Search

Recently developed inhibitors of polymerase theta (POLθ) have demonstrated synthetic lethality in BRCA-deficient tumor models. To examine the contribution of the immune microenvironment to antitumor efficacy, we characterized the effects of POLθ inhibition in immunocompetent models of BRCA1-deficient triple-negative breast cancer (TNBC) or BRCA2-deficient pancreatic ductal adenocarcinoma (PDAC). We demonstrate that genetic POLQ depletion or pharmacological POLθ inhibition induces both innate and adaptive immune responses in these models. POLθ inhibition resulted in increased micronuclei, cGAS/STING pathway activation, type I interferon gene expression, CD8+ T cell infiltration and activation, local paracrine activation of dendritic cells and upregulation of PD-L1 expression. Depletion of CD8+ T cells compromised the efficacy of POLθ inhibition, whereas antitumor effects were augmented in combination with anti-PD-1 immunotherapy. Collectively, our findings demonstrate that POLθ inhibition induces immune responses in a cGAS/STING-dependent manner and provide a rationale for combining POLθ inhibition with immune checkpoint blockade for the treatment of HR-deficient cancers.

Bioorganic & medicinal chemistry 78, 117130 (2023). doi:10.1016/j.bmc.2022.117130, PPAR gamma (PPARG) is a ligand activated transcription factor that regulates genes involved in inflammation, bone biology, lipid homeostasis, as well as a master regulator of adipogenesis and a potential lineage driver of luminal bladder cancer. While PPARG agonists lead to transcriptional activation of canonical target genes, inverse agonists have the opposite effect through inducing a transcriptionally repressive complex leading to repression of canonical target gene expression. While many agonists have been described and tested clinically, inverse agonists offer an underexplored avenue to modulate PPARG biology in vivo. Current inverse agonists lack favorable in vivo properties; herein we describe the discovery and characterization of a series of orally bioavailable 4-chloro-6-fluoroisophthalamides as covalent PPARG inverse-agonists, BAY-5516, BAY-5094, and BAY-9683. Structural studies of this series revealed distinct pre- and post-covalent binding positions, which led to the hypothesis that interactions in the pre-covalent conformation are primarily responsible for driving affinity, while interactions in the post-covalent conformation are more responsible for cellular functional effects by enhancing PPARG interactions with its corepressors. The need to simultaneously optimize for two distinct states may partially explain the steep SAR observed. Exquisite selectivity was achieved over related nuclear receptors in the subfamily due in part to a covalent warhead with low reactivity through an SNAr mechanism in addition to the specificity gained through covalent binding to a reactive cysteine uniquely positioned within the PPARG LBD. BAY-5516, BAY-5094, and BAY-9683 lead to pharmacodynamic regulation of PPARG target gene expression in vivo comparable to known inverse agonist SR10221 and represent new tools for future in vivo studies to explore their potential utility for treatment of disorders of hyperactivated PPARG including luminal bladder cancer and other disorders., Published by Elsevier, Amsterdam [u.a.]

Cancers deficient in homologous recombination (HR) repair secondary to mutations in genes such as BRCA1 or BRCA2, are dependent on alternative DNA damage response (DDR) pathways to maintain genomic integrity, rendering them susceptible to synthetic lethal targeting of these pathways. Recently, inhibitors of polymerase theta (POLθ, encoded by POLQ), the critical enzyme in microhomology-mediated end-joining (MMEJ), have been shown to be synthetic lethal with HR repair deficiency (Zhou et al. Nature Cancer 2021). Both HR and MMEJ require nucleolytic DNA end-resection to allow for DSB repair, and we have previously shown that MMEJ acts as a barrier to DNA end-resection at DSBs (Patterson-Fortin et al. Cancer Research 2022). Given the synthetic lethality between HR and MMEJ leads to unrestrained DNA end-resection generating chromosomal abnormalities and the release of nuclear DNA into the cytoplasm, we hypothesized that POLθ inhibition in HR-deficient cancers would activate the cGAS/STING innate immune response pathway and facilitate immunotherapy. To investigate the interactions of POLθ inhibition with the immune microenvironment in HR-deficient cancers, we used human cell lines and genetically modified mouse models representative of BRCA1-deficient triple-negative breast cancer (TNBC) and BRCA2-deficient pancreatic ductal adenocarcinoma (PDAC). Genetic or pharmacological inhibition of POLθ using novobiocin, a first-in-class inhibitor of the POLθ ATPase domain, induced significantly increased cytosolic dsDNA contained in micronuclei. This free DNA was sensed by the cytosolic DNA sensor cyclic GMP-AMP (cGAMP) synthetase (cGAS), increasing synthesis of cGAMP, in HR-deficient tumor cells but not in HR-proficient tumor cells. Increased cGAMP bound to and activated stimulator of interferon genes (STING), triggering phosphorylation of TBK1 and ultimately of IRF3. Activation of the cGAS/STING pathway by POLθ inhibition drove the expression of type I interferon response elements, including PD-L1. Depletion of STING by siRNA or by CRISPR abrogated this pro-inflammatory signaling and abolished the anti-tumor efficacy of novobiocin-mediated POLθ inhibition. Pharmacologic inhibition of POLθ enhanced Granzyme B+ CD8+ T-cell tumor infiltration. Importantly, antibody-mediated depletion of CD8+ T-cell severely compromised the anti-tumor efficacy of novobiocin-mediated POLθ inhibition, whereas anti-tumor activity of POLθ inhibition was augmented with the addition of either anti-PD-1 or anti-CTLA-4 antibodies. These results demonstrate that POLθ inhibition in HR-deficient cancers mediates a pro-inflammatory response in HR-deficient TNBC or PDAC tumor microenvironments, and that immune checkpoint blockade inhibition enhances the therapeutic efficacy of POLθ inhibition. Citation Format: Jeffrey Patterson-Fortin, Heta Jadhav, Constantia Pantelidou, Tin Phan, Carter Grochala, Anita K. Mehta, Jennifer L. Guerriero, Gerburg M. Wulf, Brian M. Wolpin, Ben Z. Stanger, Andrew J. Aguirre, James M. Cleary, Alan D. D'Andrea, Geoffrey I. Shapiro. Polymerase theta inhibition activates the cGAS-STING pathway and cooperates with immune checkpoint blockade in BRCA-deficient cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6190.

Recently developed inhibitors of polymerase theta (POLQ) have demonstrated synthetic lethality in pre-clinical tumor models of BRCA1 and BRCA2-deficient cancers. Since other DNA repair inhibitors, including PARP inhibitors, induce changes in the tumor immune microenvironment (Pantelidou et al., Cancer Discovery, 2019), we asked whether POLQ inhibition would cause similar alterations in preclinical models of pancreatic ductal adenocarcinoma (PDAC). Initially, we generated BRCA2-deficient cells by CRISPR-mediated deletion of the BRCA2 gene in a murine KrasLSL-G12D/+;Trp53LSL-R172H/+;Pdx1-Cre;Rosa26YFP/YFP (KPCY) PDAC cell line (Li et al., Immunity, 2018). Injection of these cells into syngeneic mice generated an aggressive immunocompetent BRCA2-deficient tumor model. We next demonstrated that novobiocin (NVB), a first-in-class inhibitor of the POLQ ATPase domain (Zhou et al., Nature Cancer, 2021), induces both innate and adaptive immune responses in this model through activation of the cGAS/STING pathway. NVB exposure resulted in upregulation of micronuclei, increased cGAS/STING pathway activation, increased type I interferon expression, increased CD8+ T-cell infiltration, and local paracrine activation of dendritic cells. NVB-mediated POLQ inhibition also upregulated PD-L1 expression, correlating with increased cGAS/STING pathway activation. These NVB-induced changes were associated with antitumor activity. Whereas depletion of CD8+ T-cells compromised the efficacy of NVB, the antitumor effects of POLQ inhibition were augmented when combined with anti-PD-1 immunotherapy. Taken together, our findings demonstrate that NVB-mediated POLQ inhibition induces immune responses in a cGAS/STING-dependent manner and provides a rationale for combining POLQ inhibition with immune checkpoint blockade for the treatment of BRCA2-deficient pancreatic cancers. Citation Format: Jeffrey Patterson-Fortin, Heta Jadhav, Constantia Pantelidou, Tin Phan, Carter Grochala, Jie Hao, Junning Wang, Elizabeth A. Andrews, Jennifer L. Guerriero, Brian M. Wolpin, Ben Z. Stanger, Andrew A. Aguirre, James M. Cleary, Alan A. D'Andrea, Geoffrey I. Shapiro. Polymerase theta inhibition synergizes with anti-PD-1 immunotherapy in BRCA2-deficient pancreatic ductal adenocarcinoma [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 C010.

Introduction: CDK4/6 inhibitor (CDK4/6i) resistance is a pressing clinical problem for patients with ER+ breast cancer who are treated with CDK4/6 inhibitors combined with hormonal therapy. Our previous work identified CDK6 overexpression as a critical determinant of acquired resistance to CDK4/6i. Bromodomain and extra-terminal domain (BET) family proteins (BRD2, BRD3, BRD4, and BRDT) play important roles in regulating the expression of oncogenes in multiple cancer types. BET inhibitors (BETi), such as JQ1, have been extensively studied for the treatment of cancers. It has been shown that BRD4 is recruited to the CDK6 gene promoter and that BET inhibition reduces CDK6 mRNA and protein expression. In this study, we investigated the potential of BET inhibition to reverse CDK4/6i resistance of ER+ breast cancer. Methods: By long-term exposure to increasing concentrations of a CDK4/6i, we developed MCF7 and T47D ER+ breast cancer cell lines with acquired resistance to several FDA-approved CDK4/6 inhibitors demonstrating marked CDK6 overexpression. Cell viability and survival assays were performed to assess the in vitro efficacy of BETi. Flow cytometry was performed to evaluate cell cycle alterations as well as cell death. Gene expression was analyzed by Western blotting and qPCR to examine cell cycle-related molecular changes. Markers of apoptosis were also evaluated by Western blotting. CDK4/6i-resistant ER+ tumors were established in immunodeficient mouse models to explore the in vivo efficacy of BETi. Results: Our data showed that JQ1 significantly reduced cell viability and survival of CDK4/6i-resistant ER+ breast cancer cells in the presence of palbociclib, a commonly used CDK4/6i for the treatment of metastatic ER+ HER2- breast cancer. JQ1 also induced marked G1 cell cycle arrest and apoptosis in CDK4/6 inhibitor-resistant MCF7 and T47D cell lines in a time and dose-dependent manner. Analyses of key molecular mediators of cell cycle progression identified CDK6 and cyclin D1 as the major targets of JQ1 in CDK4/6i-resistant cells. Furthermore, we demonstrated that JQ1 suppressed CDK6 expression at the mRNA level. Importantly, we determined that growth suppression and killing of CDK4/6i-resistant cells by BETi were largely the consequence of CDK6 downregulation by BET inhibition. We have also established CDK4/6i-resistant xenografts successfully from MCF7 cells to test the in vivo efficacy of BETi in reversing CDK4/6 inhibitor resistance. Conclusions: Our results suggest that BET inhibition has potent efficacy in reversing acquired CDK4/6i resistance of ER+ breast cancer, mainly through its inhibitory regulation of CDK6. BET inhibitors are therefore promising candidate therapeutic agents for the treatment of CDK4/6i-resistant ER+ breast cancer. Citation Format: Renyan Liu, Constantia Pantelidou, Heta Jadhav, Geoffrey Shapiro. Bromodomain inhibition reduces CDK6 expression and reverses CDK4/6 inhibitor resistance in ER+ breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5649.

Poly (ADP-ribose) polymerase (PARP) inhibitors exert their efficacy by inducing synthetic lethal effects, as well as cGAS/STING-mediated immune responses in BRCA- and other homologous recombination repair-deficient cancer cells. Here, we investigated whether the immunologic and therapeutic effects of PARP inhibition in BRCA-deficient breast cancer models could be augmented by synthetic cyclic dinucleotide agonists of STING. In in vitro experiments, combined olaparib-mediated PARP inhibition and ADU-S100-mediated STING agonism induced a greater degree of STING pathway activation and proinflammatory cytokine production compared to monotherapies in BRCA1-deficient human MDA-MB-436 cells and mouse triple-negative breast cancer cell lines derived from a genetically engineered model of BRCA-deficient breast cancer. In vivo experiments were conducted in mice bearing completely established tumors derived from the genetically engineered mouse model expanded syngeneically. In this model, the combination also induced an improved immune response compared to either monotherapy alone, evidenced by a greater degree of cytotoxic T cell recruitment and activation, and enhanced dendritic cell activation and antigen presentation. Nanostring mRNA analysis indicated that combinatorial effects were the result of augmented interferon signaling and antigen processing, as well as heightened leukocyte and dendritic cell functions. The combination markedly improved anti-tumor efficacy in vivo compared to monotherapy treatment, with evidence of complete tumor clearance and prolongation of survival. Finally, in mice treated with the combination, tumors could not be re-established upon rechallenge, indicating the induction of immunologic memory. These results support the development of combined PARP inhibition and STING agonism in BRCA-associated breast cancer. Citation Format: Heta Jadhav, Constantia Pantelidou, Aditi Kothari, Renyan Liu, Gerburg Wulf, Jennifer L. Guerriero, Geoffrey I. Shapiro. STING agonism enhances anti-tumor immune responses and therapeutic efficacy of PARP inhibition in BRCA-associated breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1288.