Scale bars=500 pm for H&E staining and 50 pm for CD68+ and CD86+ immunofluorescence, p values were calculated by one or two-way ANOVA with Tukey post hoc test. Significant p values are shown.

[0012] FIGs. 3A-3H show PTP inhibition paired with multiple rounds of chemotherapy substantially reduces tumor growth while increasing immune infiltration and macrophage activation. (FIG. 3A) B16F10 tumor volumes after treatment with vehicle or multiple rounds of cisplatin and / or BVT948 (n=6 per group). (FIG. 3B) Tumors were isolated, dissociated, and flow cytometry performed for CD45+ cells 12 days after the start of the treatment (n=4). (FIG. 3C) Clustering of CD45+ intra-tumoral immune cells harvested 12 days after the start of respective treatments as determined by scRNAseq analysis (n=4 per group). (FIG. 3D) Macrophage clusters separated by treatment arm show relatively fewer M0 / M2 macrophages in the combination treated group (n=4 per group). (FIG. 3E) Heat map of differentially expressed M1 and M2 markers in macrophages as determined by scRNAseq following tumor harvest (n=4 per group). (FIG. 3F) Signaling pathways upregulated in macrophages from the combination therapy group as compared to cisplatin monotherapy (n=4 per group). (FIG. 3G) Macrophage derived ligand signaling to immune cell receptors as determined by Cell Chat analysis (n=4 per group). (FIG. 3H) Transcriptionally inferred immune cell signaling networks in cisplatin or combination therapy treated samples (n=4 per group), p values are calculated by one or two-way ANOVA with Tukey post hoc test, p values are shown. Experiments for (FIG. 3A) were performed in duplicate.

[0013] FIGs. 4A-4H show tumor secretions suppress macrophage MyD88-dependent DAMP responsiveness. (FIG. 4A) Sections prepared from B16F10 tumors harvested 6 days after treatment start were stained for the DNA damage response marker yH2AX (n=4 per group, scale bar= 20 pm). (FIG. 4B) Pathway analysis of intra-tumoral M1 macrophages identified upregulation of multiple pro-inflammatory signaling cascades (n=4 per group). (FIG. 4C) MTT viability measurement of B16F10 tumor cells treated with increasing concentrations of cisplatin for 24 hours (n=6). qRT-PCR measurement of (FIG. 4D) wildtype or (FIG. 4E) MyD88 KO macrophage pro-inflammatory gene expression after 24 hours of transwell co-culture with B16F10 tumors cells treated with varying concentrations of cisplatin in the presence or absence of BVT948 (n=6). Tumor cells suppress M1-induced macrophage MyD88 (FIG. 4F) mRNA and (FIG. 4G) protein levels after transwell co-culture for 24 hours (n=5). (FIG. 4H) TIMER analysis of publicly availableATTORNEY DOCKET NO. 222120-2100TCGA data stratifying melanoma patient survival based on transcriptionally inferred macrophage infiltration and MyD88 expression (n=471). p values are calculated by one-way ANOVA with Tukey post hoc test. Significant p values are shown.

[0014] FIGs. 5A-5G show diverse tumors suppress Statl -mediated macrophage MyD88 expression. (FIG. 5A) TCGA analysis demonstrating positive correlation of STAT1 and MYD88 expression in melanoma patients (n=229). (FIG. 5B) Analysis of publicly available TCGA data shows melanoma patient survival stratified by high or low STAT1 expression (n=229 in each group). (FIG. 5C) SCENIC regulon analysis of intra-tumoral macrophages identifies a subset regulated by Statl and lrf2 / 7 (n=4). (FIG. 5D) Statl and phospho-Statl protein expression in M1 stimulated macrophages are suppressed by transwell co-culture with diverse tumor cells after 24 hours as shown by Western blot (n=3). (FIG. 5E) Immunofluorescent staining of M1 induced Lyz2- Cre:tdTomato macrophages for Statl when cultured in the presence or absence of B16F10 conditioned medium for 24 hours shows reduced Statl expression and nuclear translocation (representative images of n=3; scale bar=20 pm). (FIG. 5F) ChlP-PCR of Statl binding to the MyD88 promoter in the presence or absence of tumor cells (n=3). (FIG. 5G) Fludarabine treatment suppresses macrophage MyD88 expression in M1 induced macrophages comparably to coculture with B16F10 tumor cells for 24 hours (n=6). p values are calculated by one-way ANOVA with Tukey post hoc test. Significant p values are shown.

[0015] FIGs. 6A-6J show macrophage Mer activation induces suppressive ternary complex formation that limits MyD88 expression which can be rescued by PTP inhibition. (FIG. 6A) qRT- PCR measurement of MyD88 expression in M1 induced macrophages either co-cultured with B16F10 tumor cells or with 1.5 pg / mL Prosl , 200 ng / mL Gas6, or 2.5 pg / mL Gal3 after 24 hours of treatment (n=5). (FIG. 6B) Effect of siRNA knockdown of TAM ligands expressed by B16F10 cells on M1-induced macrophage MyD88 suppression as measured by qRT-PCR in the transwell assay after 24 hours (n=5). (FIG. 6C) Tumor mediated suppression of MyD88 expression occurs in M1-induced wildtype and Ax , but not Mer / _or Tyro3’ / _, macrophages after co-culture with B16F10 cells for 24 hours (n=5). (FIG. 6D) DAMP responsiveness of Mer / _macrophages are not suppressed by tumor secretions after cisplatin treatment for 24 hours in a co-culture model (n=5). (FIG. 6E) MyD88 expression of pro-inflammatory macrophages co-cultured with B16F10 cells is not rescued by treatment with 300 nM Mer / Axl / Tyro3 kinase inhibitors as measured by qRT-PCR (n=5). (FIG. 6F) MyD88 mRNA expression is not suppressed in Mer Y867F macrophages cultured in the presence of B16F10 tumor secretions for 24 hours (n=5). (FIG. 6G) A schematic representation of the MER:PTP1 b protein-protein interaction is presented. MER is shown inATTORNEY DOCKET NO. 222120-2100 surface rendering with amino acid residues within 5 A of PTP1 B shaded. PTP1B is represented as cartoon (grey) with residues having atoms within 5 A of MER shaded. (FIG. 6H) The open book representation of the interface between MER (left) and PTP1 B (right) is shown with the electrostatic surfaces rendered and residues involved in hydrogen-bonding interactions labeled. Some interactions involve mainchain atoms. (FIG. 6I) Association of Mer and Statl with PTP1b is increased after co-culture of M1 induced macrophages with B16F10 tumor cells but inhibited by treatment with the PTP inhibitor BVT948 (n=3). (FIG. 6J) Treatment with PTP inhibitors rescues MyD88 mRNA expression in inflammatory macrophages co-cultured with B16F10 tumor cells for 24 hours (n=5). p values are calculated by one-way ANOVA with Tukey post hoc test. Significant p values are shown.

[0016] FIGs. 7A-7F show the Pros1 :Mer signaling axis is activated proximal to chemotherapy induced damage and PTP inhibition rescues macrophage MyD88 and M1 gene expression in vivo. (FIG. 7A) Signaling diagram illustrating the sequence of events leading to tumor-mediated macrophage MyD88 suppression and rescue with PTP inhibition. (FIG. 7B) B16F10 tumors from mice receiving vehicle, cisplatin, BVT948, or combination therapy were harvested 6 days after treatment and immunostained for yH2AX or Prosl (n=4 per group, scale bar=500 pm for H&E, yH2AX and Prosl and 20pm for magnified yH2AX images). (FIG. 7C) Proximity ligation assay of Mer and Statl was performed on B16F10 tumor sections isolated 6 days after treatment start to determine protein-protein interactions (n=4 per group, scale bar=500 pm). (FIG. 7D) Immunofluorescent staining of MyD88 and CD68 within B16F10 tumor sections collected 6 days after treatment shows increased MyD88 expression and colocalization in BVT948 or combination therapy treated mice when compared to vehicle or cisplatin treatment (n=4, scale bar=20pm). CD45+ / CD11 b+ / F4:80+ macrophages were isolated from B16F10 tumors from each treatment group and expression of (FIG. 7E) MyD88 or (FIG. 7F) pro-inflammatory genes measured by qRT- PCR (n=4 per group), p values are calculated by one-way ANOVA with Tukey post hoc test. Significant p values are shown.

[0017] FIGs. 8A-8D show PTP inhibition improves the efficacy of radiotherapy in limiting tumor growth in multiple preclinical models. (FIG. 8A) Tumor volumes of individual B16F10 tumor bearing mice from different treatment arms (n=8) and (FIG. 8B) images of H&E-stained end-stage tumors (representative images of n=8 per group, scale bar=500 pm). (FIG. 8C) LLC tumor volumes from individual mice in different treatment arms (n=8). (FIG. 8D) H&E-stained sections from end-stage LLC tumors (representative images of n=8 per group, scale bar=500 pm).ATTORNEY DOCKET NO. 222120-2100

[0018] FIGs. 9A-9D show the efficacy of chemotherapy is improved by PTP inhibition. (FIG. 9A) Measurements of individual B16F10 tumor volumes from respective treatment groups (n=9) and (FIG. 9B) H&E-stained end-stage B16F10 tumors (representative images of n=9 per group, scale bar=500 pm) (FIG. 9C) LLC tumor volumes from individual mice in different treatment arms (n=9). (FIG. 9D) H&E-stained sections from end-stage LLC tumors (representative images of n=9 per group, scale bar=500 pm).

[0019] FIGs. 10A-10C show BVT948 treatment of early GEMM6 tumors results in some durable responses and reduces growth when combined with vemurafenib in larger tumors. (FIG. 10A) Volume measurements of GEMM6 tumors when treatment (vehicle or BVT948) was started when tumors reached 50 mm3(n=8). (FIG. 10B) Measurement of individual GEMM6 tumor volumes after treatment was started when tumors reached 200 mm3(n=7, scale bar=500 pm). (FIG. 10C) Images of H&E-stained GEMM6 tumor sections from different treatment arms (representative images of n=7) p values are calculated by two-way ANOVA with Tukey post hoc test, p values for comparisons are shown.

[0020] FIGs. 11A-11 B show PTP inhibitors have limited effects on tumor cell viability in vitro and increase angiogenesis associated pathways in vivo. (FIG. 11A) Tumor cells were treated with individual PTP inhibitors and cell viability measured by MTT assay after 24 hours (n=6, p values are calculated by one-way ANOVA with Tukey post hoc test, p values are shown). (FIG. 11B) Pathways upregulated in BVT948 versus vehicle treated panCK+ B16F10 cells as determined by Nanostring GeoMx analysis of panCK+ B16F10 cells from tumor sections from vehicle or BVT948 treated samples (n=4).

[0021] FIGs. 12A-12E show Prosl deficient tumors show reduced tumor growth after cisplatin treatment and increased immune activation. (FIG. 12A) Individual tumor volume measurements from parental B16F10 or Prosl deficient (BdP) tumors treated with vehicle or cisplatin (n=7 per arm). (FIG. 12B) Volcano plot comparison of disaggregated cells from vehicle treated BdP compared to B16F10 tumors. Upregulated pathways when comparing vehicle treated BdP cells to B16F10 with (FIG. 12C) KEGG or (FIG. 12D) Reactome databases. (FIG. 12E) CIBERSORTX deconvolution identified major intra-tumoral cellular subsets from bulk RNAseq of disaggregated tumors (n=4 per treatment arm).

[0022] FIGs. 13A-13B show encapsome and Isotype control treatment of tumor bearing mice do not significantly affect outcomes. (FIG. 13A) Treatment of B16F10 tumor bearing mice with encapsome (control for clodronate) does not reduce efficacy of combination therapy, nor doesATTORNEY DOCKET NO. 222120-2100 treatment with isotype control antibody (control for anti-CD8 antibody). (n=6) (FIG. 13B) Measurement of individual B16F10 tumor volume in different treatment arms (n=6).p values are calculated by two-way ANOVA with Tukey post hoc test, p values are shown.

[0023] FIGs. 14A-14B show combination therapy requires a sufficient number of cisplatin doses and daily BVT948 treatment. B16F10 tumor bearing mice were given 1 or 3 consecutive daily doses of cisplatin and treated either daily or every other day with BVT948 and subcutaneous B16F10 tumor volume measured and reported in aggregate (FIG. 14A) or individually (FIG. 14B) (n=4). p values are calculated by two-way ANOVA with Tukey post hoc test, p values among experimental groups are shown.

[0024] FIGs. 15A-15B show two doses of cisplatin in combination with BVT948 PTP inhibition substantially reduces B16F10 tumor growth. (FIG. 15A) Plots of individual tumor growth during vehicle, two rounds of cisplatin, BVT948, or the combination (n=8). (FIG. 15B) Images of H&E- stained tumor sections from end-stage tumors (representative images of n=8, scale bar=1 mm).

[0025] FIG. 16 shows flow cytometry gating strategy to identify live CD45+ immune cells. B16F10 tumors were isolated 12 days after treatment start, dissociated, and stained for CD45. Resulting cells were gated by size and granularity (left) and forward scatter (middle) as well as Phycoerythrin-Cyanine5 (PE-Cy5) labeled CD45 antibody and Live / Dead Am-Cyan to determine cell viability (right).

[0026] FIG. 17 shows changes in tumor immune cell composition following respective treatments. Violin plots of SingleR annotated immune cells show a general reduction of immune cells after chemotherapy which is generally rescued by addition of PTP inhibitor in the combination treatment group.

[0027] FIG. 18 shows clustering and characterization of intra-tumoral macrophages identifies M1 and M0 / M2 subsets. UMAP analysis of macrophages across all treatment arms with violin plots of specific M1 , M2, and TLR genes by cluster.

[0028] FIG. 19 shows macrophage pathway analysis identifies upregulated signaling networks for each cluster. Gene Ontology (GO) and KEGG pathway analysis identifies differentially upregulated pathways among macrophage clusters.

[0029] FIG. 20 shows macrophage signaling is affected by respective treatments. Bubble plot of transcriptionally inferred macrophage signaling to other immune cell types (n=4).ATTORNEY DOCKET NO. 222120-2100

[0030] FIG. 21 shows intra-tumoral B cell subpopulations across different treatment groups. SingleR annotation of B cell populations using the ImmGen (fine) database reference shows that cisplatin reduces B cells numbers and that in other treatment groups, B cells tend to be T3 or follicular B cells (n=4).

[0031] FIG. 22 shows the T cell population is reduced by cisplatin therapy and altered by BVT948 or combination therapy. UMAP analysis of T cells, and subsequent gene expression characterization, identifies changes in T cell populations though the subset of cytotoxic CD8+ T cells is generally limited except for following BVT948 monotherapy (n=4).

[0032] FIG. 23 shows neutrophil clusters and associated gene expression identify both pro- and anti-tumor subsets though there is a general expansion after combination therapy. UMAP and gene expression characterization identify changes in neutrophil subsets following respective therapies (n=4).

[0033] FIG. 24 shows characterization of monocyte subsets in each treatment group identifies varied effects. UMAP and gene expression characterization identifies changes in monocyte subsets following respective therapies based on markers in subsets (n=4).

[0034] FIG. 25 shows immune cell signaling is altered between treatment groups. (A) The aggregate number and strength of communications from each immune cell subset as identified by Cell Chat (n=4).

[0035] FIG. 26 shows tumor cell secretions dampen the macrophage pro-inflammatory response to TLR agonism. MyD88 or pro-inflammatory gene expression of naive or TLR agonist treated macrophages cultured in the presence or absence of B16F10 tumor cells for 24 hours, as measured by qRT-PCR (n=5, p values are calculated by one-way ANOVA with Tukey post hoc test. Significant p values are shown in figure panels).

[0036] FIG. 27 shows cisplatin treated macrophages are not M1 activated. Macrophages cultured alone in increasing concentrations of cisplatin do not show concomitant increases in Mi- associated gene expression (n=5). p values are calculated by one-way ANOVA with Tukey post hoc test. None of the comparisons were statistically significant.

[0037] FIGs. 28A-28C show BVT948 treatment restores macrophage sensitivity to tumor DAMPs. (FIG. 28A) Naive macrophages cultured with 0.2 pm-filtered B16F10 cell starvation conditioned medium show increased responsiveness to DAMPs in the presence of BVT948, as shown by qRT-PCR of pro-inflammatory genes (n=5). (FIG. 28B) Filtered conditioned mediaATTORNEY DOCKET NO. 222120-2100 collected from 10 Gy treated LLC cells increased the expression of proinflammatory mediators in murine peritoneal macrophage when paired with BVT948, as measured by qRT-PCR (n=5). (FIG. 28C) Macrophage DAMP sensitivity, as measured by qRT-PCR, when cultured in the presence of serum starvation GEMM6 conditioned medium in the presence or absence of BVT948, or with protease or benzonase treatment to remove protein or nucleic acid DAMPs, respectively (n=5). p values are calculated by one-way ANOVA with Tukey post hoc test. Significant p values among experimental groups are shown.

[0038] FIG. 29 shows tumor cell secretions do not suppress MyD88 levels of naive macrophages. MyD88 protein levels of naive macrophages cultured with tumor cells of diverse origins are not suppressed by tumor secretions (n=3). p values were calculated by one-way ANOVA with Tukey post hoc test. None of the comparisons were statistically significant.

[0039] FIG. 30 shows tumor cells do not suppress Statl expression in naive macrophages. Statl protein levels of naive macrophages co-cultured with tumor cells are not reduced after 24 hours, p values were calculated by one-way ANOVA with Tukey post hoc test. None of the comparisons were statistically significant.

[0040] FIGs. 31A-31B show Statl has two binding sites on the MyD88 promoter (proximal, distal) but tumor cell secretions do not affect association at the distal site. (FIG. 31 A) Statl binding sites within the MyD88 promoter region and ChlP-PCR primer locations. (FIG. 31 B) ChlP-qPCR of the distal Statl binding site of the MyD88 promoter in M1 induced macrophages in the presence or absence of B16F10 tumor cell secretions (n=3). p values were calculated by one-way ANOVA with Tukey post hoc test. Comparisons were not statistically significant.

[0041] FIG. 32 shows fludarabine reduces macrophage Statl expression. Fludarabine treatment of M1 induced macrophages reduces Statl mRNA expression comparably to co-culture with B16F10 tumor cells (n=7). p values are calculated by one-way ANOVA with Tukey post hoc test. Significant p values are shown.

[0042] FIG. 33 shows targeted siRNAs reduce tumor expression of Prosl or Gas6. B16F10 cells treated with targeted siRNAs show reduced mRNA expression of Mer ligands relative to untreated and non-targeting controls. (n=3). p values are calculated by one-way ANOVA with Tukey post hoc test, p values for comparisons among experimental groups are shown.

[0043] FIG. 34 shows macrophages isolated from Tyro3, Mer, and Axl knock-out mice respond equivalently to wildtype cells when stimulated with IFNy+LPS. MyD88 mRNA expression after 24ATTORNEY DOCKET NO. 222120-2100 hours of IFNy+LPS treatment was comparable between wildtype, Tyro3- / -, Mer- / -, and Axl- / - mouse macrophages (n=5). p values are calculated by one-way ANOVA with Tukey post hoc test, p values are shown.

[0044] FIGs. 35A-35C show PTP1 B-MER and PTP1 B-insulin receptor tyrosine kinase complex structures show an overlapping binding surface on PTP1 B. (FIG. 35A) The structures of PTP1 B in complex with MER (left) and insulin receptor tyrosine kinase (IRK) (right) are shown. PTP1B is shown in surface representation with residues of PTP1 B that engage with the MER and IRK shaded, respectively. MER and IRK are illustrated as cartoons. Solitary PTP1 B structures from complexes in (FIG. 35A) are shown in (FIG. 35B), left and middle. The overlay of the two surfaces is shown to the right. To show the common orientation of the two PTP1 B structures, (FIG. 35C) renders PTP1 B in cartoon mode with common coloring schemes to (A) and (B).

[0045] FIGs. 36A-36B show PTP1b inhibition prevents Mer:PTP1b:Stat1 complex formation. (FIG. 36A) Co-immunoprecipitation of Statl shows increased association of Statl with Mer and PTP1 b when M1-induced macrophages are co-cultured with B16F10 tumor cells, which is decreased with BVT948 treatment (n=3). (FIG. 36B) Western blot of transwell co-culture of M1 induced macrophages shows that different PTP inhibitors can rescue total and p-Stat1 levels (n=3). p values are calculated by one-way ANOVA with Tukey post hoc test. Significant p values are shown in figure panels.

[0046] Additional advantages of the invention will be set forth in part in the description which follows, and in part will be obvious from the description, or can be learned by practice of the invention. The advantages of the invention will be realized and attained by means of the elements and combinations particularly pointed out in the appended claims. It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the invention, as claimed.DETAILED DESCRIPTION

[0047] In one aspect, it is believed that PTP1 b inhibitors can support adaptive immune activation for both T and B cells. In a further aspect, this suggests that PTP1 b inhibition could provide a clinically actionable means of interrupting Pros'! :Mer signaling and potentially improve other aspects of the anti-tumor immune response

[0048] Disclosed herein is evidence that PTP inhibition enhances the efficacy of radio- and chemotherapy in multiple treatment refractory preclinical models, leading to 40-90% reduction inATTORNEY DOCKET NO. 222120-2100 tumor growth. In one aspect, in the context of combining chemotherapy and PTP inhibition, tumor suppression is mediated by phagocytic innate immune cells and the role for CD8+ T cells appears limited. In another aspect, mechanistically, PTP1b inhibition rescues macrophage DAMP responsiveness by blocking the Mer:Stat1 signaling axis and restoring expression of MyD88, which is an essential signal transducer for all but one TLR. In one aspect, while PTP inhibitors, like other drugs, are subject to the limitations of off-target or on-target / off-cell effects, these results suggest that combining PTP1b inhibition with radiotherapy may yield survival benefits in a tumoragnostic manner, providing a rationale for further study and future clinical trials.Methods for Treating or Preventing Cancer in a Subject

[0049] In one aspect, disclosed herein is a method for treating or preventing cancer in a subject, the method including at least the steps of:(a) administering radiotherapy to the subject; and(b) administering a PTP1 b inhibitor to the subject.

[0050] In an aspect, the PTP1b inhibitor can be selected from BVT948, PTP1 b inhibitor III, NSC87877, PTP1 b inhibitor, PTP inhibitor IV, MSI-1436, ISIS-PTP1BRX, another PTP1 b inhibitor, or any combination thereof. In one aspect, when the PTP1b inhibitor is BVT948, the PTP1b inhibitor can be administered at about 10 mg / kg of the subject’s body weight. In some aspects, the PTP1b inhibitor are administered simultaneously, or are administered sequentially.

[0051] In another aspect, the radiotherapy can be administered at a dosage of from about 6 Gy to about 10 Gy once per day for from about 1 day to about 3 days, or can be administered at a dosage of about 6 Gy, 7 Gy, 8 Gy, 9 Gy, or about 10 Gy for 1 day, 2 days, or 3 days. In an alternative aspect, the radiotherapy can be administered at lower dosages and / or over longer times as long as the total dosage of radiation adds up to from about 6 Gy to about 10 Gy (for example, 10 sessions administering 0.6 Gy each over the course of three weeks, or 20 sessions administering 0.35 Gy each over the course of six weeks). In an aspect, radiation dosage can be selected based on subject body weight, type and location of cancer, patient tolerance to treatment, and the like.

[0052] In a further aspect, a chemotherapeutic agent can optionally be administered to the subject. In one aspect, the chemotherapeutic agent can be selected from daunorubicin, dactinomycin, doxorubicin, bleomycin, mitomycin, nitrogen mustard, chlorambucil, melphalan, cyclophosphamide, 6-mercaptopurine, 6-thioguanine, cytarabine (CA), 5-fluorouracil (5-FU),ATTORNEY DOCKET NO. 222120-2100 floxuridine (5-FUdR), methotrexate (MTX), colchicine, Vincristine, vinblastine, etoposide, teniposide, cisplatin, diethylstilbestrol (DES), vemurafenib, or any combination thereof. In an aspect, when the chemotherapeutic agent is cisplatin, the chemotherapeutic agent can be administered at a dosage of about 5 mg / kg of the subject’s body weight. In an alternative aspect, when the chemotherapeutic agent is vemurafenib, the chemotherapeutic agent can be administered at a dosage of about 30 mg / kg of the subject’s body weight.

[0053] In some aspects, the chemotherapeutic agent can be cisplatin and the PTP1 b inhibitor can be BVT948; however, other combinations of chemotherapeutic agent and PTP1 b inhibitor are contemplated and should also be considered disclosed. In an aspect, the chemotherapeutic agent and the PTP1b inhibitor can be provided in separate compositions and can be administered simultaneously or sequentially. In an alternative aspect, the chemotherapeutic agent and the PTP1 b inhibitor can be provided in a single composition. In any of these aspects, the chemotherapeutic agent and the PTP1 b inhibitor can be administered orally, intravenously, intraperitoneally, or any combination thereof.

[0054] In some aspects, the method can include administering at least one immunotherapeutic agent, such as, for example, a PD-1 inhibitor, a PD-L1 inhibitor, a CTLA4 inhibitor, or any combination thereof, to the subject. Further in this aspect, the PD-1 inhibitor can be pembrolizumab, nivolumab, pidilizumab, AMP-224, AMP-514, PDR001 , or any combination thereof. In another aspect, the PD-L1 inhibitor can be atezolizumab, avelumab, durvalumab, BMS0936559, or any combination thereof. In still another aspect, the CTLA4 inhibitor can be ipilimumab, tremelimumab, or any combination thereof. In any of these aspects, the PTP1b inhibitor, the chemotherapeutic agent if used, and the additional chemotherapeutic agent can be administered simultaneously or sequentially.

[0055] In an alternative aspect, no chemotherapeutic agents are used or required in order to perform the disclosed method. Further in this aspect, the PTP1 b inhibitor and the radiotherapy are sufficient to accomplish the desired results (i.e., successful treatment of cancer, reduction of M2 macrophages in a tumor microenvironment, increasing M1 macrophages in the tumor microenvironment, or other effects as described herein).

[0056] In one aspect, the cancer can be acute lymphoblastic leukemia, acute myeloid leukemia, adrenocortical carcinoma, anal cancer, astrocytoma, basal cell carcinoma, bladder cancer, BRCA1 breast cancer, BRCA2 breast cancer, Burkitt’s lymphoma, carcinoid tumor, cervical cancer, chondroblastoma, chronic lymphocytic leukemia, chronic myelogenous leukemia, colonATTORNEY DOCKET NO. 222120-2100 cancer, cutaneous t-cell lymphoma, endometrial cancer, ependymoma, esophageal cancer, extrahepatic bile duct cancer, gallbladder cancer, glioblastoma, another high-grade glioma, a giant cell tumor of the bone, hairy cell leukemia, head and neck cancer, Hodgkin’s lymphoma, hypopharyngeal cancer, intraocular melanoma, islet cell carcinoma, Kaposi’s sarcoma, laryngeal cancer, lung carcinoma, lip and oral cavity cancer, liver cancer, medulloblastoma, melanoma, Merkel cell carcinoma, mesothelioma, multiple myeloma, nasopharyngeal cancer, neuroblastoma, non-Hodgkin’s lymphoma, non-small cell lung cancer, oropharyngeal cancer, osteosarcoma, ovarian cancer, pancreatic cancer, parathyroid cancer, penile cancer, pheochromocytoma, pituitary tumor, pleuropulmonary blastoma, primary central nervous system lymphoma, prostate cancer, rectal cancer, renal cell cancer, retinoblastoma, rhabdomyosarcoma, salivary gland cancer, secondary acute myeloid leukemia, small cell lung cancer, small intestine cancer, soft tissue sarcoma, squamous cell carcinoma, stomach cancer, testicular cancer, thymoma, thyroid cancer, uterine sarcoma, vaginal cancer, vulvar cancer, Waldenstrom’s macroglobulinemia, Wilms’ tumor, or any combination thereof. In an aspect, the cancer is melanoma or lung carcinoma.

[0057] In another aspect, the subject is a mammal such as, for example, a human, non-human primate, cat, dog, mouse, rat, hamster, guinea pig, horse, goat, cattle, swine, sheep, or rabbit.

[0058] In some aspects, the method reduces growth of a tumor compared to an otherwise identical tumor on which the method has not been performed by at least 50%, 55%, 60%, 65%, 60%, 75%, 80%, 85%, 90%, or at least 95%, wherein growth can be assessed by a method commonly known in the art such as tumor volume, tumor mass, or the like. In some aspects, the method can stop tumor progression completely.

[0059] In one aspect, the method can be repeated from about 1 time to about 3 times, or more times as needed, such as 1 , 2, 3, 4, 5, 6, 7, 8, 9, or about 10 times.

[0060] In some aspects, performing the method reduces numbers of M2 macrophages within a tumor microenvironment (TME) in the subject relative to a TME in a subject on whom the method has not been performed. In another aspect, performing the method increases numbers of M1 macrophages within a tumor microenvironment (TME) in the subject relative to a TME in a subject on whom the method has not been performed.Methods for Enhancing the Efficacy of Radiotherapy in a SubjectATTORNEY DOCKET NO. 222120-2100

[0061] In one aspect, disclosed herein are methods for enhancing the efficacy of radiotherapy in a subject having cancer, the methods including at least the step of administering a PTP1b inhibitor to the subject. In a further aspect, the PTP1b inhibitor can be selected from BVT948, PTP1 b inhibitor III, NSC87877, PTP1b inhibitor, PTP inhibitor IV, MSI-1436, ISIS-PTP1 BRX, or any combination thereof. In one aspect, when the PTP1 b inhibitor is BVT948, the PTP1 b inhibitor can be administered at about 10 mg / kg of the subject’s body weight.

[0062] In some aspects, the method can include administering at least one immunotherapeutic agent, such as, for example, a PD-1 inhibitor, a PD-L1 inhibitor, a CTLA4 inhibitor, or any combination thereof, to the subject. Further in this aspect, the PD-1 inhibitor can be pembrolizumab, nivolumab, pidilizumab, AMP-224, AMP-514, PDR001 , or any combination thereof. In another aspect, the PD-L1 inhibitor can be atezolizumab, avelumab, durvalumab, BMS0936559, or any combination thereof. In still another aspect, the CTLA4 inhibitor can be ipilimumab, tremelimumab, or any combination thereof.

[0063] In one aspect, the cancer can be acute lymphoblastic leukemia, acute myeloid leukemia, adrenocortical carcinoma, anal cancer, astrocytoma, basal cell carcinoma, bladder cancer, BRCA1 breast cancer, BRCA2 breast cancer, Burkitt’s lymphoma, carcinoid tumor, cervical cancer, chondroblastoma, chronic lymphocytic leukemia, chronic myelogenous leukemia, colon cancer, cutaneous t-cell lymphoma, endometrial cancer, ependymoma, esophageal cancer, extrahepatic bile duct cancer, gallbladder cancer, glioblastoma, another high-grade glioma, a giant cell tumor of the bone, hairy cell leukemia, head and neck cancer, Hodgkin’s lymphoma, hypopharyngeal cancer, intraocular melanoma, islet cell carcinoma, Kaposi’s sarcoma, laryngeal cancer, lung carcinoma, lip and oral cavity cancer, liver cancer, medulloblastoma, melanoma, Merkel cell carcinoma, mesothelioma, multiple myeloma, nasopharyngeal cancer, neuroblastoma, non-Hodgkin’s lymphoma, non-small cell lung cancer, oropharyngeal cancer, osteosarcoma, ovarian cancer, pancreatic cancer, parathyroid cancer, penile cancer, pheochromocytoma, pituitary tumor, pleuropulmonary blastoma, primary central nervous system lymphoma, prostate cancer, rectal cancer, renal cell cancer, retinoblastoma, rhabdomyosarcoma, salivary gland cancer, secondary acute myeloid leukemia, small cell lung cancer, small intestine cancer, soft tissue sarcoma, squamous cell carcinoma, stomach cancer, testicular cancer, thymoma, thyroid cancer, uterine sarcoma, vaginal cancer, vulvar cancer, Waldenstrom’s macroglobulinemia, Wilms’ tumor, or any combination thereof. In an aspect, the cancer is melanoma or lung carcinoma.ATTORNEY DOCKET NO. 222120-2100

[0064] In another aspect, the subject is a mammal such as, for example, a human, non-human primate, cat, dog, mouse, rat, hamster, guinea pig, horse, goat, cattle, swine, sheep, or rabbit.

[0065] In some aspects, performing the method reduces numbers of M2 macrophages within a tumor microenvironment (TME) in the subject relative to a TME in a subject on whom the method has not been performed. In another aspect, performing the method increases numbers of M1 macrophages within a tumor microenvironment (T E) in the subject relative to a TME in a subject on whom the method has not been performed.

[0066] Many modifications and other embodiments disclosed herein will come to mind to one skilled in the art to which the disclosed compositions and methods pertain having the benefit of the teachings presented in the foregoing descriptions and the associated drawings. Therefore, it is to be understood that the disclosures are not to be limited to the specific embodiments disclosed and that modifications and other embodiments are intended to be included within the scope of the appended claims. The skilled artisan will recognize many variants and adaptations of the aspects described herein. These variants and adaptations are intended to be included in the teachings of this disclosure and to be encompassed by the claims herein.

[0067] Although specific terms are employed herein, they are used in a generic and descriptive sense only and not for purposes of limitation.

[0068] 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.

[0069] Any recited method can be carried out in the order of events recited or in any other order that is logically possible. That is, unless otherwise expressly stated, it is in no way intended that any method or aspect set forth herein be construed as requiring that its steps be performed in a specific order. Accordingly, where a method claim does not specifically state in the claims or descriptions that the steps are to be limited to a specific order, it is no way intended that an order be inferred, in any respect. This holds for any possible non-express basis for interpretation, including matters of logic with respect to arrangement of steps or operational flow, plain meaning derived from grammatical organization or punctuation, or the number or type of aspects described in the specification.ATTORNEY DOCKET NO. 222120-2100

[0070] All publications mentioned herein are incorporated herein by reference to disclose and describe the methods and / or materials in connection with which the publications are cited. The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention. Further, the dates of publication provided herein can be different from the actual publication dates, which can require independent confirmation.

[0071] While aspects of the present disclosure can be described and claimed in a particular statutory class, such as the system statutory class, this is for convenience only and one of skill in the art will understand that each aspect of the present disclosure can be described and claimed in any statutory class.

[0072] It is also to be understood that the terminology used herein is for the purpose of describing particular aspects only and is not intended to be limiting. 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 the disclosed compositions and methods belong. It will be further understood that terms, such as those defined in commonly used dictionaries, should be interpreted as having a meaning that is consistent with their meaning in the context of the specification and relevant art and should not be interpreted in an idealized or overly formal sense unless expressly defined herein.

[0073] Prior to describing the various aspects of the present disclosure, the following definitions are provided and should be used unless otherwise indicated. Additional terms may be defined elsewhere in the present disclosure.Definitions

[0074] As used herein, “comprising” is to be interpreted as specifying the presence of the stated features, integers, steps, or components as referred to, but does not preclude the presence or addition of one or more features, integers, steps, or components, or groups thereof. Moreover, each of the terms “by,” “comprising,” “comprises,” “comprised of,” “including,” “includes,” “included,” “involving,” “involves,” “involved,” and “such as” are used in their open, non-limiting sense and may be used interchangeably. Further, the term “comprising” is intended to include examples and aspects encompassed by the terms “consisting essentially of” and “consisting of.” Similarly, the term “consisting essentially of” is intended to include examples encompassed by the term “consisting of.ATTORNEY DOCKET NO. 222120-2100

[0075] 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. Thus, for example, reference to “a binding site,” “a primer,” or “an anticancer agent,” include, but are not limited to, mixtures or combinations of two or more such binding sites, primers, or anticancer agents, and the like.

[0076] It should be noted that ratios, concentrations, amounts, and other numerical data can be expressed herein in a range format. 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. Ranges can be expressed herein as from “about” one particular value, and / or to “about” another particular value. Similarly, when values are expressed as approximations, by use of the antecedent “about,” it will be understood that the particular value forms a further aspect. For example, if the value “about 10” is disclosed, then “10” is also disclosed.

[0077] When a range is expressed, a further aspect includes from the one particular value and / or to the other particular value. For example, 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, e.g. the phrase “x to y” includes the range from ‘x’ to ‘y’ as well as the range greater than ‘x’ and less than ‘y’. The range can also be expressed as an upper limit, e.g. 'about x, y, z, or less’ and should be interpreted to include the specific ranges of ‘about x’, ‘about y’, and ‘about z’ as well as the ranges of ‘less than x’, less than y’, and ‘less than z’. Likewise, the phrase ‘about x, y, z, or greater’ should be interpreted to include the specific ranges of ‘about x,’ about y,’ and ‘about z’ as well as the ranges of ‘greater than x,’ greater than y, ’ and ‘greater than z.’ In addition, the phrase “about ‘x’ to ‘y’”, where ‘x’ and ‘y’ are numerical values, includes “about ‘x’ to about ‘y’”.

[0078] It is to be understood that such a range format is used for convenience and brevity, and thus, should be interpreted in a flexible manner to include not only the numerical values explicitly recited as the limits of the range, but also to include all the individual numerical values or subranges encompassed within that range as if each numerical value and sub-range is explicitly recited. To illustrate, a numerical range of “about 0.1 % to 5%” should be interpreted to include not only the explicitly recited values of about 0.1 % to about 5%, but also include individual values (e.g., about 1%, about 2%, about 3%, and about 4%) and the sub-ranges (e.g., about 0.5% toATTORNEY DOCKET NO. 222120-2100 about 1.1%; about 5% to about 2.4%; about 0.5% to about 3.2%, and about 0.5% to about 4.4%, and other possible sub-ranges) within the indicated range.

[0079] As used herein, the terms “about,” “approximate,” “at or about,” and “substantially” mean that the amount or value in question can be the exact value or a value that provides equivalent results or effects as recited in the claims or taught herein. That is, it is understood that amounts, sizes, formulations, parameters, and other quantities and characteristics are not and need not be exact, but may be approximate and / or larger or smaller, as desired, reflecting tolerances, conversion factors, rounding off, measurement error and the like, and other factors known to those of skill in the art such that equivalent results or effects are obtained. In some circumstances, the value that provides equivalent results or effects cannot be reasonably determined. In such cases, it is generally understood, as used herein, that “about” and “at or about” mean the nominal value indicated ±10% variation unless otherwise indicated or inferred. In general, an amount, size, formulation, parameter or other quantity or characteristic is “about,” “approximate,” or “at or about” whether or not expressly stated to be such. It is understood that where “about,” “approximate,” or “at or about” is used before a quantitative value, the parameter also includes the specific quantitative value itself, unless specifically stated otherwise.

[0080] As used herein, the term “effective amount” refers to an amount that is sufficient to achieve the desired modification of a physical property of the composition or material. For example, an “effective amount” of an anticancer agent refers to an amount that is sufficient to achieve the desired improvement in the property modulated by the formulation component, e.g., achieving remission or shrinkage of a tumor. The specific level in terms of wt% in a composition required as an effective amount will depend upon a variety of factors including the type and location of the cancer; patient age, weight, and gender; and other treatments concurrently being administered.

[0081] As used herein, the terms “optional” or “optionally” means that the subsequently described event or circumstance can or cannot occur, and that the description includes instances where said event or circumstance occurs and instances where it does not.

[0082] “Macrophages” as used herein refers to white blood cells that engulf and digest pathogens including cancer cells, debris, microbes, and the like. In a tumor microenvironment, macrophages can differentiate into “M1” or “M2” macrophages, where “M1” macrophages are pro-inflammatory and tend to inhibit tumor formation and growth, while “M2” macrophages are anti-inflammatory and tend to support tumor growth. In an aspect, the disclosed methods reduce numbers of M2 macrophages and / or increase numbers of M1 macrophages in a subject.ATTORNEY DOCKET NO. 222120-2100

[0083] As used herein, “PD-1” refers to “programmed cell death protein 1 ,” which is a cell surface receptor on immune system cells including T cells and B cells. In a further aspect, PD-1 is an immune checkpoint, and “PD-1 inhibitors” block PD-1 , causing the immune system to be activated to attack tumors.

[0084] As used herein, “PD-L1” refers to “programmed death-ligand 1 ,” which is a transmembrane protein that may suppress the adaptive immune system in conditions such as pregnancy, autoimmune disease, and the like, and is highly expressed in many cancers. “PD-L1” binds to PD-1 to modulate activation or inhibition on T cells and / or B cells. “PD-L1 inhibitors” block the immune checkpoint PD-L1 , allowing the subject’s immune system to attack tumor cells.

[0085] “CTLA4” refers to “cytotoxic T-lymphocyte associated protein 4,” which is a protein receptor and immune checkpoint. CTI_A4 is upregulated in T cells after activation, which is commonly seen in cancers. In some aspects, “CTLA4 inhibitors” can be used to inhibit immune system tolerance to tumors, that is, they can be used as immunotherapy agents for cancer patients.

[0086] Unless otherwise specified, temperatures referred to herein are based on atmospheric pressure (i.e., one atmosphere).

[0087] Now having described the aspects of the present disclosure, in general, the following Examples describe some additional aspects of the present disclosure. While aspects of the present disclosure are described in connection with the following examples and the corresponding text and figures, there is no intent to limit aspects of the present disclosure to this description. On the contrary, the intent is to cover all alternatives, modifications, and equivalents included within the spirit and scope of the present disclosure.ASPECTS

[0088] The present disclosure can be described in accordance with the following numbered aspects, which should not be confused with the claims.

[0089] Aspect 1 . A method for treating or preventing cancer in a subject, the method comprising:(a) administering radiotherapy to the subject; and(b) administering a PTP1 b inhibitor to the subject.ATTORNEY DOCKET NO. 222120-2100

[0090] Aspect 2. The method of aspect 1 , wherein the PTP1 b inhibitor comprises BVT948, PTP1 b inhibitor III, NSC87877, PTP1b inhibitor, PTP inhibitor IV, MSI-1436, ISIS-PTP1 BRX, or any combination thereof.

[0091] Aspect s. The method of aspect , wherein the BVT948 is administered at about 10 mg / kg of the subject’s body weight.

[0092] Aspect 4. The method of any one of aspects 1-3, wherein the PTP1 b inhibitor and the radiotherapy are administered simultaneously.

[0093] Aspect 5. The method of any one of aspects 1-3, wherein the PTP1 b inhibitor and the radiotherapy are administered sequentially.

[0094] Aspect s. The method of any one of aspects 1-5, wherein the radiotherapy is administered at a dosage of from about 6 Gy to about 10 Gy once per day for from about 1 to about 3 days.

[0095] Aspect 7. The method of any one of aspects 1-6, further comprising administering a chemotherapeutic agent to the subject.

[0096] Aspect 8. The method of aspect 7, wherein the chemotherapeutic agent comprises daunorubicin, dactinomycin, doxorubicin, bleomycin, mitomycin, nitrogen mustard, chlorambucil, melphalan, cyclophosphamide, 6-mercaptopurine, 6-thioguanine, cytarabine (CA), 5-fluorouracil (5-FU), floxuridine (5-FUdR), methotrexate (MTX), colchicine, Vincristine, vinblastine, etoposide, teniposide, cisplatin, diethylstilbestrol (DES), vemurafenib, or any combination thereof.

[0097] Aspect 9. The method of aspect 8, wherein the cisplatin is administered at a dosage of about 5 mg / kg of the subject’s body weight.

[0098] Aspect 10. The method of aspect 8, wherein the vemurafenib is administered at a dosage of about 30 mg / kg of the subject’s body weight.

[0099] Aspect 11. The method of aspect 8 or 9, wherein the chemotherapeutic agent is cisplatin and the PTP1b inhibitor is BVT948.

[0100] Aspect 12. The method of any one of aspects 8-11 , wherein the chemotherapeutic agent and the PTP1b inhibitor are provided in separate compositions.

[0101] Aspect 13. The method of aspect 12, wherein the chemotherapeutic agent and the PTP1b inhibitor are administered simultaneously or sequentially.ATTORNEY DOCKET NO. 222120-2100

[0102] Aspect 14. The method of any one of aspects 8-11 , wherein the chemotherapeutic agent and the PTP1b inhibitor are provided in a single composition.

[0103] Aspect 15. The method of any one of aspects 8-14, wherein the chemotherapeutic agent and the PTP1 b inhibitor are administered orally, intravenously, intraperitoneally, or any combination thereof.

[0104] Aspect 16. The method of any one of aspects 1-6, wherein a chemotherapeutic agent is not administered to the subject.

[0105] Aspect 17. The method of any one of aspects 1-16, further comprising at least one immunotherapeutic agent to the subject.

[0106] Aspect 18. The method of aspect 17, wherein the at least one immunotherapeutic agent comprises a PD-1 inhibitor, a PD-L1 inhibitor, a CTI_A4 inhibitor, or any combination thereof.

[0107] Aspect 19. The method of aspect 18, wherein the PD-1 inhibitor comprises pembrolizumab, nivolumab, pidilizumab, AMP-224, AMP-514, PDR001 , or any combination thereof.

[0108] Aspect 20. The method of aspect 18, wherein the PD-L1 inhibitor comprises atezolizumab, avelumab, durvalumab, BMS0936559, or any combination thereof.

[0109] Aspect 21. The method of aspect 18, wherein the CTLA4 inhibitor comprises ipilimumab, tremelimumab, or any combination thereof.

[0110] Aspect 22. The method of any one of aspects 17-21 , wherein the PTP1b inhibitor, the chemotherapeutic agent if used, and the immunotherapeutic agent are administered simultaneously or sequentially.

[0111] Aspect 23. The method of any one of aspects 1-22, wherein the cancer comprises acute lymphoblastic leukemia, acute myeloid leukemia, adrenocortical carcinoma, anal cancer, astrocytoma, basal cell carcinoma, bladder cancer, BRCA1 breast cancer, BRCA2 breast cancer, Burkitt’s lymphoma, carcinoid tumor, cervical cancer, chondroblastoma, chronic lymphocytic leukemia, chronic myelogenous leukemia, colon cancer, cutaneous t-cell lymphoma, endometrial cancer, ependymoma, esophageal cancer, extrahepatic bile duct cancer, gallbladder cancer, glioblastoma, another high-grade glioma, a giant cell tumor of the bone, hairy cell leukemia, head and neck cancer, Hodgkin’s lymphoma, hypopharyngeal cancer, intraocular melanoma, islet cell carcinoma, Kaposi’s sarcoma, laryngeal cancer, lung carcinoma, lip and oral cavity cancer, liverATTORNEY DOCKET NO. 222120-2100 cancer, medulloblastoma, melanoma, Merkel cell carcinoma, mesothelioma, multiple myeloma, nasopharyngeal cancer, neuroblastoma, non-Hodgkin’s lymphoma, non-small cell lung cancer, oropharyngeal cancer, osteosarcoma, ovarian cancer, pancreatic cancer, parathyroid cancer, penile cancer, pheochromocytoma, pituitary tumor, pleuropulmonary blastoma, primary central nervous system lymphoma, prostate cancer, rectal cancer, renal cell cancer, retinoblastoma, rhabdomyosarcoma, salivary gland cancer, secondary acute myeloid leukemia, small cell lung cancer, small intestine cancer, soft tissue sarcoma, squamous cell carcinoma, stomach cancer, testicular cancer, thymoma, thyroid cancer, uterine sarcoma, vaginal cancer, vulvar cancer, Waldenstrom’s macroglobulinemia, Wilms’ tumor, or any combination thereof.

[0112] Aspect 24. The method of aspect 23, wherein the cancer is melanoma or lung carcinoma.

[0113] Aspect 25. The method of any one of aspects 1-24, wherein the subject is a mammal.

[0114] Aspect 26. The method of aspect 25, wherein the mammal is a human, non-human primate, cat, dog, mouse, rat, hamster, guinea pig, horse, goat, cattle, swine, sheep, or rabbit.

[0115] Aspect 27. The method of any one of aspects 1-26, wherein the method reduces growth of a tumor compared to an otherwise identical tumor on which the method has not been performed by at least 50%.

[0116] Aspect 28. The method of aspect 27, wherein the method reduces growth of a tumor compared to an otherwise identical tumor on which the method has not been performed by at least 90%.

[0117] Aspect 29. The method of aspect 27, wherein the method stops tumor progression.

[0118] Aspect 30. The method of any one of aspects 1-29, wherein the method is repeated from 1 to 3 times.

[0119] Aspect 31. The method of any one of aspects 1-30, wherein performing the method reduces numbers of M2 macrophages within a tumor microenvironment (TME) in the subject relative to a TME in a subject on whom the method has not been performed.

[0120] Aspect 32. The method of any one of aspects 1-31 , wherein performing the method increases numbers of M1 macrophages within a tumor microenvironment (TME) in the subject relative to a TME in a subject on whom the method has not been performed.

[0121] Aspect 33. A method for enhancing efficacy of radiotherapy in a subject having cancer, the method comprising: administering a PTP1b inhibitor to the subject.ATTORNEY DOCKET NO. 222120-2100

[0122] Aspect 34. The method of aspect 33, wherein the PTP1b inhibitor comprises BVT948, PTP1 b inhibitor III, NSC87877, PTP1 b inhibitor, PTP inhibitor IV, MSI-1436, ISIS-PTP1 BRX, another PTP1 b inhibitor, or any combination thereof.

[0123] Aspect 35. The method of aspect 34, wherein the BVT948 is administered at about 10 mg / kg of the subject’s body weight.

[0124] Aspect 36. The method of any one of aspects 33-35, further comprising at least one immunotherapeutic agent to the subject.

[0125] Aspect 37. The method of aspect 36, wherein the at least one immunotherapeutic agent comprises a PD-1 inhibitor, a PD-L1 inhibitor, a CTI_A4 inhibitor, or any combination thereof.

[0126] Aspect 38. The method of aspect 37, wherein the PD-1 inhibitor comprises pembrolizumab, nivolumab, pidilizumab, AMP-224, AMP-514, PDR001 , or any combination thereof.

[0127] Aspect 39. The method of aspect 37, wherein the PD-L1 inhibitor comprises atezolizumab, avelumab, durvalumab, BMS0936559, or any combination thereof.

[0128] Aspect 40. The method of aspect 37, wherein the CTLA4 inhibitor comprises ipilimumab, tremelimumab, or any combination thereof.

[0129] Aspect 41. The method of any one of aspects 33-40, wherein the cancer comprises acute lymphoblastic leukemia, acute myeloid leukemia, adrenocortical carcinoma, anal cancer, astrocytoma, basal cell carcinoma, bladder cancer, BRCA1 breast cancer, BRCA2 breast cancer, Burkitt’s lymphoma, carcinoid tumor, cervical cancer, chondroblastoma, chronic lymphocytic leukemia, chronic myelogenous leukemia, colon cancer, cutaneous t-cell lymphoma, endometrial cancer, ependymoma, esophageal cancer, extrahepatic bile duct cancer, gallbladder cancer, glioblastoma, another high-grade glioma, a giant cell tumor of the bone, hairy cell leukemia, head and neck cancer, Hodgkin’s lymphoma, hypopharyngeal cancer, intraocular melanoma, islet cell carcinoma, Kaposi’s sarcoma, laryngeal cancer, lung carcinoma, lip and oral cavity cancer, liver cancer, medulloblastoma, melanoma, Merkel cell carcinoma, mesothelioma, multiple myeloma, nasopharyngeal cancer, neuroblastoma, non-Hodgkin’s lymphoma, non-small cell lung cancer, oropharyngeal cancer, osteosarcoma, ovarian cancer, pancreatic cancer, parathyroid cancer, penile cancer, pheochromocytoma, pituitary tumor, pleuropulmonary blastoma, primary central nervous system lymphoma, prostate cancer, rectal cancer, renal cell cancer, retinoblastoma, rhabdomyosarcoma, salivary gland cancer, secondary acute myeloid leukemia, small cell lungATTORNEY DOCKET NO. 222120-2100 cancer, small intestine cancer, soft tissue sarcoma, squamous cell carcinoma, stomach cancer, testicular cancer, thymoma, thyroid cancer, uterine sarcoma, vaginal cancer, vulvar cancer, Waldenstrom’s macroglobulinemia, Wilms’ tumor, or any combination thereof.

[0130] Aspect 42. The method of aspect 41, wherein the cancer is melanoma or lung carcinoma.

[0131] Aspect 43. The method of any one of aspects 33-42, wherein the subject is a mammal.

[0132] Aspect 44. The method of aspect 43, wherein the mammal is a human, non-human primate, cat, dog, mouse, rat, hamster, guinea pig, horse, goat, cattle, swine, sheep, or rabbit.

[0133] Aspect 45. The method of any one of aspects 33-44, wherein the method is repeated from 1 to 3 times.

[0134] Aspect 46. The method of any one of aspects 33-45, wherein performing the method reduces numbers of M2 macrophages within a tumor microenvironment (TME) in the subject relative to a TME in a subject on whom the method has not been performed.

[0135] Aspect 47. The method of any one of aspects 33-46, wherein performing the method increases numbers of M1 macrophages within a tumor microenvironment (TME) in the subject relative to a TME in a subject on whom the method has not been performed.EXAMPLES

[0136] 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 the compounds, compositions, articles, devices and / or methods claimed herein are made and evaluated, and are intended to be purely exemplary of the disclosure and are not intended to limit the scope of what the inventors regard as their disclosure. 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 or is at ambient temperature, and pressure is at or near atmospheric.Example 1 : Pairing PTP Inhibition with Radio- or Chemotherapy Reduced Tumor Growth in Multiple Preclinical Models

[0137] To determine whether PTP inhibition would improve the efficacy of cytotoxic therapy for checkpoint blockade refractory cancers, mice bearing B16F10 or LLC tumors were treated with vehicle, traditional monotherapy (radio- or chemotherapy), PTP inhibitor (BVT948 , 10 mg / kg, IP, once daily), or the combination (FIGs. 1A and 1D).ATTORNEY DOCKET NO. 222120-2100

[0138] B16F10 tumor bearing mice treated with radiotherapy (6 Gy, local, once daily for 3 days) showed a 48% reduction in tumor growth relative to vehicle controls, while combining radiotherapy and BVT948 caused a 78% decrease (FIGs. 1B and 8A-8D). In a proof-of-concept experiment, growth of subcutaneous Lewis Lung Carcinoma tumors, which are highly aggressive and have a different mutational burden and immune repertoire than B16F10, showed a 40% growth reduction following radiotherapy while combination treatments effectively stopped tumor progression over the course of the study (FIGs. 1C and 8A-8D). In both models, PTP inhibition alone had limited (B16F10, ns) or minor (LLC, 19% reduction, ns) effects (FIGs. 1B-1C), respectively.

[0139] To determine whether PTP inhibition would also improve the efficacy of chemotherapy, B16F10 or LLC tumor bearing mice were treated with vehicle, chemotherapy (cisplatin, 5mg / kg, IP, once daily for 3 days), BVT948, or the combination. Neither cisplatin nor BVT948 monotherapy reduced tumor growth in either model (FIGs. 1 E-1 F), suggesting chemotherapeutic resistance and limited direct anti-tumor effects of PTP inhibition, respectively. In both models, combination therapy reduced tumor growth by 40-60% (FIGs. 1 E-1 F and 9A-9D), indicating that PTP inhibition promotes therapeutic efficacy in a cytotoxic treatment dependent manner.

[0140] The GEMM6 (BrafV600E, Pten- / - ) melanoma model is a relatively slower growing cancer and is susceptible to vemurafenib because of the engineered Braf V600E mutation intrinsic to ~60% of human tumors . While the mode of action differs between vemurafenib and cisplatin, both agents cause tumor cell death and DAMP release. In a preliminary experiment, mice bearing smaller GEMM6 tumors (50 mm3) were treated with BVT948 alone. This led to complete tumor regression in 3 of 8 mice, which were immune to rechallenge one month later (FIG. 10A), indicating immunologic memory. The other mice on the study progressed with 5 of 8 showing substantially delayed tumor growth and 2 showing signs of toxicity (FIG. 10A). This was the only indication of compound toxicity in any of the studies conducted, even after daily BVT948 treatment as long as 2 months. Mice bearing larger GEMM6 tumors (200 mm3) treated with combination therapy showed a 47% reduction in tumor growth compared to vehicle and monotherapy treatments, which were statistically equivalent (FIGs. 1G-1 H and 10B-10C).

[0141] While the effects of PTP inhibition were not pronounced in vivo, there were relatively minor changes in tumor cell line viability when treated with PTP inhibitors in vitro, with some compounds promoting cell proliferation and others having the opposite effect. Results were cell line and / or compound specific (FIG. 11 A).ATTORNEY DOCKET NO. 222120-2100

[0142] To determine whether PTP inhibition had other effects on tumor cells in vivo, spatial transcriptomic analysis of panCK+ / CD45- B16F10 cells from vehicle or BVT948 treated tumors was performed. BVT948 treatment led to upregulation of angiogenesis-associated pathways, but other changes were limited (FIG. 11 B).

[0143] Taken together, the results indicate that pairing PTP inhibition with traditional therapeutics can improve treatment efficacy with limited toxicity.Example 2: Activation of the Tumor-Prosl Macrophage Axis Limits the Immune Response to Chemotherapy

[0144] PTP1b inhibition prevents tumor-secreted Prosl from activating Mer to suppress the macrophage pro-inflammatory response in vitro. To determine whether this signaling axis plays a role in limiting the immune response during cytotoxic therapy in vivo, a two-pronged approach was taken.

[0145] Mice bearing parental B16F10 or Prosi-deficient (BdP) tumors were treated with vehicle or cisplatin. While cisplatin treatment did not reduce B16F10 tumor growth, BdP tumor growth was reduced by 63±10% (mean ±SEM) (FIGs. 2A and 12A). RNAseq analysis of disaggregated whole end-stage tumors identified broad changes in gene expression among treatment groups (FIG. 2B). Comparison of cisplatin treated BdP to B16F10 tumors showed increased immune activation marker expression (FIG. 2C) and signaling (FIGs. 2D-2E). Similar results were observed in the comparison of vehicle treated BdP to B16F10 tumors (FIGs. 12B-12D). CIBERSORTX deconvolution also identified macrophages, B cells, and NK cells as predominant intra-tumoral immune populations in respective treatment groups (FIG. 12E)

[0146] Others have demonstrated that PTP1 b genetic deletion or inhibition increases the CD8+ T cell anti-tumor response , . To determine whether macrophages and / or CD8+ T cells potentiate the effects of PTP inhibition in the context of chemotherapy, macrophages and other phagocytes (e.g., dendritic cells , monocytes ) or CD8+ T cells were depleted using clodronate or an anti-CD8 antibody, respectively. Clodronate treatment eliminated the benefits of combination therapy while anti-CD8 antibody treatment had little effect (FIGs. 2F-2G). As expected, clodronate reduced the number of intra-tumoral CD68+ macrophages, as well as expression of the proinflammatory marker CD86 (FIG. 2H). Neither encapsome nor isotype control antibody treatment had significant effects on combination therapy (FIGs. 13A-13B).ATTORNEY DOCKET NO. 222120-2100

[0147] Taken together, the results indicate that the effects of combination therapy are immune mediated, as opposed to being an effect of PTP inhibition on cancer cell intrinsic repair mechanisms. They also highlight the importance of the innate immune response in potentiating the effect combination therapy.Example 3: Pairing PTP Inhibition with Chemotherapy Increases Macrophage Activation, Intra-Tumoral Immune Infiltration, and Survival

[0148] One potential benefit of increasing the efficacy of chemotherapy is that it may be possible to reduce the number of doses given to patients, thereby improving adherence to prescribed treatment regimens and / or patient quality of life. To determine whether PTP inhibition could reduce the number of cisplatin doses necessary, or whether daily BVT948 administration was essential for the effect in the B16F10 model, the dosing schedule for combination therapy was varied. While the initial combination therapy regimen was efficacious in reducing tumor growth by 44%, decreasing the number of days of cisplatin treatment from 3 to 1 , or administering BVT948 every other day, ablated the effect (FIGs. 14A-14B). This suggests that 1) there is a threshold amount of tumor cell death and DAMP release necessary to sufficiently activate macrophages and that 2) daily PTP inhibitor treatment is necessary to prevent macrophage inactivation.

[0149] It was therefore hypothesized that multiple rounds of chemotherapy would sustain DAMP release and further improve outcomes. B16F10 melanoma bearing mice were treated with two rounds of cisplatin, once daily for 3 days, spaced three days apart (FIGs. 3A and 15A-15B). This more intensive treatment regimen was successful in reducing tumor growth by 65% while combination therapy effectively prevented tumor progression during the study (FIGs. 3A and 15A- 15B).

[0150] To better understand the effects of the improved combination therapy on macrophages and other immune cells, single cell RNA sequencing (scRNAseq) was performed. In preparation for scRNAseq, CD45+ immune cells were isolated from B16F10 tumors treated with vehicle, two rounds of cisplatin, daily BVT948, or the combination. Flow cytometry showed that while cisplatin monotherapy substantially reduced immune infiltration, combination therapy led to a 6±1.0-fold increase in intra-tumoral CD45+ cells when compared to cisplatin treatment (FIG. 3B, gating strategy - FIG. 16). Notably, Uniform Manifold Approximation and Projection (UMAP) clustering (FIG. 3C) and cell content (FIG. 17) analysis showed globally reduced immune infiltration following cisplatin treatment though there was less of a decrease in the macrophage populationATTORNEY DOCKET NO. 222120-2100 as compared to other cell subsets. Combination therapy bolstered the infiltration of most immune subsets (FIG. 17).

[0151] Cluster (FIGs. 3D and 18) and pathway (FIG. 19) analysis of macrophage subsets identified two major sub-populations among all treatment groups including pro-wound healing (MO / M2) and pro-inflammatory (M1) polarized cells. While there were more MO / M2 macrophages in the vehicle and monotherapy treated groups, the ratio of polarized macrophages was skewed towards M1 following combination therapy (FIG. 3D). Differential gene expression analysis in macrophage populations showed an increase in inflammatory gene expression after combination therapy with a concomitant decrease in the expression of wound healing associated genes (FIG. 3E). Pathways associated with inflammation, cytokine production, and chemokine-mediated cellular migration were also upregulated in macrophages from combination treated mice relative to cisplatin monotherapy (FIG. 3F).

[0152] Further analysis of cell-cell communications showed that combination therapy increased the number and types of macrophage derived signals to other immune cell subsets (FIGs. 3G and 20), including increases in recruitment (e.g., Ccl / Ccr) and activation (e.g., Tnf-superfamily) signals.

[0153] PTP1b inhibition can have an activating effect on B cells and T cells. Analysis of SingleR annotated B cells subsets did not identify robust changes in developmental state among treatment groups and populations from each arm were largely antigen naive (FIG. 21). Cluster and gene expression analysis of T cells (FIG. 22) identified a relatively small proportion of cytotoxic effector CD8+ T cells, which was increased with BVT948 treatment. However, combination therapy further reduced the number of effector CD8+ T cells. After combination therapy, other immune subsets showed mixed pro-inflammatory and pro-tumor groups including neutrophils and monocytes (FIGs. 23-24, respectively). Dendritic cells were one of the least well-represented innate immune populations, even following combination therapy (FIG. 17).

[0154] Visualization of inferred cell-cell communications were performed using the Cell Chat analytical package . Combination therapy led to a generalized increase in innate immune cell-cell signaling (depicted by increases in line weights from one immune cell subset to another) as compared to cisplatin monotherapy (FIGs. 3H and 11). Interestingly, the signaling to and from T and B cells did not appear to change substantially between cisplatin or combination therapy treated groups (FIG. 3H), further suggesting limited activation.Example 4: Macrophage Responsiveness to Tumor DAMPs is MyD88-MediatedATTORNEY DOCKET NO. 222120-2100

[0155] Cisplatin and other platinum-based chemotherapies induce double stranded DNA breaks and subsequent expression of yH2AX, a biomarker for the DNA damage response . A 4.0±0.7- fold increase in yH2AX protein expression was observed in tumor sections from cisplatin treated mice as compared to vehicle controls (FIG. 4A). Visualization at higher magnification showed yH2AX in peri-nuclear and extracellular spaces after cisplatin treatment (FIG. 4A) indicative of DAMP release.

[0156] Bioinformatic analysis identified a number of pathways upregulated in intra-tumoral pro- inflammatory macrophages, including TLRs (FIG. 4B) that recognize DAMPS. To determine whether tumor secretions (e.g., Prosl) reduce TLR activation in a simplified model, a transwell system was used in which B16F10 cells were co-cultured with macrophages treated with individual TLR agonists to mimic DAMPs. In most cases, tumor secretions limited TLR activation and downstream pro-inflammatory gene expression (FIG. 26). In all cases, pro-inflammatory gene expression was positively correlated with MyD88 expression (FIG. 26). It was therefore posited that tumor suppression of macrophage MyD88 could limit responsiveness to the spectrum of DAMPS.

[0157] A simplified ex vivo co-culture system was developed to test the effects of chemotherapy released DAMPs on macrophage activation in the presence of tumor secretions. To establish an in vitro dose of chemotherapy sufficient to induce DAMP release, B16F10 cells were treated with increasing concentrations of cisplatin and viability measured by MTT assay. Treating B16F10 cells with 100 pM or 500 pM cisplatin reduced cell survival by 29% and 64%, respectively (FIG. 4C). To test the effects of tumor secretions on DAMP-mediated activation, tissue resident (peritoneal) macrophages were cultured on a 0.4 pm pore size permeable membrane in the same well as B16F10 tumor cells. In the co-culture model, even at concentrations of cisplatin that induced robust tumor cell death, limited macrophage activation was observed relative to untreated controls (FIG. 4D). However, co-treatment with BVT948 restored the macrophage inflammatory activation following chemotherapy-mediated DAMP release, even at relatively low (50 pM) concentrations of cisplatin (FIG. 4D). To determine whether BVT948 rescue of DAMP responsiveness was MyD88 dependent, the transwell assay was repeated with macrophages isolated from MyD88 knock-out mice. Neither cisplatin nor BVT948 could restore inflammatory gene expression in the absence of macrophage MyD88 (FIG. 4E), highlighting that MyD88 is essential for DAMP mediated activation. It is important to note that cisplatin treatment of macrophages alone was not sufficient to induce macrophage DAMP / TLR activation (FIG. 27),ATTORNEY DOCKET NO. 222120-2100 likely because macrophages are not proliferating at an appreciation rate sufficient for cisplatin to induce robust cell death.

[0158] In alternative in vitro assays, BVT948 also improved macrophage M1 activation in the presence of tumor DAMP conditioned medium. In these models, tumor cells were either serum starved or treated with radiotherapy, the conditioned DAMP containing medium collected, 0.2 pm filtered, and added to naive macrophages in the presence or absence of BVT948. After 24 hours, macrophage expression of M1 -associated markers was increased when treated with BVT948, as determined by qRT-PCR (FIGs. 28A-28B). Further, to determine whether protein or nucleic acid DAMPs contribute more substantially to macrophage activation in the presence of BVT948, conditioned medium from serum starved tumor cells was treated with protease or benzonase to selectively remove proteins or nucleic acids, respectively. In the presence of BVT948, macrophage activation in response to DAMP conditioned medium was increased, though degradation of either protein or nucleic acid DAMPs limited the M1 response (FIG. 28C).

[0159] As MyD88 was essential for DAMP signaling transduction (FIG. 4E) and reduced by tumor secretions during TLR agonism (FIG. 26), it was hypothesized MyD88 suppression may be a generalized mechanism to limit macrophage TLR activation. In a transwell assay, macrophages were M1-stimulated using the TLR4 agonist LPS and IFNy and co-cultured with syngeneic tumor lines including melanoma (B16F10, GEMM6), lung (LLC), pancreatic (KPC2713, KPPC4548, KPPC4394) or breast (EO771) cancer cells. After 24 hours in co-culture, macrophage MyD88 mRNA and protein expression were analyzed by qRT-PCR (FIG. 4F) or Western blot analysis (FIG. 4G), respectively. In all cases, M1-stimulation increased MyD88 mRNA and protein levels which were in turn reduced to naive levels in the presence of tumor cell secretions. It should also be noted that co-culture of naive macrophages with tumor cells did not further reduce MyD88 protein levels (FIG. 29). This implies that macrophages must be M1 -stimulated in order for tumor- mediated suppression to occur.

[0160] To ascertain whether MyD88 suppression could have clinical implications, TIMER analysis of publicly available TCGA data with the XCell algorithm was used to determine whether intra- tumoral macrophage MyD88 expression is associated with patient survival. Melanoma patients with transcriptionally inferred increases in macrophage content with higher MyD88 expression had a significantly increased survival rate as compared to other groups (FIG. 4H). This finding suggests that pharmacologically rescuing macrophage MyD88 expression within human tumorsATTORNEY DOCKET NO. 222120-2100 may provide a survival benefit, warranting further study into mechanisms governing macrophage MyD88 expression.Example 5: Suppression of Macrophage MyD88 is Facilitated by Tumor-Mediated Inhibition of Statl

[0161] Pathway analysis of the M1 macrophage cluster identified several potential pro- inflammatory signaling mediators that could regulate MyD88 including the Jak-Stat pathway (FIG> 4B). Statl has been described as a positive regulator of MyD88 expression and can be inhibited by tumor cells . Analysis of publicly available TCGA melanoma patient data also showed that intra-tumoral STAT1 is positively correlated with MYD88 expression (FIG. 5A) and patient survival (FIG. 5B). SCENIC analysis also identified a macrophage population transcriptionally regulated by Statl (FIG. 5C) and I rf2 / 7, which are involved in TLR and inflammatory signaling.

[0162] To first determine whether a correlation exists between macrophage Statl and MyD88 expression, a transwell co-culture system was utilized. In this model, a panel of murine tumor cell lines were co-cultured with pro-inflammatory peritoneal macrophages and changes in macrophage phospho- and total-Statl protein levels characterized by Western blot analysis. Interestingly, co-culture reduced macrophage total and phospho-Statl to naive levels (FIG. 5D). However, tumor cells co-cultured with naive macrophages did not significantly reduce Statl expression (FIG. 30), demonstrating context dependence of suppression.

[0163] Because reduced Statl dephosphorylation is associated with decreased nuclear translocation, M1 induced Lyz2-Cre:tdTomato macrophages were cultured in the presence or absence of B16F10 conditioned medium and immunostained for Statl . Corroborating Western blot findings, tumor conditioned medium reduced macrophage Statl expression by 88% and nuclear translocation was halved by the presence of B16F10 secretions (FIG. 5E).

[0164] Statl has proximal and distal MyD88 promoter binding sites (FIG. 31A). Chromatin immunoprecipitation (ChIP) qPCR was used to determine how co-culture affected Statl binding to the MyD88 promoters. While association at the distal site was unchanged after co-culture (FIG. 31 B), binding at the proximal site was reduced by 379±0.9-fold as compared to M1-induced controls (FIG. 5F).

[0165] To further demonstrate that Statl suppression leads to reduced MyD88 expression, M1 induced macrophages were treated with fludarabine, a clinically approved STAT1 inhibitor.ATTORNEY DOCKET NO. 222120-2100Fludarabine suppressed M1 induced macrophage Statl levels (FIG. 32) and reduced MyD88 expression comparably to B16F10 cells (FIG. 5G).ATTORNEY DOCKET NO. 222120-2100Example 6: A Mer, PTP1b, and Statl Ternary Complex Facilitates MyD88 Suppression but is Disrupted by PTP Inhibition

[0166] Tumor-secreted Prosl limits the efficacy of chemotherapy (FIGs. 2A-2H) presumptively by preventing the macrophage response to DAMPs (FIGs. 4A-4H) through suppression of Statl - mediated MyD88 expression (FIGs. 5A-5G). However, the mechanistic linkage between the Prosl receptor, Statl , PTP1 b, and MyD88 remained unclear. Further, as Prosl is part of a larger family of Tyro3 / Axl / Mer (TAM) receptor ligands, whether this form of immunosuppression is redundant or characteristic of other ligands in the family was yet to be determined.

[0167] To identify the roles of respective TAM ligands, gain- and loss-of-function approaches were used. Addition of exogenous Prosl to M1 stimulated macrophages suppressed MyD88 expression comparably to transwell co-culture with B16F10 cells. However, treatment of exogenous Gas6 or Gal3 had limited effects (FIG. 6A). Conversely, pre-treating B16F10 cells with siRNA targeting Prosl or Gas6 prior to co-culture showed that reducing tumor cell Prosl expression prevented MyD88 suppression (FIG. 6B). The efficiency of each siRNA knockdown exceeded 97% for each target (FIG. 33).

[0168] Of Tyro3, Axl, and Mer, Mer is the most highly expressed on tissue resident macrophages, followed by Axl and Tyro313. Each of the receptors has been implicated in immune suppression. Before addressing questions specifically on Mer signaling, it was important to identify the potential roles of other receptors in the setting of MyD88 suppression. A transwell system was utilized in which B16F10 tumor cells were co-cultured with M1-induced Mer, Tyro3, or Axl deficient macrophages and MyD88 expression assayed. Neither Mer nor Tyro3 knockout macrophages could suppress MyD88 in the co-culture model (FIG. 6C). However, Axl deficient macrophages showed MyD88 suppression comparable to wildtype macrophages (FIG. 6C), demonstrating that Axl is not essential in this process. Importantly, M1 stimulation of the three KO macrophages led to equivalent MyD88 expression (FIG. 34), limiting the possibility that differences are due to KO macrophage hypersensitivity.

[0169] In addition, when Mer KO macrophages were transwell co-cultured with tumor cells and treated with cisplatin, pro-inflammatory gene expression was rescued (FIG. 6D), similar to the effect of BVT948 addition (FIG. 4D), further demonstrating a role for Mer in the suppression of DAMP responsiveness.

[0170] After establishing ligand: receptor interactions, the next question to address was which structural motifs of the receptors facilitated suppression. The kinase domains of the TAMATTORNEY DOCKET NO. 222120-2100 receptors have been targeted pharmacologically, with some of the drugs in early-stage clinical trials. While there is contradictory evidence on the role of the Mer kinase domain in suppression of TLR agonism, TAM kinase inhibitors were tested to determine whether they could restore macrophage MyD88 expression comparably to genetic deletion. Surprisingly, when transwell cocultured M1 macrophages were treated with 300 nM TAM kinase inhibitors (UNC2371 , BMS777607, or LDC1267), none of the compounds restored MyD88 expression (FIG. 6E), implying a Mer kinase-independent mechanism.

[0171] Because Mer is expressed at 300-fold higher levels than Tyro3 on tissue resident macrophages, Mer structural motifs that may mediate the effect were a focus. Georgescu and colleagues, and later Mahajan and Earp, described another Mer domain in which amino acid Y867 was involved in NF-kB signaling and the binding of adapter proteins, including Grb2, Shc1 , and Vav1. CRISPR / Cas9 was utilized to generate a mouse model (C57BL / 6 background) bearing the Y867F mutation, similar to an in vitro mutation utilized by Georgescu, et al. Macrophages isolated from Mer Y867F mice were M1-stimulated and co-cultured with B16F10 tumors cells. Similar to the Mer KO macrophages, ablation of the adapter binding site prevented MyD88 suppression (FIG. 6F).

[0172] A role for PTP1b as a downstream mediator of Mer signaling was identified. PTP1b has also been implicated in regulation of Statl phosphorylation , which suggested there may be a link between Mer and Statl signaling and play a part in suppressing DAMP responsiveness. Therefore, structural modeling was used to determine the potential surfaces involved in MER:PTP1 B interaction. Previously solved structures of MER and PTP1 B were docked together with ClusPro 2.0. The balanced, electrostatic-favored, and hydrophobic-favored scoring schemes each converge to the same docked structure, representing the top cluster of structures set away from the rest of the cluster packs. In the docked complex between PTP1 B and MER (FIG. 6G) the interface is composed of a mixture of hydrogen bonding, electrostatic, and hydrophobic interactions and is comprised of a predicted buried surface area between MER and PTP1 B of 1338 and 1232 A2, respectively. A representation of the electrostatic surface potential and hydrogen bonding interactions is shown in FIG. 6H. The complex is reminiscent of the interaction between PTP1 B and the insulin receptor tyrosine kinase domain (IRK) [PDB id: 2B4S], For this reason, the two complex structures were aligned via the PTP1B domains. The alignment showed that MER and IRK share an overlapping binding surface on PTP1 B (FIGs. 35A-35C). Based on the positioning of the MER:PTP1 B interaction and importance of the Mer adapter binding site inATTORNEY DOCKET NO. 222120-2100MyD88 suppression, it is possible that an adapter protein may facilitate the interactions in a context dependent manner.

[0173] To determine whether Mer:PTP1 b:Stat1 complexes are formed during tumor-meditated suppression, immunoprecipitation of PTPIb was conducted on naive, M1 , or transwell co-cultured M1 macrophages. Co-culture increased association of PTP1b with Mer and Statl , 2.14±0.29-fold and 9.80±3.80-fold, respectively (FIG. 6I). Addition of BVT948 during co-culture reduced association of PTP1 b with Mer and Statl (FIG. 61). Presumably, the PTP inhibitor compound interfered with PTP1 b docking with Mer. To confirm complex formation, the experiment was repeated with Statl as the immunoprecipitated target with probes for Mer and PTP1b. Findings were comparable in that B16F10 secretions caused increased association of Mer, PTP1b, and Statl , which was reversed upon treatment with BVT948 (FIG. 36A).

[0174] PTP inhibitors vary in their selectivity and off-target effects. Therefore, multiple inhibitors that target PTP1 b were assayed to determine if they could restore MyD88 expression and for their effect on Statl . M1 -induced macrophages were co-cultured with B16F10 tumor cells and treated with BVT948, PTP inhibitor, or NSC87877. After 24 hours, MyD88 expression was assayed by qRT-PCR and, for all compounds tested, inhibition restored MyD88 mRNA levels (FIG. 6J). In addition, inhibitor treatment increased phospho- and total-Statl , as well as MyD88, protein levels (FIG. 36B).Example 7: PTP Inhibition Counteracts Pros1 :Mer Signaling and Rescues Macrophage Activation During Chemotherapy

[0175] Based on collective in vivo and ex vivo observations, a model signaling diagram was developed (FIG. 7A). Within the tumor, chemo- or radiotherapy should induce tumor cell death and the subsequent release of DAMPs, which promote the macrophage pro-inflammatory response (FIG. 7A). However, upon tumor Prosl -mediated Mer activation, ternary complex formation is induced, leading to downstream suppression of the TLR adapter protein MyD88. With reduced MyD88, macrophages are less able to respond to DAMP-activating TLR signaling, creating a more tumor permissive milieu (FIG. 7A). By blocking the Mer:PTP1b interaction using BVT948, MyD88 expression is rescued, allowing macrophages to respond to tumor derived DAMPS (FIG. 7A).

[0176] This process of events is observed in vivo following cisplatin treatment. In areas immediately adjacent to DAMP release, which is indicated by extracellular yH2AX staining, tumor cells upregulated the expression of Prosl (FIG. 7B). Proximal to Prosl expression, theATTORNEY DOCKET NO. 222120-2100 interactions between Mer and Statl were increased (FIG. 7C), as visualized by Proximity Ligation Assay . However, after combination therapy there was a reduction in Mer: Statl interactions, which were also observed ex vivo (FIG. 61). Treatment with BVT948 increased MyD88 protein in CD68+ macrophages (FIG> 7D) as well as mRNA levels in CD45+ / Cd11b+ / F4:80+ macrophages flow sorted from tumors (FIG. 7E), particularly in the context of combination treatment. Further, macrophage expression of M1-associated genes, including IL1 , IL6, and iNOS were increased following rescue of MyD88 expression (FIG. 7F).Example 8: Discussion

[0177] Herein, an innate immune checkpoint utilized by tumor cells proximal to damaged areas that limits local macrophage DAMP responsiveness is described. Tumor-secreted Prosl actives the macrophage Mer receptor in a kinase-independent manner to induce ternary complex formation with PTP1b and Statl . By limiting Statl activation, either through dephosphorylation or sequestration outside the nucleus, Statl -mediated MyD88 expression is reduced, curtailing TLR signaling. The ability to suppress macrophage MyD88 expression appears to be conserved among different murine tumor cell models and may be a generalized mechanism to limit TLR activation after cytotoxic therapy.

[0178] By pharmacologically inhibiting PTP1 b, the Mer:PTP1b:Stat1 ternary complex is disrupted, MyD88 expression rescued, and macrophage DAMP sensitivity restored. Increasing M1 polarization within the macrophage population causes generalized expansion of intra-tumoral immune subsets and substantial reductions in tumor growth in multiple preclinical models.

[0179] Unlike other studies, a pronounced anti-tumor role for cytotoxic CD8+ T cells after PTP inhibition was not observed; this has previously been shown to increase the efficacy of other therapies like anti-PD-1 and GVAX31. One likely explanation is that chemotherapy substantially decreased the number of CD8+ effector T cells within the tumor, though macrophage numbers were less affected. In this immune repertoire limited context, remaining macrophages were sufficient to potentiate the larger anti-tumor immune response. Interestingly, even after immune infiltration was largely restored by combination therapy, the number of cytotoxic CD8+ T cells did not show a commensurate increase. This suggests the effect of macrophage reactivation was propagated in a manner largely independent of CD8 T cells. It is therefore unclear whether addition of adaptive checkpoint blockade strategies (e.g., anti-PD-1 / PD-L1 and / or anti-CTLA4) could further improve the efficacy of combination therapy, which is an area for future study.ATTORNEY DOCKET NO. 222120-2100

[0180] PTP1b inhibitors are already in clinical trials, including MSI-1436 (Phase I, NCT00806338), PTP1b-Rx (Phase II, NCT00330330), and ABBV-CLS-484 (Phase I, NCT04777994). With reasonable toxicity profiles, these agents or others may have novel usage in combination with existing cytotoxic treatment regimens. Many human tumors express PROS1 which can dampen the innate immune response. DAMPs are also intrinsic to most, if not all, cancers, suggesting that PTP1b inhibition may be a generalized means of improving the efficacy of multiple forms of therapy and providing a rationale for future combinatorial clinical trials.

[0181] In summary, PTP1b inhibition, when paired with radiotherapy or chemotherapy, can substantially reduce tumor growth in multiple preclinical models. Improved efficacy is driven, in part, by restoring the macrophage inflammatory response to DAMPs and the subsequent activation of other immune cell subsets. Because of favorable toxicity profiles for existing PTP1 b inhibitors, there is a rationale for future clinical trials to explore efficacy of combination therapy in cancer patients.Example 9: Materials and Methods

[0182] Study approval. All animal studies were performed in accordance with UAB and UNC Institutional Animal Care and Use Committee guidelines after institutional protocol approval.

[0183] Tumor implantation, treatment, and harvest. For animal studies, 105luciferase-tagged B16F10 melanoma cells, 106GEMM6 melanoma cells or 2x106LCC cells were implanted subcutaneously into the right flank of C57BL / 6J mice (000664, The Jackson Laboratory) of both sexes in roughly equal numbers. Tumor bearing mice were randomly assigned to study arms and received intraperitoneal injections of Cisplatin (5 mg / kg, Tocris), BVT948 (10 mg / kg, Tocris), Vemurafenib (30 mg / kg, Tocris), or described combinations. For macrophage depletion, mice were treated with clodrosome (50 mg / kg) or Encapsome (50 mg / kg) intraperitoneally (Encapsula NanoSciences) 2 days before tumor implantation followed by repeated inoculation (25 mg / kg) every 6 days. For lymphocyte depletion, mice were treated intraperitoneally with anti-mCD8 antibody (10 mg / kg) or an lgG2a isotype control (10 mg / kg) (Invivogen) 2 days before tumor implantation, repeating this inoculation every 7 days. For radiotherapy experiments, mice received 6 Gy of local radiotherapy once daily for 3 days. Unless otherwise indicated, tumors were harvested when they reached 1.5 cm in any direction.

[0184] Generation of transgenic mice. The Lyz2-Cre:R26RtdTomato mouse line was generated as previously described. In brief, Lyz2-Cre mice (004781, The Jackson Laboratory) were crossed with lineage reporter R26RtdTomato mice (007908, The Jackson Laboratory). MyD88-KO andATTORNEY DOCKET NO. 222120-2100Mer-KO mice were obtained from The Jackson Laboratory (009088 and 011122, respectively). Tyro3-KO and Axl-KO mice were obtained from the colony of the Earp Lab and previously described. MerTK Y867F mouse line was generated using CRISPR / Cas9 genome editing by the UAB Transgenic and Genetically Engineered Models Core.

[0185] Peritoneal macrophage isolation and culture. Peritoneal macrophages were isolated from 8-week-old C57BL / 6J mice (male or female, in roughly equal numbers). Lavage of macrophages was performed by injection of PBS (Gibco) into the peritoneal cavity followed by collection of injected fluid. After collection, cells were centrifuged at 1200 rpm for 5 minutes, resuspended in DMEM high glucose, 10% FBS, 1% penicillin-streptomycin (Gibco) and plated. After 2 hours, adherent cells were washed with PBS and maintained in DMEM / F12 supplemented with 10% FBS, 1 % penicillin-streptomycin and 20 ng / mL of M-CSF (Biolegend) for 2 days at 37 °C with 5% CO2. Macrophages were treated for 24 hours with 50 ng / mL LPS (Sigma Millipore), 100 ng / mL IFNy (BioLegend), 300 ng / mL PAM3CSK4 (InvivoGen), 107cells / mL heat killed Listeria monocytogenes (HKLM) (InvivoGen), 100 ng / mL Flagellin Salmonella typhimurium standard (InvivoGen), 10 ng / mL FSL1 (InvivoGen), 2.5 pg / mL ssRNA40 (InvivoGen), 2.5 pg / mL CL 075 (Tocris), 5 pM ODN1826, 50 pM Fludarabine (SelleckChem), 50 nM Momelotinib (SelleckChem), 5 pM BVT948 (Sigma Millipore), 200 pM PTP1b inhibitor III (Santa Cruz Biotechnology Inc.), 5 pM NSC87877 (Santa Cruz Biotechnology Inc.), 50 pM PTP1 b inhibitor (Cayman), 1 mM PTP Inhibitor IV (Santa Cruz Biotechnology Inc.), 1 pM TSC401 (Tocris), 300 nM UNC2371 (SelleckChem), 300 nM BMS777607 (SelleckChem), 300 nM LDC1267 (SelleckChem) 1.5 pg / mL recombinant Prosl (R&D Systems), 200 ng / mL recombinant Gas6 (R&D Systems), 2.5 pg / mL recombinant Gal3 (R&D Systems) or 10-500 pM Cisplatin (Tocris).

[0186] Cell culture and treatment. B16F10 (CRL-6475, ATCC), Lewis lung carcinoma (LLC) (CRL-1642, ATCC), KPC2713 (Dr. Yuliya Pylayeva-Gupta Laboratory, UNC) were cultured in DMEM high glucose supplemented with 10% FBS, 1% penicillin-streptomycin (Gibco). GEMM6 (Dr. Alisha Holtzhausen, UNC), KPPC4662 (Dr. Yuliya Pylayeva-Gupta laboratory, UNC), KPPC4548 (Yuliya Pylayeva-Gupta laboratory, UNC) and E0771 (CRL-3461 , ATCC) were maintained in RPMI 1640 supplemented with 10% PBS, 1% penicillin / streptomycin (Gibco) while PyMT cells (CRL-3279, ATCC) were cultured in Ham’s F-12K with 10% FBS, 1 % penicillin- streptomycin (Gibco). Cells were periodically assayed for mycoplasma contamination (ThermoFisher). Transwell assays were performed in 0.4 pm polyester membrane insert culture plates (Corning). 500,000 cells of each line were plated per well in DMEM / F12 supplemented with 10% FBS, 1% penicillin-streptomycin and 20 ng / mL of M-CSF for culture with mouseATTORNEY DOCKET NO. 222120-2100 macrophages. Forthe radiotherapy in vitro experiment, 10,000,000 LLC cells were irradiated once with a 6 Gy dose and then cultured for 1 day. Conditioned media were then collected, 0.2 pm filtered and added to primary macrophages for 24 hours.

[0187] siRNA transfection assay. Non-targeting (Dharmacon), Prosl (Dharmacon) and Gas6 (Dharmacon) siRNAs were transfected into B16F10 cells using Lipofectamine RNAiMAX reagent (ThermoFisher) according to manufacturer’s instructions; adding fresh DM EM high glucose media supplemented with 10% FBS and 1 % penicillin-streptomycin (Gibco) 5 hours after the start of the experiment. 2 days after the start of transfection, B16F10 cells were resuspended with 0.05% Trypsin (Gibco) and used for transwell experiments.

[0188] DAMP characterization assay. B16F10 or GEMM6 cells were resuspended in DMEM media deficient of FBS and amino acids, with 1% penicillin-streptomycin (Gibco) and incubated for 3 days. Conditioned media was collected, 0.2 pm filtered and incubated with 0.35 U / mL proteinase K-agarose beads (Sigma Millipore) or 40 U / mL of benzonase endonuclease (Sigma Millipore) for 4 hours at 37 °C with rotation when indicated. Conditioned media was diluted twofold with DMEM / F12 with 10% FBS, 1 % penicillin-streptomycin (Gibco) and 20 ng / mL of M-CSF (Biolegend), supplemented with 5 pM BVT948 (Sigma Millipore) and added to primary macrophages for 24 hours.

[0189] MTT. B16F10, GEMM6 or LLC cells (3,000) were seeded in 96-well plates (Corning) and treated with 5 pM NSC87877 (Santa Cruz Biotechnology Inc.), 50 pM PTP1b inhibitor (Cayman), 2 pM MSI-1436 (MedChemExpress) or 10-500 pM Cisplatin (Cayman) for 24 hours. After treatment, cells were incubated with 0.5 mg / mL of 3-(4,5-dimethylthiazol-2-yl)-2,5- diphenyltetrazolium bromide (MTT) (Invitrogen) for4h at 37 °C. Formazan crystals were dissolved with isopropanol (Fisher Scientific) containing 4mM HCI (Fisher Scientific) and 10% NP-40 (ThermoFisher). Optical density was measured at 570 nm using a plate reader (BioTek).

[0190] qRT-PCR. RNA was isolated using the RNeasy Plus Mini Kit (Qiagen) and cDNA prepared using the Reverse Transcription System (Promega). qRT-PCR was performed using the PowerUp™ SYBR™ Green Master Mix (Applied Biosystems) on a QuantStudio Pro Real-time PCR system (Applied Biosystems) with the following cycling parameters: 95 °C for 10 min, 50 cycles of 95 °C for 10 seconds followed by 60 °C for 1 min. Melt curve analysis was also performed. Expression fold change was calculated using the 2-AACTmethod .

[0191] Immunohistochemistry and immunofluorescence analysis. Immunohistochemistry was carried out on formalin-fixed, paraffin-embedded 4 pm sections using primary antibodies againstATTORNEY DOCKET NO. 222120-2100 yH2AX (#9718S, Cell Signaling Technology) or Prosl (MBS8503666, MyBioSource) and the rabbit specific HRP / DAB IHC Detection Kit-Micro-polymer (ab236469, Abeam). Immunofluorescence was carried out using primary antibodies against CD68 (#97778, Cell Signaling Technology), CD86 (105001, BioLegend), CD206 (AF2535, R&D Systems) or MyD88 (#4283S, Cell Signaling Technology), and Alexa-fluor-594 or Alexa-fluor-647 conjugated secondary antibodies. Sections were imaged using a BZ-X810 microscope (Keyence) and quantified using Keyence analysis software. When required, staining of multiple markers on a single slide was performed by staining and imaging the first antigen, followed by stripping, and re-probing for a different antigen. During the stripping process, slides were incubated with a 2% SDS (Bio-Rad), 0.8% beta-mercaptoethanol (Millipore Sigma), 62.5 mM Tris-HCI (Fisher Scientific) buffer for 30 min at 56 °C, and washed with PBS four times, 15 minutes each, before being incubated with the antibody for the second target.

[0192] Proximate ligation (PLA) assay. The PLA assay was performed as described previously . Briefly, antigen retrieval was performed for formalin-fixed, paraffin-embedded 4 pm sections using pre-heated Trilogy buffer (922P-09, Millipore Sigma) for 30 min. Slices were then permeabilized with 0.5% Triton X-100 (BP151-100, Fisher Scientific) and incubated with antibodies against Mer (AF591 , R&D Systems) and Statl (9172S, Cell Signaling Technology). Protein-protein interactions were visualized using the Duo-Link In situ Red Starter kit Goat / Rabbit (DUO92105, Millipore Sigma). Sections were then imaged using a BZ-X810 microscope (Keyence) and quantified using the Keyence software.

[0193] Immunocytochemistry. After treatments, murine macrophages were fixed with 4% formaldehyde (Electron Microscopy Sciences) for 15 minutes at room temperature and permeabilized with acetone (Fisher Scientific) for 10 minutes at -20 °C. Samples were blocked using BlockAid blocking solution (ThermoFisher), incubated with primary antibodies to MyD88 (4283S, Cell Signaling), CD86 (105001 , Biolegend) or total STAT1 (9172S, Cell Signaling) followed by Alexa Fluor 488-labeled secondary antibody (A-21206 or A-21210, ThermoFisher), then mounted with SlowFade Gold Antifade with DAPI (Invitrogen). Images were taken with using a BZ-X810 fluorescence microscope (Keyence). Fluorescence intensity was measured with BZ- X800 software while colocalization was analyzed using ImageJ software (NIH) with JACoP plugin.

[0194] Coimmunoprecipitation and Western blot. Coimmunoprecipitation was performed using protease A / G agarose beads (Santa Cruz Biotechnology Inc.) and antibodies for PTP1b (Abeam) or STAT1 (Cell Signaling). Samples were incubated for 3 days at 4 °C with rotation and washedATTORNEY DOCKET NO. 222120-2100 three times with RIPA buffer before loading on 10% Mini-PROTEAN TGX stain-free gels (BioRad). After electrophoresis using the Mini-PROTEAN Tetra System (Bio-Rad), proteins were transferred to a 0.2 pm nitrocellulose Trans-Blot Mini Turbo Transfer Pack (Bio-Rad) using a Trans-Blot Turbo Transfer System (Bio-Rad). Membranes were blocked with 5% BSA (Fisher Scientific) for 1 hour and assayed with antibodies against MyD88 (4283S, Cell Signaling), total STAT1 (9172S, Cell Signaling), phospho-Tyrosine 701 STAT1 (9167S, Cell Signaling), phospho- Serine 727 STAT1 (8826S, Cell Signaling), PTP1 b (ab252928, Abeam) Mer (14-5751-82, ThermoFisher) or fB-tubulin (2128S, Cell Signaling). Detection was performed with ECL HPR- linked anti-rabbit antibody (7074S, Cell Signaling) and the Clarity-Max Western ECL substrate (Bio-Rad). Blots were imaged with the ChemiDoc MP Imaging System (Bio-Rad) and quantitated using Imaged software (NIH).

[0195] ChIP assay. ChIP was performed as previously described with modifications. Briefly, 3-5 million cells were PBS-washed and crosslinked with formaldehyde (1%), followed by quenching with 0.125 M glycine. After washing with cold PBS, cells were suspended in lysis buffer (50 mM HEPES pH 7.5, 140 mM NaCI, 1 mM EDTA, 10% glycerol, 0.5% NP-40, 0.25% TritonX-100, and protease inhibitors) and subjected to sonication to shear the chromatin DNA into sizes ranging from 200 to 500 bp. The fragmented chromatin was centrifuged at 15,000 rpm for 15 min at 4 °C. The supernatant was incubated with STAT1 antibody (Cell Signaling) and Protein G Dynabeads magnetic beads (Invitrogen) at 4 °C overnight. After washing six times with RIPA wash buffer (50 mM Hepes-KOH, pH 7.6, 500 mM LiCI, 1 mM EDTA, 1 % NP-40, and 0.7% Na-Deoxycholate) and once with TE buffer (10 mM Tris-HCI pH 8.0, 1 mM EDTA, and 50 mM NaCI), chromatin was eluted from beads in elution buffer (50 mM Tris-HCI pH 8.0, 10 mM EDTA, and 1 % SDS). After reverse crosslinking, ChIP DNA was purified using DNA purification columns (Zymo Research) and subjected to downstream quantitative real-time PCR analysis (ChlP-qPCR).

[0196] Flow Cytometry. Tumors were disaggregated after resection with 1 mg / mL of collagenase IV (Millipore Sigma), 0.1 mg / mL of hyaluronidase V (Millipore Sigma) and 20 U / mL of DNasel (Millipore Sigma) for 2 hours at 37 °C. Tumor lysates were then blocked for 15 min with a CD16 / CD32 antibody (BD Biosciences) and stained for 20 min with eBioscience cell viability dye (65-0866-14, Invitrogen), brilliant violet 421 CD45, APC CD11b and Alexa Fluor 488 F4 / 80 antibodies (147719, 101212 and 123120 respectively, Biolegend) in PBS supplemented with 2% FBS. 1 ,000 CD45+ / CD11b+ / F4:80+ macrophages were sorted using a BD FACSAria Cell sorter (BD Biosciences) into PBS. qRT-PCR was then performed with the macrophage lysate as described.ATTORNEY DOCKET NO. 222120-2100

[0197] Single-cell preparation and transcriptomic analysis. All sequencing experiments were performed at the UAB Flow Cytometry and Single Cell Core Facility using Chromium Single Cell 3’ Reagent Kits (10x Genomics). Following cell harvest, staining, 50,000 CD45+ cells were sorted into PBS. After viability determination, cells were loaded onto the Chromium 3’ Chip (v3.1). Chromium 3’ chips containing cell suspensions, Single Cell 3’ Gel Beads and Partitioning Oil were loaded into the Chromium Single Cell Controller (10x Genomics) to generate Gel Bead-in- EMulsion (GEMs) droplets. Next, mRNA, cell-hashing antibody- derived tags (HTOs) were reverse transcribed, barcoded, and purified using a Dynabeads MyOne SILANE bead cleanup mixture (Invitrogen). The cDNA was amplified by PCR according to the Chromium Single Cell 3’ Reagent Kits protocol with primers added for HTOs (1 mL of 0.1 mlVI, 5'- GTGACTGGAGTTCAGACGTGTGCTC-3', SEQ ID NO. 10). Amplified cDNA was size separated with SPRI beads (Beckman Coulter) into <300 nt fragments containing HTOs and >300 nt fragments containing mRNA-templated cDNA. cDNA sequencing libraries were generated according to manufacturer’s instructions (10x Genomics). Purified HTO sequencing libraries were amplified to append P5 and P7 adapter sequences compatible with the Illumina Flow Cell using a common SI-PCR forward primer (10x Genomics Single Cell 3P). Sequencing libraries were pooled. Libraries were sequenced on an Illumina NovaSeq instrument and paired-end reads according to instructions from 10x Genomics (UAB Genomics Core). Bowtie Version 1.1.1 was used with parameter -e80, and reads were aligned to Ensembl release 85 Mus musculus GRCm38 reference. Each library was filtered to include only abundant barcodes (>500 total counts). Next, putatively stressed or dying cells were excluded based on mitochondrial genes, and normalization of data was carried out. Scrublet was used to identify clusters of cell doublets that co-expressed marker genes of distinct cell types. Sample independent graph-based clustering to remove cells expressing transcripts for committed cells followed by differentially expressed gene identification and pathway analysis was performed using Partek Flow software (Partek Inc.) or Seurat. Cellular annotation was performed using the SingleR package. Cell-cell communication analysis was performed using the Cell Chat analytical package using the R interface. The SCENIC package was used to identify regulons in the macrophage population.

[0198] RNAseq and analysis. End-stage B16F10 or BdP tumors were disaggregated, lysed, and mRNA collected using the RNeasy Plus Mini Kit (Qiagen). RNA quality was assayed with a spectrophotometer (Implen) and sequencing libraries were generated using NEBNext UltraTM RNA Library Prep Kit for the Illumina system (New England Biolabs). Library preparations were then sequenced using the HiSeq XTEN platform (Illumina). Raw data was first processed throughATTORNEY DOCKET NO. 222120-2100 fastp software to eliminate adapter, poly-N and low-quality reads. Paired-end clean reads were aligned to the reference genome using the Spliced Transcripts Alignment to a Reference (STAR) software (Illumina). R package clusterProfile was used for KEGG and Reactome enrichment analysis. CibersortX analysis was carried out using the CibersortX website.

[0199] Spatial transcriptomic analysis. Tumor sections were labeled with Alexa fluor 532 pan-CK and alexa fluor 594 CD45 antibodies (NBP2-33200AF532 and NBP1-44763AF594 respectively, Novus Biologicals) and pan-CK+ / CD45- regions were processed using the NanoString GeoMx platform, sequencing conducted, and data analyzed in R Studio using the “GeomxTools” package available on Bioconductor.

[0200] Modeling protein-protein interactions. Coordinates for Mer (PDBID: 7AAZ) and PTP1b (PDBID: 7LFO) were acquired from the RCSB Protein Data Bank . Incomplete side-chains in Mer were manually built with COOT . Molecular docking of the Mer and PTP1b was performed with ClusPro 2.0. The final model was solvated and energy minimized with YASARA . Electrostatic parameters were calculated with PDB2PQR and APBS . Images of structural models were generated with PyMOL

[0201] Survival plots. Analysis of the correlation between MYD88, PROS1 and STAT 1 expression and melanoma patient survival was performed using the Gene Expression Profiling Interactive Analysis (GEPIA) tools and TIMER2.0.

[0202] Statistical analysis. Statistical analysis was performed using GraphPad Prism software (GraphPad Software Inc.). A p value less than 0.05 was considered as statistically significant. Graphs display the mean ± SEM for each experimental group. The number of repetitions performed to arrive at the total sample size (n) is detailed in each figure legend.

[0203] It should be emphasized that the above-described embodiments of the present disclosure are merely possible examples of implementations set forth for a clear understanding of the principles of the disclosure. Many variations and modifications may be made to the abovedescribed embodiment(s) without departing substantially from the spirit and principles of the disclosure. All such modifications and variations are intended to be included herein within the scope of this disclosure and protected by the following claims.REFERENCES1. Alam MS. Proximity Ligation Assay (PLA). Curr Protoc Immunol. 2018 Nov;123(1):e58. doi: 10.1002 / cpim.58. Epub 2018 Sep 20. PMID: 30238640; PMCID: PMC6205916.ATTORNEY DOCKET NO. 222120-2100 Aran D, et al. xCell: digitally portraying the tissue cellular heterogeneity landscape. Genome Biol. 2017 Nov 15;18(1):220. doi: 10.1186 / s13059-017-1349-1. PMID: 29141660; PMCID: PMC5688663. Asano T. Drug Resistance in Cancer Therapy and the Role of Epigenetics. 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Claims

ATTORNEY DOCKET NO. 222120-2100CLAIMSWhat is claimed is:

1. A method for treating or preventing cancer in a subject, the method comprising:(a) administering radiotherapy to the subject; and(b) administering a PTP1 b inhibitor to the subject.

2. The method of claim 1 , wherein the PTP1b inhibitor comprises BVT948, PTP1b inhibitor III, NSC87877, PTP1b inhibitor, PTP inhibitor IV, MSI-1436, ISIS-PTP1 BRX, or any combination thereof.

3. The method of claim 2, wherein the BVT948 is administered at about 10 mg / kg of the subject’s body weight.

4. The method of claim 1 , wherein the PTP1b inhibitor and the radiotherapy are administered simultaneously.

5. The method of claim 1 , wherein the PTP1b inhibitor and the radiotherapy are administered sequentially.

6. The method of claim 1 , wherein the radiotherapy is administered at a dosage of from about 6 Gy to about 10 Gy once per day for from about 1 to about 3 days.

7. The method of claim 1 , further comprising at least one immunotherapeutic agent to the subject.

8. The method of claim 7, wherein the at least one immunotherapeutic agent comprises a PD-1 inhibitor, a PD-L1 inhibitor, a CTLA4 inhibitor, or any combination thereof.

9. The method of claim 8, wherein the PD-1 inhibitor comprises pembrolizumab, nivolumab, pidilizumab, AMP-224, AMP-514, PDR001 , or any combination thereof.

10. The method of claim 8, wherein the PD-L1 inhibitor comprises atezolizumab, avelumab, durvalumab, BMS0936559, or any combination thereof.

11. The method of claim 8, wherein the CTLA4 inhibitor comprises ipilimumab, tremelimumab, or any combination thereof.

12. The method of claim 1 , wherein the cancer comprises acute lymphoblastic leukemia, acute myeloid leukemia, adrenocortical carcinoma, anal cancer, astrocytoma, basal cell carcinoma, bladder cancer, BRCA1 breast cancer, BRCA2 breast cancer, Burkitt’s lymphoma, carcinoid tumor, cervical cancer, chondroblastoma, chronic lymphocytic leukemia, chronic myelogenousATTORNEY DOCKET NO. 222120-2100 leukemia, colon cancer, cutaneous t-cell lymphoma, endometrial cancer, ependymoma, esophageal cancer, extrahepatic bile duct cancer, gallbladder cancer, glioblastoma, another highgrade glioma, a giant cell tumor of the bone, hairy cell leukemia, head and neck cancer, Hodgkin’s lymphoma, hypopharyngeal cancer, intraocular melanoma, islet cell carcinoma, Kaposi’s sarcoma, laryngeal cancer, lung carcinoma, lip and oral cavity cancer, liver cancer, medulloblastoma, melanoma, Merkel cell carcinoma, mesothelioma, multiple myeloma, nasopharyngeal cancer, neuroblastoma, non-Hodgkin’s lymphoma, non-small cell lung cancer, oropharyngeal cancer, osteosarcoma, ovarian cancer, pancreatic cancer, parathyroid cancer, penile cancer, pheochromocytoma, pituitary tumor, pleuropulmonary blastoma, primary central nervous system lymphoma, prostate cancer, rectal cancer, renal cell cancer, retinoblastoma, rhabdomyosarcoma, salivary gland cancer, secondary acute myeloid leukemia, small cell lung cancer, small intestine cancer, soft tissue sarcoma, squamous cell carcinoma, stomach cancer, testicular cancer, thymoma, thyroid cancer, uterine sarcoma, vaginal cancer, vulvar cancer, Waldenstrom’s macroglobulinemia, Wilms’ tumor, or any combination thereof.

13. The method of claim 12, wherein the cancer is melanoma or lung carcinoma.

14. The method of claim 1 , wherein the subject is a mammal.

15. The method of claim 14, wherein the mammal is a human, non-human primate, cat, dog, mouse, rat, hamster, guinea pig, horse, goat, cattle, swine, sheep, or rabbit.

16. The method of claim 1 , wherein the method reduces growth of a tumor compared to an otherwise identical tumor on which the method has not been performed by at least 50%.

17. The method of claim 16, wherein the method reduces growth of a tumor compared to an otherwise identical tumor on which the method has not been performed by at least 90%.

18. The method of claim 16, wherein the method stops tumor progression.

19. The method of claim 1 , wherein the method is repeated from 1 to 3 times.

20. The method of claim 1 , wherein performing the method reduces numbers of M2 macrophages within a tumor microenvironment (TME) in the subject relative to a TME in a subject on whom the method has not been performed.

21. The method of claim 1 , wherein performing the method increases numbers of M1 macrophages within a tumor microenvironment (TME) in the subject relative to a TME in a subject on whom the method has not been performed.ATTORNEY DOCKET NO. 222120-210022. A method for enhancing efficacy of radiotherapy in a subject having cancer, the method comprising: administering a PTP1 b inhibitor to the subject.

23. The method of claim 22, wherein the PTP1b inhibitor comprises BVT948, PTP1b inhibitor III, NSC87877, PTP1 b inhibitor, PTP inhibitor IV, MSI-1436, ISIS-PTP1BRX, another PTP1 b inhibitor, or any combination thereof.

24. The method of claim 23, wherein the BVT948 is administered at about 10 mg / kg of the subject’s body weight.

25. The method of claim 22, further comprising at least one immunotherapeutic agent to the subject.

26. The method of claim 25, wherein the at least one immunotherapeutic agent comprises a PD- 1 inhibitor, a PD-L1 inhibitor, a CTLA4 inhibitor, or any combination thereof.

27. The method of claim 26, wherein the PD-1 inhibitor comprises pembrolizumab, nivolumab, pidilizumab, AMP-224, AMP-514, PDR001 , or any combination thereof.

28. The method of claim 26, wherein the PD-L1 inhibitor comprises atezolizumab, avelumab, durvalumab, BMS0936559, or any combination thereof.

29. The method of claim 26, wherein the CTI.A4 inhibitor comprises ipilimumab, tremelimumab, or any combination thereof.

30. The method of claim 22, wherein the cancer comprises acute lymphoblastic leukemia, acute myeloid leukemia, adrenocortical carcinoma, anal cancer, astrocytoma, basal cell carcinoma, bladder cancer, BRCA1 breast cancer, BRCA2 breast cancer, Burkitt’s lymphoma, carcinoid tumor, cervical cancer, chondroblastoma, chronic lymphocytic leukemia, chronic myelogenous leukemia, colon cancer, cutaneous t-cell lymphoma, endometrial cancer, ependymoma, esophageal cancer, extrahepatic bile duct cancer, gallbladder cancer, glioblastoma, another highgrade glioma, a giant cell tumor of the bone, hairy cell leukemia, head and neck cancer, Hodgkin’s lymphoma, hypopharyngeal cancer, intraocular melanoma, islet cell carcinoma, Kaposi’s sarcoma, laryngeal cancer, lung carcinoma, lip and oral cavity cancer, liver cancer, medulloblastoma, melanoma, Merkel cell carcinoma, mesothelioma, multiple myeloma, nasopharyngeal cancer, neuroblastoma, non-Hodgkin’s lymphoma, non-small cell lung cancer, oropharyngeal cancer, osteosarcoma, ovarian cancer, pancreatic cancer, parathyroid cancer, penile cancer, pheochromocytoma, pituitary tumor, pleuropulmonary blastoma, primary central nervous system lymphoma, prostate cancer, rectal cancer, renal cell cancer, retinoblastoma,ATTORNEY DOCKET NO. 222120-2100 rhabdomyosarcoma, salivary gland cancer, secondary acute myeloid leukemia, small cell lung cancer, small intestine cancer, soft tissue sarcoma, squamous cell carcinoma, stomach cancer, testicular cancer, thymoma, thyroid cancer, uterine sarcoma, vaginal cancer, vulvar cancer, Waldenstrom’s macroglobulinemia, Wilms’ tumor, or any combination thereof.

31. The method of claim 30, wherein the cancer is melanoma or lung carcinoma.

32. The method of claim 22, wherein the subject is a mammal.

33. The method of claim 32, wherein the mammal is a human, non-human primate, cat, dog, mouse, rat, hamster, guinea pig, horse, goat, cattle, swine, sheep, or rabbit.

34. The method of claim 22, wherein the method is repeated from 1 to 3 times.

35. The method of claim 22, wherein performing the method reduces numbers of M2 macrophages within a tumor microenvironment (T E) in the subject relative to a TME in a subject on whom the method has not been performed.

36. The method of claim 22, wherein performing the method increases numbers of M1 macrophages within a tumor microenvironment (T E) in the subject relative to a TME in a subject on whom the method has not been performed.

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