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The protein JAK2 is a prototypical member of the Janus kinase family, and mediates signals from numerous cytokine receptors. The constitutively active V617F mutant of JAK2 is prevalent in many bone marrow disorders, blood cancers, and autoimmune diseases, and is an important drug target. Structures have been determined for each of the four individual domains making up JAK2, and for certain pairs of these domains, but no structure of full-length JAK2 is available, and thus the mechanisms underlying JAK2 regulation and the aberrant activity of the V617F mutant have been incompletely understood. Here we propose structural models of full-length JAK2 in both its active and inactive forms. Construction of these models was informed by long-timescale molecular dynamics simulations. Subsequent mutagenesis experiments showed that mutations at the putative interdomain interfaces modulated JAK2 activity. The models provide a structural basis for understanding JAK2 autoinhibition and activation, and suggest that the constitutive activity of the V617F mutant may arise from a dual effect of destabilizing the inactive conformation and stabilizing the active conformation.

FGFR2 fusions, amplifications, and mutations are oncogenic drivers that occur across multiple tumor types. Clinical efficacy observed with pan-FGFR inhibitors has validated the driver status of FGFR2 in FGFR2 fusion-positive intrahepatic cholangiocarcinoma (ICC), however, FGFR1-mediated toxicities (hyperphosphatemia, tissue mineralization) and the emergence of on-target FGFR2 resistance mutations limit the efficacy of pan-FGFR inhibitors. To overcome these limitations, we designed RLY-4008, a potent and highly selective, FGFR2 inhibitor. Despite significant investment in traditional structure-based drug design, selective targeting of FGFR2 has not been achieved. We leveraged differences in conformational dynamics between FGFR2 and other FGFR isoforms observed through molecular dynamics simulations to enable the design of RLY-4008. RLY-4008 inhibits FGFR2 with low nanomolar potency and demonstrates > 200-fold selectivity over FGFR1, and > 80- and > 5000-fold selectivity over FGFR3 and FGFR4, respectively, in biochemical assays. Additionally, RLY-4008 demonstrates high kinome selectivity for FGFR2 against a panel of > 400 human kinases. RLY-4008 has strong activity against primary and acquired FGFR2 resistance mutations in cellular assays, and potent antiproliferative effects on FGFR2-altered human tumor cell lines. In vivo, RLY-4008 demonstrates dose-dependent FGFR2 inhibition and induces regression in multiple human xenograft tumor models, including FGFR2 fusion-positive ICC, gastric, and lung cancers, FGFR2-amplified gastric cancer, and FGFR2-mutant endometrial cancer. Strikingly, RLY-4008 induces regression in an FGFR2 fusion-positive ICC model harboring the FGFR2V564F gatekeeper mutation and an endometrial cancer model harboring the FGFR2N549K mutation, two mutations that drive clinical progression on current pan-FGFR inhibitors. In the FGFR2V564F model, pan-FGFR inhibitors are ineffective, even at maximally tolerated doses. Notably, treatment of these tumors with RLY-4008 induces rapid regression and restores body weight. In rat and dog toxicology studies, RLY-4008 is well tolerated and is not associated with hyperphosphatemia or tissue mineralization at exposures significantly above those required to induce regression in all models. In contrast to pan-FGFR inhibitors, RLY-4008 is highly selective for FGFR2 and demonstrates strong activity against FGFR2 resistance mutations, suggesting that RLY-4008 may have broader therapeutic potential via preventing and overcoming therapeutic resistance. Together, these data and the favorable pharmaceutical properties of RLY-4008 strongly support its clinical development in FGFR2-altered tumors. Citation Format: Jessica Casaletto, Dejan Maglic, B. Barry Toure, Alex Taylor, Heike Schoenherr, Brandi Hudson, Kamil Bruderek, Songping Zhao, Patrick O'Hearn, Nastaran Gerami-Moayed, Demetri Moustakas, Roberto Valverde, Lindsey Foster, Hakan Gunaydin, Pelin Ayaz, Dina Sharon, Donald Bergstrom, James Watters. RLY-4008, a novel precision therapy for FGFR2-driven cancers designed to potently and selectively inhibit FGFR2 and FGFR2 resistance mutations [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1455.