Resistance to RET-inhibition in RET-rearranged NSCLC is mediated by reactivation of RAS/MAPK signaling

Sarah K. Nelson-Taylor, Anh T. Le, Minjae Yoo, Laura Schubert, Katie M. Mishall, Andrea Doak, Marileila Varella-Garcia, Aik-Choon Tan, Robert C. Doebele

Research output: Contribution to journalArticle

13 Citations (Scopus)

Abstract

Oncogenic rearrangements in RET are present in 1%–2% of lung adenocarcinoma patients. Ponatinib is a multi-kinase inhibitor with low-nanomolar potency against the RET kinase domain. Here, we demonstrate that ponatinib exhibits potent antiproliferative activity in RET fusion–positive LC-2/ad lung adenocarcinoma cells and inhibits phosphorylation of the RET fusion protein and signaling through ERK1/2 and AKT. Using distinct dose escalation strategies, two ponatinib-resistant LC-2/ ad cell lines, PR1 and PR2, were derived. PR1 and PR2 cell lines retained expression, but not phosphorylation of the RET fusion and lacked evidence of a resistance mutation in the RET kinase domain. Both resistant lines retained activation of the MAPK pathway. Next-generation RNA sequencing revealed an oncogenic NRAS p.Q61K mutation in the PR1 cell. PR1 cell proliferation was preferentially sensitive to siRNA knockdown of NRAS compared with knockdown of RET, more sensitive to MEK inhibition than the parental line, and NRAS dependence was maintained in the absence of chronic RET inhibition. Expression of NRAS p.Q61K in RET fusion expressing TPC1 cells conferred resistance to ponatinib. PR2 cells exhibited increased expression of EGFR and AXL. EGFR inhibition decreased cell proliferation and phosphorylation of ERK1/2 and AKT in PR2 cells, but not LC-2/ad cells. Although AXL inhibition enhanced PR2 sensitivity to afatinib, it was unable to decrease cell proliferation by itself. Thus, EGFR and AXL cooperatively rescued signaling from RET inhibition in the PR2 cells. Collectively, these findings demonstrate that resistance to ponatinib in RET-rearranged lung adenocarcinoma is mediated by bypass signaling mechanisms that result in restored RAS/MAPK activation.

Original languageEnglish
Pages (from-to)1623-1633
Number of pages11
JournalMolecular Cancer Therapeutics
Volume16
Issue number8
DOIs
Publication statusPublished - 2017 Aug 1
Externally publishedYes

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Phosphotransferases
Phosphorylation
Cell Proliferation
RNA Sequence Analysis
Cell Line
Mutation
Mitogen-Activated Protein Kinase Kinases
Small Interfering RNA
ponatinib
Adenocarcinoma of lung
Proteins
BIBW 2992

ASJC Scopus subject areas

  • Oncology
  • Cancer Research

Cite this

Nelson-Taylor, S. K., Le, A. T., Yoo, M., Schubert, L., Mishall, K. M., Doak, A., ... Doebele, R. C. (2017). Resistance to RET-inhibition in RET-rearranged NSCLC is mediated by reactivation of RAS/MAPK signaling. Molecular Cancer Therapeutics, 16(8), 1623-1633. https://doi.org/10.1158/1535-7163.MCT-17-0008

Resistance to RET-inhibition in RET-rearranged NSCLC is mediated by reactivation of RAS/MAPK signaling. / Nelson-Taylor, Sarah K.; Le, Anh T.; Yoo, Minjae; Schubert, Laura; Mishall, Katie M.; Doak, Andrea; Varella-Garcia, Marileila; Tan, Aik-Choon; Doebele, Robert C.

In: Molecular Cancer Therapeutics, Vol. 16, No. 8, 01.08.2017, p. 1623-1633.

Research output: Contribution to journalArticle

Nelson-Taylor, SK, Le, AT, Yoo, M, Schubert, L, Mishall, KM, Doak, A, Varella-Garcia, M, Tan, A-C & Doebele, RC 2017, 'Resistance to RET-inhibition in RET-rearranged NSCLC is mediated by reactivation of RAS/MAPK signaling', Molecular Cancer Therapeutics, vol. 16, no. 8, pp. 1623-1633. https://doi.org/10.1158/1535-7163.MCT-17-0008
Nelson-Taylor, Sarah K. ; Le, Anh T. ; Yoo, Minjae ; Schubert, Laura ; Mishall, Katie M. ; Doak, Andrea ; Varella-Garcia, Marileila ; Tan, Aik-Choon ; Doebele, Robert C. / Resistance to RET-inhibition in RET-rearranged NSCLC is mediated by reactivation of RAS/MAPK signaling. In: Molecular Cancer Therapeutics. 2017 ; Vol. 16, No. 8. pp. 1623-1633.
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AU - Mishall, Katie M.

AU - Doak, Andrea

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N2 - Oncogenic rearrangements in RET are present in 1%–2% of lung adenocarcinoma patients. Ponatinib is a multi-kinase inhibitor with low-nanomolar potency against the RET kinase domain. Here, we demonstrate that ponatinib exhibits potent antiproliferative activity in RET fusion–positive LC-2/ad lung adenocarcinoma cells and inhibits phosphorylation of the RET fusion protein and signaling through ERK1/2 and AKT. Using distinct dose escalation strategies, two ponatinib-resistant LC-2/ ad cell lines, PR1 and PR2, were derived. PR1 and PR2 cell lines retained expression, but not phosphorylation of the RET fusion and lacked evidence of a resistance mutation in the RET kinase domain. Both resistant lines retained activation of the MAPK pathway. Next-generation RNA sequencing revealed an oncogenic NRAS p.Q61K mutation in the PR1 cell. PR1 cell proliferation was preferentially sensitive to siRNA knockdown of NRAS compared with knockdown of RET, more sensitive to MEK inhibition than the parental line, and NRAS dependence was maintained in the absence of chronic RET inhibition. Expression of NRAS p.Q61K in RET fusion expressing TPC1 cells conferred resistance to ponatinib. PR2 cells exhibited increased expression of EGFR and AXL. EGFR inhibition decreased cell proliferation and phosphorylation of ERK1/2 and AKT in PR2 cells, but not LC-2/ad cells. Although AXL inhibition enhanced PR2 sensitivity to afatinib, it was unable to decrease cell proliferation by itself. Thus, EGFR and AXL cooperatively rescued signaling from RET inhibition in the PR2 cells. Collectively, these findings demonstrate that resistance to ponatinib in RET-rearranged lung adenocarcinoma is mediated by bypass signaling mechanisms that result in restored RAS/MAPK activation.

AB - Oncogenic rearrangements in RET are present in 1%–2% of lung adenocarcinoma patients. Ponatinib is a multi-kinase inhibitor with low-nanomolar potency against the RET kinase domain. Here, we demonstrate that ponatinib exhibits potent antiproliferative activity in RET fusion–positive LC-2/ad lung adenocarcinoma cells and inhibits phosphorylation of the RET fusion protein and signaling through ERK1/2 and AKT. Using distinct dose escalation strategies, two ponatinib-resistant LC-2/ ad cell lines, PR1 and PR2, were derived. PR1 and PR2 cell lines retained expression, but not phosphorylation of the RET fusion and lacked evidence of a resistance mutation in the RET kinase domain. Both resistant lines retained activation of the MAPK pathway. Next-generation RNA sequencing revealed an oncogenic NRAS p.Q61K mutation in the PR1 cell. PR1 cell proliferation was preferentially sensitive to siRNA knockdown of NRAS compared with knockdown of RET, more sensitive to MEK inhibition than the parental line, and NRAS dependence was maintained in the absence of chronic RET inhibition. Expression of NRAS p.Q61K in RET fusion expressing TPC1 cells conferred resistance to ponatinib. PR2 cells exhibited increased expression of EGFR and AXL. EGFR inhibition decreased cell proliferation and phosphorylation of ERK1/2 and AKT in PR2 cells, but not LC-2/ad cells. Although AXL inhibition enhanced PR2 sensitivity to afatinib, it was unable to decrease cell proliferation by itself. Thus, EGFR and AXL cooperatively rescued signaling from RET inhibition in the PR2 cells. Collectively, these findings demonstrate that resistance to ponatinib in RET-rearranged lung adenocarcinoma is mediated by bypass signaling mechanisms that result in restored RAS/MAPK activation.

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