Abstract 290: NF1 loss modulates cellular fitness in KIT-mutant gastrointestinal stromal tumor (GIST)

Abstract

Abstract Patients with inoperable GIST obtain substantial clinical benefit from imatinib and other KIT/PDGFRA inhibitors. However, secondary resistance develops in 90% of patients within 18-24 months. Reactivation of oncogenic signaling pathways at time of progression is most often due to secondary mutation in KIT or PDGFRA, but downstream RAS or BRAF mutations are occasionally alternative mechanisms of KIT/PDGFRA-inhibitor resistance. We identified deleterious NF1 mutations in 3 KIT-mutant GIST samples from patients progressing on imatinib. In genomic scale loss-of-function shRNA genomic screens, designed to identify genes involved in imatinib resistance, a shRNA targeting NF1 was the most enriched shRNA in imatinib-treated GIST cells out of 54,000 shRNAs (NF1 gene score 29/11,238). To evaluate the contribution of NF1 loss to KIT-mutant GIST resistance, NF1-deficient GIST models were generated from 2 patient-derived, KIT-mutant GIST cell lines, GIST882 and GIST-T1, by sequential cotransfection with Cas9 and 2 independent NF1-targeting CRISPR-sgRNAs. Complete loss of NF1 protein was observed after 5-10 passages due to on-target heterogeneous genomic deletion at the NF1 locus. Orthogonal validations were obtained using stable shRNA-mediated NF1 knockdown with similar results in the same GIST cell lines. NF1-deleted cells retained expression of mutant KIT but were resistant to imatinib (IC50 196 and 500nM, significantly increased from baseline 34 and 110nM, for GIST-T1 and GIST882, respectively). Notably, after interruption of imatinib treatment, NF1-deficient GIST cells showed reduced proliferation (69% and 77% growth reduction at day 7 off imatinib, for GIST-T1 and GIST882, t test p<0.001) despite continuous expression of activated KIT and downstream signaling effectors. In conclusion, acquired NF1 loss induces TKI resistance in KIT-mutant GIST cells but results in reduced cellular fitness in the absence of KIT-inhibitor treatment. These findings suggest that NF1-deficient GIST subclones that expand on imatinib treatment can be destabilized by intermittently discontinuing KIT-inhibitor therapy. Citation Format: Adrian Marino-Enriquez, Bin Li, Nacef Bahri, Alexandra Lauria, Yuexiang Wang, Jonathan A. Fletcher. NF1 loss modulates cellular fitness in KIT-mutant gastrointestinal stromal tumor (GIST). [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 290.