(P 335) FOCAL ADHESION KINASE (FAK) IS A THERPEUTIC TARGET IN CHROMOSOME 8 GAIN-ASSOCIATED MALIGNANT PERIPHERAL NERVE SHEATH TUMORS (MPNST)

Objective: Malignant peripheral nerve sheath tumors (MPNST) are aggressive sarcomas often associated with Neurofibromatosis type 1 (NF1), a genetic syndrome driven by overactivation of the RAS pathway. Mitogen-activated protein kinase kinase inhibitors (MEKi) target downstream components of the RAS pathway and have been approved for NF1 patients with plexiform neurofibromas (PN). However, MPNST often develop resistance to MEKi through several compensatory mechanisms. Our lab recently discovered the critical role of chromosome 8 (Chr8) genes in MPNST. The Chr8 gene FAK (focal adhesion kinase, also known as PTK2) is overexpressed in various tumor types, suggesting its importance in tumor development and progression. Recently, the combination of defactinib, a FAKi, and avutometinib, a Raf/MEKi, was granted FDA orphan drug designation for ovarian cancer. In this study, we aim to evaluate FAK as a therapeutic target for MPNST and investigate the FAKi and Raf/MEKi combination.

Methods: A CRISPR gRNA screen was used to identify the Chr8 genes involved in MPNST survival. The effect of FAK shRNA knockdown and a FAK inhibitor (FAKi), alone or combined with a Rafi/MEKi, in MPNST cells was assessed using IncuCyte proliferation and apoptosis assays. Synergy was determined by SynergyFinder software. RNA and protein expression of FAK and its downstream targets were assessed by qPCR and Wes SimpleWestern™ analyses. The efficacy of a FAKi, both as a monotherapy and in combination with a Rafi/MEKi, was evaluated in PDX mouse models of MPNST with Chr8 gain.

Results: The CRISPR gRNA screen identified 57 genes on Chr8 important for MPNST cell survival, including FAK. Knockdown of FAK by shRNA in MPNST cells inhibited cell proliferation in vitro and reduced tumorigenesis in vivo. Consistently, the FAKi defactinib significantly inhibited the proliferation in three MPNST cell-lines, JW23.3, JH-2-002, and MPNST-724 (IC50-values: 1.72-4.65μM). Moreover, the combination of defactinib and avutometinib was additive or synergistic in all three cell-lines. Avutometinib induced compensatory activation of FAK, while defactinib/avutometinib combination decreased phosphorylation of FAK, ERK1/2, and STAT3, and augmented cleavage of Caspase-3 and PARP-1, using WES western system. FAKi significantly decreased tumor growth, and combination of FAKi with Raf/MEKi reduced tumor growth more that either drug alone in Chr8-gain MPNST PDX models. Notably, complete tumor shrinkage was observed in 4 of 6 mice after three weeks of combination treatment in the MN2 PDX model.

Conclusion: In summary, FAK plays a critical role in MPNST pathogenesis. These results demonstrate the potential therapeutic efficacy of a FAKi, both alone and in combination with a Raf/MEKi, for the treatment of MPNST.