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The recent discovery and clinical validation of KRAS inhibitors (KRASi) has ushered in a new therapeutic approach to directly address the previously undruggable mutant KRAS-driven cancers. Unfortunately, as with other oncogene-directed therapies, acquired resistance to KRASi has been observed that is partially attributed to secondary mutations in KRAS and/or mutations or fusions in other mitogen-activated protein kinase (MAPK) signaling genes. Emerging evidence suggests that amplification of oncogenes on acentric extrachromosomal DNA (ecDNA) might constitute an important mechanism of rapid resistance to receptor tyrosine kinase (RTK) and MAPK targeting therapies. Therefore, we investigated whether resistance to KRASi monotherapy is mediated through an ecDNA mechanism. We previously noted focal gene amplification of KRAS and MET in recurrent tumors isolated from patients treated with the selective KRASG12C inhibitor, adagrasib. Analysis of next-generation sequencing (NGS) data from a patient tumor revealed evidence of circular DNA structures encompassing the KRAS locus. Consistent with this finding, interphase FISH analysis indicated the presence of KRAS amplifications on ecDNA in this adagrasib-treated patient tumor. To further interrogate this biology, we examined a genetically modified CT26 murine tumor model harboring KRASG12C alteration. Analogous to the clinical observations, both adagrasib as well as the recently approved KRASi sotorasib, induced transient tumor regressions that subsequently resumed growth following several weeks of continuous therapy. Strikingly, whole-genome sequencing data from isolated recurrent tumors revealed focal amplification of KRASG12C and metaphase FISH confirmed high levels of KRASG12C on ecDNA as compared to vehicle-treated samples. Further mechanistic studies are underway. Collectively, these observations implicate ecDNA as an important mediator of resistance to KRASi monotherapy and reinforce the critical need for novel therapeutic strategies to address ecDNA oncogene amplification-driven cancers. Citation Format: Ryan J Hansen, Steven Horton, Julie Wiese, BellJohn Bibay, E.Lorena Mora-Blanco, Nam-Phuong Nguyen, Lars D Engstrom, David M Briere, Andres Calinisan, Peter Olson, Mark M Awad, Andrew J Aguirre, Jason Christiansen, Christian Hassig, Shailaja Kasibhatla. Detection of KRAS amplification on extrachromosomal DNA (ecDNA) upon acquired resistance to KRASG12C inhibitors [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2021 Oct 7-10. Philadelphia (PA): AACR; Mol Cancer Ther 2021;20(12 Suppl):Abstract nr LBA005.

Depending on genetic and other factors, tumor cells exhibit replication stress (RS) to various extents. Low levels of RS contribute to genomic instability, an enabling hallmark of cancer, whereas high levels may be cytotoxic. The DNA damage response (DDR) checkpoint kinase 1, Chk1, is essential for managing RS by stabilizing replication forks, abrogating origin firing, pausing the cell cycle, and fostering homologous recombination DNA repair. Consequently, tumor cells become highly reliant on Chk1 to manage RS and maintain proliferation and survival. Certain chemotherapeutic drugs, including nucleoside analogs, DNA cross-linkers, and topoisomerase inhibitors, are effective extrinsic inducers of RS. Here we demonstrate that the potent, selective oral Chk1 inhibitor, SRA737, strongly synergizes with a range of extrinsic inducers of RS to kill tumor cells in vitro and in vivo. We find that clinically used drugs including the DNA crosslinking agents mitomycin C and cisplatin, as well as the nucleoside analog gemcitabine, exacerbate cellular RS markers (e.g. γ-H2A.X and p-RPA) and synergize with SRA737 to increase tumor cell death in vitro. Combination indices (CI) at various doses across a cell line panel were 0.46, 0.54, and 0.25 for mitomycin C, cisplatin, and gemcitabine, respectively. Similar results were observed with both type I and type II topoisomerase inhibitors (SN-38 and etoposide). Notably, gemcitabine potentiates SRA737 in several bladder cancer cell lines including 5637, TCCSUP, and J82 cells, as well as in human primary bladder cancer patient 3D organoids. Consistent with the observed cytotoxicity, the combination of SRA737 and gemcitabine induces γ-H2A.X and p-RPA 3- to 5-fold over gemcitabine alone in tumor xenografts. Moreover, the combination inhibits tumor growth by 64% following administration of both agents at 100 mg/kg in a gemcitabine-insensitive patient-derived xenograft (PDX) model of bladder cancer. Importantly, SRA737 also synergizes with subtherapeutic doses of gemcitabine (40 mg/kg) in xenograft models of colorectal adenocarcinoma and osteosarcoma, indications where gemcitabine is not standard of care. These findings support the clinical development of SRA737 in combination with gemcitabine in bladder cancer, as well as the broader application of SRA737 in combination with extrinsic inducers of RS. Citation Format: Ryan J. Hansen, Bryan Strouse, Kenna Anderes, Gregg Smith, Christian Hassig. The Chk1 inhibitor, SRA737, demonstrates chemical synthetic lethality with replication stress-inducing agents, including low-dose gemcitabine, in preclinical models of cancer [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr B181.

O(6)-Benzylguanine (O(6)-BG), a potent inactivator of the DNA repair protein O(6)-alkylguanine-DNA alkyltransferase (AGT), is presently in clinical trials combined with alkylating agents that modify the O(6) position of DNA guanine residues, i.e., 1,3-bis(2-chloroethyl)-1-nitrosourea and temozolomide. Previous work demonstrated that O(6)-BG also enhances the cytotoxicity of cyclophosphamide, ifosfamide, and nitrogen mustards in Chinese hamster ovary cells. We have extended this study to include other clinically relevant agents that form interstrand and intrastrand cross-links including cisplatin and carboplatin. Pretreatment of a series of head and neck tumor cell lines (i.e., SQ20b, JSQ3, SCC25, SCC35, and SCC61), Chinese hamster ovary cells, and HT29 human colon tumor cells with O(6)-BG (100 micro M for 2 h before treatment and 2 h during treatment) resulted in a 2-fold decrease in the ED(50) of cisplatin and a concomitant increase in the percentage of cells undergoing apoptosis. The enhancement was independent of AGT activity. Similar enhancement was observed with carboplatin, but no enhancement was seen in AGT-deficient cell lines with radiation or temozolomide, demonstrating the dependence of the effect on bifunctional, cross-linking agents. Furthermore, levels of platinum on DNA after treatment with cisplatin increased 1.4-fold in SQ20b cells and 4.5-fold in JSQ3 cells immediately after treatment with O(6)-BG plus cisplatin and remained elevated for 48 h. Consistent with greater cytotoxicity and apoptosis is the approximately 2-fold higher amount of DNA damage when cells are treated with O(6)-BG plus cisplatin compared with cisplatin alone. Modulation of cisplatin therapy with O(6)-BG might improve the prognosis of patients with head and neck, ovarian, testicular, or lung cancer who are treated with this drug.