Resistance to RET-Inhibition in RET-Rearranged NSCLC Is Mediated By Reactivation of RAS/MAPK Signaling

Abstract

Oncogenic rearrangements in RET are present in 1-2% of lung adenocarcinoma (LAD) patients. Ponatinib is a multi-kinase inhibitor with low-nanomolar potency against the RET kinase domain. Here, we demonstrate that ponatinib exhibits potent anti-proliferative activity in RET fusion positive LC-2/ad LAD 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 to 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 LAD is mediated by bypass signaling mechanisms that result in restored RAS/MAPK activation.

An Acquired NRAS Q61K Mutation in BRAF V600E-Mutant Lung Adenocarcinoma Resistant to Dabrafenib Plus Trametinib

“…Interestingly, whereas NRAS mutations are only rarely identified as potential driver mutations in NSCLC, they have also been implicated in acquired resistance to targeted therapies in NSCLC including EGFR and RET inhibition. [10][11][12] Our finding is also in line with previous observations implicating NRAS Q61K in resistance to both single-agent BRAF inhibition as well as combination BRAF/MEK inhibition in melanoma. 8,9,13 In the case of combination BRAF and MEK inhibition in NSCLC, it is not clear if this mutation leads to resistance by overcoming MAPK pathway inhibition, for example, by activating compensatory signaling though other RAF family members, or by activating the PI3K pathway or by a combination of these two mechanisms.…”

Inhibitor repurposing reveals ALK, LTK, FGFR, RET and TRK kinases as the targets of AZD1480

“…Ponatinib (0.1 μM; Pon.) was used as a positive control for CCDC6-RET inhibition [ 52 ]. (D) RCS-F@F cells were treated with AZD1480 for 4 hours before FGF2, and analyzed for FRS2 and ERK phosphorylation (p).…”

Novel targeted therapeutics for MEN2

“…vandetanib, Carlomagno et al 2004). A recent work reported NRAS mutations in ponatinib-resistant RETrearranged NSCLC cells (Nelson-Taylor et al 2017). It is also unknown whether sequential targeted therapies with multiple RET inhibitors will improve patients OS.…”