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 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.

Figure 1. Ponatinib inhibits RET and decreases cell proliferation of LC-2/ad cells

(A) Cell viability of LC-2/ad (blue), TPC1 (pink), and H2228 (green) cells treated with increasing doses of ponatinib for 72 hours. Error bars: mean ± SEM for 3 triplicate experiments (n=9). (B) Western blot analysis of LC-2/ad cells treated with increasing doses of ponatinib for 2 hours. Lysates were analyzed via western blotting with the antibodies indicated, with P-TYR used to assess P-RET.

Figure 2. PR1 and PR2 cells are resistant to ponatinib

(A) Dose escalation schema for the derivation of PR1 and PR2 derivatives of the LC-2/ad cell line. (B) Cell viability of LC-2/ad (blue), PR1 (pink), and PR2 (green) cells treated with increasing doses of ponatinib for 72 hours. Error bars: mean ± SEM for 3 triplicate experiments (n=9). (C) Western blot analysis of untreated LC-2/ad, PR1 and PR2 cells. (D) Western blot analysis of PR1 and PR2 cells removed from ponatinib for 24 hours prior to 2 hours treatment with increasing doses of ponatinib. Lysates were analyzed via western blotting with the antibodies indicated, with P-TYR used to assess P-RET.

Figure 3. NRAS Q61K drives resistance in the PR1 cell line

(A) DNA sequencing of NRAS revealed the PR1 cell line is heterozygous for an A to C single base pair substitution responsible for the NRAS p.Q61K mutation. This mutation was not found in either LC-2/ad cells or PR2 cells. (B) LC-2/ad cells were transfected with siRNA targeting RET, NRAS, RET and NRAS, or a non-targeted control. Cell viability was measured after 7 days using the CyQuant Direct Cell Proliferation Assay and normalized to the non-targeting control. Error bars: mean ± SEM for 3 triplicate experiments (n=9). (C) Western blot analysis of LC-2/ad cells transfected under the same conditions as (B). Lysates were collected 72 hours after transfection and analyzed via Western blotting with the antibodies indicated. (D) PR1 cells were transfected with siRNA targeting RET, NRAS, RET and NRAS, or a non-targeted control. Cell viability was measured after 7 days using the CyQuant Direct Cell Proliferation Assay and normalized to the non-targeting control. Error bars: mean ± SEM for 3 triplicate experiments (n=9). (E) Western Blot analysis of PR1 cells transfected under the same conditions as (D). Lysates were collected 72 hours after transfection and analyzed via western blotting with the antibodies indicated. (F) Cell viability of LC-2/ad (blue) and PR1 (pink) cells (which had been removed from ponatinib for 24 hours) treated with increasing doses of trametinib for 72 hours at which point cell viability was measured using the CyQuant Direct Cell Proliferation Assay. Error bars: mean ± SEM for 3 triplicate experiments (n=9).

Figure 4. EGFR regulates MAPK and PI3K signaling and proliferation in the PR2 cell line

(A) Western Blot analysis of LC-2/ad and PR2 cells treated with a DMSO control, 10 nM ponatinib, 500 nM afatinib, or 10 nM ponatinib and 500 nM ponatinib for 4 hours. Lysates were analyzed via Western blotting with the antibodies indicated, with P-TYR used to assess P-RET. (B) Cell viability of LC-2/ad (blue) and PR2 (green) cells treated with increasing doses of afatinib for 72 hours. Error bars: mean ± SEM for 3 triplicate experiments (n=9).

Figure 5. AXL inhibition enhances sensitivity to EGFR inhibition in the PR2 cell line

(A) Cell viability of PR2 cells treated with increasing doses of afatinib plus DMSO (green), 500 nM cabozantinib (pink), or 500 nM foretinib (blue) for 72 hours. Error bars: mean ± SEM for 3 triplicate experiments (n=9). (B) Cell viability of PR2 cells transfected with siRNA targeting AXL (siAXL) or a non-targeting control (siNT) for 24 hours and subsequently treated with increasing doses afatinib for 72 hours. Error bars: mean ± SEM for 3 triplicate experiments (n=9). (C) Western blot analysis of LC-2/ad and PR2 cells serum starved for 2 hours with DMSO (-) or 500 nM afatinib (+) b and then treated with vehicle (-) or 100 ng/ml EGF (+) for 15 minutes. Lysates were analyzed via Western blotting with the antibodies indicated. (D) Western blot analysis of LC-2/ad and PR2 cells serum starved for 2 hours with DMSO (-) or 500 nM foretinib (+) and then treated with vehicle (-) or 100 ng/ml GAS6 (+) for 30 minutes. Lysates were analyzed via western blotting with the antibodies indicated.

Figure 6. LC-2/ad cells are primed to utilize AXL and EGFR signaling when RET is inhibited

(A) LC-2/ad cells were treated with 10 nM ponatinib for the 0, 4, 24, and 48 hours. Lysates were analyzed via Western blotting with the antibodies indicated. (B) Cell viability of LC-2/ad cells treated with increasing doses of ponatinib plus vehicle control (blue), 100 ng/mL EGF (gold), 100 ng/mL GAS6 (pink), or 100 ng/mL EGF + 100 ng/mL GAS6 (green) for 72 hours. Error bars: mean ± SEM for 3 triplicate experiments (n=9).