Citations

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 DAC combinations serve as frontline therapies for AML. DAC forms DNA-DNMT1 crosslinks and activates the ATR-CHK1-WEE1 DDR. Combinations of DNA-protein crosslinking agents with ATR, CHK1, or WEE1 inhibitors (i) have shown efficacy in solid tumors. This study evaluates the cytotoxic synergy of DAC plus ATRi, CHK1i, or WEE1i in AML. Public datasets were queried for mRNA expression and dependency. Commercial cell lines (U937, KG1a, Molm13, MV-4-11) were genomically validated. Molm13 TP53-/- clones were generated via CRISPR. Cells were cultured in complete RPMI. To treat, cells were cultured for 48 h in RPMI with drugs or diluent (0.2% v/v DMSO). Protein expression was evaluated by Western blotting (WB). Apoptosis was assayed by flow cytometry with annexin V or propidium iodide. Primary AML samples were obtained at study centers after informed consent. Isolated mononuclear cells (Ficoll) were used in colony forming unit (CFU) assays with DMSO or drug for 12-16 days. Synergy was determined by median-effect (CalcuSyn) where a combination index <1 is synergistic and >1 antagonistic. DNMT1 expression (TCGA) was highest in AML vs all other tumor types (p<0.001). In CRISPR screens (DepMap), AML cell lines were increasingly dependent on DNMT1, ATR, CHK1, and WEE1 (ANOVA p<0.001). WB of AML cell lines treated with DAC (25 - 200 nM) for 24 h showed phosphorylations of H2AX and CHK1 S296/S345 that were most pronounced in TP53MT lines. In apoptosis assays, ATRi, CHK1i, and WEE1i were synergistic with clinical concentrations of DAC (Table 1). In isogenic Molm13 lines, TP53 loss conferred DAC resistance that was overcome with combination treatment. Selective CHK1i were generally more synergistic than CHK1/2i, consistent with a CHK1 DDR. CFU assays of primary AML samples also showed synergy between DAC and ATRi, CHK1i, and especially WEE1i. Table 1. Synergy between decitabine and replication checkpoint inhibitors in preclinical models of AML. Cell line data are derived from subdiploid apoptosis assays. U937 data were replicated using annexin V. Prexasertib showed no activity in KG1a cells (suggesting drug efflux) requiring the use of the alternate CHK1 inhibitor, rabusertib. Abbreviations: ATR, ataxia telangiectasia and Rad3 related; CHK1/2, checkpoint kinase 1 and/or 2; CI, combination index; Conc, concentration; DNMT1, DNA methylatransferase 1; IQR, interquartile range; MT, mutated; ND, not determined; SD, standard deviation; WT, wild type. Drug Target Conc. (nM) Molm13 (TP53WT) Molm13 (TP53-/-) U937 (TP53G187fs*/-) KG1a (TP53V225fs*/-) Primary AML (TP53WT orTP53MT) Units Decitabine DNMT1 6.25 – 200 25 ± 11 350 ± 260 310 ± 180 6.1 ± 1.6 ND EC50, nM (mean, SD) Ceralasertib ATR 250 – 500 0.93 (0.66 – 1.24) 0.90 (0.84 – 0.96) 0.15 (0.08 – 0.21) 0.74 (0.63 – 1.02) 0.66 (0.43 – 2.43) CI with decitabine (median, IQR) Camonsertib ATR 10 – 100 0.27 (0.19 – 0.59) 0.24 (0.22 – 0.34) 0.34 (0.21 – 0.39) ND ND Prexasertib CHK1/2 1.0 – 6.0 0.89 (0.82 – 1.06) 0.78 (0.68 – 0.90) 0.95 (0.93 – 1.08) ND 0.88 (0.29 – 1.28) MK-8776 CHK1 250 – 1, 000 0.57 (0.48 – 0.80) 0.76 (0.73 – 1.19) 0.89 (0.57 – 1.39) ND ND Rabusertib CHK1 600 – 1, 000 ND ND ND 0.51 (0.48 – 0.56) ND Adavosertib WEE1 100 – 400 0.70 (0.63 – 1.11) 0.99 (0.78 – 1.14) 0.67 (0.60 – 0.74) 0.67 (0.37 – 0.93) 0.65 (0.43 – 0.88) DAC synergizes with ATRi, CHK1i, and WEE1i in AML. Future work will test these combinations in vivo and incorporate BH3 mimetics. Citation Format: Clifford M. Csizmar, Antoine N. Saliba, Olivia K. Rossman, Kevin L. Peterson, X. Wei Meng, B. Douglas Smith, Gabriel Ghiaur, Jonathan A. Webster, Mrinal M. Patnaik, Aref Al-Kali, Scott H. Kaufmann. Decitabine (DAC) induces a DNA damage response (DDR) and synergizes with replication checkpoint inhibitors in acute myeloid leukemia (AML) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 1170.

Abstract Zinc finger (ZNF) motifs are some of the most frequently occurring domains in the human genome. It was only recently that ZNF proteins emerged as key regulators of genome integrity in mammalian cells. In this study, we report a new role for the Krüppel-type ZNF-containing protein ZNF432 as a novel poly(ADP-ribose) (PAR) reader that regulates the DNA damage response. We show that ZNF432 is recruited to DNA lesions via DNA- and PAR-dependent mechanisms. Remarkably, ZNF432 stimulates PARP-1 activity in vitro and in cellulo. Knockdown of ZNF432 inhibits phospho-DNA-PKcs and increases RAD51 foci formation following irradiation. Moreover, purified ZNF432 preferentially binds single-stranded DNA and impairs EXO1-mediated DNA resection. Consequently, the loss of ZNF432 in a cellular system leads to resistance to PARP inhibitors while its overexpression results in sensitivity. Taken together, our results support the emerging concept that ZNF-containing proteins can modulate PARylation, which can be embodied by the pivotal role of ZNF432 to finely balance the outcome of PARPi response by regulating homologous recombination.

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.

326 Background: Review initial outcomes for patients enrolled on the University of Florida Proton Therapy Institute PC01 protocol for patients with unresectable pancreatic cancer. Methods: The protocol received Institutional Review Board (IRB) approval in 7/2007. The first patient was enrolled in 4/2010. The accrual target was 66 patients to test if the Serious Adverse Event (SAE) ratecould be reduced from 15% (expected) to less than 5%. Protocol therapy consisted of proton therapy to a planning target volume (PTV) dose of 59.4 Cobalt Gray Equivalent (CGE) at 1.8CGE per fraction with concomitant oral Capecitabine (1000mg PO BID 5 days/week on radiation treatment days). Only gross disease was targeted. Results: 12 patients were enrolled. 1 patient died of a gunshot wound 5 days after starting treatment so only 11 patients are reported. Median follow up from start of treatment: 13 months (range 2 to 21 months). Median age: 68 (range 51 to 86) years. Gender: Males 5, Females 6. Race/Ethnicity: 9 White, 2 Hispanic. Overall survival: 90% at 6 months and 60% at 12 months. Freedom from progression: 80% at 6 months and 53% at one year. Local control: 100% at 6 months and 86% at 12 months. Freedom from metastasis: 80% at 6 months and 67% at 12 months.No patient experienced any grade 3 or greater toxicity during treatment or the follow up period. Grade 2 toxicity was limited to a single patient experiencing grade 2 fatigue. Median weight loss over the course of treatment was 1.7 (range: loss of 5.7 to gain of 4.9) Kg. 4 patients were deemed to have had an adequate radiographic response to radiotherapy so as to justify surgical exploration. Conclusions: Proton therapy to 59.4 CGE with concomitant Capecitabine was well tolerated with no grade 2 or greater gastrointestinal toxicities. 4 of 11 patients achieved a radiographic response allowing for attempted surgical resection. The study was closed to accrual on 9/1/2013 due to recognition that slow accrual made achievement of the primary study endpoint unlikely. At the same time, the lack of any meaningful gastrointestinal toxicity suggests significant opportunities for treatment intensification when proton therapy is used in this setting. Clinical trial information: NCT00685763.