Immune checkpoint inhibitors (ICI) have changed the treatment paradigm for many cancers but have not shown benefit in prostate cancer. Chronic inflammation contributes to the immunosuppressive prostate tumor microenvironment and is associated with poor response to ICIs. The primary source of inflammatory cytokine production is the inflammasome. In this study, we identify the proviral integration site for Moloney murine leukemia virus (PIM) kinases as regulators of inflammasome activation in tumor-associated macrophages (TAM). The analysis of clinical data from a cohort of patients with treatment-naïve, hormone-responsive prostate cancer revealed that tumors from patients with high PIM1/2/3 displayed an immunosuppressive tumor microenvironment characterized by high inflammation and a high density of repressive immune cells, most notably TAMs. Macrophage-specific knockout of PIM reduced tumor growth in syngeneic models of prostate cancer. Transcriptional analyses indicated that eliminating PIM from macrophages enhanced the adaptive immune response and increased cytotoxic immune cells. Combined treatment with PIM inhibitors and ICIs synergistically reduced tumor growth. Immune profiling revealed that PIM inhibitors sensitized prostate cancer tumors to ICIs by increasing tumor suppressive TAMs and increasing the activation of cytotoxic T cells. Our data implicate macrophage PIM as a driver of inflammation that limits ICI potency and provide preclinical evidence that PIM inhibitors are an effective strategy to improve the ICI efficacy in prostate cancer.

211 Background: The Proviral Integration site for Moloney murine leukemia virus ( PIM1, 2, and 3) kinases are frequently overexpressed in prostate cancer (PC) and associated with aggressiveness and immune evasion. PIM overexpression increases macrophage infiltration and survival in in vivo models of PC. Here, we characterized the association of PIM1/2/3 triplet-combined High versus Low with PC immune signatures on real-world patients' clinical presentation. Methods: 44 patients of treatment-naive metastatic hormone-sensitive PC (mHSPC) patients (pts) samples were analyzed by next-generation sequencing (592, NextSeq; WES, NovaSeq), (WTS; NovaSeq) (Caris Life Sciences, Phoenix, AZ). PC with PIM1/2/3-high(H) (N=28) and -low(L) (N=16) expressions were classified by top and bottom quartile, respectively. Pathway enrichment was determined by GSEA (Broad Inst). Immune cell fractions were calculated by deconvolution of WTS using Quantiseq. Statistical significance was determined by Mann-Whitney U and adjusted for multiple comparisons (p<0.05). Continuous and categorical variables were evaluated using t-tests or Kruskal-Wallis Rank sum tests and Chi-square or Fisher’s exact tests, respectively. Results: mHSPC pts median ages were 67.28 (SD 10.65), 88% White, 44% non-Hispanic, ECOG <2 95% and other demographics showed no differences between PIM-H and L. PIM-H vs L tumors presentation was 48 vs 35% Gleason >9, 82 vs 57% T>3, 76 vs 69% N1, and 92 vs 81% M1, all p>0.05. Median PSA levels at diagnosis were 66.0 [14.0-450.7] vs 24.0 [6.6-95.0] by PIM-H and L, p=0.14. PIM-H had a higher median MAPK activation score compared to PIM-L PC (0.85 vs -1.38, q<0.05). PIM-H PC exhibited higher PSA (548.2 vs 213.2) and AR (118.6 vs 50.8) expression, all q<0.05. PIM-H had enrichment of protein secretion signaling pathway (NES: 1.2; FDR <0.25). PIM-H had higher expression of immunostimulatory ( IL1β, TNF, IL-2, and TNFSF13, FC: 1.9-3.1, p<0.05) and hypoxia-related genes ( HIF1⍺, ARNT, VEGFA, PDGFB, LDHA, NDRG1, SLC2A, PGK1, and DDIT4, FC: 1.3-2.4, p<0.05). PIM-H had higher expression of MHC class I (HLA-A, -B, -C, TAP1, TAP2, B2M, FC: 1.9-2.2, all p<0.05) and MHC class II (HLA-DPA1, -DRB1, -DPB2, -DQB2, -DBP1, -DQB1 FC: 1.8-3.2, all p<0.05) genes. PIM-H had increased infiltration of B cells, M2 MØ and NK cells (all p <=0.05). PIM-H PC had a higher T cell inflamed score (70 vs -138, p<0.05). Conclusions: These data indicate a strong association of PIM expression with increased MAPK activation score, T cell inflamed score, inflammatory, PSA, AR, MHC class I and MHC class II gene expression, and differential immune cell infiltration. However, this did not significantly translate to a worse clinical presentation of mHSPC. A better understanding of these differences with additional research may provide a rationale for tailored therapeutic approaches for PIM-expressing mHSPC.

Abstract Prostate cancer is the second leading cause of cancer-related deaths in American men. While prostate cancer patients typically respond to androgen-deprivation therapy, androgen receptor signaling inhibitor (ARSI), and/or taxane chemotherapy, patients inevitably develop resistance, and the disease progresses to an untreatable and lethal form known as castrate resistant prostate cancer (CRPC). Immunotherapy has changed the treatment paradigm for many types of cancer; however, immune checkpoint inhibitors have not shown clinical benefits in CRPC. Clinical and preclinical evidence indicates that this lack of efficacy can largely be attributed to the immunosuppressive tumor microenvironment. Tumor associated macrophages (TAMs) secrete factors that promote tumor progression and suppress antitumor immunity, making them a promising therapeutic target. The Proviral Integration site for Moloney murine leukemia virus (PIM) kinases are serine/threonine kinases that are overexpressed in prostate cancer. PIM regulates many signaling pathways that promote cell proliferation and survival. However, how PIM kinase alters the prostate tumor immune microenvironment and impacts immunotherapy resistance is not well understood. We analyzed primary prostate and metastatic lymph node samples from treatment-naive metastatic hormone-sensitive prostate cancer patients. Prostate cancer samples with PIM1/PIM2/PIM3-high and -low expression were classified by top and bottom quartile, respectively. PIM high tumors had higher expression of immunostimulatory genes (IL1β, TNF, and TNFSF13), increased infiltration of M2 macrophages, B cells, and NK Cells, and a higher T cell inflamed score compared to PIM-low tumors. Our results demonstrate that PIM overexpressing prostate tumors display increased inflammation and infiltration of immunosuppressive immune cells, including M2 macrophages. Due to the changes observed in inflammation, we hypothesized that PIM kinase may regulate macrophage inflammatory signaling. We treated bone marrow derived macrophages with a PIM kinase inhibitor and showed that PIM inhibition suppresses inflammasome signaling and the release of the pro-inflammatory cytokine, IL-1β. Chronic inflammation can lead to the recruitment of TAMs, further promoting resistance to immune checkpoint inhibitors. Utilizing a syngeneic mouse model of prostate cancer, we demonstrated that PIM inhibition in combination with immune checkpoint blockade synergistically decreased tumor growth. Furthermore, immunoprofiling demonstrated that combination treatment enhances T cell activity. Overall, our results suggest that PIM kinase plays an important role in regulating the prostate tumor immune microenvironment and that PIM kinase may be a potential target to enhance the efficacy of immunotherapy for the treatment of prostate cancer. Citation Format: Amber N. Clements, Kai Sutterby, Sachin Kumar Deshmukh, Sharon Wu, Joanne Xiu, Alex Farrell, Milan Radovich, Chadi Nabhan, Elisabeth I. Heath, Rana R. McKay, Alejandro Recio-Boiles, Noel A. Warfel. PIM kinases alter the prostate tumor immune microenvironment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6875.

e17080 Background: The Proviral Integration site for Moloney murine leukemia virus (PIM) kinases, a family of pro-survival kinases ( PIM1, PIM2, and PIM3), are frequently overexpressed in prostate cancer (PC) and associated with PC aggressiveness and immune evasion. Preclinical data indicate that PIM1 overexpression increases macrophage infiltration and survival using in vivo models of PC. Here, we characterized the association of PIM1, PIM2, and PIM3 with PC immune signatures. Methods: 77 primary prostate and 11 metastatic lymph node samples from treatment-naive metastatic hormone-sensitive PC pts were analyzed by DNA next-generation sequencing (592-gene, NextSeq; WES, NovaSeq) and Whole Transcriptome Sequencing (WTS; NovaSeq) (Caris Life Sciences, Phoenix, AZ). PC with PIM1/PIM2/PIM3-high(H) and -low(L) expression were classified by top and bottom quartile, respectively. Androgen receptor (AR) signature and Neuroendocrine Prostate Cancer (NEPC) score were calculated based on the expression level of previously defined gene signatures (Hieronymus et al. 2006, Beltran et al. 2016). Pathway enrichment was determined by GSEA (Broad Inst). Immune cell fractions were calculated by deconvolution of WTS using Quantiseq. Statistical significance was determined by chi-square and Mann-Whitney U (p < 0.05) and adjusted for multiple comparisons (q < 0.05). Results: PIM1/PIM2/PIM3-H PC had a higher median MAPK activation score (MPAS) compared to PIM1/PIM2/PIM3-L PC (0.9, 1.3, 0 vs -1.6, -1.4, -1.1 respectively, all p < 0.05). There was no difference in AR signature or NEPC score between PIM-H and PIM-L PC. PIM3-H PC exhibited higher PSA expression (3.15-fold, p < 0.05). PIM1/PIM2-H PC had higher AR expression (1.9 and 3.5-fold, respectively, all p < 0.05). PIM2/PIM3-H PC had enrichment of PI3K/AKT/mTOR, IL2/STAT5, IL6/JAK/STAT3, KRAS, TGFβ, NOTCH, hypoxia signaling, and ROS, P53, OXPHOS pathway and EMT (NES: 1.3-1.5, all FDR < 0.25). PIM1/PIM2-H PC had higher expression of immunostimulatory genes ( IL1β, TNF, and TNFSF13, FC: 1.1-3.1, p < 0.05). PIM1/PIM2/PIM3-H PC had higher expression of MHC class I ( HLA-A, HLA-B, HLA-C, B2M, FC: 1.3-2.5, all p < 0.05) and MHC class II ( HLA-DPA1, HLA-DRB1, HLA-DPB2, TAP1, TAP2, FC: 1.1-3.2, all p < 0.05) genes. PIM3-H PC had increased infiltration of M2 macrophages (8% vs 5.2%) and NK cells (5.2% vs 3.3%), while PIM2-H PC had increased Tregs (1.7% vs 0%) (all p < 0.05). Moreover , PIM1/PIM2/PIM3-H PC had a high T cell inflamed score compared to PIM1/PIM2/PIM3-L PC (66, 80, 56 vs -143, -157, -136, respectively, all p < 0.05). Conclusions: Our data suggest a strong association between PIM expression and increased MAPK activation score, T cell inflamed score, inflammatory, MHC class I and MHC class II gene expression, and differential immune cell infiltration. A better understanding of these differences will provide a rationale for tailored therapeutic approaches for PIM-expressing PC.