LAUSR

The neurotrophic tyrosine receptor kinases (NTRKs) are a family of neuronal transmembrane receptors that signal through several pathways including JAK/STAT, PI3K/AKT, and MEK/ERK to promote proliferation, differentiation, and survival. Although much of the literature has focused on the importance of these receptors in neuronal development, over the past decade, gene fusions containing NTRK family members have been implicated in driving tumor growth in salivary gland, breast, lung, colon, and neuronal cancers. Moreover, expression of NTRK2 and NTRK3 has been reported in lymphoid and myeloid malignancies (Li, Z et al. Blood, 2009). Interestingly, NTRK3 expression has been associated with poorer prognosis. Despite these initial studies, the role of NTRK receptors largely remains under-investigated in the setting of hematologic malignancies. Herein, we describe somatic mutations in NTRK2 and NTRK3 that we identified using next-generation sequencing of primary leukemia patient samples. We evaluated changes in downstream signaling driven by these mutations in our previously validated Ba/F3 transformation model system. Lastly, we demonstrate that selective inhibition of NTRK2 and NTRK3 with entrectinib, a well-validated NTRK inhibitor currently in clinical trials for NTRK-fusion positive cancers, can inhibit proliferation and induce apoptosis of NTRK transformed cells. We found two point mutations in NTRK2 that were transforming — one in the extracellular domain (A203T) and one in the juxtamembrane domain (R458G). Similarly, we found two point mutations that were transforming in NTRK3. One was in the extracellular domain (E176D) and the other within the transmembrane domain (L449F). Immunoblot results suggest that expression of total and phosphorylated NTRK2 and NTRK3 is increased in mutant-transformed Ba/F3 cells relative to wild-type cells. Furthermore, NTRK2 and NTRK3 mutant-driven cells exhibited enhanced phosphorylation of AKT, SRC, and ERK compared to wild-type cells. While phosphorylation of STAT3 was significantly increased in Ba/F3 cells transfected with both NTRK3 mutants, it was increased only in one of the NTRK2 mutants (A203T). Upon treating NTRK mutant-transformed cells with entrectinib (0, 5, 25, and 100 nM), we observed a dose-dependent decrease in total and phosphorylated NTRK2 and NTRK3 in all mutants except for NTRK2 mutant R458G. With R458G, total and phosphorylated NTRK2 expression decreased up to 25 nM of entrectinib but that effect was diminished at 100 nM. Taken together, our data demonstrates that mutations in NTRK2 and NTRK3 have transformative potential to promote downstream survival signaling and leukemogenesis. More importantly, these activated pathways can be pharmacologically attenuated using entrectinib. Our findings contribute to ongoing efforts to understand molecular changes that drive hematological malignancies and could potentially alter current treatment modalities. Citation Format: Sunil K. Joshi, Kristin Qian, Jacob P. Wagner, Monika A. Davare, Cristina E. Tognon, Brian J. Druker. Transforming NTRK2 and NTRK3 mutations as potential drivers of leukemia [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 970.