Flatten, Karen S.

Abstract Despite long-standing interest in combining topoisomerase I (TOP1) poisons with PARP inhibitors (PARPis), the mechanistic basis for the variable and often unpredictable response to this strategy remains unclear. Early preclinical studies suggested that PARP inhibition amplifies TOP1 poison-induced DNA damage, implying broad therapeutic potential; however, emerging translational and clinical data—particularly in ovarian cancer—indicate that synergy is context-dependent and governed by tumor-intrinsic features that have not been fully defined.Here we assessed determinants of response in five HR proficient ovarian cancer (OC) patient-derived xenografts (PDXs) previously treated with the SN-38 prodrug etirinotecan pegol (NKTR-102) in combination with the PARPi rucaparib (C. Nitschmann, Clin. Cancer Res. 22(2) Suppl. B50, 2016). Although all models were refractory to single-agent rucaparib, 50% exhibited enhancement of NKTR-102-induced tumor regression with the addition of rucaparib. Neither PARP1 abundance nor extent of rucaparib-mediated poly(ADP-ribose) suppression correlated with therapeutic benefit. Instead, sensitization was restricted to PDXs intrinsically responsive to NKTR-102 and was strongly associated with high levels of NKTR-102-induced TOP1-DNA covalent complexes. In further analysis, 2 of 3 resistant PDXs exhibited high levels of the ABC transporter ABCG2 despite lack of previous therapy, revealing a previously underappreciated mechanism of de novo resistance that limits effective TOP1 inhibitor activity independent of homologous recombination status. Notably, NKTR-102 response did not track with platinum/taxane sensitivity, highlighting the distinct molecular circuitry governing TOP1 poison susceptibility.Collectively, these findings demonstrate that rucaparib can potentiate NKTR-102-induced regression in tumors capable of forming abundant TOP1-DNA covalent complexes. This work identifies TOP1-DNA complex trapping and lack of ABCG2 expression as mechanistic predictors of benefit, providing an updated framework for selecting patients most likely to respond to TOP1 poison/PARPi combinations and challenging the long-standing assumption that PARP inhibition can universally resensitize tumors to TOP1 poisons. Citation Format: Annapoorna Venkatachalam, Caroline C. Nitschmann, Karen S. Flatten, Rachel Hurley, Xiaonan Hou, Cristina Correia, Paula A. Schenider, Krista Goergen, Matthew Maurer, Ethan P. Heinzen, Ann L. Oberg, S John Weroha, Paul Haluska, Scott H. Kaufmann, Elizabeth M. Swisher. TOP1-DNA protein complex as a predictive biomarker for NKTR-102/Rucaparib combination therapy in PARP-inhibitor resistant ovarian cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(8_Suppl):Abstract nr LB364.

Abstract BRCA2 mutations contribute to the pathogenesis and treatment sensitivity of a subset of ovarian, breast, prostate, and pancreatic cancers. When these cancers become therapy resistant, secondary mutations that restore the BRCA2 open reading frame are found in half the cases, but other causes of resistance remain incompletely understood. Here, we identified translational readthrough of a premature termination codon (PTC) as a cause of resistance to poly(ADP-ribose) polymerase inhibitors (PARPis) and cisplatin in cells derived from the BRCA2-mutated ovarian cancer line PEO1 by PARPi selection. Despite persistence of the signature 4965C > G (p.Y1655X) BRCA2 mutation, low-level expression of full-length BRCA2 protein was detectable in these cells by immunoblotting and tandem mass spectrometry. Either BRCA2 knockdown or gene interruption 5′ or 3′ to the PTC restored treatment sensitivity, implicating BRCA2 in the resistance. Reporter assays demonstrated UAG-selective readthrough in the resistant clones but not parental cells. Moreover, custom searching of global proteomic data indicated readthrough of stop codons, particularly UAGs, in additional proteins in the resistant clones. Finally, multi-omic analysis identified multiple changes in the nonsense-mediated decay and termination machineries that favor readthrough. Accordingly, the present results identify PTC readthrough as a potential mechanism of drug resistance in cells with BRCA2 nonsense mutations.

Abstract Background: The kinases ataxia-telangiectasia-mutated-and-Rad3-related kinase (ATR), checkpoint kinase 1 (CHK1) and WEE1 participate in the response to replication stress. Inhibitors of ATR, WEE1, and CHK1 are being tested for antineoplastic activity as monotherapies and in combination with chemotherapy. Although these agents impair cell cycle checkpoints, fork stabilization, origin firing, and HR, little is known about the apoptotic pathways that are engaged and the critical mediators activating cell death mechanisms. The present study utilizes high-grade serous ovarian cancer (HGSOC) cell lines and patient-derived xenografts (PDXs) to understand the predominant apoptotic players and assess changes involved in the resistance setting after treatment with these inhibitors. Methods: HGSOC cell lines were examined for responses to ceralasertib (ATRi), prexasertib (CHK1i), or adavosertib (WEE1i) using colony forming assays, immunoblotting, and assays for apoptosis. To characterize the cell death networks after prexasertib treatment, RNA-seq analysis was performed in two representative cell lines. Mechanisms involved in acquired resistance and downstream changes in cell fate were evaluated by generating prexasertib- resistant HGSOC cell lines. Results: Irrespective of the homologous recombination (HR) status, ovarian cell lines, and PDXs underwent cell death during prolonged exposure at clinically achievable concentrations. CDC25A-CDK2 signaling was predictive of responsiveness in vitro and in vivo. The subsequent apoptotic response was c-Jun N-terminal kinases (JNK) dependent and involved engagement of mitochondrial pathways rather than the TNFa-induced death receptor-mediated apoptosis we recently described in acute leukemia (H. Ding, N. Vincelette, et al., Cancer Res. 81:2666-2678, 2021). Additionally, ovarian lines selected for CHK1i resistance failed to die in response to either ATRi or WEE1i but were sensitive to front-line therapy options, including carboplatin and PARP inhibitor. Conclusions: Unlike AML, death receptor-mediated apoptosis was not observed; instead, the primary mechanism of cell killing in HGSOC lines involves activation of the mitochondrial apoptotic pathway. Our findings identify the key cell death-initiating events that serve as measures of response to replication checkpoint inhibitors in ovarian cancer. Acquired resistance to these therapies is multifactorial and mainly involves failure to exacerbate the replication stress response. Citation Format: Annapoorna Venkatachalam, Cristina Correia, Kevin L. Peterson, Xiaonan Hou, Annabella R. Strathman, Paula A Schneider, Karen S. Flatten, Laura N. Duffield, Cordelia D. McGehee, Melissa C. Larson, Xue Meng, Nicole D Vincelette, Ann L. Oberg, Hu Li, John Weroha, Scott H Kaufmann. Transcriptomic insights reveal role of BCL2 proteins in replication checkpoint inhibitor response [abstract]. In: Proceedings of the AACR Special Conference on Ovarian Cancer; 2023 Oct 5-7; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(5 Suppl_2):Abstract nr B012.

AbstractBackgroundDespite recent approval of several new agents, relapsed acute lymphoblastic leukemia (ALL) remains challenging to treat. Sapanisertib (MLN0128/TAK‐228) is an oral TORC1/2 inhibitor that exhibited preclinical activity against ALL.MethodsWe conducted a single‐arm multi‐center Phase II study of sapanisertib monotherapy (3 mg orally daily of the milled formulation for 21 days every 28 days) in patients with ALL through the Experimental Therapeutics Clinical Trials Network (NCI‐9775).ResultsSixteen patients, 15 of whom were previously treated (median 3 prior lines of therapy), were enrolled. Major grade 3–4 non‐hematologic toxicities included mucositis (3 patients) and hyperglycemia (2 patients) as well as hepatic failure, seizures, confusion, pneumonitis, and anorexia (1 patient each). Grade >2 hematological toxicity included leukopenia (3), lymphopenia (2), thrombocytopenia, and neutropenia (1). The best response was stable disease in 2 patients (12.5%), while only 3 patients (19%) were able to proceed to Cycle 2. Pharmacokinetic analysis demonstrated drug exposures similar to those observed in solid tumor patients. Immunoblotting in serially collected samples indicated limited impact of treatment on phosphorylation of mTOR pathway substrates such as 4EBP1, S6, and AKT.ConclusionIn summary, single‐agent sapanisertib had a good safety profile but limited target inhibition or efficacy in ALL as a single agent. This trial was registered at ClinicalTrials.gov as NCT02484430.

Abstract Background: The kinases ataxia-telangiectasia-mutated-and-Rad3-related (ATR), checkpoint kinase 1 (CHK1) and WEE1 participate in the response to replication stress. Inhibitors of ATR, WEE1 and CHK1 are being tested for antineoplastic activity as monotherapies and in combination with chemotherapy. Although these agents impair cell cycle checkpoints, fork stabilization, origin firing and homologous recombination (HR), little is known about the apoptotic pathways that are engaged and the critical mediators activating cell death mechanisms. The present study utilizes high grade serous ovarian cancer (HGSOC) cell lines and patient derived xenografts (PDXs) to identify the primary mechanism of cell death in ovarian cancer and assess changes involved in the resistance setting after treatment with these inhibitors. Methods: A panel of HGSOC cell lines was examined for responses to ceralasertib (ATRi), prexasertib (CHK1i) or adavosertib (WEE1i) using colony forming assays, immunoblotting and assays for apoptosis. Mechanisms involved in acquired resistance and downstream changes in cell fate were evaluated by generating prexasertib-resistant HGSOC cell lines. Subsequent NGS analysis was performed in the sensitive and resistant pairs. Results: Irrespective of the homologous recombination (HR) status, ovarian cell lines and PDXs underwent cell death during prolonged exposure at clinically achievable concentrations. Treatment of ovarian cancer cell lines with ATRi, CHK1i or WEE1i activated the replication checkpoint, consistent with previously published reports. The subsequent apoptotic response involved PMAIP1 and BCL2L11 upregulation rather than the TNFa-induced death receptor-mediated apoptosis we recently described in acute leukemia (Cancer Res. PMID: 33414171). Additionally, ovarian lines selected for CHK1i resistance failed to die in response to either ATRi or WEE1i. Conclusions: Prexasertib, ceralasertib, and adavosertib exhibit monotherapy activity in both HR deficient and proficient HGSOC cell lines and PDX models. Unlike AML, death receptor mediated apoptosis was not observed; instead, the primary mechanism of cell killing in HGSOC lines involves activation of the mitochondrial apoptotic pathway. Cross-resistance of these DNA damage repair modulators is likely multifactorial and mainly involves diminished replication stress response. Citation Format: Annapoorna Venkatachalam, Kevin L. Peterson, Cristina Correia, Karen S. Flatten, Xianon Hou, Paula A. Schneider, Emily Balczewski, Cordelia McGehee, Rachel M. Hurley, Xue W. Meng, Chance Sine, Rameen Shah, Nicole Vincelettte, Husheng Ding, Hu Li, Saravut (John) Weroha, Scott H. Kaufmann. CHK1 inhibitor prexasertib induces NOXA-dependent apoptosis in ovarian 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 1832.

The poly(ADP-ribose) binding protein CHFR regulates cellular responses to mitotic stress. The deubiquitinase UBC13, which regulates CHFR levels, has been associated with better overall survival in paclitaxel-treated ovarian cancer. Despite the extensive use of taxanes in the treatment of ovarian cancer, little is known about expression of CHFR itself in this disease. In the present study, tissue microarrays containing ovarian carcinoma samples from 417 women who underwent initial surgical debulking were stained with anti-CHFR antibody and scored in a blinded fashion. CHFR levels, expressed as a modified H-score, were examined for association with histology, grade, time to progression (TTP) and overall survival (OS). In addition, patient-derived xenografts from 69 ovarian carcinoma patients were examined for CHFR expression and sensitivity to paclitaxel monotherapy. In clinical ovarian cancer specimens, CHFR expression was positively associated with serous histology (p = 0.0048), higher grade (p = 0.000014) and higher stage (p = 0.016). After correction for stage and debulking, there was no significant association between CHFR staining and overall survival (p = 0.62) or time to progression (p = 0.91) in patients with high grade serous cancers treated with platinum/taxane chemotherapy (N = 249). Likewise, no association between CHFR expression and paclitaxel sensitivity was observed in ovarian cancer PDXs treated with paclitaxel monotherapy. Accordingly, differences in CHFR expression are unlikely to play a major role in paclitaxel sensitivity of high grade serous 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 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.

Abstract Introduction: Topoisomerase I is a nuclear enzyme that relaxes torsional strain in DNA by nicking one DNA strand, allowing rotation, then resealing the DNA backbone. This is an important enzyme in replication and transcription, and therefore has been targeted by anti-cancer agents such as camptothecins. These agents intercalate into DNA at the topoisomerase I active site and inhibit the religation step catalyzed by the enzyme, causing increased topoisomerase I-DNA complexes that impede advancing replication forks or transcription complexes, leading to DNA damage and cell death. Platinating agents also stabilize topoisomerase I-DNA covalent complexes. The topotecan/carboplatin combination has recently shown promise in combination with the PARP inhibitor veliparib in aggressive and transformed myeloproliferative neoplasms (MPNs) and chronic myelomonocytic leukemia (K Pratz et al., Clin Cancer Res. 23:899, 2017). Given that both classes of agents have a narrow therapeutic window, it is important to try to predict the sensitivity/resistance of these agents in malignancies such as acute myeloid leukemia (AML) and transformed MPNs. Some of mechanisms of drug resistance that have been described are decreased numbers of drug-stabilized topoisomerase I-DNA complexes due to drug efflux, diminished topoisomerase I expression or topoisomerase I mutation. Thus, being able to detect and quantify these DNA-protein complexes may provide insight into drug sensitivity. Methods: Using a unique monoclonal antibody that detects topoisomerase I-DNA covalent complexes (A. Patel et al., Nucleic Acids Res. 44:2816, 2016), immunofluorescence techniques were used to quantify these topoisomerase I-DNA complexes in AML cell lines (SET2, HEL, K562, U937, ML2, MV-4-11, THP1, HL60, ML1, and Molm13) and clinical AML samples. To verify the fidelity of the anti-topoisomerase I-DNA covalent complex antibody (TopIcc), siRNA knockdown methods were utilized with confirmatory immunofluorescence and Western blotting. Slot blots were additionally used to assess the presence of these complexes in AML cell lines. Western blots were performed to assess topoisomerase I levels in AML cell lines. Topotecan drug sensitivity analysis was performed by using an MTS assay. Results: With treatment of the topoisomerase I inhibitor, topotecan, there was an increase in the percentage of leukemia cells positive for the topoisomerase I-DNA covalent complexes. The degree of increase in complexes varied across cell lines and patient samples. To ensure we were detecting TopIcc by immunofluorescence, we performed a topoisomerase-I siRNA knockdown to confirm the specificity of the antibody for TopIcc's. Slot blots on multiple AML cell lines confirmed the presence of topoisomerase-DNA covalent complexes. The Western blot of all the AML cell lines, ordered from low to high TopIcc revealed increasing amounts of topoisomerase I. The MTS assay revealed lower topotecan IC50 values in three of the cell lines with higher complex formation, including Molm13, ML1 and ML2 (IC50 6-8 nM compared to 13-46 nM in the other cell lines). Conclusions: Based on recent clinical results, there has been renewed interested in topoisomerase I inhibitors, especially for aggressive and transformed MPNs. Topoisomerase I-DNA complex repair/downregulation has been a hallmark of drug resistance to topoisomerase I inhibitors. Therefore, detecting and quantifying these complexes in treated samples has been hypothesized to provide insight into the drug sensitivity, which is particularly important in camptothecins and platinating agents given their narrow therapeutic windows. Immunodetection techniques using an anti-TopIcc antibody may help predict sensitivity of AML to topoisomerase I inhibitors and will be used as a correlative biomarker study in an upcoming phase II randomized study of topotecan/carboplatin with or without veliparib or placebo in advanced myeloproliferative disorders and chronic myelomonocytic leukemia (ClinicalTrials.gov NCT03289910). Disclosures Pratz: AbbVie: Consultancy, Research Funding; Agios: Research Funding; Astellas: Consultancy, Research Funding; Boston Scientific: Consultancy; Millenium/Takeda: Research Funding. Kaufmann:Mayo Clinic: Patents & Royalties: Co-inventor on a patent held by Mayo Clinic for the use of the anti-TOP1cc antibody as a diagnostic reagent.

Abstract BACKGROUND: Acute myeloid leukemia (AML) is an aggressive leukemia with 5-year overall survival of 20-25%. The major reason for treatment failure in AML is resistance to chemotherapy. Thus, there is an urgent need for identification of novel therapeutic agents for AML. Neoplastic cells, including AML, have dysfunctional redox regulation that results in increased reactive oxygen species (ROS). Accumulation of ROS leads to oxidation of free and incorporated nucleotides, leading to DNA damage and cell death. MTH1 is a nudix family hydrolase that sanitizes the oxidized nucleotide pool to prevent incorporation of these damaged bases in the DNA. MTH1 is thought to be non-essential for normal cells but crucial for neoplastic cells in order to avoid incorporation of oxidized dNTPs into DNA, thereby evading DNA damage and cell death. Whether MTH1 inhibitors have any activity against AML is not known. METHODS: Neoplastic myeloid cell lines HL-60, HEL, K562, KG1A, ML1, MV-4-11, SET2, and U937 were treated with varying concentrations of TH588 for a total of 48 hours. In experiments using the pan-caspase inhibitor Q-VD-OPh (Qvd), cells were pre-treated with 5µM Qvd for 1 hour followed by TH588. Cells were washed and stained with annexin, propidium iodide (PI), or MitoTracker (Life Technologies, Carlsbad, CA) for flow cytometry. To evaluate the potential impact of MTH1 inhibition on chemorefractory AML, HL-60/VCR cells were treated with vehicle control or TH588 in culture medium with or without vincristine (1µg/ml). Percentage apoptosis was calculated by normalizing to vehicle only control. With IRB approval, bone marrow aspirate samples were obtained from patients with untreated AML or healthy controls. Mononuclear cells were analyzed using colony-forming unit (CFU) assays. The total number of erythroid (CFU-E) and myeloid (CFU-G, CFU-GM) colonies containing ≥50 cells were read on day 14 and reported as percentage colonies compared to vehicle control. RESULTS: TH588 induced dose-dependent cell death in each of the neoplastic cell lines tested except HEL. In particular, treatment with TH588 resulted in a dose-dependent increase in the number of cells undergoing apoptosis as indicated by annexin V and/or PI staining (IC50 3.1-21.3µM, Figure 1). Pre-treatment with Qvd significantly inhibited TH588-induced cell death in all the cell lines studied except KG1A and SET2, suggesting a caspase-dependent mechanism of cell death. In further studies, cells treated with TH588 exhibited decreased MitoTracker staining; and Qvd pretreatment increased the number of MitoTrackerLow cells at the same time apoptotic cells decreased, suggesting that mitochondrial damage is upstream of caspase activation in TH588-induced apoptosis. Treatment with TH588 not only induced apoptosis in HL-60/VCR cells, but also facilitated further apoptosis in cells co-treated with vincristine and TH588 (Figure 2). Treatment with TH588 also diminished colony formation in a primary AML sample (IC50 6µM, Figure 3). Analysis of additional primary AML samples is ongoing. DISCUSSION: Our results show that the MTH1 inhibitor TH588 induces apoptosis in most neoplastic myeloid cells. MTH1 causes mitochondrial damage that, in turn, leads to caspase-dependent apoptosis in these cells. In HL-60/VCR cells representing chemorefractory phenotype, TH588 induces apoptosis as a single agent and resensitizes cells to vincristine. Moreover, TH588 significantly diminished colony formation in primary AML ex vivo. Further preclinical and possible clinical study of this class of agent appears warranted. Figure 1. Induction of cell death by MTH1 inhibitor TH588 in neoplastic myeloid cell lines. Figure 1. Induction of cell death by MTH1 inhibitor TH588 in neoplastic myeloid cell lines. Figure 2. TH588 induces apoptosis in HL-60/VCR cells and resensitizes cells to vincristine. Figure 2. TH588 induces apoptosis in HL-60/VCR cells and resensitizes cells to vincristine. Figure 3. TH588 significantly diminished colony formation in primary AML ex vivo indose-dependent manner. Figure 3. TH588 significantly diminished colony formation in primary AML ex vivo indose-dependent manner. Disclosures No relevant conflicts of interest to declare.