Supplementary Figures 1-8 Fig. S1: Cell viability of LC-2/ad, PR1, and PR2 cells treated with alectinib or cabozantinib. Fig. S2: RET break-apart FISH analysis of LC-2/ad, PR1, PR2, and H2228. Fig. S3: Phosphatase inhibitor treatment of PR1 and PR2 restores phospho-RET. Fig S4: NRAS Q61K increases NRAS GTP loading and induces RET inhibitor resistance in TPC1 cells. Fig S5: PR1 cells remain resistant to ponatinib after withdrawal from chronic ponatinib treatment. Fig S6: PR2 cells have lost RET signaling dependence. Fig S7: AXL signaling contributes to EGFR-mediated resistance to ROS1 inhibitors in HCC78-TAER cells. Fig S8: PR2 cells demonstrate plasticity when withdrawn from chronic ponatinib and display dual dependence on RET and EGFR. Supplementary Tables 1-3 Table S1: Normalized gene expression of RTKs in LC-2/ad, PR1 and PR2 cells. Table S2: Normalized gene expression of RTK ligands in LC-2/ad, PR1 and PR2 cells. Table S3: Normalized gene expression of protein tyrosine phosphatases in LC-2/ad, PR1, and PR2 cell lines.
ARTICLE 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. Mol Cancer Ther; 16(8); 1623–33. ©2017 AACR.
