Karnitz, Larry M.

Poly(ADP-ribose) polymerase (PARP) inhibitors (PARPis) are an important therapy for high-grade serous ovarian cancer (HGSOC). However, PARPi resistance frequently emerges, necessitating previously unrecognized approaches to improve HGSOC responses. Here, we showed that the anaplastic lymphoma kinase (ALK) inhibitor brigatinib enhances PARPi activity in HGSOC cells by disrupting an adaptive survival mechanism orchestrated by Fos-related antigen 1 (FRA1) in response to PARPi. This effect of brigatinib occurred through an ALK-independent pathway, wherein brigatinib induced a dual blockade of focal adhesion kinase (FAK) and EPH receptor A2 (EPHA2) tyrosine kinases, leading to the suppression of protein kinase B (Akt) and extracellular-regulated kinase (ERK) signaling accompanied by disruption of a phosphorylation event crucial for FRA1 protein stability. Moreover, in HGSOC patient-derived xenograft (PDX) models, brigatinib and PARPi combination therapy induced tumor regression and improved overall survival compared with PARPi alone, particularly in models with high FAK and EPHA2. These findings support dual targeting of FAK and EPHA2 as a strategy to achieve effective and durable PARPi responses and identify a promising biomarker-based combinatorial approach using brigatinib and PARPi for HGSOC, particularly the subset characterized by high FAK and EPHA2.

Abstract PARP inhibitors (PARPis) are now a standard of care for high-grade serous ovarian cancer (HGSOC), but resistance to these drugs poses a significant concern, necessitating the exploration of novel treatment strategies. Here we investigated the mechanism underlying enhanced PARPi activity in HGSOC cells following treatment with brigatinib—a second-generation anaplastic lymphoma kinase (ALK) inhibitor approved for treating lung cancer. Our previous work has shown that brigatinib is a dual inhibitor of tyrosine kinases FAK and EPHA2 with subsequent additive reduction in downstream PI3K/AKT and MAPK/ERK cell survival signaling. Suppression of these pathways conferred PARPi sensitivity to HGSOC cell lines and patient-derived xenograft (PDX) models, particularly in those with high FAK and EPHA2. To dissect the mechanism linking the FAK/EPHA2 – AKT/ERK pathway to PARPi synergy, we focused on proteomics approaches which revealed that FRA1—an Activator Protein 1 (AP-1) transcription factor known to be downstream of AKT/ERK—and its targets were decreased by brigatinib treatment. In subsequent phosphoproteomics approaches and cell-based confirmatory studies, a phosphorylation crucial for FRA1 stability was found to be disrupted, leading to reduction in total FRA1 protein levels and synergy with PARPis. To explore how FRA1 mediates response to PARPis, immunoblotting analysis revealed that PARPis increased the abundance of FRA1 in HGSOC cells at early timepoints, indicating potential involvement in an adaptive survival response. Follow-up pharmacologic and gene silencing techniques demonstrated that brigatinib still reduces FRA1 in PARPi-treated cells, and FRA1 depletion induces apoptosis when combined with PARPi to levels comparable to brigatinib treatment. Overall, our studies unveil a beneficial ALK-independent effect of brigatinib that may offer new avenues for considering its repurposing as a HGSOC therapy through disruption of a PARPi-induced adaptive survival mechanism. Citation Format: Julie R. Duffield, Xiaonan Hou, Benjamin W. Wilson, Anjali Prasad, Iman K. McKeon-Makki, Amelia M. Huehls, Xinyan Wu, Cristina Correia, Melissa C. Larson, Fergus J. Couch, Ann L. Oberg, Scott H. Kaufmann, Larry M. Karnitz, Arun Kanakkanthara. A PARP-inhibitor-induced early adaptive survival response is tackled through depletion of FRA1 by brigatinib in high-grade serous ovarian carcinoma [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Ovarian Cancer Research; 2025 Sep 19-21; Denver, CO. Philadelphia (PA): AACR; Cancer Res 2025;85(18_Suppl):Abstract nr A024.

Abstract PARP inhibitors (PARPis) have now become a standard of care for high-grade serous ovarian cancer (HGSOC), especially for those with defects in homologous recombination (HR) DNA repair. However, the innate and acquired PARPi resistance in HGSOC poses a significant concern, necessitating the exploration of novel treatment options and strategies. Here we investigated the potential of combining brigatinib—a second-generation anaplastic lymphoma kinase (ALK) inhibitor approved for treating lung cancer—with PARPis as way to enhance PARPi activity in HGSOC. Clonogenic cell survival assays revealed that brigatinib synergizes with PARPis in HR-proficient and -deficient HGSOC cell lines. In subsequent proteomics approaches and cell-based confirmatory studies, we found that brigatnib suppressed tyrosine kinases FAK and EphA2 with reduction in downstream signaling. Follow-up pharmacologic and gene-silencing techniques demonstrated that dual FAK and EphA2 inhibition additively reduces PI3K/Akt and MAPK/ERK cell survival signaling to levels conferring PARPi sensitivity. Additionally, the efficacy of this therapeutic approach on tumor size reduction was assessed in HGSOC patient-derived xenograft (PDX) models, with combination treatment inducing tumor regression more effectively than either agent alone. Overall, our studies unveil a beneficial ALK-independent effect of brigatinib that may offer new avenues for considering its repurposing as a HGSOC therapy. Citation Format: Julie R. Duffield, Xiaonan Hou, Iman K. McKeon-Makki, Amelia M. Huehls, Benjamin W. Wilson, Anjali Prasad, Xinyan Wu, Scott H. Kaufmann, Larry M. Karnitz, John J. Weroha, Arun Kanakkanthara. Brigatinib induces synergy with PARP inhibitors through dual inhibition of FAK and EphA2 in high-grade serous ovarian carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4538.

Abstract ZC3H18 is a multifunctional regulator of gene expression that is lost in a subset of high-grade serous ovarian cancers (HGSOC). Our prior studies have established that the loss of ZC3H18 diminishes BRCA1 levels, leading to the disruption of homologous recombination DNA repair (HR) and heightened sensitivity of HGSOC cells to PARP inhibitors. Here we show an additional facet to ZC3H18's impact, showing that its loss also induces a reprogramming of energy metabolism in HGSOC. Our findings demonstrate that the depletion of ZC3H18 results in reduced mRNA and protein levels of the NAD+-biosynthetic enzyme, NMNAT1, in HGSOC cells. Mechanistic studies uncovered that ZC3H18 occupies the NMNAT1 promoter, facilitating NMNAT1 transcription by recruiting CDK12 onto the promoter. Consistent with this mechanism, loss of ZC3H18 led to a reduction in CDK12 levels on the NMNAT1 promoter, diminishing NMNAT1 abundance and subsequently causing a decline in cellular NAD levels. These observations unveil a previously unknown role for ZC3H18 in HGSOC, shedding light on its involvement in the regulation of energy metabolism. Importantly, our findings provide valuable insights into potential therapeutic pathways that leverage the metabolic changes linked to ZC3H18 deficiency. Citation Format: Benjamin Wilson, Natalie Wilson, Xianfeng Chen, Shuwen Zhang, Chen Wang, Larry M. Karnitz, Scott H. Kaufmann, Arun Kanakkanthara. ZC3H18: A novel regulator of NAD+ metabolism in high-grade serous ovarian cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4447.

Abstract PARP inhibitors (PARPi) have activity in homologous recombination (HR) repair-deficient, high-grade serous ovarian cancers (HGSOC). However, even responsive tumors develop PARPi resistance, highlighting the need to delay or prevent the appearance of PARPi resistance. Here, we showed that the ALK kinase inhibitor ceritinib synergizes with PARPis by inhibiting complex I of the mitochondrial electron transport chain, which increases production of reactive oxygen species (ROS) and subsequent induction of oxidative DNA damage that is repaired in a PARP-dependent manner. In addition, combined treatment with ceritinib and PARPi synergized in HGSOC cell lines irrespective of HR status, and a combination of ceritinib with the PARPi olaparib induced tumor regression more effectively than olaparib alone in HGSOC patient-derived xenograft (PDX) models. Notably, the ceritinib and olaparib combination was most effective in PDX models with preexisting PARPi sensitivity and was well tolerated. These findings unveil suppression of mitochondrial respiration, accumulation of ROS, and subsequent induction of DNA damage as novel effects of ceritinib. They also suggest that the ceritinib and PARPi combination warrants further investigation as a means to enhance PARPi activity in HGSOC, particularly in tumors with preexisting HR defects. Significance: The kinase inhibitor ceritinib synergizes with PARPi to induce tumor regression in ovarian cancer models, suggesting that ceritinib combined with PARPi may be an effective strategy for treating ovarian cancer.

Abstract Acquired PARP inhibitor (PARPi) resistance in BRCA1- or BRCA2-mutant ovarian cancer often results from secondary mutations that restore expression of functional protein. RAD51C is a less commonly studied ovarian cancer susceptibility gene whose promoter is sometimes methylated, leading to homologous recombination (HR) deficiency and PARPi sensitivity. For this study, the PARPi-sensitive patient-derived ovarian cancer xenograft PH039, which lacks HR gene mutations but harbors RAD51C promoter methylation, was selected for PARPi resistance by cyclical niraparib treatment in vivo. PH039 acquired PARPi resistance by the third treatment cycle and grew through subsequent treatment with either niraparib or rucaparib. Transcriptional profiling throughout the course of resistance development showed widespread pathway level changes along with a marked increase in RAD51C mRNA, which reflected loss of RAD51C promoter methylation. Analysis of ovarian cancer samples from the ARIEL2 Part 1 clinical trial of rucaparib monotherapy likewise indicated an association between loss of RAD51C methylation prior to on-study biopsy and limited response. Interestingly, the PARPi resistant PH039 model remained platinum sensitive. Collectively, these results not only indicate that PARPi treatment pressure can reverse RAD51C methylation and restore RAD51C expression, but also provide a model for studying the clinical observation that PARPi and platinum sensitivity are sometimes dissociated.

Abstract Platinum-based DNA cross-linking agents are widely used anti-cancer drugs. Tumor resistance to platinum compounds is a major determinant of patient survival, including in high grade serous ovarian cancer (HGSOC). Remarkably, despite >30 years of literature on platinum responses in human cancer, none of these findings is used clinically as a predictive biomarker to stratify patients for platinum resistance, nor exploited therapeutically to treat platinum-resistant disease. Thus, understanding mechanisms of platinum resistance is an urgent goal, both to identify predictive biomarkers of platinum response (to spare patients with platinum-resistant tumors futile platinum therapy) and to develop efficacious therapies for platinum-resistant disease. To better understand mechanisms underlying platinum resistance in HGSOC, we performed comprehensive, dynamic (+/-carboplatin), multiomic profiling of DNA, RNA, protein and post-translational modifications (phosphorylation, ubiquitination, acetylation) to identify the cellular networks that respond to platinum treatment and associate with platinum resistance in three HGSOC intra-patient cell line pairs (PEA1S/PEA2R, PEO1S/PEO4R, PEO14S/PEO23R). The cell line pairs were derived from ascites or pleural effusions (Langdon et al., 1988) from three patients both before (PEA1S, PEO1S, PEO14S) and after (PEA2R, PEO4R, PEO23R, respectively) their tumors became clinically platinum resistant (i.e., in vivo development of resistance). The molecular profiles revealed extensive responses to carboplatin and differential responses between platinum-sensitive and platinum-resistant cells. Higher oxidative phosphorylation and fatty acid oxidation (FAO) pathway expression were observed in the platinum-resistant cells, which was further validated in patient-derived xenograft (PDX) models. We show that pharmacologic inhibition or CRISPR knockout of CPT1A, which represents a rate limiting step of FAO, sensitize HGSOC cells to platinum. Thus, FAO, and CPT1A in particular, represent a candidate therapeutic target to overcome platinum resistance in HGSOC. Citation Format: Hong Wang, Dongqing Hugang, Shrabanti Chowdhury, Sara Savage, Richard Ivey, Jacob Kennedy, Jeffrey Whiteaker, Chenwei Lin, Xiaonan Hou, Catherine Huntoon, Uliana Voytovich, Zahra Shire, Qing Yu, Steven Gygi, Andrew Hoofnagle, Zachary Herbert, Anna Calinawan, Larry Karnitz, S. John Weroha, Scott Kaufmann, Bing Zhang, Pei Wang, Michael Birrer, Amanda Paulovich. Multiomic analysis identifies CPT1A and fatty acid oxidation as a potential therapeutic target in platinum-refractory high grade serous ovarian cancer [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 1426.

Abstract Tumors with defective homologous recombination (HR) DNA repair are more sensitive to chemotherapies that induce lesions repaired by HR as well as PARP inhibitors (PARPis). However, these therapies have limited activity in HR-proficient cells. Accordingly, agents that disrupt HR may be a means to augment the activities of these therapies in HR-proficient tumors. Here we show that VLX600, a small molecule that has been in a phase I clinical trial, disrupts HR and synergizes with PARPis and platinum compounds in ovarian cancer cells. We further found that VLX600 and other iron chelators disrupt HR, in part, by inhibiting iron-dependent histone lysine demethylases (KDM) family members, thus blocking recruitment of HR repair proteins, including RAD51, to double-strand DNA breaks. Collectively, these findings suggest that pharmacologically targeting KDM family members with VLX600 may be a potential novel strategy to therapeutically induce HR defects in ovarian cancers and correspondingly sensitize them to platinum agents and PARPis, two standard-of-care therapies for ovarian cancer.

Abstract Although inhibitors of the kinases CHK1, ATR, and WEE1 are undergoing clinical testing, it remains unclear how these three classes of agents kill susceptible cells and whether they utilize the same cytotoxic mechanism. Here we observed that CHK1 inhibition induces apoptosis in a subset of acute leukemia cell lines in vitro, including TP53-null acute myeloid leukemia (AML) and BCR/ABL–positive acute lymphoid leukemia (ALL), and inhibits leukemic colony formation in clinical AML samples ex vivo. In further studies, downregulation or inhibition of CHK1 triggered signaling in sensitive human acute leukemia cell lines that involved CDK2 activation followed by AP1-dependent TNF transactivation, TNFα production, and engagement of a TNFR1- and BID-dependent apoptotic pathway. AML lines that were intrinsically resistant to CHK1 inhibition exhibited high CHK1 expression and were sensitized by CHK1 downregulation. Signaling through this same CDK2–AP1–TNF cytotoxic pathway was also initiated by ATR or WEE1 inhibitors in vitro and during CHK1 inhibitor treatment of AML xenografts in vivo. Collectively, these observations not only identify new contributors to the antileukemic cell action of CHK1, ATR, and WEE1 inhibitors, but also delineate a previously undescribed pathway leading from aberrant CDK2 activation to death ligand–induced killing that can potentially be exploited for acute leukemia treatment. Significance: This study demonstrates that replication checkpoint inhibitors can kill AML cells through a pathway involving AP1-mediated TNF gene activation and subsequent TP53-independent, TNFα-induced apoptosis, which can potentially be exploited clinically.