Selpercatinib Resistance in RET Fusion Positive NSCLC Mediated by MAPK Pathway Reactivation
Poster #: 142
Session/Time: A
Author:
Sajin Marcus Cyr, BS, MS
Mentor:
Romel Somwar, BS, PhD
Research Type: Basic Science
Abstract
INTRODUCTION:
RET (REarranged during Transfection) is a proto-oncogene that lies on chromosome 10q11.21. It encodes a single-pass receptor tyrosine kinase (RTK) whose signaling regulates the growth and development of the nervous system and other tissues. Importantly, ligand binding and dimerization with other regulators across the cell membrane regulate its signaling and kinase activity. In cancer, oncogenic RET kinases result from either somatic mutations in the kinase domain or fusions of that domain with other genes at its 5' end. These partner genes typically contribute a protein-protein interaction motif that allows RET to dimerize and thus remain constitutively active. These RET fusions are primary oncogenes in cancers of multiple histologies including papillary thyroid cancer and lung adenocarcinomas (LUAD). Currently, the only clinically approved treatment for patients with RET-dependent cancers are the RET inhibitors selpercatinib (any histology) and pralsetinib (lung and thyroid cancers), both of which have been shown to result in improvement of overall survival. Resistance to RET therapy, however, develops invariably and coincidentally with activating mutations in RAS, RAF (upstream activators of MEK1/2 and ERK1/2) or other alterations. Still, the mechanism(s) by which the MAPK pathway adapts to drug treatment and its role in dampening therapeutic response has not been explored in lung cancer. Consequently, we aimed to systematically characterize the involvement of the MAPK pathway in driving RET therapy resistance and develop a viable therapeutic strategy to overcome it. Methodology: We examined MSKCC molecular diagnostic data to identify patients with RET fusion and MAPK pathway alterations. We generated cell lines from both RET fusion LUAD patient samples and lentiviral mediated expression of RET fusion cDNAs. Oncogenic KRAS (G12D) was expressed under the control of a doxycycline-inducible promoter in a RET fusion-driven cell line. Protein phosphorylation was determined via Western blotting in cells treated with selpercatinib for 0,3,6,12, 24 and 48 hours. Cell growth was determined using a viability dye.
RESULTS:
We found multiple patients with RET fusions and activating KRAS or BRAF mutations. Expression of KRASG12D in cells with RET fusion reduced sensitivity to selpercatinib. Additionally, treatment of RET fusion-driven cell lines with selpercatinib resulted in rapid downregulation of growth and survival signals (AKT, ERK1/2, MEK1/2, S6) and subsequent reactivation of the MAPK pathway (ERK1/2 and MEK1/2) within 12 hours. Concomitantly, negative MAPK pathway regulator (eg. DUSP4/6, SPRY2, SPRED1) expression was depressed, likely accounting for the rebound of MAPK phosphorylation. Additionally, the transcription factor capicua (CIC), which suppresses negative MAPK regulator expression, remained localized to the nucleus following selpercatinib treatment. Furthermore, growth of RET fusion-driven cell lines was reduced by inhibitors of MEK1/2 (binimetinib), ERK1/2 (ulixertinib) and RAS (RMC6236) and combinations of these drugs with selpercatinib were synergistic in blocking cell growth.
CONCLUSION:
Lung adenocarcinoma cell lines with RET fusion adapt to RET therapy by rapidly reactivating the MAPK pathway, likely via CIC-mediated suppression of negative MAPK pathway regulators. Combinations of selperacatinib with other inhibitors at different nodes within the MAPK pathway represents an effective therapeutic strategy to improve response in patients afflicted with RET fusion NSCLC.
RET (REarranged during Transfection) is a proto-oncogene that lies on chromosome 10q11.21. It encodes a single-pass receptor tyrosine kinase (RTK) whose signaling regulates the growth and development of the nervous system and other tissues. Importantly, ligand binding and dimerization with other regulators across the cell membrane regulate its signaling and kinase activity. In cancer, oncogenic RET kinases result from either somatic mutations in the kinase domain or fusions of that domain with other genes at its 5' end. These partner genes typically contribute a protein-protein interaction motif that allows RET to dimerize and thus remain constitutively active. These RET fusions are primary oncogenes in cancers of multiple histologies including papillary thyroid cancer and lung adenocarcinomas (LUAD). Currently, the only clinically approved treatment for patients with RET-dependent cancers are the RET inhibitors selpercatinib (any histology) and pralsetinib (lung and thyroid cancers), both of which have been shown to result in improvement of overall survival. Resistance to RET therapy, however, develops invariably and coincidentally with activating mutations in RAS, RAF (upstream activators of MEK1/2 and ERK1/2) or other alterations. Still, the mechanism(s) by which the MAPK pathway adapts to drug treatment and its role in dampening therapeutic response has not been explored in lung cancer. Consequently, we aimed to systematically characterize the involvement of the MAPK pathway in driving RET therapy resistance and develop a viable therapeutic strategy to overcome it. Methodology: We examined MSKCC molecular diagnostic data to identify patients with RET fusion and MAPK pathway alterations. We generated cell lines from both RET fusion LUAD patient samples and lentiviral mediated expression of RET fusion cDNAs. Oncogenic KRAS (G12D) was expressed under the control of a doxycycline-inducible promoter in a RET fusion-driven cell line. Protein phosphorylation was determined via Western blotting in cells treated with selpercatinib for 0,3,6,12, 24 and 48 hours. Cell growth was determined using a viability dye.
RESULTS:
We found multiple patients with RET fusions and activating KRAS or BRAF mutations. Expression of KRASG12D in cells with RET fusion reduced sensitivity to selpercatinib. Additionally, treatment of RET fusion-driven cell lines with selpercatinib resulted in rapid downregulation of growth and survival signals (AKT, ERK1/2, MEK1/2, S6) and subsequent reactivation of the MAPK pathway (ERK1/2 and MEK1/2) within 12 hours. Concomitantly, negative MAPK pathway regulator (eg. DUSP4/6, SPRY2, SPRED1) expression was depressed, likely accounting for the rebound of MAPK phosphorylation. Additionally, the transcription factor capicua (CIC), which suppresses negative MAPK regulator expression, remained localized to the nucleus following selpercatinib treatment. Furthermore, growth of RET fusion-driven cell lines was reduced by inhibitors of MEK1/2 (binimetinib), ERK1/2 (ulixertinib) and RAS (RMC6236) and combinations of these drugs with selpercatinib were synergistic in blocking cell growth.
CONCLUSION:
Lung adenocarcinoma cell lines with RET fusion adapt to RET therapy by rapidly reactivating the MAPK pathway, likely via CIC-mediated suppression of negative MAPK pathway regulators. Combinations of selperacatinib with other inhibitors at different nodes within the MAPK pathway represents an effective therapeutic strategy to improve response in patients afflicted with RET fusion NSCLC.