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247 Selective inhibitors of nuclear export (SINE) block the expression of DNA damage repair proteins and sensitize cancer cells to DNA damage therapeutic agents
Poster Session – DNA Repair Modulation (including PARP, CHK, ATR, ATM) Conclusions: These studies reveal no evidence for an allosteric trapping mechanism and indicate that all PARP inhibitors examined trap PARP1 via catalytic inhibition. The potency of PARP inhibitors with respect to trapping and catalytic inhibition is linearly correlated in biochemical systems. In cells, trapping potency is related to concentrations required for potentiation in animal models. Detection of PARP trapping in cells requires supraphysiologic conditions that exhaust cellular NAD+ and PAR, exceed concentrations required to elicit synergism and are not tolerated in vivo. In addition trapping potency appears to be inversely correlated with tolerability. Quantitation of the degree of trapping that is tolerable and is required for therapeutic benefit is under active investigation. Disclosures: All authors are employees of AbbVie. The design, study conduct, and financial support for this research were provided by AbbVie. AbbVie participated in the interpretation of data, review, and approval of the publication. 247 POSTER Selective inhibitors of nuclear export (SINE) block the expression of DNA damage repair proteins and sensitize cancer cells to DNA damage therapeutic agents T. Kashyap1 , M. Crochiere1 , S. Friedlander1 , B. Klebanov1 , W. Senapedis1 , E. Baloglu1 , D. del Alamo1 , S. Tamir1 , T. Rashal1 , D. McCauley1 , R. Carlson1 , M. Kauffman1 , S. Shacham1 , Y. Landesman1 . 1 Karyopharm Therapeutics, Natick MA, USA Background: SINE is a family of small-molecule drugs that inhibit Exportin 1 (XPO1/CRM1) mediated nuclear export, resulting in retention of major tumor suppressor proteins (TSPs) such as p53, FOXO, pRB and IkB and subsequently in specific cancer cell death. Selinexor is the clinical SINE compound currently in human phase I/II clinical trials in patients with solid and hematological malignancies. The goal of this study was to evaluate the effects of selinexor on DNA repair mechanisms and to test the cytotoxic effects of combining selinexor with DNA damaging agent on hematological and solid tumor. Methods: Whole protein cell lysates from solid and hematological cancer cell lines treated with selinexor with or without agents that induce DNA damage were analyzed in Reverse Phase Protein Arrays (RPPA), immunoblots and quantitative PCR. Selinexor treated cells from solid and hematological cancer lines were analyzed by immunofluorescence to evaluate DNA damage. Non-small cell lung cancer A549 Xenografts were treated with the selinexor (5 mg/kg) and radiation (3 Gy) alone or in combination and tumor growth was evaluated for 28 days. Results: Treatment of solid and hematological cancer cell lines with selinexor did not induce DNA damage in cancer cells but reduced the expression of DNA damage repair proteins: MSH2, MSH6, PMS2, MLH1, Rad51, CHK1 and FOXM1. Selienxor regulates the expression of CHK1, RAD51, MSH2, MSH6 and MLH1 on the transcriptional levels and PMS2 expression on the posttranslational level. There was a trend between the degree of DNA-damage-repair-protein reduction to selinexor sensitivity. Knock down of Chk1 alone, induced cytotoxicity whereas silencing of the other DNA repair proteins did not affect cell viability. Selinexor treatment following exposure to DNA damaging agents like doxorubicin and idarubicin inhibited the repair mechanism of DNA damage caused by these agents and resulted in synergistic cell killing as measured by induction of PARP and Caspase 3 cleavage. In vivo, selinexor (5 mg/kg) and radiation (3 Gy) decreased xenograft tumor size of the non-small cell lung cancer A549 by 15% and 43% respectively, relative to vehicle whereas combination of selinexor and radiation resulted in a 96% tumor decrease. Conclusion: Selinexor reduce the DNA repair mechanism in solid and hematological cancer cell lines and combination of selinexor with agents that cause DNA damage induces cancer cell death that is superior to each therapy alone. These data suggest that such a combination treatment is predicted to result with synergistic therapeutic outcome in cancer patients. 248 POSTER Use of ATR inhibitor in combination with topoisomerase I inhibitor kills cancer cells by disabling DNA replication initiation and fork elongation R. Josse´ 1 , S.E. Martin2 , R. Guha2 , P. Ormanoglu2 , T. Pfister3 , J. Morris4 , J. Doroshow4 , Y. Pommier1 . 1 NCI, Developmental Therapeutic Branch, Bethesda MD, USA; 2 NCATS, Division of Preclinical Innovation, Rockville MD, USA; 3 Leidos Biomedical Research Inc, Laboratory of Human Toxicology and Pharmacology, Frederick MD, USA; 4 DTP-DCTD, Drug synthesis and Chemistry Branch, Bethesda MD, USA Camptothecin and its derivatives, topotecan and irinotecan are specific topoisomerase I inhibitors and potent anticancer drugs. These agents produce well-characterized double-strand breaks upon collision of replication forks with topoisomerase I cleavage complexes. In an attempt to
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develop novel drugs combinations, we conducted a synthetic lethal siRNA screening using a library that targets nearly 7000 human genes. Depletion of ATR, the main transducer of replication stress-induced DNA damage response came at the top candidate gene with synthetic lethality with camptothecin. Validation studies showed that ATR siRNA exacerbated cytotoxic response to both camptothecin and the indenoisoquinoline LMP400 (indotecan), a novel topoisomerase inhibitor in clinical trial. Inhibition of ATR by the recently developed specific inhibitor VE-821 induced synergistic antiproliferative activity when combined with either topoisomerase inhibitor. Cytotoxicity induced by the combination with LMP-400 was greater than with camptothecin. Using single cell analysis and DNA fiber spread, we show that VE-821 abrogates the S-phase checkpoint, and restores origin firing and replication fork progression in cells treated with camptothecin or LMP-400. Moreover, the combination of topoisomerase inhibitors with VE-821 inhibited the phosphorylation of ATR and ATR-mediated Chk1 phosphorylation, while strongly inducing gH2AX. Single cell analysis revealed that the gH2AX pattern changed over time from well-defined focus to a pan-nuclear staining. The change in gH2AX pattern can be useful as a predictive biomarker to evaluate the efficacy of therapy. The key implication of our work is the mechanistic rationale it provides to evaluate the combination of topoisomerase I inhibitors with ATR inhibitors. 249 POSTER Preclinical efficacy of the PARP inhibitor rucaparib (CO338/AG014699/PF-01367338) in pancreatic cancer models with homologous recombination deficiencies (HRD) L. Robillard1 , K. Lin1 , P.P. Lopez-Casas2 , M. Hidalgo2 , T.C. Harding1 . 1 Clovis Oncology, San Francisco CA, USA; 2 CNIO, Madrid, Spain Rucaparib is an oral, potent, small molecule inhibitor of poly (ADP-ribose) polymerase (PARP) being developed for the maintenance treatment of platinum-sensitive ovarian cancer in patients with homologous recombination deficient (HRD) tumors, including those with BRCA1 and BRCA2 mutations. Mutations in BRCA and other homologous recombination HR pathway genes are frequently observed in other tumor types including breast and pancreatic (Alexandrov et al., 2013; Nature). Here, we investigated the efficacy of rucaparib as single agent in pancreatic cancer (PC) cell lines and xenograft models with HRD. Sensitivity to rucaparib was determined in a panel of PC lines (SU86.86, Panc-1, MIA PaCa-2, Panc 10.05) following siRNA knock-down of HR genes (ATM, ATR, BRCA1, BRCA2, PALB2 and RAD51C) to model gene mutations frequently observed in PC. BRCA1, BRCA2, PALB2 and RAD51C siRNA knockdownshowed synthetic lethality (30% of control GI50 ) in the majority of PC cell lines examined and correlated with the induction of HRD as assessed by gH2AX and RAD51 foci formation. As a complement to the cell line data we examined the efficacy of rucaparib in 3 BRCA2 mutant patient-derived xenograft (PDX) PC models (Oncotest, GmbH): PAXF_1876 (BRAF, PTEN, HRAS), PAXF_2005 (KRAS, TP53) and PAXF_2094 (KRAS). Models were selected on BRCA2 frameshift or nonsense mutations that should be functionally deleterious. Rucaparib administration to animals bearing preestablished tumors at 150 mg/kg/day BID (modeled Phase 2/3 dose in mice) resulted in significant monotherapy activity in all models examined with a 44, 96 and 67% reduction in tumor growth at 28 days post-dosing for PAXF_1876, 2005 and 2094, respectively. Expansion of rucaparib preclinical efficacy data beyond BRCA2 mutated PDX models is currently on-going in a PALB2 mutated PDX PC model (JH033; Villarroel et al., 2010; Mol. Ca. Ther.). In addition, the activity of rucaparib was examined in a BRCA2 mutant pancreatic ductal adenocarcinoma patient following FOLFIRINOX progression in a Phase I study (NCT01482715) was shown to be consistent with preclinical observations; a 56% reduction in the patient’s target lesions and a PFS of 6.4 months was observed with no significant toxicity. A Phase 2 study of rucaparib in patients with pancreatic cancer and a known deleterious BRCA mutation is currently on-going (NCT02042378). These findings support the hypothesis that pancreatic cancers exhibiting HRD are sensitive to rucaparib inhibition.
Thursday 20 November 2014
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develop novel drugs combinations, we conducted a synthetic lethal siRNA screening using a library that targets nearly 7000 human genes. Depletion of ATR, the main transducer of replication stress-induced DNA damage response came at the top candidate gene with synthetic lethality with camptothecin. Validation studies showed that ATR siRNA exacerbated cytotoxic response to both camptothecin and the indenoisoquinoline LMP400 (indotecan), a novel topoisomerase inhibitor in clinical trial. Inhibition of ATR by the recently developed specific inhibitor VE-821 induced synergistic antiproliferative activity when combined with either topoisomerase inhibitor. Cytotoxicity induced by the combination with LMP-400 was greater than with camptothecin. Using single cell analysis and DNA fiber spread, we show that VE-821 abrogates the S-phase checkpoint, and restores origin firing and replication fork progression in cells treated with camptothecin or LMP-400. Moreover, the combination of topoisomerase inhibitors with VE-821 inhibited the phosphorylation of ATR and ATR-mediated Chk1 phosphorylation, while strongly inducing gH2AX. Single cell analysis revealed that the gH2AX pattern changed over time from well-defined focus to a pan-nuclear staining. The change in gH2AX pattern can be useful as a predictive biomarker to evaluate the efficacy of therapy. The key implication of our work is the mechanistic rationale it provides to evaluate the combination of topoisomerase I inhibitors with ATR inhibitors. 249 POSTER Preclinical efficacy of the PARP inhibitor rucaparib (CO338/AG014699/PF-01367338) in pancreatic cancer models with homologous recombination deficiencies (HRD) L. Robillard1 , K. Lin1 , P.P. Lopez-Casas2 , M. Hidalgo2 , T.C. Harding1 . 1 Clovis Oncology, San Francisco CA, USA; 2 CNIO, Madrid, Spain Rucaparib is an oral, potent, small molecule inhibitor of poly (ADP-ribose) polymerase (PARP) being developed for the maintenance treatment of platinum-sensitive ovarian cancer in patients with homologous recombination deficient (HRD) tumors, including those with BRCA1 and BRCA2 mutations. Mutations in BRCA and other homologous recombination HR pathway genes are frequently observed in other tumor types including breast and pancreatic (Alexandrov et al., 2013; Nature). Here, we investigated the efficacy of rucaparib as single agent in pancreatic cancer (PC) cell lines and xenograft models with HRD. Sensitivity to rucaparib was determined in a panel of PC lines (SU86.86, Panc-1, MIA PaCa-2, Panc 10.05) following siRNA knock-down of HR genes (ATM, ATR, BRCA1, BRCA2, PALB2 and RAD51C) to model gene mutations frequently observed in PC. BRCA1, BRCA2, PALB2 and RAD51C siRNA knockdownshowed synthetic lethality (30% of control GI50 ) in the majority of PC cell lines examined and correlated with the induction of HRD as assessed by gH2AX and RAD51 foci formation. As a complement to the cell line data we examined the efficacy of rucaparib in 3 BRCA2 mutant patient-derived xenograft (PDX) PC models (Oncotest, GmbH): PAXF_1876 (BRAF, PTEN, HRAS), PAXF_2005 (KRAS, TP53) and PAXF_2094 (KRAS). Models were selected on BRCA2 frameshift or nonsense mutations that should be functionally deleterious. Rucaparib administration to animals bearing preestablished tumors at 150 mg/kg/day BID (modeled Phase 2/3 dose in mice) resulted in significant monotherapy activity in all models examined with a 44, 96 and 67% reduction in tumor growth at 28 days post-dosing for PAXF_1876, 2005 and 2094, respectively. Expansion of rucaparib preclinical efficacy data beyond BRCA2 mutated PDX models is currently on-going in a PALB2 mutated PDX PC model (JH033; Villarroel et al., 2010; Mol. Ca. Ther.). In addition, the activity of rucaparib was examined in a BRCA2 mutant pancreatic ductal adenocarcinoma patient following FOLFIRINOX progression in a Phase I study (NCT01482715) was shown to be consistent with preclinical observations; a 56% reduction in the patient’s target lesions and a PFS of 6.4 months was observed with no significant toxicity. A Phase 2 study of rucaparib in patients with pancreatic cancer and a known deleterious BRCA mutation is currently on-going (NCT02042378). These findings support the hypothesis that pancreatic cancers exhibiting HRD are sensitive to rucaparib inhibition.