No abstract

T-cell based immunotherapies that produce durable and sometimes curative responses in other malignancies have failed in pancreatic ductal adenocarcinoma (PDAC) due to poor T cell infiltration and tumor immunogenicity. We and others have demonstrated that induction of cellular senescence and its accompanying senescence-associated secretory phenotype (SASP) can be a powerful way to enhance T cell infiltration into tumors and reactivate a different type of innate Natural Killer (NK) cell anti-tumor immunity that can mediate tumor control. Here, we found that the pancreas tumor microenvironment (TME) suppresses NK cell surveillance following therapy-induced senescence (TIS) with MEK and CDK4/6 inhibitor treatment through EZH2-mediated repression of pro-inflammatory SASP genes. Genetic or pharmacological inhibition of EZH2 or its methyltransferase activity enhanced the production of pro-inflammatory SASP factors and led to NK and T cell infiltration and immune-mediated tumor control in transplanted and genetically engineered PDAC mouse models. Mechanistically, EZH2 suppression induced expression of SASP factors CCL2 and CXCL9/10 necessary for lymophocyte chemotaxis into the pancreas TME. EZH2 levels were also associated with reduced NK cell numbers, SASP expression, and overall survival in a PDAC patient cohort. Taken together these results demonstrate that EZH2 mediates repression of the pro-inflammatory SASP in the pancreas TME, and that EZH2 blockade in combination with senescence-inducing therapies could be a powerful means to reactivate absent NK and T cell surveillance in PDAC to achieve immune-mediated tumor responses. Citation Format: Loretah Chibaya, Katherine C. Murphy, Yvette Lopez Diaz, Kelly D. DeMarco, Haibo Liu, Sneha Gopalan, Melissa Faulkner, Junhui Li, John P. Morris IV, Yu-jui Ho, Janelle Simon, Wei Luan, Amanda Kulick, Elisa de Stanchina, Karl Simin, Lihua J. Zhu, Thomas G. Fazzio, Scott W. Lowe, Marcus Ruscetti. EZH2 inhibition remodels the inflammatory senescence-associated secretory phenotype and promotes immune surveillance in PDAC [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer; 2022 Sep 13-16; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2022;82(22 Suppl):Abstract nr C015.

Most prostate cancers are immunologically “cold” with poor tumor immunogenicity and a lack of cytotoxic T cells, and as such are generally unresponsive to immune checkpoint blockade (ICB) therapies that can reactivate T cell immunity and lead to durable and curative responses in other treatment-refractory solid tumors. We recently demonstrated that therapeutic induction of a durable growth arrest phenotype known as cellular senescence in the setting of pancreatic cancer can be novel approach to make “cold” tumors “hot” and sensitize them to anti-PD-1 ICB (Ruscetti et al. Cell 2020). This senescence-driven immunological effect is mediated through induction of immunomodulatory cell surface molecules, including MHC-I expression required for antigen presentation to T cells, and activation of the senescence-associated secretory phenotype (SASP), a collection of pleiotropic cytokines, chemokines, and other growth factors that can remodel the surrounding tumor-immune landscape. While this acute SASP activation can promote cytotoxic CD8+ T and Natural Killer (NK) cell anti-tumor immunity in some contexts, chronic SASP can alternatively lead to immune suppression and tumor cell invasion and metastasis that contributes to relapse after therapy. Here we set out to characterize the senescence-inducing capacity of chemotherapies, DNA repair inhibitors, and cell cycle targeting agents implicated in cellular senescence and used in the treatment of castration-resistant prostate cancer (CRPC), and the impact of their subsequent SASPs on immune responses and therapy outcomes. Using a suite of genetically-defined murine prostate cancer cell lines and an in vitro pipeline to rapidly define senescence phenotypes following treatment, we found that many commonly used cancer therapeutics can induce cellular senescence, with chemotherapies, Aurora kinase inhibitors, and CDK inhibitors producing the most robust senescence-induced cell cycle arrest across conditions. Prostate tumor cells harboring compound tumor suppressor losses such as Pten, p53, and Rb1 were more susceptible to therapeutic senescence induction. Strikingly, only a subset of therapies that induce senescence-associated growth arrest produced an inflammatory SASP and induction of activating NK cell ligands and antigen presentation and processing genes necessary for NK and T cell immunity. Moreover, whereas most SASPs induced NK cell activity in vitro, they also polarized macrophages toward a pro-tumorigenic and immune suppressive M2 phenotype that may limit their anti-tumor efficacy. CDK inhibitors produced a SASP that could activate lymphocytes without inducing suppressive myeloid populations in MYC-driven tumors, suggesting their use may be a promising approach to target prostate cancers harboring the currently “undruggable” MYC oncogene. Collectively, our studies demonstrate that induction of senescence and its non-cell autonomous arm, the SASP, could be a unique approach to treat CRPC and create new immunological dependencies that can be potentially harnessed for prostate cancer immunotherapy. Citation Format: Lin Zhou, Katherine C. Murphy, Jarin Snyder, Kelly D. DeMarco, Boyang Ma, Marcus Ruscetti. Leveraging therapy-induced senescence for prostate cancer immunotherapy [abstract]. In: Proceedings of the AACR Special Conference: Advances in Prostate Cancer Research; 2023 Mar 15-18; Denver, Colorado. Philadelphia (PA): AACR; Cancer Res 2023;83(11 Suppl):Abstract nr B035.

The advent of immunotherapies such as immune checkpoint blockade (ICB) has greatly expanded the therapeutic options available to patients with treatment-refractory solid tumors. However, patients with castration-resistant prostate cancer (CRPC) generally remain unresponsive to ICB therapy due to an immunologically “cold” prostate tumor microenvironment (TME) with limited accessibility to cytotoxic lymphocytes such as Natural Killer (NK) and T cells that can mediate potent anti-tumor immunity. During the course of tumor evolution, CRPCs acquire many genomic alterations and copy number changes that can influence therapy outcomes. Yet the impact of such genetic alterations on the immune TME and response to ICB remains largely unknown, in part due to the lack of models to functionally study them in an immune competent setting. To this end, we previously developed a novel electroporation-based genetically engineered mouse model (EPO-GEMM) to rapidly generate genetically defined prostate tumors de novo within an intact prostate TME (Leibold*, Ruscetti* et al., Cancer Discovery 2020). Here, we applied the EPO-GEMM approach to characterize the immune landscapes of distinct genetic subtypes of CRPC and subsequently identify and target tumor intrinsic mediators of immune suppression for prostate cancer immunotherapy. Using transposon systems to overexpress the MYC oncogene and CRISPR/Cas9 vectors to inactivate tumor suppressor genes (TSGs) commonly altered in CRPC (Pten, P53, Rb1, Apc), we generated a suite of genetically distinct prostate cancer-bearing EPO-GEMMs. Histological analysis revealed genotype-specific immune infiltrates, with MYC-driven tumors generally lacking cytotoxic CD8+ T cells and harboring increased numbers of regulatory T cells (Tregs). A similar correlation was also observed between MYC levels and cytotoxic lymphocyte suppression in CRPC patient samples. In particular, tumors with compound MYC overexpression and p53 loss (MP) displayed drastic NK cell suppression. Cytokine and RNA-seq profiling of primary EPO-GEMM CRPCs, tumor-derived cell lines, and isogenic Myc-CaP cells engineered with TSG losses revealed that MP alterations led to not only inhibition of inflammatory cytokine and interferon signaling and antigen presentation/processing gene expression, but also induction of VEGF signaling and increased angiogenesis in the prostate TME. Treatment of MP tumors with a VEGFR2 antibody to block VEGF signaling significantly reduced tumor growth and increased NK and CD8+T cell infiltration and activation, indicating a phenotypic switch from a traditionally “cold” TME. Our results demonstrate that the genetic configuration of prostate cancer shapes its surrounding TME. Additionally, targeting VEGF signaling may be a mechanism to overcome ICB resistance in prostate cancer. More broadly, we believe that by understanding the tumor intrinsic mechanisms driving immune suppression, we can identify rational immunotherapy combinations for CRPC based on the genetic fingerprint of a patient’s tumor for precision medicine. Citation Format: Katherine C. Murphy, Kelly DeMarco, Yvette Lopez-Diaz, Lin Zhou, Marcus Ruscetti. Identifying and targeting the genetic determinants of immune suppression and immunotherapy failure in prostate cancer [abstract]. In: Proceedings of the AACR Special Conference: Advances in Prostate Cancer Research; 2023 Mar 15-18; Denver, Colorado. Philadelphia (PA): AACR; Cancer Res 2023;83(11 Suppl):Abstract nr A078.

T cell-activating immunotherapies that produce durable and even curative responses in some malignancies have failed in pancreatic ductal adenocarcinoma (PDAC) due to rampant immune suppression and poor tumor immunogenicity. We and others have demonstrated that induction of cellular senescence and its accompanying senescence-associated secretory phenotype (SASP) can be an effective approach to activate not only T cell but also cytotoxic Natural Killer (NK) cell-mediated anti-tumor immunity. Here we found that the pancreas tumor microenvironment (TME) suppresses NK and T cell surveillance following therapy-induced senescence through EZH2-mediated repression of pro-inflammatory SASP genes. Genetic or pharmacological inhibition of EZH2 or its methyltransferase activity stimulated the production of pro-inflammatory SASP chemokines CCL2 and CXCL9/10 that led to enhanced NK and T cell infiltration and tumor eradication in preclinical PDAC mouse models. EZH2 activity was also associated with suppression of SASP-associated inflammatory chemokines and cytotoxic lymphocyte immunity and reduced overall survival in a PDAC patient cohort. These results demonstrate that EZH2 mediates epigenetic repression of the pro-inflammatory SASP in the pancreas TME, and that EZH2 blockade in combination with senescence-inducing therapies could be a powerful means to potentiate NK and T cell surveillance in PDAC to achieve immune-mediated tumor control.