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IKBKE inhibitor in combination with a MEK inhibitor in KRAS mutant colorectal and non-small cell lung cancer models
S146 Poster abstracts
Poster Session – Molecular targeted agents II, Thursday 1 December 2016
Conclusions: ALM301 sensitised p53 DBD mutant OAC cell lines to hypoxia-induced apoptosis. ALM301 also sensitised OAC cell lines to chemotherapies used in standard neo-adjuvant treatment of OAC. Together these preclinical findings support a potential clinical role for Akt inhibitors in the treatment of OAC. However, given the context specific effect observed in this study, further investigations are required to determine the mechanisms underlying ALM301-induced sensitisation to 5-FU and CDDP which could inform a biomarker strategy for patient stratification. Conflict of interest: Ownership: Gerald Gavory is employed by Almac Discovery; Timothy Harrison is employed by Almac Discovery and Queen’s University Belfast; Richard Kennedy is employed as the Medical Director for Almac Diagnostics; Nuala McCabe is employed by Almac diagnostics. 446 Poster (Board P125) Anti-tumor activity of a TBK1/IKBKE inhibitor in combination with a MEK inhibitor in KRAS mutant colorectal and non-small cell lung cancer models S. Stinson1 , J. He1 , D. Hollenback1 , J. Jia1 , J. Kaplan1 , C. Venkataramani1 , D. Babusis1 , F. Guevara2 , T. Nelson2 , J. Cavanaugh2 , H. Asahina2 , A. Ray1 , E. Sicinska2 , C. Fuchs2 , D. Barbie2 , K.K. Wong2 , K. Ng2 , D. Dornan1 . 1 Gilead Sciences, Inc., Research, Foster City, USA; 2 Dana-Farber Cancer Institute, Medical Oncology, Boston, USA Background: One third of all human cancers have a mutated RAS allele and, regardless of indication, they are generally refractory to standard of care therapies. The clinical activity of agents that target downstream of RAS, such as MEK inhibitors, have limited single agent activity and tumors become refractory to therapy by pathway reactivation. This emphasizes the need for combination therapy to treat RAS-driven cancers. TANK Binding Kinase 1 (TBK1) is required for the RAS-dependent transformation of murine embryonic fibroblasts and directly binds RALB, a key effector pathway downstream of RAS signaling. KRAS-dependent cancer cell lines are sensitive to TBK1 knockdown. I-Kappa-B Kinase Epsilon (IKBKE), a functionally similar homolog, can promote an autocrine cytokine loop that contributes to proliferation. Together, TBK1 and IKBKE, can promote RASdriven tumorigenesis. Methods: The activity of a novel and potent dual TBK1/IKBKE inhibitor was evaluated in clonogenic growth assays using KRAS mutant or KRAS WT cancer cell lines. The TBK1/IKBKE inhibitor, a MEK inhibitor, and the combination the two were evaluated in a patient-derived KRAS mutant colorectal cancer (CRC) xenograft model and a KrasWT/LSL-G12D ;p53flox/flox genetically engineered mouse model (GEMM) of non-small cell lung cancer (NSCLC). The dose of the MEK inhibitor was chosen to match reported clinical exposure levels of the compound. The primary endpoints included tumor burden, overall survival, and pharmacokinetics. Results: In vitro, KRAS mutant cells were more sensitive to TBK1/IKBKE or MEK inhibition than KRAS WT cells. In KRAS mutant cells, inhibition of MEK resulted in TBK1 pathway activation as determined by Western blot for pTBK1. In the CRC patient-derived xenograft, the combination of the novel TBK1/IKBKE inhibitor and a MEK inhibitor resulted in significant tumor growth inhibition and an increase in overall survival compared to MEK inhibitor alone. In the NSCLC GEMM, the combination of TBK1/IKBKE inhibitor and MEK inhibitor resulted in significant tumor regression compared to MEK inhibitor alone. Conclusion: These data suggest that the combination of a TBK1/IKBKE inhibitor with a MEK inhibitor may be beneficial in KRAS mutant CRCs and NSCLCs. Conflict of interest: Ownership: All Gilead employees are stock holders. Corporate-sponsored Research: Kwok-Kin Wong, David Barbie, and Kimmie Ng are funded by Gilead Sciences for pre-clinical research. 447 Poster (Board P126) TP53 gain of function mutations identified by tumor sequencing performed in the context of a community based personalized medicine cancer program C.W. Drescher1 , A.B. Berry1,2 , J.D. Beatty1 , D. Xu2 , X. Liu1 , M. Zhang2 , K. Keith2 , J.D. Scanlan3 , J.M. Pagel1 , P.J. Gold1 , D. Markowitz1 , T.L. Benkers4 , C. Bonham1 , M. Tameishi1 , T.D. Brown1 . 1 Swedish Cancer Institute, Swedish Cancer Institute, Seattle, USA; 2 CellNetix Pathology and Laboratories, Molecular Pathology, Seattle, USA; 3 Swedish Medical Center, The Swedish Center for Research and Innovation, Seattle, USA; 4 Swedish Neuroscience Institute, Ben & Catherine Ivy Center, Seattle, USA Background: Roughly one-half of all human cancers contain a TP53 mutation. Most TP53 mutations are missense mutations that lead to loss of the normal p53 oncosuppressive functions. However, at least some mutants demonstrate oncogenic properties by way of gain of function
(GOF) mechanisms. The existence of functional gains of certain tumor p53 mutations has important implications for cancer prognosis and therapy. Methods: An Institutional Review Board (IRB) approved prospective registration protocol was activated in Sep, 2014 with the objective of establishing a centralized longitudinal clinical, molecular phenotypic, and research data repository for newly diagnosed patients (pts) presenting for care at a community based, tertiary care cancer research program. The protocol includes profiling of tumor tissue to identify mutations in 68 cancer associated genes including exons 4−6 and 8−10 of TP53 using a next generation sequencing (NGS) assay. Exon 7 was not included in the NGS assay for technical reasons. TP53 sequence results were classified as either wild type (WT), variant of undetermined significance (VUS) or mutated (mt). Eleven mutations (P151S, R175H, R248Q, R248W, R273C, R273H, R273L, G245S, H179L, R175S, and R273P) were classified as GOF mutations based on published evidence of oncogenic activity using invitro or in-vivo model systems. The two GOF mutations localized to exon 7 (R248Q and R248W) were identified using a PCR-based approach. Results: As of Jan 14, 2016, 468 pts had been enrolled, completed sequencing and had baseline and follow-up clinical-pathologic data available for analysis. TP53 sequence results for these pts are summarized in Table 1. Overall 47 pts (10%) were identified as having GOF mutations. The frequency of GOF mutations varied by disease site (X2 = 37.8 p < 0.001); these results were driven largely by the absence of GOF mutations in pts with breast cancer. GOF mutations in decreasing frequency were: R273C, H or L (n = 18), R175H (n = 15), R248W or Q (n = 13) and P151S (n = 1). Three of the four R273L occurred in pts with NSCLC and accounted for 60% of GOF mutations in NSCLC pts. Table 1.
Breast CNS Colorectal (CR) Non-CR GI NSCLC Ovary Other
# % # % # % # % # % # % # %
mt GOF
mt non-GOF
VUS
WT
Total
0 0 12 16 9 11.5 10 15.8 5 10.9 3 9.1 8 7.1
10 16.4 16 21.3 23 29.5 18 28.6 16 34.8 11 33.3 12 10.7
2 3.3 8 10.7 2 3.6 3 4.8 0 0 0 0 5 4.5
49 80.3 39 52 44 56.4 32 50.8 25 54.3 19 57.6 87 77.7
61 75 78 63 46 33 112
For CR and non-CR GI cancers combined pts with GOF mutations were more likely to have died from disease (X2 = 7.3; p = 0.026) and Kaplan– Meier survival analysis showed they had shortened mean survival (MantelCox Log Rank X2 = 9.252; p = 0.01). Conclusions: Our data from a clinical cohort provide preliminary estimates of the frequency of TP53 GOF mutations by disease site and highlight the importance of taking into account the TP53 mutational profile when considering therapeutic strategies for TP53 mutated cancers. No conflict of interest. 448 Poster (Board P127) Novel pan sigma receptor modulator exerts strong apoptotic effect in in vitro primary 3D-cell cultures of human glioblastoma S. Pignatta1 , C. Arienti1 , M. Zanoni1 , A. Zamagni1 , S. Collina2 , L. Tosatto3 , M. Cortesi1 , E. Nigrisoli4 , D. Bartolini4 , M. Faedi5 , M. Bonafe` 6 , A. Tesei1 . 1 Drug Discovery & Radiobiology Unit, Biosciences Laboratory, Istituto Scientifico Romagnolo per lo Studio e la Cura dei Tumori IRST IRCCS, Oncology Department, Meldola, Italy; 2 University of Pavia, Department of Drug Sciences, Pavia, Italy; 3 M. Bufalini Hospital of Cesena, Neurosurgery Unit, Cesena, Italy; 4 Pathology Unit, M. Bufalini Hospital of Cesena, Department of Clinical Pathology, Cesena, Italy; 5 Istituto Scientifico Romagnolo per lo Studio e la Cura dei Tumori IRST IRCCS, Department of Medical Oncology, Meldola, Italy; 6 University of Bologna, Department of Experimental, Diagnostic and Specialty Medicine − DIMES, Bologna, Italy Background: Human glioblastoma (hGBM) is the most common and feared primary malignant brain tumor. Preventive measures, such as lifestyle changes, are ineffective in averting this neoplastic disease. Early diagnosis and treatment do not improve outcomes, precluding the utility of screening for GBM. Virtually, all GBM patients relapse within 7−10 months of the end of first-line treatments. For these reasons, the choice of treatment should be personalized and new therapeutic approaches
Poster Session – Molecular targeted agents II, Thursday 1 December 2016
Conclusions: ALM301 sensitised p53 DBD mutant OAC cell lines to hypoxia-induced apoptosis. ALM301 also sensitised OAC cell lines to chemotherapies used in standard neo-adjuvant treatment of OAC. Together these preclinical findings support a potential clinical role for Akt inhibitors in the treatment of OAC. However, given the context specific effect observed in this study, further investigations are required to determine the mechanisms underlying ALM301-induced sensitisation to 5-FU and CDDP which could inform a biomarker strategy for patient stratification. Conflict of interest: Ownership: Gerald Gavory is employed by Almac Discovery; Timothy Harrison is employed by Almac Discovery and Queen’s University Belfast; Richard Kennedy is employed as the Medical Director for Almac Diagnostics; Nuala McCabe is employed by Almac diagnostics. 446 Poster (Board P125) Anti-tumor activity of a TBK1/IKBKE inhibitor in combination with a MEK inhibitor in KRAS mutant colorectal and non-small cell lung cancer models S. Stinson1 , J. He1 , D. Hollenback1 , J. Jia1 , J. Kaplan1 , C. Venkataramani1 , D. Babusis1 , F. Guevara2 , T. Nelson2 , J. Cavanaugh2 , H. Asahina2 , A. Ray1 , E. Sicinska2 , C. Fuchs2 , D. Barbie2 , K.K. Wong2 , K. Ng2 , D. Dornan1 . 1 Gilead Sciences, Inc., Research, Foster City, USA; 2 Dana-Farber Cancer Institute, Medical Oncology, Boston, USA Background: One third of all human cancers have a mutated RAS allele and, regardless of indication, they are generally refractory to standard of care therapies. The clinical activity of agents that target downstream of RAS, such as MEK inhibitors, have limited single agent activity and tumors become refractory to therapy by pathway reactivation. This emphasizes the need for combination therapy to treat RAS-driven cancers. TANK Binding Kinase 1 (TBK1) is required for the RAS-dependent transformation of murine embryonic fibroblasts and directly binds RALB, a key effector pathway downstream of RAS signaling. KRAS-dependent cancer cell lines are sensitive to TBK1 knockdown. I-Kappa-B Kinase Epsilon (IKBKE), a functionally similar homolog, can promote an autocrine cytokine loop that contributes to proliferation. Together, TBK1 and IKBKE, can promote RASdriven tumorigenesis. Methods: The activity of a novel and potent dual TBK1/IKBKE inhibitor was evaluated in clonogenic growth assays using KRAS mutant or KRAS WT cancer cell lines. The TBK1/IKBKE inhibitor, a MEK inhibitor, and the combination the two were evaluated in a patient-derived KRAS mutant colorectal cancer (CRC) xenograft model and a KrasWT/LSL-G12D ;p53flox/flox genetically engineered mouse model (GEMM) of non-small cell lung cancer (NSCLC). The dose of the MEK inhibitor was chosen to match reported clinical exposure levels of the compound. The primary endpoints included tumor burden, overall survival, and pharmacokinetics. Results: In vitro, KRAS mutant cells were more sensitive to TBK1/IKBKE or MEK inhibition than KRAS WT cells. In KRAS mutant cells, inhibition of MEK resulted in TBK1 pathway activation as determined by Western blot for pTBK1. In the CRC patient-derived xenograft, the combination of the novel TBK1/IKBKE inhibitor and a MEK inhibitor resulted in significant tumor growth inhibition and an increase in overall survival compared to MEK inhibitor alone. In the NSCLC GEMM, the combination of TBK1/IKBKE inhibitor and MEK inhibitor resulted in significant tumor regression compared to MEK inhibitor alone. Conclusion: These data suggest that the combination of a TBK1/IKBKE inhibitor with a MEK inhibitor may be beneficial in KRAS mutant CRCs and NSCLCs. Conflict of interest: Ownership: All Gilead employees are stock holders. Corporate-sponsored Research: Kwok-Kin Wong, David Barbie, and Kimmie Ng are funded by Gilead Sciences for pre-clinical research. 447 Poster (Board P126) TP53 gain of function mutations identified by tumor sequencing performed in the context of a community based personalized medicine cancer program C.W. Drescher1 , A.B. Berry1,2 , J.D. Beatty1 , D. Xu2 , X. Liu1 , M. Zhang2 , K. Keith2 , J.D. Scanlan3 , J.M. Pagel1 , P.J. Gold1 , D. Markowitz1 , T.L. Benkers4 , C. Bonham1 , M. Tameishi1 , T.D. Brown1 . 1 Swedish Cancer Institute, Swedish Cancer Institute, Seattle, USA; 2 CellNetix Pathology and Laboratories, Molecular Pathology, Seattle, USA; 3 Swedish Medical Center, The Swedish Center for Research and Innovation, Seattle, USA; 4 Swedish Neuroscience Institute, Ben & Catherine Ivy Center, Seattle, USA Background: Roughly one-half of all human cancers contain a TP53 mutation. Most TP53 mutations are missense mutations that lead to loss of the normal p53 oncosuppressive functions. However, at least some mutants demonstrate oncogenic properties by way of gain of function
(GOF) mechanisms. The existence of functional gains of certain tumor p53 mutations has important implications for cancer prognosis and therapy. Methods: An Institutional Review Board (IRB) approved prospective registration protocol was activated in Sep, 2014 with the objective of establishing a centralized longitudinal clinical, molecular phenotypic, and research data repository for newly diagnosed patients (pts) presenting for care at a community based, tertiary care cancer research program. The protocol includes profiling of tumor tissue to identify mutations in 68 cancer associated genes including exons 4−6 and 8−10 of TP53 using a next generation sequencing (NGS) assay. Exon 7 was not included in the NGS assay for technical reasons. TP53 sequence results were classified as either wild type (WT), variant of undetermined significance (VUS) or mutated (mt). Eleven mutations (P151S, R175H, R248Q, R248W, R273C, R273H, R273L, G245S, H179L, R175S, and R273P) were classified as GOF mutations based on published evidence of oncogenic activity using invitro or in-vivo model systems. The two GOF mutations localized to exon 7 (R248Q and R248W) were identified using a PCR-based approach. Results: As of Jan 14, 2016, 468 pts had been enrolled, completed sequencing and had baseline and follow-up clinical-pathologic data available for analysis. TP53 sequence results for these pts are summarized in Table 1. Overall 47 pts (10%) were identified as having GOF mutations. The frequency of GOF mutations varied by disease site (X2 = 37.8 p < 0.001); these results were driven largely by the absence of GOF mutations in pts with breast cancer. GOF mutations in decreasing frequency were: R273C, H or L (n = 18), R175H (n = 15), R248W or Q (n = 13) and P151S (n = 1). Three of the four R273L occurred in pts with NSCLC and accounted for 60% of GOF mutations in NSCLC pts. Table 1.
Breast CNS Colorectal (CR) Non-CR GI NSCLC Ovary Other
# % # % # % # % # % # % # %
mt GOF
mt non-GOF
VUS
WT
Total
0 0 12 16 9 11.5 10 15.8 5 10.9 3 9.1 8 7.1
10 16.4 16 21.3 23 29.5 18 28.6 16 34.8 11 33.3 12 10.7
2 3.3 8 10.7 2 3.6 3 4.8 0 0 0 0 5 4.5
49 80.3 39 52 44 56.4 32 50.8 25 54.3 19 57.6 87 77.7
61 75 78 63 46 33 112
For CR and non-CR GI cancers combined pts with GOF mutations were more likely to have died from disease (X2 = 7.3; p = 0.026) and Kaplan– Meier survival analysis showed they had shortened mean survival (MantelCox Log Rank X2 = 9.252; p = 0.01). Conclusions: Our data from a clinical cohort provide preliminary estimates of the frequency of TP53 GOF mutations by disease site and highlight the importance of taking into account the TP53 mutational profile when considering therapeutic strategies for TP53 mutated cancers. No conflict of interest. 448 Poster (Board P127) Novel pan sigma receptor modulator exerts strong apoptotic effect in in vitro primary 3D-cell cultures of human glioblastoma S. Pignatta1 , C. Arienti1 , M. Zanoni1 , A. Zamagni1 , S. Collina2 , L. Tosatto3 , M. Cortesi1 , E. Nigrisoli4 , D. Bartolini4 , M. Faedi5 , M. Bonafe` 6 , A. Tesei1 . 1 Drug Discovery & Radiobiology Unit, Biosciences Laboratory, Istituto Scientifico Romagnolo per lo Studio e la Cura dei Tumori IRST IRCCS, Oncology Department, Meldola, Italy; 2 University of Pavia, Department of Drug Sciences, Pavia, Italy; 3 M. Bufalini Hospital of Cesena, Neurosurgery Unit, Cesena, Italy; 4 Pathology Unit, M. Bufalini Hospital of Cesena, Department of Clinical Pathology, Cesena, Italy; 5 Istituto Scientifico Romagnolo per lo Studio e la Cura dei Tumori IRST IRCCS, Department of Medical Oncology, Meldola, Italy; 6 University of Bologna, Department of Experimental, Diagnostic and Specialty Medicine − DIMES, Bologna, Italy Background: Human glioblastoma (hGBM) is the most common and feared primary malignant brain tumor. Preventive measures, such as lifestyle changes, are ineffective in averting this neoplastic disease. Early diagnosis and treatment do not improve outcomes, precluding the utility of screening for GBM. Virtually, all GBM patients relapse within 7−10 months of the end of first-line treatments. For these reasons, the choice of treatment should be personalized and new therapeutic approaches