Prexasertib has activity in BRCA1-deficient ovariain cancer cells that have restored replication fork stability and acquired PARP inhibitor resistance

Sensitivity of BRCA1-deficient RPE cells with acquired olaparib resistance to prexasertib and lack of effect of monotherapy pre-treatment on the synergistic cytotoxicity between prexasertib and olaparib in TOV112D cells

Prexasertib exhibits monotherapy activity in olaparib-resistant HGSOC PDX models

Characterization of high-grade serous ovarian cancer models

Supplementary Figures and Methods // Supplementary Figure 1: The DDB2 proteo-probe assay measures cellular nucleotide excision repair (NER) capacity. Low magnification (20X) images of wild-type (WT) or mutant ERCC2-complemented XP cells before, 5 minutes after, and 150 minutes after UV exposure (see Figure 1 for high magnification [63X] images). DDB2 proteoprobe binding to UV-induced (6,4)-photoproducts is visualized using an anti-FLAG antibody (red). Nuclei are stained with DAPI (blue). ERCC2 expression is confirmed with an anti-Myc antibody (green). R683W is a common germline pathogenic allele, D312N is a common non-pathogenic germline allele, and G607A is a mutation identified in a cisplatin responsive tumor. Supplementary Figure 2: Sensitivity profiles for WT and mutant ERCC2-complemented cells for non-cisplatin agents. A. Carboplatin. B. Doxorubicin (Adriamycin) is a DNA intercalating agents that results in creation of double-strand DNA breaks. C. Rucaparib is a poly(ADP-ribose) polymerase inhibitor that demonstrates enhanced toxicity in homologous recombination (HR)-deficient cells. D. Ionizing radiation (IR). R683W is a common pathogenic germline allele in patients with the xeroderma pigmentosum complementation group D (XPD). D312N is a common, non-pathogenic germline ERCC2 allele in the general population. Y14C and G607A are helicase domain mutations identified in cisplatin responsive patients and are associated with NER loss and cisplatin sensitivity (Figure 2). Supplementary Figure 3: ERCC2 mutations identified in cisplatin non-responders are functionally deleterious. A. Cisplatin sensitivity curves for WT or mutant ERCC2-complemented cells. Whereas WT ERCC2 rescues cisplatin sensitivity, the sensitivity profiles for S44L and S246F, identified in two cisplatin non-responders from the FCCC cohort, are similar to mock-infected cells. B. The S44L and S246F mutants also fail to rescue sensitivity to UV sensitivity. C. Immunoblot showing expression of FLAG-tagged WT or mutant ERCC2. Supplementary Figure 4: Identification of an ERCC2 E86Q mutation in matched pre- and post-treatment tumor samples. The E86Q mutation was not identified in the pre-treatment sample by the standard mutation-calling algorithm due to the low number of reads, but was later identified by "force-calling" after the E86Q mutation was identified in the post-treatment sample. Correcting for tumor purity, the E86Q mutation was inferred to be clonal in both the pre- and post-treatment samples. Supplementary Figure 5: Phylogenetic tree showing relationship between pre- and post-chemotherapy tumors from a patient with an ERCC2 E86Q mutation. The pre- and post-chemotherapy tumors share several mutations, indicating a common ancestor. Shared mutations in other select cancer genes are noted. Supplementary Figure 6: CRISPR/Cas9 technology was used to create a KU19-19 cell line harboring an in-frame deletion at the endogenous ERCC2 allele. A. An sgRNA guide was designed to target the region near T484, a common site of ERCC2 mutations in MIBC tumors. B. Integrated Genomics Viewer (IGV) screenshot showing sequence of WT ERCC2 allele in KU19-19 parental cell line. C. The CRISPR-edited cell line KE1 harbors an in-frame deletion at T484. Supplementary Figure 7: An ERCC2 mutation confers an NER defect in a bladder cancer cell line. Low magnification (20X) images from a DDB2 proteoprobe assay comparing KU19-19 (WT ERCC2) and KE1 (mutant ERCC2) cell lines (see Figure 4 for high magnification [63X] images). Whereas the KU19-19 cell line efficiently resolves DDB2 foci, the foci persist in NER-deficient KE1 cells. Supplementary Figure 8: KU19-19 (WT ERCC2) and KE1 (mutant ERCC2) cell lines have similar sensitivity to the PARP inhibitor olaparib. Supplementary Figure 9: Overall survival for patients with ERCC2 wild-type (blue) versus mutant (red) tumors in the updated TCGA bladder tumor cohort (Robertson et al. Cell 2017; 171:1-17). There was no significant difference in survival between the groups (P=0.095). This figure was created using cBioPortal (Gao et al. Sci Signaling 2013; 6(269):pl1 and Cerami et al. Cancer Discov 2012; 2(5):401).

Body weights of HGSOC tumor-bearing mice treated with prexasertib or prexasertib/olaparib

Supplementary Figure Legends

RAD51 and gamma-H2AX staining in response to olaparib in HGSOC PDX models

Cyclin E expression in ovarian cancer cell lines

AbstractPurpose: PARP inhibitors are approved for the treatment of high-grade serous ovarian cancers (HGSOC). Therapeutic resistance, resulting from restoration of homologous recombination (HR) repair or replication fork stabilization, is a pressing clinical problem. We assessed the activity of prexasertib, a checkpoint kinase 1 (CHK1) inhibitor known to cause replication catastrophe, as monotherapy and in combination with the PARP inhibitor olaparib in preclinical models of HGSOC, including those with acquired PARP inhibitor resistance. Experimental Design: Prexasertib was tested as a single agent or in combination with olaparib in 14 clinically annotated and molecularly characterized luciferized HGSOC patient-derived xenograft (PDX) models and in a panel of ovarian cancer cell lines. The ability of prexasertib to impair HR repair and replication fork stability was also assessed. Results: Prexasertib monotherapy demonstrated antitumor activity across the 14 PDX models. Thirteen models were resistant to olaparib monotherapy, including 4 carrying BRCA1 mutation. The combination of olaparib with prexasertib was synergistic and produced significant tumor growth inhibition in an olaparib-resistant model and further augmented the degree and durability of response in the olaparib-sensitive model. HGSOC cell lines, including those with acquired PARP inhibitor resistance, were also sensitive to prexasertib, associated with induction of DNA damage and replication stress. Prexasertib also sensitized these cell lines to PARP inhibition and compromised both HR repair and replication fork stability. Conclusions: Prexasertib exhibits monotherapy activity in PARP inhibitor–resistant HGSOC PDX and cell line models, reverses restored HR and replication fork stability, and synergizes with PARP inhibition.

Synthetic lethality-an interaction between two genetic events through which the co-occurrence of these two genetic events leads to cell death, but each event alone does not-can be exploited for cancer therapeutics1. DNA repair processes represent attractive synthetic lethal targets, because many cancers exhibit an impairment of a DNA repair pathway, which can lead to dependence on specific repair proteins2. The success of poly(ADP-ribose) polymerase 1 (PARP-1) inhibitors in cancers with deficiencies in homologous recombination highlights the potential of this approach3. Hypothesizing that other DNA repair defects would give rise to synthetic lethal relationships, we queried dependencies in cancers with microsatellite instability (MSI), which results from deficient DNA mismatch repair. Here we analysed data from large-scale silencing screens using CRISPR-Cas9-mediated knockout and RNA interference, and found that the RecQ DNA helicase WRN was selectively essential in MSI models in vitro and in vivo, yet dispensable in models of cancers that are microsatellite stable. Depletion of WRN induced double-stranded DNA breaks and promoted apoptosis and cell cycle arrest selectively in MSI models. MSI cancer models required the helicase activity of WRN, but not its exonuclease activity. These findings show that WRN is a synthetic lethal vulnerability and promising drug target for MSI cancers.

AbstractPurpose: DNA-damaging agents comprise the backbone of systemic treatment for many tumor types; however, few reliable predictive biomarkers are available to guide use of these agents. In muscle-invasive bladder cancer (MIBC), cisplatin-based chemotherapy improves survival, yet response varies widely among patients. Here, we sought to define the role of the nucleotide excision repair (NER) gene ERCC2 as a biomarker predictive of response to cisplatin in MIBC. Experimental Design: Somatic missense mutations in ERCC2 are associated with improved response to cisplatin-based chemotherapy; however, clinically identified ERCC2 mutations are distributed throughout the gene, and the impact of individual ERCC2 variants on NER capacity and cisplatin sensitivity is unknown. We developed a microscopy-based NER assay to profile ERCC2 mutations observed retrospectively in prior studies and prospectively within the context of an institution-wide tumor profiling initiative. In addition, we created the first ERCC2-deficient bladder cancer preclinical model for studying the impact of ERCC2 loss of function. Results: We used our functional assay to test the NER capacity of clinically observed ERCC2 mutations and found that most ERCC2 helicase domain mutations cannot support NER. Furthermore, we show that introducing an ERCC2 mutation into a bladder cancer cell line abrogates NER activity and is sufficient to drive cisplatin sensitivity in an orthotopic xenograft model. Conclusions: Our data support a direct role for ERCC2 mutations in driving cisplatin response, define the functional landscape of ERCC2 mutations in bladder cancer, and provide an opportunity to apply combined genomic and functional approaches to prospectively guide therapy decisions in bladder cancer. See related commentary by Grivas, p. 907

Selective killing of cancer cells while minimizing damage to healthy tissues is the goal of clinical radiation therapy. This therapeutic ratio can be improved by image-guided radiation delivery and selective radiosensitization of cancer cells. Here, we have designed and tested a novel trimodal theranostic nanoparticle made of bismuth and gadolinium for on-site radiosensitization and image contrast enhancement to improve the efficacy and accuracy of radiation therapy. We demonstrate in vivo magnetic resonance (MR), computed tomography (CT) contrast enhancement, and tumor suppression with prolonged survival in a non-small cell lung carcinoma model during clinical radiation therapy. Histological studies show minimal off-target toxicities due to the nanoparticles or radiation. By mimicking existing clinical workflows, we show that the bismuth-gadolinium nanoparticles are highly compatible with current CT-guided radiation therapy and emerging MR-guided approaches. This study reports the first in vivo proof-of-principle for image-guided radiation therapy with a new class of theranostic nanoparticles.

Abstract ERCC2 is a core member of the nucleotide excision repair (NER) pathway, a highly conserved and remarkably versatile DNA repair pathway responsible for repairing intrastrand DNA adducts created by genotoxic agents such as UV irradiation and platinum-based chemotherapies. Recent large-scale genomic efforts have shown that somatic ERCC2 missense mutations are present in approximately 20% of all primary muscle-invasive bladder cancers (MIBC). We previously showed that ERCC2 mutations are associated with treatment response and overall survival in MIBC patients treated with cisplatin-based chemotherapy. Initial functional studies on a subset of the observed ERCC2 mutations suggest that the mutations confer loss of normal cellular NER capacity. However, sequencing of additional MIBC cohorts has revealed that mutations occur across the ERCC2 gene, and the functional effects of the majority of these mutations remain unknown. In order to understand the functional landscape of ERCC2 mutations in MIBC, we have developed a high-throughput fluorescence-based assay to test the functional consequences of mutations in ERCC2 and other NER genes on cellular NER capacity. We apply this approach to all observed ERCC2 mutations across three published MIBC cohorts and find that the majority of ERCC2 mutations result in complete or near-complete loss of cellular NER. In addition, by correlating our functional results with available clinical data, we find interesting examples of cases in which ERCC2 status and cisplatin response are decoupled, highlighting the importance of using functional data to complement genomic and clinical endpoints in the search for reliable predictive biomarkers. This abstract is also being presented as Poster A29. Citation Format: Kent Mouw, Jean-Bernard Lazaro, Alexis Damish, Elizaveta Reznichenko, Zoe Frazier, David Liu, Jaegil Kim, Paz Polak, Levi Garraway, Gad Getz, Jonathan Rosenberg, Eliezer Van Allen, Alan D'Andrea. Somatic ERCC2 mutations, nucleotide excision repair (NER) function, and cisplatin response in muscle-invasive bladder cancer (MIBC) [abstract]. In: Proceedings of the AACR Special Conference on DNA Repair: Tumor Development and Therapeutic Response; 2016 Nov 2-5; Montreal, QC, Canada. Philadelphia (PA): AACR; Mol Cancer Res 2017;15(4_Suppl):Abstract nr PR18.

Somatic mutations in the nucleotide excision repair (NER) gene ERCC2 are present in ∼20% of primary muscle-invasive bladder cancers (MIBC). Our group and others have shown that ERCC2 mutations are associated with improved pathologic response and survival in patients receiving cisplatin-based treatment, suggesting that ERCC2 mutations may confer loss of NER capacity and drive cisplatin sensitivity in this context. Large-scale sequencing efforts are now identifying dozens of additional ERCC2 mutations in MIBC and other tumor types; however, mutations are distributed across the ERCC2 gene, making it difficult to predict their functional significance. To understand the functional landscape of ERCC2 mutations in MIBC, we developed a high-throughput, fluorescence-based microscopy assay and used it to characterize a series of clinically-observed ERCC2 mutations. The assay uses a red fluorescence protein (RFP)-tagged DDB2 proteoprobe to measure cellular NER capacity. DDB2 is the NER protein that binds tightly and specifically to UV-induced pyrimidine dimers. ERCC2-deficient cells complemented with WT ERCC2 or a clinically-observed ERCC2 mutant are plated in 24- or 96-well format, exposed to UV irradiation, and then treated with the DDB2 proteoprobe. Immediately following UV treatment, all cells form RFP-DDB2 foci at sites of UV-induced DNA damage. In cells with functional NER, the RFP-DDB2 foci are resolved as the lesions are repaired, whereas foci persist in cells with NER loss. NER capacity is calculated as the fraction of cells that resolve RFP-DDB2 foci two hours after UV exposure. As proof of principle, we used the assay to test all ERCC2 mutants observed across 3 clinical MIBC cohorts (∼40 ERCC2 mutants in total) and correlated the results with available clinical data. In most cases, clinical and functional data were concordant: ERCC2 mutations were present in patients with robust cisplatin response and were unable to support NER in vitro. However, we also found cases in which clinical cisplatin response and in vitro NER capacity were decoupled, including examples of deleterious ERCC2 mutations in cisplatin non-responders and non-deleterious ERCC2 mutations in cisplatin responders. Although large-scale sequencing efforts have identified thousands of protein-coding mutations across tumors, studying the impact of these mutations on tumor behavior and treatment response will require reliable high-throughput functional assays. In MIBC, somatic ERCC2 mutations are associated with improved response to cisplatin-based treatment. Here, we use a novel high-throughput assay to map the functional landscape of ERCC2 mutations in MIBC and show that the majority of ERCC2 mutations result in loss of cellular NER activity. Together, these data suggest that ERCC2 mutation status may be a useful biomarker to identify patients who are ideal candidates for bladder-sparing treatment with concurrent cisplatin-based chemoradiotherapy.

Despite the development of novel drugs, alkylating agents remain an important component of therapy in multiple myeloma (MM). DNA repair processes contribute towards sensitivity to alkylating agents and therefore we here evaluate the role of nucleotide excision repair (NER), which is involved in the removal of bulky adducts and DNA crosslinks in MM. We first evaluated NER activity using a novel functional assay and observed a heterogeneous NER efficiency in MM cell lines and patient samples. Using next-generation sequencing data, we identified that expression of the canonical NER gene, excision repair cross-complementation group 3 ( ERCC3 ), significantly impacted the outcome in newly diagnosed MM patients treated with alkylating agents. Next, using small RNA interference, stable knockdown and overexpression, and small-molecule inhibitors targeting xeroderma pigmentosum complementation group B (XPB), the DNA helicase encoded by ERCC3 , we demonstrate that NER inhibition significantly increases sensitivity and overcomes resistance to alkylating agents in MM. Moreover, inhibiting XPB leads to the dual inhibition of NER and transcription and is particularly efficient in myeloma cells. Altogether, we show that NER impacts alkylating agents sensitivity in myeloma cells and identify ERCC3 as a potential therapeutic target in MM.