LAUSR

Chromosomal rearrangements involving the rearranged during transfection (RET) tyrosine kinase occur in 1-2% of NSCLCs. This results in the expression of a constitutively active fusion kinase that promotes cancer cell growth, proliferation and survival. In lung cancer, RET is most commonly fused to KIF5B, but can have many other 59 partners such as CCDC6, NCOA, TRIM33 and TRIM24. Clinical trials with several multi-kinase inhibitors, which potently inhibit RET, have had low response rates between 18-53%. Novel, more highly specific RET inhibitors are now in clinical development. Collectively, the clinical trial data suggest that patients with KIF5B-RET rearrangements have exceptionally low responses to RET inhibitors in relation to non-KIF5B-RET fusions. In order to understand the role of RET 59 partners in regulating response to RET inhibitors we have generated two novel KIF5B-RET+ patient-derived cell lines, CUTO22 (K23;R12) and CUTO32 (K15;R12). We have confirmed that each of these cell lines contain the KIF5B-RET fusion and express the RET fusion protein. MTS proliferation assays have revealed that the CUTO22 cell line is very sensitive to the highly specific RET inhibitor, CEP-32496/RXDX-105, and the multi-kinase inhibitor ponatinib. Conversely, the CUTO32 cell line is dramatically resistant to both RXDX-105 and ponatinib. Phospho-RET (pRET) is inhibited by RXDX-105 and ponatinib in a dose-dependent manner in both the CUTO22 and CUTO32 cell line. Interestingly, despite successful RET inhibition, downstream signaling in pERK and pAKT remains active. In a time-course experiment, pRET is inhibited for up to 24 hours while pERK and pAKT is sustained. Additionally, knockdown of RET with siRNA did not reduce downstream signaling activation in CUTO22 or CUTO32 cells. Combined inhibition of possible bypass pathways, EGFR or mTOR, did not significantly enhance sensitivity to RET inhibitors in CUTO22 and CUTO32 cell lines. To isolate the role of the 59 partners in response to RET inhibitors we have utilized CRISPR/Cas9 technology to generate Kif5b-Ret and Trim24-Ret rearrangements in Ba/F3 cells. We have confirmed with PCR or RT-PCR that these cell lines harbor the intended Ret fusions and express the RET protein. Both the Kif5b-Ret+ and Trim24-Ret+ Ba/F3 cell lines are highly sensitive to multiple RET inhibitors. In conclusion, these data suggest that RET inhibitors successfully inhibit the target protein, but not downstream signaling, suggesting that KIF5B-RET may have unique co-alterations or reliance on alternative pathways to promote growth and proliferation. Citation Format: Laura Schubert, Anh T. Le, Andrea E. Doak, Robert C. Doebele. Novel KIF5B-RET+ NSCLC cell lines demonstrate differential responses to RET inhibitors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1842.