Constitutively activated pY705-STAT3 is oncogenic and prevalent in many tumor types, and has been shown to be driven in large part by constitutive autocrine and paracrine cytokine signaling. JAK kinases are critical mediators of cytokine signaling, and JAK1 has been shown to play an essential role downstream of IL-6 family cytokines, the upregulation of which has been documented in many tumor types. A JAK1 selective inhibitor should provide a therapeutic means of suppressing Y705 phosphorylation of STAT3 without incurring the hematological toxicity associated with inhibition of JAK2 and JAK3 activity, allowing for more intensive dosing and greater combinability with other agents, particularly those with hematological toxicities. We have developed highly selective inhibitors of JAK1 and used them to address the hypothesis that JAK1 is the primary driver of STAT3 mediated signaling in cancer cells. The compounds are highly selective in enzyme assays, with 1000-fold selectivity for JAK1 vs. other JAK family members, and minimal activity against other kinases at 1uM. In cell lines, they are over 500-fold selective for JAK1 vs. JAK2, measured using assays specific for JAK1-mediated STAT3 vs. JAK2-mediated STAT5 phosphorylation. In vivo, the compounds are also highly selective for JAK1 mediated signaling, measured by comparing inhibition of STAT3 phosphorylation in H1975 subcutaneous xenografts vs. inhibition of STAT5 phosphorylation in a disseminated TEL-JAK2 transformed BaF3 model. At doses where inhibition of STAT3 phosphorylation was greater than 90%, only minimal inhibition of STAT5 phosphorylation was observed. In a wide variety of human tumor lines (CRC, NSCLC, pancreatic, DLBCL) with a variety of genetic backgrounds (K-ras, b-raf, PI3K, EGFR mutant, PTEN null), complete knockdown of pSTAT3 (Y705) was observed with JAK1 selective compounds, at concentrations achievable in vivo. These effects are consistent with experiments where inhibition of Y705 phosphorylation of STAT3 was achieved with a JAK1-specific siRNA. At doses where in vivo activity was observed, the compounds had only minimal effect in an erythropoietin-driven mouse model of polycythemia and extramedullary erythropoiesis. These data support the hypotheses that in a variety of tumor types, pSTAT3-mediated signaling is dependent on JAK1 kinase activity, and that a selective JAK1 inhibitor can suppress STAT3 activation at doses that avoid the hematological toxicity associated with JAK2 and JAK3 inhibition.

Citation Format: Richard Woessner, Geraldine Bebernitz, Kirsten Bell, Robert Caccese, Nakpangi Johnson, Jason Kettle, Caroline Rivard, Patricia Schroeder, Qibin Su, Haixia Wang, Minwei Ye, Weijia Zheng, Michael Zinda, Paul Lyne, Melissa Vasbinder, Dennis Huszar, Claudio Chuaqui. Highly selective JAK1 kinase inhibitors suppress Y705-mediated STAT3 activation in a wide range of tumor types, with reduced hematological toxicity. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 931. doi:10.1158/1538-7445.AM2013-931