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XLSX file - 132K, Table S2: Effects of EPZ-6438 on Gene Expression in EZH2 Mutant Lymphoma Cells.

The SWI/SNF complex is a major regulator of gene expression and is increasingly thought to play an important role in human cancer, as evidenced by the high frequency of subunit mutations across virtually all cancer types. We previously reported that in preclinical models, malignant rhabdoid tumors, which are deficient in the SWI/SNF core component INI1 (SMARCB1), are selectively killed by inhibitors of the H3K27 histone methyltransferase EZH2. Given the demonstrated antagonistic activities of the SWI/SNF complex and the EZH2-containing PRC2 complex, we investigated whether additional cancers with SWI/SNF mutations are sensitive to selective EZH2 inhibition. It has been recently reported that ovarian cancers with dual loss of the redundant SWI/SNF components SMARCA4 and SMARCA2 are characteristic of a rare rhabdoid-like subtype known as small-cell carcinoma of the ovary hypercalcemic type (SCCOHT). Here, we provide evidence that a subset of commonly used ovarian carcinoma cell lines were misdiagnosed and instead were derived from a SCCOHT tumor. We also demonstrate that tazemetostat, a potent and selective EZH2 inhibitor currently in phase II clinical trials, induces potent antiproliferative and antitumor effects in SCCOHT cell lines and xenografts deficient in both SMARCA2 and SMARCA4. These results exemplify an additional class of rhabdoid-like tumors that are dependent on EZH2 activity for survival. Mol Cancer Ther; 16(5); 850–60. ©2017 AACR.

The EZH2 small-molecule inhibitor tazemetostat (EPZ-6438) is currently being evaluated in phase II clinical trials for the treatment of non-Hodgkin lymphoma (NHL). We have previously shown that EZH2 inhibitors display an antiproliferative effect in multiple preclinical models of NHL, and that models bearing gain-of-function mutations in EZH2 were consistently more sensitive to EZH2 inhibition than lymphomas with wild-type (WT) EZH2. Here, we demonstrate that cell lines bearing EZH2 mutations show a cytotoxic response, while cell lines with WT-EZH2 show a cytostatic response and only tumor growth inhibition without regression in a xenograft model. Previous work has demonstrated that cotreatment with tazemetostat and glucocorticoid receptor agonists lead to a synergistic antiproliferative effect in both mutant and wild-type backgrounds, which may provide clues to the mechanism of action of EZH2 inhibition in WT-EZH2 models. Multiple agents that inhibit the B-cell receptor pathway (e.g., ibrutinib) were found to have synergistic benefit when combined with tazemetostat in both mutant and WT-EZH2 backgrounds of diffuse large B-cell lymphomas (DLBCL). The relationship between B-cell activation and EZH2 inhibition is consistent with the proposed role of EZH2 in B-cell maturation. To further support this, we observe that cell lines treated with tazemetostat show an increase in the B-cell maturation regulator, PRDM1/BLIMP1, and gene signatures corresponding to more advanced stages of maturation. These findings suggest that EZH2 inhibition in both mutant and wild-type backgrounds leads to increased B-cell maturation and a greater dependence on B-cell activation signaling. Mol Cancer Ther; 16(11); 2586–97. ©2017 AACR.

PDF file - 811K, Fig. S1. Effects of EPZ-6438 on Gene Promoter H3K27 Methylation, Recruitment of PRC2 Components to Gene Promoters, and Cell Cycle, in WSU-DLCL2 Cells. Fig. S2: Effects of EPZ-6438 on Gene Expression in EZH2 Mutant Lymphoma Cell Lines. Fig. S3: Pharmacokinetic Profiles of EPZ-6438 in Rats and Mice. Fig. S4: EPZ-6438 Compound Levels in Plasma and WSU-DLCL2 Xenograft Tumor Homogenates from Mice Dosed for 7 or 28 Days. Fig. S5: In vivo Effects of EPZ-6438 in Lymphoma Xenograft Models. Fig. S6: Body Weights of Mice during Xenograft Efficacy Studies.

PDF file - 25K, Table S1: LCC Values for EPZ-6438 for WSU-DLCL2 Human Lymphoma Cells Dosed Either Continuously or After Compound Washout.

Table S1: Conditions and seeding densities of all cell lines used in studies; Table S2. Single agent IC50 values for agents tested for 3 days in 96-well plates as described in Materials and Methods; Figure S1. Tazemetostat dosing reduced H3K27me3 in tumor xenografts and is tolerated by SCID mice; Figure S2: CD40L has little effect on growth of DLBCL cell lines; Figure S3. SU-DHL-5 cells were treated for 4 days with TAZ (0.1 to 1 µM) followed by addition of 500 ng/mL CD40L at the indicated time points (1-60 minutes; Figure S4. An ABC-DLBCL gene signature (1) is up-regulated following tazemetostat treatment in KARPAS-422 (EZH2 mutant GCB), Farage (EZH2 WT GCB), SU-DHL-5 (EZH2 WT GCB), TMD8 (EZH2 WT ABC) and ABC vs. GCB gene signature was applied to RNAseq data from each cell line with and without tazemetostat treatment.; Figure S5: CD40L, but not IL-21 induces PRDM1 in combination with tazemetostat (TAZ); Figure S6. A CD40 responsive gene set (2) is up-regulated following tazemetostat treatment in KARPAS-422 (EZH2 mutant GCB), Farage (EZH2 WT GCB), SU-DHL-5 (EZH2 WT GCB), TMD8 (EZH2 WT ABC) and Basso_CD40¬_SIGNALING_UP gene signature was applied to RNAseq data from each cell line with and without tazemetostat treatment; Figure S7. Model for tazametostat mechanism of action in DLBCL

PDF File -1 MB, Supplemental data is to further support the statements made it the main text. Captions: Supplemental Table 1: Cell Lines used in the CRISPR pooled screen. Supplemental Table 2: Table of ovarian lines screened for tazemetostat sensitivity along with mutational and protein status for SMARCA2, SMARCA4, and ARID1A. Supplemental Table 3: p-values of COV434 SCCOHT cell line treated with tazemetostat. Supplemental Table 4: p-values of JHOC-5 clear cell ovarian cell line treated with tazemetostat. Supplemental Table 5: p-values of cell cycle analysis after tazemetostat treatment. Supplemental Table 6: p-values of apoptosis analysis after tazemetostat treatment. Supplemental Table 7: LogP scores for SWI/SNF components in ovarian lines +/- tazemetostat treatment. Supplemental Figure 1: Baseline measurement of histone 3 lysine 27 trimethyl mark and EZH2. Supplemental Figure 2a: Growth curves of SCCOHT and ovarian cancer cell lines treated with tazemetostat. Supplemental Figure 2b: Growth curves of SCCOHT and ovarian cancer cell lines treated with EPZ-007210. Supplemental Figure 3: Reduction in H3K27me3 mark after treatment. Supplemental Figure 4a: Sensitivity to PLK1 knockout as pan-essential in CRISPR pooled screen. Supplemental Figure 4b: Control sgRNAs from CRISPR pooled screen. Supplemental Figure 5: TOV112D xenograft tumor growth inhibition, tumor volume, and methyl mark. Supplemental Figure 6: body weights of mice treated with tazemetostat. Supplemental Figure 7: measured blood plasma levels of tazemetostat from xenograft mice.

Mutations within the catalytic domain of the histone methyltransferase EZH2 have been identified in subsets of patients with non-Hodgkin lymphoma (NHL). These genetic alterations are hypothesized to confer an oncogenic dependency on EZH2 enzymatic activity in these cancers. We have previously reported the discovery of EPZ005678 and EPZ-6438, potent and selective S-adenosyl-methionine-competitive small molecule inhibitors of EZH2. Although both compounds are similar with respect to their mechanism of action and selectivity, EPZ-6438 possesses superior potency and drug-like properties, including good oral bioavailability in animals. Here, we characterize the activity of EPZ-6438 in preclinical models of NHL. EPZ-6438 selectively inhibits intracellular lysine 27 of histone H3 (H3K27) methylation in a concentration- and time-dependent manner in both EZH2 wild-type and mutant lymphoma cells. Inhibition of H3K27 trimethylation (H3K27Me3) leads to selective cell killing of human lymphoma cell lines bearing EZH2 catalytic domain point mutations. Treatment of EZH2-mutant NHL xenograft-bearing mice with EPZ-6438 causes dose-dependent tumor growth inhibition, including complete and sustained tumor regressions with correlative diminution of H3K27Me3 levels in tumors and selected normal tissues. Mice dosed orally with EPZ-6438 for 28 days remained tumor free for up to 63 days after stopping compound treatment in two EZH2-mutant xenograft models. These data confirm the dependency of EZH2-mutant NHL on EZH2 activity and portend the utility of EPZ-6438 as a potential treatment for these genetically defined cancers. Mol Cancer Ther; 13(4); 842–54. ©2014 AACR.

Supplementary Figure Legend

Gene list for Fig.5B, C, D

Acquired and intrinsic resistance to receptor tyrosine kinase inhibitors (RTKi) represents a major hurdle in improving the management of clear cell renal cell carcinoma (ccRCC). Recent reports suggest that drug resistance is driven by tumor adaptation via epigenetic mechanisms that activate alternative survival pathways. The histone methyl transferase EZH2 is frequently altered in many cancers, including ccRCC. To evaluate its role in ccRCC resistance to RTKi, we established and characterized a spontaneously metastatic, patient-derived xenograft model that is intrinsically resistant to the RTKi sunitinib, but not to the VEGF therapeutic antibody bevacizumab. Sunitinib maintained its antiangiogenic and antimetastatic activity but lost its direct antitumor effects due to kinome reprogramming, which resulted in suppression of proapoptotic and cell-cycle–regulatory target genes. Modulating EZH2 expression or activity suppressed phosphorylation of certain RTKs, restoring the antitumor effects of sunitinib in models of acquired or intrinsically resistant ccRCC. Overall, our results highlight EZH2 as a rational target for therapeutic intervention in sunitinib-resistant ccRCC as well as a predictive marker for RTKi response in this disease. Cancer Res; 77(23); 6651–66. ©2017 AACR.

Supplemental data on additional models to support the role of EZH2 in RTKi resistance

Supplementary Figures 1-4 from PDK1 Attenuation Fails to Prevent Tumor Formation in PTEN-Deficient Transgenic Mouse Models

BackgroundPARP7 is a mono-ART that is upregulated in response to cellular stress (e.g., viral infection, cigarette smoke), and suppresses the Type I interferon (IFN) response following cytosolic nucleic acid sensing. RBN-2397 is a first-in-class PARP7 inhibitor, inducing cancer cell autonomous and immune stimulatory effects in preclinical models through enhanced Type I IFN signaling in cancer cells. Moreover, RBN-2397 induces CD8 T cell-dependent tumor-specific immune memory in an immunocompetent mouse cancer model.1 RBN-2397 is currently being tested in an ongoing Phase I clinical study (NCT04053673).2 Here we aimed to compare biomarker results from preclinical models and patient samples.MethodsIn preclinical models, interferon-stimulated gene (ISG) expression was assessed by qPCR, NanoString, or ELISA. Plasma CXCL10 from patients was measured by MSD while ISG expression in PBMCs was measured by NanoString. Baseline and on-treatment patient tumor biopsies were analyzed by NanoString, CD8/GZMB IHC, and MIBI-TOF to characterize immune changes in the tumor microenvironment.ResultsRBN-2397 potently restored tumoral Type I IFN signaling in preclinical models as demonstrated by increases in ISGs, namely CXCL10, which were not observed in non-tumor tissue (e.g. spleen, PBMCs). In peripheral blood from patients treated with RBN-2397, neither plasma nor PBMC CXCL10 increased more than 2-fold over baseline. Expression of 42 ISGs was not consistently induced in a dose-dependent manner in PBMCs. However, in tumor types of interest (e.g. cancers of the upper aerodigestive tract), CXCL10 expression increased 1.5 to 8-fold, with similar effects observed for a subset of ISGs in 5 evaluable paired biopsy samples.Confirming preclinical studies [1], up to 8-fold increases in CD8 T cell infiltration along with induction of granzyme B expression were observed in 4 of 5 paired patient tumor biopsies by immunohistochemistry. Using the MIBI-TOF technology, we observed up to 50-fold increases in intratumoral activated T cells as well as monocytes and M1 macrophages, most strikingly in two NSCLC patients.ConclusionsInhibition of PARP7 with RBN-2397 restores tumor-intrinsic Type I IFN signaling in preclinical models leading to enhanced adaptive immunity, resulting in CD8 T cell-dependent durable tumor regressions. These observations are mirrored in samples from patients treated with RBN-2397 in that pharmacodynamic effects of RBN-2397 were preferentially observed in tumor tissue relative to the periphery, including an increase in immune infiltration into the tumor microenvironment. These data provide evidence for induction of an adaptive immune response and confirm the tumor-intrinsic, immunomodulatory mechanism of action of RBN-2397 in patients.ReferencesGozgit, et al. PARP7 negatively regulates the type I interferon response in cancer cells and its inhibition triggers antitumor immunity. Cancer Cell 2021; In press.Falchook, et al. A first-in-human phase 1 study of a novel PARP7 inhibitor RBN-2397 in patients with advanced solid tumors. ASCO 2021; oral presentation.

The type 2 cytokines IL-4 and IL-13, which share use of an IL-4 receptor alpha chain and its nuclear induction of the transcription factor STAT6, are crucial in elicitation and maintenance of allergic conditions that include asthma. Prior work has shown a physical and functional association of STAT6 with PARP14, an ADP-ribosyl monotransferase. Moreover, elimination of all PARP14 expression by gene targeting led to altered recall antibody responses and attenuation of ovalbumin-specific allergic lung inflammation with no apparent health issues for mice lacking this protein. However, an unanswered question is whether or not inhibition of the catalytic function has any biological consequence since PARP14 has multiple functional domains apart from the portion that catalyzes ADP-ribosylation. As reported separately, iterative structural analyses and medicinal chemistry fostered the generation of a compound, RBN2759, that is highly selective in its inhibition of PARP14 with negligible impact on other members of the PARP gene family. We show here that administration of this compound to mice previously sensitized to the allergenAlternaria alternataachieved biochemically active levels and altered physiological responses to the antigen. These results show for the first time that in vivo administration of a specific inhibitor of the ADP-ribosyltransferase activity encoded by PARP14 is sufficient to alter biological responses. Specifically, the orally absorbable pharmaceutical compound decreased allergen-induced mucus, blunted the induced increases in circulating IgE, and prevented suppression of IgG2a. We conclude that the catalytic activity can contribute to pathogenesis in allergic processes and propose that other biological endpoints that depend on ADP-ribosylation by PARP14 can be targeted using selective inhibition.

Targeting cytosolic nucleic acid sensing pathways to activate the Type I interferon (IFN) response is an emerging therapeutic strategy being explored in oncology. The PARP family consists of seventeen enzymes that regulate fundamental biological processes including response to cellular stress. PARP7 (TIPARP) is a stress-induced mono-ART that catalyzes the transfer of a single unit of ADP-ribose onto substrates (MARylation) to regulate their function and plays a role in suppressing the Type I IFN response in tumor cells (Gozgit 2021 Cancer Cell). RBN-2397 is the first potent and selective small molecule inhibitor of PARP7 catalytic function. To investigate the cell autonomous effects of PARP7 inhibition, we performed a cell line screen to identify PARP7 dependent cancer cell lines. We found that treatment of a subset of lines across several cancers led to a robust decrease in cell viability. Additionally, dosing of tumor bearing mice led to complete regressions in NCI-H1373 lung cancer xenografts. To investigate the mechanism of action (MOA) leading to decreased cell viability, we treated NCI-H1373 cells with RBN-2397 and found accumulation of cells in the G0/G1 phase of the cell cycle indicative of a cell cycle arrest. This arrest in NCI-H1373 cells was associated with the induction of senescence and increased mRNA expression of senescence associated secretory phenotype (SASP) genes. To evaluate the in vivo MOA, we performed an NCI-H1373 xenograft study and collected tumors after 7 days of RBN-2397 treatment. PARP7 inhibition led to decreased expression of Ki67, and increased expression of P21 and cleaved caspase-3, suggesting decreased proliferation and increased apoptosis. Increased expression of SASP genes was also observed in RBN-2397 treated tumors. Finally, we investigated transcriptional changes after RBN-2397 treatment by RNA sequencing. In addition to the effects observed in Type I IFN signaling, we also observed differential expression of genes associated with other pathways including autophagy and energy metabolism. Further evaluation of key autophagy proteins revealed that RBN-2397 affects autophagy flux and leads to a decrease in the oxygen consumption rate of cells and reduced ATP production from the mitochondria, suggesting that a change in energy metabolism may be related to the tumor intrinsic effect of RBN-2397. In summary, we show treatment of cancer cells with RBN-2397 not only leads to activation of tumor cell IFN signaling, but also causes G1 arrest and senescence, and changes in cancer cell autophagy and energy metabolism. In vivo, RBN-2397 treatment leads to complete tumor regressions in xenografts accompanied by decreased proliferation and increased apoptosis of tumor cells. RBN-2397 is currently being evaluated in the clinic as single agent in selected cancer types (NCT04053673) and in combination with anti-PD-1 therapies. Citation Format: Jennifer R. Molina, Joseph M. Gozgit, Melissa M. Vasbinder, Ryan P. Abo, Kaiko kunii, Kristy G. Kuplast-Barr, Bin Gui, Sunaina P. Nayak, Elena Minissale, Kerren K. Swinger, Tim J. Wigle, Alvin Z. Lu, Danielle J. Blackwell, Christina R. Majer, Yue Ren, Ellen Bamberg, Mario Niepel, Jan-Rung Mo, William D. Church, Ahmed S. Mady, Jeff Song, Zacharenia A. Varsamis, Luke Utley, Patricia E. Rao, Timoty J. Mitchison, Kevin W. Kuntz, Victoria M. Richon, Kristen McEachern, Heike Keilhack. PARP7 inhibitor RBN-2397 increases tumoral IFN signaling leading to various tumor cell intrinsic effects and tumor regressions in mouse models [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 2154.

Background: PARP7 is a mono-ART that is upregulated in response to cellular stress (e.g., viral infection, cigarette smoke), and suppresses the Type I interferon (IFN) response following cytosolic nucleic acid sensing. RBN-2397 is a first-in-class PARP7 inhibitor, inducing cancer cell autonomous and immune stimulatory effects in preclinical models through enhanced Type I IFN signaling in cancer cells. Moreover, RBN-2397 induces CD8 T cell-dependent tumor-specific immune memory in an immunocompetent mouse cancer model [1]. RBN-2397 is currently being tested in an ongoing Phase I clinical study (NCT04053673) [2]. Here we present evidence of proof of mechanism in the paired biopsies of tumors from Phase 1 patients. Methods: Plasma CXCL10 from patients was measured by MSD while ISG expression in PBMCs was measured by NanoString. Baseline and on-treatment patient tumor biopsies were analyzed by NanoString, CD8/GZMB IHC, and MIBI-TOF to characterize immune changes in the tumor microenvironment. Results: In peripheral blood from patients treated with RBN-2397, neither plasma nor PBMC CXCL10 increased more than 2-fold over baseline. Expression of 42 ISGs was not consistently induced in a dose-dependent manner in PBMCs. However, in tumor types of interest (e.g., cancers of the upper aerodigestive tract), CXCL10 expression increased, with similar effects observed for a subset of ISGs in multiple evaluable paired biopsy samples. Confirming preclinical studies [1], increases in CD8 T cell infiltration along with induction of granzyme B expression were observed in several evaluable paired patient tumor biopsies by immunohistochemistry. Using the MIBI-TOF technology, we observed up to 50-fold increases in intratumoral activated T cells as well as monocytes and M1 macrophages, most strikingly in two NSCLC patients. Conclusions: In patients treated with RBN-2397 pharmacodynamic effects were preferentially observed in tumor tissue relative to the periphery, including an increase in immune infiltration into the tumor microenvironment. These data provide evidence for induction of an adaptive immune response and confirm the tumor-intrinsic, immunomodulatory mechanism of action of RBN-2397 in patients. References: 1. Gozgit et al. PARP7 negatively regulates the Type I interferon response in cancer cells and its inhibition triggers antitumor immunity. Cancer Cell. 2021 2. Falchook et al. A First-In-Human Phase 1 Study of a Novel PARP7 Inhibitor RBN-2397 in Patients with Advanced Solid Tumors. ASCO 2021 oral presentation Citation Format: Kristy Kuplast-Barr, Melissa L. Johnson, Manish R. Patel, Timothy A. Yap, Gerald S. Falchook, Patricia LoRusso, Ryan Abo, Chang Liu, Erika L. Manyak, Lisa Cleary, Viviana Bozon, Sudha Parasuraman, Heike Keilhack, Kristen McEachern. RBN-2397, a novel, potent, and selective PARP7 inhibitor, induces tumor-intrinsic type I interferon responses and adaptive immunity in patient tumors [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 1836.

PARP7 is a monoPARP that catalyzes the transfer of single units of ADP-ribose onto substrates to change their function. Here, we identify PARP7 as a negative regulator of nucleic acid sensing in tumor cells. Inhibition of PARP7 restores type I interferon (IFN) signaling responses to nucleic acids in tumor models. Restored signaling can directly inhibit cell proliferation and activate the immune system, both of which contribute to tumor regression. Oral dosing of the PARP7 small-molecule inhibitor, RBN-2397, results in complete tumor regression in a lung cancer xenograft and induces tumor-specific adaptive immune memory in an immunocompetent mouse cancer model, dependent on inducing type I IFN signaling in tumor cells. PARP7 is a therapeutic target whose inhibition induces both cancer cell-autonomous and immune stimulatory effects via enhanced IFN signaling. These data support the targeting of a monoPARP in cancer and introduce a potent and selective PARP7 inhibitor to enter clinical development.

Summary PARP14 has been implicated by genetic knockout studies to promote protumor macrophage polarization and suppress the antitumor inflammatory response due to its role in modulating interleukin-4 (IL-4) and interferon-γ signaling pathways. Here, we describe structure-based design efforts leading to the discovery of a potent and highly selective PARP14 chemical probe. RBN012759 inhibits PARP14 with a biochemical half-maximal inhibitory concentration of 0.003 μM, exhibits >300-fold selectivity over all PARP family members, and its profile enables further study of PARP14 biology and disease association both in vitro and in vivo. Inhibition of PARP14 with RBN012759 reverses IL-4-driven protumor gene expression in macrophages and induces an inflammatory mRNA signature similar to that induced by immune checkpoint inhibitor therapy in primary human tumor explants. These data support an immune suppressive role of PARP14 in tumors and suggest potential utility of PARP14 inhibitors in the treatment of cancer.

Genomic instability in cancer cells leads to cellular stress through the accumulation of aberrant nucleic acid species in the cytosol. We have shown that PARP7, a monoPARP, is a negative regulator of cytosolic nucleic acid sensing in cancer cells. RBN-2397 is a potent and selective PARP7 inhibitor that induces antitumor immunity in preclinical models and is currently being evaluated in a Phase I clinical trial. In our preclinical investigations, we found that in a subset of cancer cell lines, such as NCI-H1373, inhibition of PARP7 triggers Type I IFN release, STAT1 phosphorylation, and growth arrest. In contrast, other cell lines, for example, HARA, do not mount an IFN-response upon PARP7 inhibition, even though they are responsive to transfection of exogenous nucleic acids and PARP7 is expressed and enzymatically active. To investigate the underlying mechanism of PARP7 inhibition and to determine the drivers of the differential sensitivity across cell lines we performed arrayed CRISPR knockout screens, targeting approximately 240 genes in the nucleic acid sensing and IFN signaling pathways, in the presence and absence of PARP7 inhibition. Our arrayed screens confirmed multiple hits from a previous genome-wide pooled synthetic/lethal CRISPR dropout screen. For example, targeting genes in the cGAS/STING pathway conferred resistance to PARP7 inhibition in the NCI-H1373 responder cells, suggesting a critical dependence on this sensing pathway. In the PARP7 inhibitor-resistant HARA cells, deletion of components of innate immune-signaling (such as AIM2 and ADAR1), the NF-κB pathway, and genes involved in autophagy sensitized the cells to PARP7 inhibition. We further delineated the function of PARP7 by comparing the effects of the CRISPR perturbation across different cellular readouts such as STAT1 phosphorylation, IFN release, and proliferation. With our work, we shed light on the mechanism by which PARP7 acts as a critical suppressor of the innate immune response. Our findings demonstrate both redundancy and crosstalk between different nucleic acid-sensing pathways and may explain why some cell lines are resistant to PARP7 inhibition. Citation Format: Bin Gui, Ryan Abo, Patrick Flynn, Alvin Z. Lu, Jan-Rung Mo, Joseph M. Gozgit, Melissa M. Vasbinder, Zacharenia A. Varsamis, Andrew Santospago, Victoria M. Richon, Kevin W. Kuntz, Heike Keilhack, Timothy J. Mitchison, Mario Niepel. Investigating the mechanism of PARP7 inhibition in type I interferon signaling by arrayed CRISPR screening [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1021.

Targeting cytosolic nucleic acid sensing pathways and the Type I interferon (IFN) response is an emerging therapeutic strategy being explored in oncology. The PARP family consists of seventeen enzymes that regulate fundamental biological processes including response to cellular stress. In contrast to PARP1, PARP7 (TIPARP) is a monoPARP that catalyzes the transfer of single units of ADP-ribose onto substrates (MARylation) to change their function and plays a role in suppressing the Type I IFN response. RBN-2397 selectively inhibits PARP7 compared to the approved PARP1 inhibitors and demonstrates > 50-fold selectivity for inhibition of PARP7 over all PARP family members as measured by biochemical assays. The inhibition of PARP1-mediated ADP-ribosylation has been well-characterized for several PARP1 inhibitors using a cellular hydrogen peroxide-induced PARylation assay. Here, we show that RBN-2397 inhibits PARP7-dependent MARylation with an IC50 of 2 nM exhibiting a 300-fold window over PARP1-driven PARylation. Using the mouse CT26 cell line, we showed that RBN-2397, but not the PARP1 inhibitor olaparib, induced Type I IFN signaling demonstrated by STAT1 phosphorylation. The effect on pSTAT1 was phenocopied by PARP7 knockout (KO). To further demonstrate specificity, we show that simultaneous KO of PARP7 prevented any additional increase of STAT1 phosphorylation by RBN-2397; however, KO of PARP1 had no effect on the induction of Type I IFN signaling by RBN-2397. We had previously reported that RBN-2397 dosing of CT26 tumor bearing immune competent BALB/c mice led to complete and durable tumor regressions which could be reversed by interfering with tumor-derived IFN signaling (1). Here we show that in contrast, RBN-2397 showed modest activity with no tumor regressions in CT26-tumor bearing immunodeficient NOG mice. To assess which immune cell populations are involved in the antitumor effects of RBN-2397, CT26 tumor-bearing BALB/c mice were depleted of CD4 T, CD8 T or NK cells. Depletion of CD4 T or NK cells had no effect on RBN-2397 antitumor activity; however, depletion of CD8 T cells significantly reversed the effects of RBN-2397, suggesting that CD8 T cells are responsible for much of the antitumor immunity induced by RBN-2397. We have discovered and developed RBN-2397, a first-in-class, potent and selective inhibitor of PARP7. We show RBN-2397 restores Type I IFN signaling in cancer cells and that this is an on-target effect of inhibiting the catalytic activity of PARP7 and not PARP1. We further show that the adaptive immune response was required for the antitumor effects of RBN-2397. RBN-2397 is the first agent to enter clinical trials that targets this tumor-intrinsic vulnerability, and a Phase I clinical trial is underway (NCT04053673). (1) AACR Jun 22-24, 2020: Cancer Res 2020;80 (16 Suppl): Abstract nr 3405. Citation Format: Joseph M. Gozgit, Melissa M. Vasbinder, Ryan P. Abo, Kaiko Kunii, Kristy G. Kuplast-Barr, Bin Gui, Alvin Z. Lu, Jennifer R. Molina, Elena Minissale, Kerren K. Swinger, Tim J. Wigle, Danielle J. Blackwell, Christina R. Majer, Yue Ren, Mario Niepel, Ellen Bamberg, Jan-Rung Mo, David Church, Jeff Song, Luke Utley, Patricia E. Rao, Timothy J. Mitchison, Kevin W. Kuntz, Victoria M. Richon, Heike Keilhack. RBN-2397: A potent and selective small molecule inhibitor of PARP7 that induces tumor-derived antitumor immunity dependent on CD8 T cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 48.

PARP7 (TIPARP) is a monoPARP which catalyzes the transfer of single units of ADP-ribose onto substrates to change their function. Its expression is upregulated during cellular stress, including viral infection or through the activation of the aryl hydrocarbon receptor after exposure to cigarette smoke. We and others have shown that PARP7 activity suppresses the Type I interferon (IFN) response following activation by cytosolic nucleic acid sensing pathways. RBN-2397 is a first-in class PARP7 inhibitor, which induces cancer cell autonomous and immune stimulatory effects in preclinical models through enhanced Type I IFN signaling in cancer cells. Here we describe the presence of PARP7 genomic amplification with corresponding increased mRNA expression across select cancers. Elevated PARP7 expression or amplification may identify cancer patients that could derive benefit from treatment with RBN-2397. In characterizing PARP7 copy number and mRNA expression from The Cancer Genome Atlas (TCGA) database, we found the presence of PARP7 copy number amplification in a subset of tumor types, particularly those of squamous histology, as well as ovarian cancer that corresponded to higher mRNA expression levels. High PARP7 expression correlated with poor survival in squamous cancers, while it had no effect on survival in ovarian cancer. Interestingly, tumor types with high PARP7 expression also expressed higher levels of baseline interferon stimulated genes (ISGs). This parallels our findings that cancer cell lines with higher ISGs at baseline are more responsive to PARP7 inhibition. To explore PARP7 copy number variations (CNVs) in advanced cancer patients, we queried the FoundationCore® (Foundation Medicine, Inc) database. Similar to TCGA, squamous cancers as well as ovarian, breast, and pancreatic ductal adenocarcinoma (PDAC) were among the tumor types with PARP7 amplifications. Moreover, PARP7 was found to be amplified both on the background of chromosome 3q arm-level amplifications as well as focally. Congruent to our analysis of PARP7 amplifications, we evaluated PARP7 mRNA expression in both squamous and non-squamous non-small cell lung carcinoma (NSCLC), breast cancer, and PDAC primary tumor samples. Using a validated RNAscope ISH probe set, we analyzed over 1,000 patient samples and found that PARP7 was more highly expressed in tumor cells relative to corresponding normal tissues. Overall, there were varying levels of PARP7 expression across samples with higher expression levels of PARP7 in tumor cells, compared to stroma, across all cancers examined. Our analyses describing the presence of PARP7 amplifications as well as high expression levels in several tumor types including NSCLC, breast, and PDAC provides evidence for the therapeutic relevance of PARP7 inhibition and highlights potential patient selection strategies to identify those patients more likely to benefit from RBN-2397 treatment. Citation Format: Jodie Wong, Kristy Kuplast-Barr, Ryan P. Abo, Anupriya S. Kulkarni, Linda T. Nieman, Azfar Neyaz, Katherine H. Xu, Radwa Sharaf, Lee A. Albacker, David T. Ting, Heike Keilhack, Kristen A. McEachern. Elevated PARP7 expression in select cancers identifies a target population for RBN-2397 therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 381.

CD38 is an ADP-ribosyl cyclase that converts NAD+ to ADP-ribose (ADPR) or cyclic ADPR (cADPR) and nicotinamide. The enzyme can exist in either an ecto- or endo-catalytic orientation with different sub-cellular localization, and therefore can regulate internal and external NAD+ pools. Both NAD+ and cADPR can impact T cell fitness and effector function, and CD38 has been shown to be increased in settings of chronic T cell activation. CD38 can mediate the non-canonical generation of the immune suppressive adenosine by catabolizing extracellular NAD+ resulting in immunosuppression in the microenvironment. Upon immune checkpoint inhibitor (ICI) therapy, CD38 is upregulated on cancer cells to drive ICI resistance. Therefore CD38, through its catalytic activity, has been implicated in tumor immune suppression and ICI resistance. Genetic knockout of CD38 has been shown to prevent tumor growth and improve T cell fitness. Here, we describe the effects of CD38 inhibition using a small molecule inhibitor on these key metabolites in various cellular and tumor models. RBN013209 is a potent and selective small molecule inhibitor of CD38 catalytic function. We demonstrate that inhibition of CD38 with RBN013209 prevents conversion of extracellular NAD+ to ADPR or cADPR in cancer cell lines and PBMCs. Similarly, RBN013209 inhibited intracellular CD38 activity and elevated intracellular NAD+ levels in cultured human primary T cells. Oral administration of RBN013209 to naïve mice resulted in dose-dependent elevation of NAD+ and reduction of ADPR in various tissues such as spleen and liver. We next assessed the expression of CD38 protein by immunohistochemistry following ICI treatment in various syngeneic cancer models to select a model for efficacy studies. We observed increases in CD38 expression on tumor cells and infiltrating immune cells in MC38 colon cancer and B16-F10 and Cloudman S91 melanoma models. In the MC38 tumor model, we observed single agent antitumor activity with RBN013209 that was associated with changes in NAD+ and ADPR. In B16-F10 tumor-bearing mice, we observed antitumor activity with RBN013209 in combination with anti-PD-L1 therapy. To evaluate CD38 as a biomarker in clinical samples, we assessed and confirmed the tumor expression of CD38 protein from lung, prostate and kidney cancer patients. Here, we show that inhibition of CD38 with a small molecule affects both intra- and extra-cellular CD38 activity and modulates key metabolites playing an important role in immunomodulation. Further, our data indicate that CD38 is increased by ICI treatment and that inhibition of CD38 can lead to antitumor activity. Citation Format: Prashant Shambharkar, Danielle J. Blackwell, Melissa M. Vasbinder, Laurie B. Schenkel, Kaiko Kunii, Jenkins L. Lemera, Kristy G. Kuplast-Barr, Yue Ren, Ellen Bamberg, W. David Church, Christina R. Majer, Luke Utley, Kristen McEachern, Mario Niepel, Tim J. Wigle, Kevin W. Kuntz, Victoria M. Richon, Heike Keilhack, Joseph M. Gozgit. Small molecule inhibitor of CD38 modulates its intra- and extracellular functions leading to antitumor activity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1344.

PARP14 is an interferon-stimulated gene that is overexpressed in multiple tumor types and has been shown to promote the pro-tumor M2 polarization of macrophages and support Th2/Th17 signaling in models of allergic airway disease. PARP14 is a large 203 kDa protein that possesses a catalytic domain responsible for the transfer of mono-ADP-ribose to its substrates, three macrodomains that bind mono-ADP-ribose, a WWE domain that serves as a binding module for poly-ADP-ribose, and an RNA recognition motif. We have previously shown that the potent and reversible enzymatic inhibitor, RBN012759 (IC50 < 0.003 μM, 300-fold selective over monoPARPs, > 1,000-fold selective over polyPARPs), links PARP14 catalytic inhibition with suppression of the antitumor immune response in human primary macrophages and human kidney cancer explants. While this catalytic inhibitor of PARP14 was able to suppress IL-4-driven pro-tumor gene expression in macrophages, it is unknown what roles the non-enzymatic biomolecular recognition motifs play in the biological function of PARP14. To further understand this, we describe a heterobifunctional small molecule, RBN012811, based on a catalytic inhibitor of PARP14 that binds in the enzyme's NAD+-binding site and recruits the E3 ligase cereblon to ubiquitinate PARP14 and selectively target it for degradation. RBN012811 has a IC50 of 0.01 μM against PARP14 in a biophysical assay and is at least 200-fold selective over all other PARPs. In KYSE-270 cancer cells, RBN012811 has a half-maximal degradation concentration (DC50) of 0.005 μM and it does not cause degradation of other PARP enzymes. In human primary macrophages PARP14 degradation by RBN012811 led to a dose-dependent decrease of IL-10 release induced by IL-4 stimulation. Our data demonstrates that RBN012811 is a useful tool to enable further exploration of the role of PARP14 in inflammation and cancer. Citation Format: Tim Wigle, Yue Ren, Jennifer Molina, Danielle Blackwell, Laurie Schenkel, Kerren Swinger, Anne Cheug, Ryan Abo, Elena Minissale, Alvin Lu, Christina Majer, William Church, Bryan Dorsey, Mario Niepel, Nicholas Perl, Kristy Kuplast-Barr, Kristen McEachern, Melissa Vasbinder, Heike Keilhack, Kevin Kuntz. Targeted degradation of PARP14 Using a heterobifunctional small molecule [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1348.

Acquired and intrinsic resistance to receptor tyrosine kinase inhibitors (RTKi) represents a major hurdle in improving the management of clear cell renal cell carcinoma (ccRCC). Recent reports suggest that drug resistance is driven by tumor adaptation via epigenetic mechanisms that activate alternative survival pathways. The histone methyl transferase EZH2 is frequently altered in many cancers, including ccRCC. To evaluate its role in ccRCC resistance to RTKi, we established and characterized a spontaneously metastatic, patient-derived xenograft model that is intrinsically resistant to the RTKi sunitinib, but not to the VEGF therapeutic antibody bevacizumab. Sunitinib maintained its antiangiogenic and antimetastatic activity but lost its direct antitumor effects due to kinome reprogramming, which resulted in suppression of proapoptotic and cell-cycle–regulatory target genes. Modulating EZH2 expression or activity suppressed phosphorylation of certain RTKs, restoring the antitumor effects of sunitinib in models of acquired or intrinsically resistant ccRCC. Overall, our results highlight EZH2 as a rational target for therapeutic intervention in sunitinib-resistant ccRCC as well as a predictive marker for RTKi response in this disease. Cancer Res; 77(23); 6651–66. ©2017 AACR.

The EZH2 small-molecule inhibitor tazemetostat (EPZ-6438) is currently being evaluated in phase II clinical trials for the treatment of non-Hodgkin lymphoma (NHL). We have previously shown that EZH2 inhibitors display an antiproliferative effect in multiple preclinical models of NHL, and that models bearing gain-of-function mutations in EZH2 were consistently more sensitive to EZH2 inhibition than lymphomas with wild-type (WT) EZH2. Here, we demonstrate that cell lines bearing EZH2 mutations show a cytotoxic response, while cell lines with WT-EZH2 show a cytostatic response and only tumor growth inhibition without regression in a xenograft model. Previous work has demonstrated that cotreatment with tazemetostat and glucocorticoid receptor agonists lead to a synergistic antiproliferative effect in both mutant and wild-type backgrounds, which may provide clues to the mechanism of action of EZH2 inhibition in WT-EZH2 models. Multiple agents that inhibit the B-cell receptor pathway (e.g., ibrutinib) were found to have synergistic benefit when combined with tazemetostat in both mutant and WT-EZH2 backgrounds of diffuse large B-cell lymphomas (DLBCL). The relationship between B-cell activation and EZH2 inhibition is consistent with the proposed role of EZH2 in B-cell maturation. To further support this, we observe that cell lines treated with tazemetostat show an increase in the B-cell maturation regulator, PRDM1/BLIMP1, and gene signatures corresponding to more advanced stages of maturation. These findings suggest that EZH2 inhibition in both mutant and wild-type backgrounds leads to increased B-cell maturation and a greater dependence on B-cell activation signaling. Mol Cancer Ther; 16(11); 2586–97. ©2017 AACR.

The SWI/SNF complex is a major regulator of gene expression and is increasingly thought to play an important role in human cancer, as evidenced by the high frequency of subunit mutations across virtually all cancer types. We previously reported that in preclinical models, malignant rhabdoid tumors, which are deficient in the SWI/SNF core component INI1 (SMARCB1), are selectively killed by inhibitors of the H3K27 histone methyltransferase EZH2. Given the demonstrated antagonistic activities of the SWI/SNF complex and the EZH2-containing PRC2 complex, we investigated whether additional cancers with SWI/SNF mutations are sensitive to selective EZH2 inhibition. It has been recently reported that ovarian cancers with dual loss of the redundant SWI/SNF components SMARCA4 and SMARCA2 are characteristic of a rare rhabdoid-like subtype known as small-cell carcinoma of the ovary hypercalcemic type (SCCOHT). Here, we provide evidence that a subset of commonly used ovarian carcinoma cell lines were misdiagnosed and instead were derived from a SCCOHT tumor. We also demonstrate that tazemetostat, a potent and selective EZH2 inhibitor currently in phase II clinical trials, induces potent antiproliferative and antitumor effects in SCCOHT cell lines and xenografts deficient in both SMARCA2 and SMARCA4. These results exemplify an additional class of rhabdoid-like tumors that are dependent on EZH2 activity for survival. Mol Cancer Ther; 16(5); 850–60. ©2017 AACR.

Poly-ADP-ribose polymerases (PARPs) are a family of enzymes responsible for transferring individual or chains of ADP-ribose subunits to substrate targets as a type of post-translational modification. PARPs regulate a wide variety of important cellular processes, ranging from DNA damage repair to antiviral response. However, most research to date has focused primarily on the polyPARPs, which catalyze the formation of ADP-ribose polymer chains, while the monoPARPs, which transfer individual ADP-ribose monomers, have not been studied as thoroughly. This is partially due to the lack of robust assays to measure mono-ADP-ribosylation in the cell. In this study, the recently developed MAR/PAR antibody has been shown to detect mono-ADP-ribosylation in cells, enabling the field to investigate the function and therapeutic potential of monoPARPs. In this study, the antibody was used in conjunction with engineered cell lines that overexpress various PARPs to establish a panel of assays to evaluate the potencies of literature-reported PARP inhibitors. These assays should be generally applicable to other PARP family members for future compound screening efforts. A convenient and generalizable workflow to identify and validate PARP substrates has been established. As an initial demonstration, aryl hydrocarbon receptor was verified as a direct PARP7 substrate and other novel substrates for this enzyme were also identified and validated. This workflow takes advantage of commercially available detection reagents and conventional mass spectrometry instrumentation and methods. Ultimately, these assays and methods will help drive research in the PARP field and benefit future therapeutics development.

The dynamic structure of histones and DNA, also known as chromatin, is regulated by two classes of enzymes: those that mediate covalent modifications on either histone proteins or DNA and those that use the energy generated by ATP hydrolysis to mechanically alter chromatic structure. Both classes of enzymes are often found in large protein complexes. In this review, we describe two such complexes: polycomb repressive complex 2 (PRC2), with the protein methyltransferase EZH2 as its catalytic subunit, and the ATP-dependent chromatin remodeler switch/sucrose non-fermentable (SWI/SNF). EZH2 catalyzes the methylation of lysine 27 on histone H3, a covalent chromatin modification that is associated with repressed heterochromatin. The catalytic activity of SWI/SNF, in contrast, leads to a state of open chromatin associated with active transcription. In this review, we discuss the biochemical properties of both complexes, outline the principles of their regulation, and describe their opposing roles in normal development, which can be perturbed in disease settings such as cancer.

Posttranslational methylation of histones plays a critical role in gene regulation. Misregulation of histone methylation can lead to oncogenic transformation. Enhancer of Zeste homologue 2 (EZH2) methylates histone 3 at lysine 27 (H3K27) and abnormal methylation of this site is found in many cancers. Tazemetostat, an EHZ2 inhibitor in clinical development, has shown activity in both preclinical models of cancer as well as in patients with lymphoma or INI1-deficient solid tumors. Herein we report the structure-activity relationships from identification of an initial hit in a high-throughput screen through selection of tazemetostat for clinical development. The importance of several methyl groups to the potency of the inhibitors is highlighted as well as the importance of balancing pharmacokinetic properties with potency.

PARPs (poly-ADP-ribose polymerases) are a family of enzymes that regulate a wide variety of important cellular processes including cellular stress signaling pathways implicated in inflammation and cancer. PARP14 is a member of the monoPARP sub-family of PARPs, which catalyze transfer of a single ADP ribose unit (mono-ADP-ribosylation or MARylation) to their substrates, unlike their polyPARP counterparts which construct chains of ADP ribose units (poly-ADP-ribosylation or PARylation). PARP14 is an interferon-stimulated gene (ISG) that is overexpressed in tumors compared to normal tissues and has been implicated by genetic knockout studies to promote pro-tumor macrophage polarization and suppress antitumor inflammatory response due to its role in modulating IL-4 and IFN-γ signaling pathways. Efforts to further explore and validate the role of PARP14 catalytic activity in these pathways have been hampered by a lack of potent, highly selective PARP14 inhibitors. Here we describe the discovery of the first such chemical probe, RBN012759, which inhibits PARP14 with an IC50 of 0.003 µM and exhibits >300-fold selectivity over all mono- and polyPARP family members. Medicinal chemistry efforts that began with an unselective micromolar screening hit were enabled by a detailed understanding of the PARP14 and broader PARP family binding pockets. X-ray co-crystal structures of RBN012759 and key early analogs bound to PARP14 clearly illustrate the origins of their potency and selectivity. RBN012759 is a cell permeable, soluble probe that demonstrates robust, dose-dependent stabilization of endogenous PARP14 and inhibition of MARylation in primary human macrophages. RBN012759 reverses IL-4 driven (pro-tumor) gene expression in macrophages, confirming published data generated with PARP14 KO and supporting an immune suppressive role of PARP14 in tumors. Moreover, we demonstrate that PARP14 inhibition in primary human tumor explants can induce an inflammatory mRNA signature similar to immune checkpoint therapy. Summary: Structure-based design of the first potent and highly selective inhibitor of PARP14, RBN012759, is described. The discovery of this chemical probe enabled exploration of the role of PARP14 in macrophage polarization and inflammatory pathways. RBN012759 reverses IL-4 driven gene expression in macrophages and induces an inflammatory mRNA signature in human tumor explants, data which support an immune suppressive role of PARP14 in tumors. Citation Format: Laurie Schenkel, Jennifer Molina, Kerren Swinger, Ryan Abo, Danielle Blackwell, Anne Cheung, William Church, Kristy Kuplast-Barr, Alvin Lu, Elena Minissale, Mario Niepel, Melissa Vasbinder, Tim Wigle, Victoria Richon, Heike Keilhack, Kevin Kuntz. A potent and selective PARP14 inhibitor decreases pro-tumor macrophage function and elicits inflammatory responses in tumor explants [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1038.

Targeting cytosolic nucleic acid sensing pathways and the Type I interferon (IFN) response is an emerging therapeutic strategy being explored in oncology. PARP7 is a member of the monoPARP class of enzymes, which catalyze the transfer of single units of ADP-ribose onto substrates to change their function. PARP7 expression is increased by cellular stress and aromatic hydrocarbons, and the PARP7 gene is amplified in cancers, especially in those of the upper aerodigestive tract. PARP7 has also been reported to negatively regulate the Type I IFN response by interacting with TBK1 during viral infection. Herein, we identify PARP7 as a novel negative regulator of cytosolic nucleic acid sensing in tumor cells. RBN-2397, is a potent and selective small molecule inhibitor of PARP7 catalytic function. We identified a subset of cancers exhibiting dependency on PARP7 for proliferation and found that cell lines with higher baseline expression of interferon stimulated genes were more sensitive. We further show that inhibition of PARP7 by RBN-2397 restores Type I IFN signaling as demonstrated by the induction of STAT1 phosphorylation and up-regulation of genes enriched for Type I IFN signaling in NCI-H1373 lung cancer cells. We examined the antitumor effects of once daily orally administered RBN-2397 in SCID mice with subcutaneous NCIH1373 xenograft tumors and observed a dose-dependent effect of RBN-2397 on tumor growth, with regressions at dose levels ≥30 mg/kg. To evaluate the antitumor immune response in vivo, we administered RBN-2397 to CT26 tumor-bearing, immunocompetent BALB/c mice, and observed significant tumor growth inhibition at all dose levels with complete and durable regressions in a subset of mice. All of these tumor-free mice rejected a challenge of injected CT26 cells, but were able to develop 4T1 tumors, demonstrating induction of tumor-specific adaptive immune memory. The antitumor effects of RBN-2397 were further enhanced when combined with an immune checkpoint inhibitor, anti-PD1. Using CRISPR-Cas9 to knockout either TBK1 or IFNAR1 in CT26 cells, we demonstrated that RBN-2397 antitumor immunity is dependent on the effects of tumor-derived Type I interferon on immune cells. Here, we show for the first time that cancer cells use PARP7 to suppress the Type I IFN response to cytosolic nucleic acids. We have discovered and developed RBN-2397, a first-in-class, potent and selective inhibitor of PARP7. We show RBN-2397 restores Type I IFN signaling in the tumor, causes complete tumor regressions and adaptive immunity in murine models. RBN-2397 is the first agent to enter clinical trials that targets this tumor-intrinsic vulnerability. Citation Format: Joseph M. Gozgit, Melissa M. Vasbinder, Ryan P. Abo, Kaiko Kunii, Kristy G. Kuplast-Barr, Bin Gui, Alvin Z. Lu, Kerren K. Swinger, Tim J. Wigle, Danielle J. Blackwell, Christina R. Majer, Yue Ren, Mario Niepel, Zacharenia A. Varsamis, Sunaina P. Nayak, Ellen Bamberg, Jan-Rung Mo, William Church, Jeff Song, Luke Utley, Patricia E. Rao, Timothy J. Mitchison, Kevin W. Kuntz, Victoria M. Richon, Heike Keilhack. PARP7 negatively regulates the type I interferon response in cancer cells and its inhibition leads to tumor regression [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3405.

Mono(ADP-ribosylation) (MARylation) and poly(ADP-ribosylation) (PARylation) are post-translational modifications deposited on multiple amino acids by PARP enzymes using nicotinamide adenine dinucleotide (NAD+) as the ADP-ribose donating substrate. While there are approved drugs and clinical trials on-going for inhibitors of the polyPARP enzymes that deposit poly(ADP-ribose) (specifically PARP1 and PARP2 inhibitors), monoPARP enzymes that deposit mono(ADP-ribose) are only recently gaining recognition for their role in cellular stress signaling, inflammation and cancer. However, there is a lack of chemical probes to study their function in cells and in vivo. An important first step to generating chemical probes for monoPARPs is to develop screening assays to enable determination of potency and selectivity of inhibitors during the hit finding and lead optimization phases. The development of enzyme assays is complicated by the fact that the substrates for the majority of the monoPARPs are unknown, and even for those with identified substrates, it is uncertain how they engage their substrates. Here we describe the development of robust high-throughput biochemical and cellular monoPARP assays that overcome the lack of knowledge around the substrates and construction of a family-wide screening panel. We highlight derivatized microplates that activate the enzymes to self-MARylate in dissociation enhanced lanthanide fluorescence assays (DELFIA), antibodies that recognize MARylation in in-cell western (ICW) and immunofluorescence (IF) assays, and NAD+-competitive molecular probes that are used to develop in vitro time-resolved fluorescence resonance energy transfer (TR-FRET) and cellular NanoLuc bioluminescence resonance energy transfer (NanoBRET) probe displacement assays. Additionally, we employ several methods to characterize inhibitor binding kinetics. These assays have been used in high-throughput screening campaigns of up to 500,000 compounds, as well as in the development of potent and selective inhibitors of multiple monoPARP enzymes including RBN012759, a tool compound for PARP14 that inhibits in vitro with an IC50 of 3 nM and in cells using with an IC50 of 9 nM, and is 300-fold selective over all other PARP enzymes. Citation Format: Tim J. Wigle, Danielle J. Blackwell, Laurie B. Schenkel, Yue Ren, William D. Church, Hetvi J. Desai, Kerren K. Swinger, Andrew G. Santospago, Christina R. Majer, Alvin Z. Lu, Mario Niepel, Nicholas R. Perl, Melissa M. Vasbinder, Heike Keilhack, Kevin W. Kuntz. A bespoke screening platform to study mono(ADP-ribosylation) [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 506.

RBN-2397: A first-in-class PARP7 inhibitor targeting a newly discovered cancer vulnerability in stress-signaling pathways PARP7 is a monoPARP that catalyzes the transfer of single units of ADP-ribose onto substrates to change their function (MARylation). PARP7 expression is increased by cellular stresses, including aromatic hydrocarbons and the PARP7 gene is amplified in cancers, especially in those of the upper aerodigestive tract. PARP7 has also been reported to negatively regulate the Type I interferon (IFN) response by interacting with TBK1 during viral infection. As part of our drug discovery efforts to identify inhibitors of PARP7, we utilized structure-based drug design to optimize an unselective monoPARP inhibitor identified by screening Ribon's internal compound collection of PARP inhibitors. Further optimization of potency and physicochemical properties led to the discovery of RBN-2397, a potent and selective small molecule inhibitor of PARP7 catalytic function. A co-crystal structure of RBN-2397 demonstrated binding of the compound in the NAD+-binding pocket. Binding to cellular PARP7 is demonstrated by the ability of RBN-2397 to displace an active site probe in a NanoBRET assay. Functionally, RBN-2397 leads to the inhibition of MARylation of multiple intracellular proteins in PARP7-overexpressing SK-MES-1 cells. We identified a subset of cancers exhibiting dependency on PARP7 for proliferation. Cell lines with higher baseline expression of interferon stimulated genes are more sensitive to RBN-2397 in proliferation assays. We further show that inhibition of PARP7 by RBN-2397 restores Type I IFN signaling as demonstrated by the induction of STAT1 phosphorylation and upregulation of genes enriched for Type I IFN signaling in NCI-H1373 lung cancer cells. Oral dosing of RBN-2397 results in durable, complete tumor regression in a NCI-H1373 lung cancer xenograft and induces tumor-specific adaptive immune memory in an immunocompetent mouse cancer model that is dependent on tumor-derived Type I IFN signaling. Herein, we describe the discovery of the small molecule PARP7 inhibitor RBN-2397, the first therapeutic agent targeting PARP7 to enter clinical trials, and the first disclosure of the inhibitor. We demonstrate PARP7 is a novel therapeutic target and inhibition of PARP7 by RBN-2397 induces both cancer cell autonomous and immune stimulatory effects via enhanced IFN signaling. Citation Format: Melissa M. Vasbinder, Joseph M. Gozgit, Ryan P. Abo, Kaiko Kunii, Kristy G. Kuplast-Barr, Bin Gui, Alvin Z. Lu, Kerren K. Swinger, Tim J. Wigle, Danielle J. Blackwell, Christina R. Majer, Yue Ren, Mario Niepel, Zacharenia A. Varsamis, Sunaina P. Nayak, Ellen Bamberg, Jan-Rung Mo, W David Church, Jeff Song, Luke Utley, Patricia E. Rao, Timothy J. Mitchison, Kevin W. Kuntz, Victoria M. Richon, Heike Keilhack. RBN-2397: A first-in-class PARP7 inhibitor targeting a newly discovered cancer vulnerability in stress-signaling pathways [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr DDT02-01.

The poly-ADP-ribose polymerases (PARPs) are a family of 17 enzymes with conserved catalytic domains. They regulate a wide variety of important cellular processes including cellular stress signaling pathways implicated in inflammation and cancer. Much of the PARP research has been dedicated to the four polyPARPs (PARP1, 2, 5a, and 5b) which transfer poly-ADP-ribose chains on their target proteins. In particular, the critical role of PARP1/2 in DNA damage response and repair has been studied extensively, leading to effective cancer therapy. However, the majority of PARPs are monoPARPs, which transfer a single ADP-ribose to their target proteins. Recently, several of these family members have emerged in the literature as playing cancer-specific roles. While focused studies of individual monoPARPs are ongoing, a broad integrated in silico survey of the complete PARP family has yet to be done. Thus, we set out to characterize the molecular features of PARPs and their role in human cancer by mining the deep collection of publicly available molecular data from primary cancer, normal tissue samples and cancer cell lines. We designed and executed in silico analyses with the data available in the largest cancer public datasets, The Cancer Genome Atlas (TCGA) and the Cancer Dependency Map (DepMap). We explored standard oncogene hypotheses for all the PARPs, including mutational hotspots, copy-number variations, tumor mRNA overexpression, survival associations to genomic or expression variation, and cancer cell line dependency. Notably, two of the monoPARPs, PARP7 and PARP10, were found to be frequently amplified in multiple cancer types. Further analyses were aimed to identify significant bivariate relationships between PARP molecular features (e.g., expression and methylation) and other cancer-related biomarkers, such as tumor mutation burden or microsatellite instability. For gene expression data, we specifically determined associations between PARP mRNA levels and inferred tumor immune cell types from bulk RNA-sequencing, which implicated five monoPARPs having expression variation strongly associated with the tumor immune cell contexture in ten or more cancers. We also explored patterns of gene co-expression among the PARPs themselves and against the full genome. Our results provide the first pan-cancer in silico characterization of the PARP family, revealing a broad molecular and potential mechanistic diversity among the PARPs across cancer. Notwithstanding the lack of traditional oncogenic features, such as mutational hotspots, in the PARPs, our analyses highlight several monoPARPs with potential oncogenic roles and further support our focus of targeting these in the clinic. Citation Format: Ryan P. Abo, Mario Niepel, Heike Keilhack. A multi-omic characterization of PARP enzymes in cancer to identify novel monoPARP drug targets [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4381.

Inhibitors of the protein methyltransferase Enhancer of Zeste Homolog 2 (EZH2) may have significant therapeutic potential for the treatment of B cell lymphomas and other cancer indications. The ability of the scientific community to explore fully the spectrum of EZH2-associated pathobiology has been hampered by the lack of in vivo-active tool compounds for this enzyme. Here we report the discovery and characterization of EPZ011989, a potent, selective, orally bioavailable inhibitor of EZH2 with useful pharmacokinetic properties. EPZ011989 demonstrates significant tumor growth inhibition in a mouse xenograft model of human B cell lymphoma. Hence, this compound represents a powerful tool for the expanded exploration of EZH2 activity in biology.

Summary Background Activating enhancer of zeste homolog 2 (EZH2) mutations or aberrations of the switch/sucrose non-fermentable (SWI/SNF) complex (eg, mutations or deletions of the subunits INI1 or SMARCA4) can lead to aberrant histone methylation, oncogenic transformation, and a proliferative dependency on EZH2 activity. In this first-in-human study, we aimed to investigate the safety, clinical activity, pharmacokinetics, and pharmacodynamics of tazemetostat, a first-in-class selective inhibitor of EZH2. Methods We did an open-label, multicentre, dose-escalation, phase 1 study using a 3 + 3 design with planned cohort expansion at the two highest doses below the maximally tolerated dose. The study was done at two centres in France: Institut Gustave Roussy (Villejuif, Val de Marne) and Institut Bergonie (Bordeaux, Gironde). Eligible patients had relapsed or refractory B-cell non-Hodgkin lymphoma or an advanced solid tumour and were older than 18 years, with Eastern Cooperative Oncology Group performance status of 0 or 1, and adequate end-organ function. Tazemetostat was administered orally from 100 mg twice daily to 1600 mg twice daily in 28-day cycles. The primary endpoint was to establish the maximum tolerated dose or recommended phase 2 dose of tazemetostat, as determined by dose-limiting toxicities, laboratory values, and other safety or pharmacokinetic measures in cycle one according to local investigator assessment. Safety was assessed in patients who received at least one dose of tazemetostat; antitumour activity was assessed in the intention-to-treat population. This study is registered with ClinicalTrials.gov, number NCT01897571. The phase 1 part of the study is complete, and phase 2 is ongoing. Findings Between June 13, 2013, and Sept 21, 2016, 64 patients (21 with B-cell non-Hodgkin lymphoma, and 43 with advanced solid tumours) received doses of tazemetostat. The most common treatment-related adverse events, regardless of attribution, were asthenia (21 [33%] of 64 treatment-related events), anaemia (nine [14%]), anorexia (four [6%]), muscle spasms (nine [14%]), nausea (13 [20%]), and vomiting (six [9%]), usually grade 1 or 2 in severity. A single dose-limiting toxicity of grade 4 thrombocytopenia was identified at the highest dose of 1600 mg twice daily. No treatment-related deaths occurred; seven (11%) patients had non-treatment-related deaths (one at 200 mg twice daily, four at 400 mg twice daily, and two at 1600 mg twice daily). The recommended phase 2 dose was determined to be 800 mg twice daily. Durable objective responses, including complete responses, were observed in eight (38%) of 21 patients with B-cell non-Hodgkin lymphoma and two (5%) of 43 patients with solid tumours. Interpretation Tazemetostat showed a favourable safety profile and antitumour activity in patients with refractory B-cell non-Hodgkin lymphoma and advanced solid tumours, including epithelioid sarcoma. Further clinical investigation of tazemetostat monotherapy is ongoing in phase 2 studies in adults and a phase 1 study for children, which are currently enrolling patients who have B-cell non-Hodgkin lymphoma and INI1-negative or SMARCA4-negative tumours. Funding Epizyme and Eisai.

Cell proliferation assays are an important component of small molecule inhibitor screens for cancer therapies. An important but often overlooked variable involves the timing and timeframe of inhibitor treatment. Whereas many traditional chemotherapeutics kill or inhibit proliferation on the timeframe of hours or in a few days of treatment, more targeted therapies that affect other cancer-relevant pathways, including differentiation or cell stress responses, can take longer, often several days to weeks to impact cellular growth and survival. Many poly(ADP-ribose) polymerases (PARPs) are involved in cellular stress pathways; therefore, phenotypic effects of PARP inhibition are often only observed with long-term inhibitor treatment. Here we summarize several assays for analyzing long-term proliferation of both adherent and suspension cells, relying either on growth in two-dimensional tissue culture or on systems than enable growth in 3D.

Cell proliferation assays are an important component of small molecule inhibitor screens for cancer therapies. An important but often overlooked variable involves the timing and timeframe of inhibitor treatment. Whereas many traditional chemotherapeutics kill or inhibit proliferation on the timeframe of hours or in a few days of treatment, more targeted therapies that affect other cancer-relevant pathways, including differentiation or cell stress responses, can take longer, often several days to weeks to impact cellular growth and survival. Many poly(ADP-ribose) polymerases (PARPs) are involved in cellular stress pathways; therefore, phenotypic effects of PARP inhibition are often only observed with long-term inhibitor treatment. Here we summarize several assays for analyzing long-term proliferation of both adherent and suspension cells, relying either on growth in two-dimensional tissue culture or on systems than enable growth in 3D.

EZH2 and EZH1 are protein methyltransferases (PMTs) responsible for histone H3, lysine 27 (H3K27) methylation. Trimethylation of H3K27 (H3K27me3) is a hallmark of many cancers, including non-Hodgkin lymphoma (NHL). Heterozygous EZH2 point mutations at Tyr641, Ala677, and Ala687 have been observed in NHL. The Tyr641 mutations enhance activity on H3K27me2 but have weak or no activity on unmethylated H3K27, whereas the Ala677 and Ala687 mutations use substrates of all methylation states effectively. It has been proposed that enzymatic coupling of the wild-type and mutant enzymes leads to the oncogenic H3K27me3 mark in mutant-bearing NHL. We show that coupling with the wild-type enzyme is needed to achieve H3K27me3 for several mutants, but that others are capable of achieving H3K27me3 on their own. All forms of PRC2 (wild-type and mutants) display kinetic signatures that are consistent with a distributive mechanism of catalysis.

Activation of the phosphoinositide 3-kinase pathway is commonly observed in human prostate cancer. Loss of function of phosphatase and tensin homolog (PTEN) is associated with the activation of AKT and mammalian target of rapamycin (mTOR) in many cancer cell lines as well as in other model systems. However, activation of mTOR is also dependent of kinases other than AKT. Here, we show that activation of mTOR is not dependent on AKT in a prostate-specific PTEN-deficient mouse model of prostate cancer. Pathway bifurcation of AKT and mTOR was noted in both mouse and human prostate tumors. We demonstrated for the first time that cotargeting mTOR and AKT with ridaforolimus/MK-8669 and M1K-2206, respectively, delivers additive antitumor effects in vivo when compared to single agents. Our preclinical data suggest that the combination of AKT and mTOR inhibitors might be more effective in treating prostate cancer patients than current treatment regimens or either treatment alone.

The SWI/SNF complex is a major regulator of gene expression and is increasingly thought to play an important role in human cancer, as evidenced by the high frequency of subunit mutations across virtually all cancer types. We previously reported that in preclinical models, malignant rhabdoid tumors, which are deficient in the SWI/SNF core component INI1 (SMARCB1), are selectively killed by inhibitors of the H3K27 histone methyltransferase EZH2. Given the demonstrated antagonistic activities of the SWI/SNF complex and the EZH2-containing PRC2 complex, we investigated whether additional cancers with SWI/SNF mutations are sensitive to selective EZH2 inhibition. It has been recently reported that ovarian cancers with dual loss of the redundant SWI/SNF components SMARCA4 and SMARCA2 are characteristic of a rare rhabdoid-like subtype known as small-cell carcinoma of the ovary hypercalcemic type (SCCOHT). Here, we provide evidence that a subset of commonly used ovarian carcinoma cell lines were misdiagnosed and instead were derived from a SCCOHT tumor. We also demonstrate that tazemetostat, a potent and selective EZH2 inhibitor currently in phase II clinical trials, induces potent antiproliferative and antitumor effects in SCCOHT cell lines and xenografts deficient in both SMARCA2 and SMARCA4. These results exemplify an additional class of rhabdoid-like tumors that are dependent on EZH2 activity for survival.

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Tazemetostat (EPZ-6438) is a selective inhibitor of the histone methyltransferase EZH2 and currently in clinical development for non-Hodgkin lymphoma and genetically defined tumors.Tazemetostat was tested against the Pediatric Preclinical Testing Program (PPTP) solid tumor xenografts using a dose of 400 mg/kg administered twice daily by oral gavage for 28 days. H3K27me3:H3 ratios were determined in control and treated tumors.Tazemetostat induced significant differences in event-free survival (EFS) distribution compared with control in nine of 30 (30%) of the xenografts studied. Significant differences in EFS distribution were observed in five of seven (71%) rhabdoid tumor xenograft lines compared with four of 23 (17%) nonrhabdoid xenograft lines (chi-square [χTazemetostat demonstrated significant antitumor activity in rhabdoid tumor models but showed no consistent activity against any other histology. Tazemetostat reduced H3K27me3 levels irrespective of tumor response. Further preclinical testing to evaluate tazemetostat in combination with other anticancer agents is warranted.