Abstract A23: Type II JAK2 inhibitor NVP-CHZ868 has potent activity in JAK2-dependent B-cell acute lymphoblastic leukemias (B-ALLs) in vivo.
Authors: Li Loretta S. , Kopp Nadja , Wu Shuo-Chieh , Zwet Jordy Van Der , Layer Jacob V. , Weigert Oliver , Christie Amanda L. , Christodoulou Alexandra N. , Liu Huiyun , Yoda Akinori , Radimerski Thomas , Weinstock David M. 
Published: August 2015

Approximately 10% of B-ALLs harbor CRLF2 rearrangements and have a poor prognosis. Although these leukemias are addicted to JAK2 signaling, ATP-competitive type I JAK2 inhibitors have limited activity (Weigert et al. J Exp Med 2012). This may result from heterodimerization of JAK2 with other JAK family members (Koppikar et al. Nature 2012). Type II inhibitors bind JAK2 in the inactive conformation and may have non-cross resistance with type I inhibitors. In Ba/F3 cells dependent on CRLF2 and the gain-of-function allele JAK2 R683G, the type II JAK2 inhibitor NVP-CHZ868 was more potent (IC50 21nM) than the type I inhibitors NVP-BSK805 (IC50 443nM) and NVP-BVB808 (IC50 111nM). Unlike type I inhibitors, CHZ868 completely abrogated JAK2 and STAT5 phosphorylation. In addition, the JAK2 Y931C allele that confers 4-6-fold resistance to BSK805 and BVB808 did not affect the IC50 of CHZ868. We assessed in vivo efficacy of CHZ868 in mice transplanted with transgenic (CRLF2/JAK2 R683G/Cdkn2a-/- or CRLF2/JAK2 R683G/Pax5+/-/Ts1Rhr) or primary human CRLF2-rearranged B-ALLs. Mice treated for 5-6 days with CHZ868 (30mg/kg/day PO) had significant reductions in spleen size compared to control mice and complete loss of phospho-STAT5 in residual leukemia cells. In both murine leukemias and human xenografts, CHZ868 prolonged survival compared to controls (p&lt;0.001), but all mice ultimately became moribund from B-ALL. To study mechanisms of resistance to type II JAK2 inhibitors, we screened a randomly mutagenized JAK2 R683G library expressed in Ba/F3-CRLF2 cells for clones resistant to the type II inhibitor NVP-BBT594. All (&gt;30) clones sequenced harbored the same JAK2 L884P mutation. Ba/F3 cells expressing CRLF2 with JAK2 R683G/L884P had 14-fold resistance to CHZ868 (R683G IC50 16nM; R683G/L884P IC50 231nM). JAK2 L884P is homologous to an EGFR L747P activating mutation (He et al. Clin Cancer Res 2012), which destabilizes the P-loop and C-helix portion of the kinase domain. Next-generation sequencing of JAK2 from splenocytes of mice that progressed on CHZ868 treatment did not identify L884P or other missense mutations at &gt;1% frequency, suggesting in vivo treatment failure was not due to JAK2 mutation. To improve CHZ868 efficacy, we tested for synergy with multiple chemotherapy agents in MHH-CALL4 cells, which harbor a CRLF2/IGH rearrangement and JAK2 I682F mutation. Among the tested agents, dexamethasone was highly synergistic with CHZ868. In mice transplanted with CRLF2/JAK2 R683G/Pax5+/-/Ts1Rhr murine B-ALL, a 14-day course of CHZ868 prolonged survival compared to vehicle (p&lt;0.0001) or dexamethasone (p&lt;0.01), and the combination prolonged survival beyond CHZ868 monotherapy (p&lt;0.0001). In summary, the type II JAK2 inhibitor CHZ868 potently kills JAK2-dependent B-ALL, overcomes genetic resistance to type I inhibitors, and synergizes with dexamethasone in vitro and in vivo. Thus, combination strategies using dexamethasone with type II JAK2 inhibitors merit testing in patients with relapsed/refractory, JAK2-dependent B-ALL.

Citation Format: Loretta S. Li, Nadja Kopp, Shuo-Chieh Wu, Jordy Van Der Zwet, Jacob V. Layer, Oliver Weigert, Amanda L. Christie, Alexandra N. Christodoulou, Huiyun Liu, Akinori Yoda, Thomas Radimerski, David M. Weinstock. Type II JAK2 inhibitor NVP-CHZ868 has potent activity in JAK2-dependent B-cell acute lymphoblastic leukemias (B-ALLs) in vivo. [abstract]. In: Proceedings of the AACR Special Conference on Hematologic Malignancies: Translating Discoveries to Novel Therapies; Sep 20-23, 2014; Philadelphia, PA. Philadelphia (PA): AACR; Clin Cancer Res 2015;21(17 Suppl):Abstract nr A23.