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The identification of JAK2 mutations in patients with myeloproliferative neoplasms (MPN) led to the clinical development of JAK2 inhibitors, and the JAK1/2 inhibitor ruxolitinib has been approved for the treatment of myelofibrosis (MF). Although clinically tested JAK inhibitors improve MPN-associated splenomegaly and systemic symptoms, they do not significantly reduce the MPN clone in most MPN patients.We previously demonstrated that MPN cells can acquire persistence to ruxolitinib and other type I JAK inhibitors which bind the active conformation of JAK2, and that JAK2 inhibitor persistence is associated with reactivation of JAK-STAT signaling and with heterodimerization between activated JAK2 and JAK1/TYK2, consistent with activation of JAK2 in trans by other JAK kinases. We have now extended our studies to other type I JAK inhibitors in clinical development, including CYT387, BMS911543 and SAR302503. In each case we see the same mechanism of persistence as observed with ruxolitinib, with transactivation of JAK2 by other JAK kinases. Most importantly, we found that MPN cells which were persistent to one JAK inhibitor were insensitive to the other JAK inhibitors, suggesting that the mechanisms which limit overall efficacy of ruxolitinib will limit the efficacy of other JAK inhibitors in clinical development. All JAK inhibitors in clinical development are type I inhibitors that interact with and inhibit the active confirmation of the JAK2 kinase. We hypothesized that novel, type II JAK inhibitors that interact with and inhibit JAK2 in the inactive conformation might retain activity in JAK inhibitor persistent cells and show increased efficacy in murine MPN models. We therefore characterized the efficacy of NVP-CHZ868, a novel type II JAK inhibitor, in MPN cells and in murine MPN models. CHZ868 potently inhibited proliferation of cells expressing the JAK2V617F mutation or the TEL-JAK2 fusion. We found that JAK2/MPL-mutant cell lines were universally sensitive to NVP-CHZ868. CHZ868 treatment of JAK2-mutant SET2 cells induced a higher degree of apoptosis compared to ruxolitinib. Signaling studies demonstrated that CHZ868 more potently attenuated JAK-STAT signaling in JAK2/MPL-mutant cells, with suppression of JAK2 phosphorylation consistent with a type II mechanism of kinase inhibition. We next investigated the ability of CHZ868 to inhibit the proliferation and signaling of MPN cells that had acquired persistence to type I JAK inhibitors. Type II inhibition with CHZ868 completely suppressed JAK-STAT signaling in type I JAK inhibitor-persistent cells, and prevented heterodimeric activation of JAK2 by JAK1 and TYK2. Most importantly, JAK2/MPL-mutant cells which were insensitive to type I JAK inhibitors remained highly sensitive to CHZ868, demonstrating that type I JAK inhibitor persistence does not confer resistance to type II inhibitors. We next evaluated the efficacy of CHZ868 in murine models of JAK2/MPL-mutant MPN. CHZ868 showed significant activity in conditional knock-in and bone marrow transplant (BMT) models of Jak2V617F-induced polycythemia vera, with normalization of hematocrit, reversal of stem/progenitor expansion, normalization of splenomegaly/splenic architecture, and reversal of bone marrow fibrosis. CHZ868 demonstrated similar activity in the MPLW515L BMT model of MF, with normalization of blood counts, stem/progenitor expansion, spleen weights, and extramedullary hematopoiesis in vivo. Most importantly, CHZ868 resulted in significant reductions of mutant allele burden (mean allele burden reduction 49%) in the Jak2V617F model. We observed analogous reductions in allele burden in the Jak2V617F and MPLW515L BMT models, consistent with disease modifying activity. Taken together, our data demonstrate that a spectrum of type I JAK inhibitors induce JAK inhibitor persistence, by a similar mechanism of JAK2 transactivation as observed with ruxolitinib. By contrast, type II JAK inhibition with CHZ868 remains highly active in JAK inhibitor persistent cells, and shows increased activity in murine MPN models. These data demonstrate that novel JAK inhibitors can increase target inhibition and therapeutic efficacy and should be pursued as an approach to improve outcomes for MPN patients. Figure 1 Figure 1. Figure 2 Figure 2. Disclosures Koppikar: Amgen: Employment. Sellers:Novartis: Employment. Hofmann:Novartis: Employment. Baffert:Novartis: Employment. Gaul:Novartis: Employment. Radimerski:Novartis: Employment. Levine:Novartis: Consultancy, Grant support Other.

Approximately 10% of B-ALLs harbor CRLF2 rearrangements, which may portend a poor prognosis. Although these leukemias are addicted to JAK2 signaling, ATP-competitive type I JAK2 inhibitors have limited activity in vitro or in vivo (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, which may overcome this resistance. When assayed in MHH-CALL4 cells harboring a CRLF2/IGH rearrangement and JAK2 I682F mutation, the type II JAK2 inhibitors NVP-BBT594 and NVP-CHZ868 were 10-35-fold more potent than the type I JAK2 inhibitors NVP-BSK805 and NVP-BVB808. Similarly, in Ba/F3 cells dependent on CRLF2 and the gain-of-function allele JAK2 R683G, the IC50 for CHZ868 was 5-20-fold lower than the IC50s for BSK805 and BVB808. Unlike type I inhibitors, which induce paradoxical hyperphosphorylation of JAK2, CHZ868 completely blocks JAK2 and STAT5 phosphorylation. In addition, the JAK2 Y931C allele that confers 4-6-fold resistance to BSK805 and BVB808 did not alter sensitivity to CHZ868. CHZ868 abrogates STAT5 phosphorylation in Ba/F3 cells expressing CRLF2 with JAK2 R683G/Y931C while BVB808 does not. CHZ868 is the first type II JAK2 inhibitor amenable to in vivo use. We assessed its efficacy in mice transplanted with transgenic (CRLF2/JAK2 R683G/Cdkn2a-/- or CRLF2/JAK2 R683G/Pax5+/-/Ts1Rhr) or primary human CRLF2-rearranged B-ALLs. Splenocytes from patient-derived xenografts (PDXs) treated with CHZ868 in vivo for 3 days are more primed for apoptosis as demonstrated by a 2-6-fold EC50 reduction for PUMA permeabilizing activity compared to vehicle. Transcriptional profiling of splenocytes from CHZ868-treated PDXs revealed downregulation of critical survival pathways including E2F1, STAT3, and AKT-mediated signaling. Of note, 2 of the most downregulated genes are STAT targets, PIM1 and Myc. Mice treated for 5-6 days with CHZ868 had significant reductions in spleen size and complete loss of phospho-STAT5 in residual leukemia cells. In both murine leukemias and human xenografts, CHZ868 prolonged survival compared to controls (p30 clones sequenced harbored the same JAK2 L884P mutation. Ba/F3 cells expressing CRLF2 with JAK2 R683G/L884P displayed cross-resistance to CHZ868, while sensitivity to type I inhibitors was not affected. Structural modeling of the JAK2 JH1 domain suggested that L884P alters the binding pocket for type II inhibitors. JAK2 L884P is homologous to an EGFR L747P activating mutation, which destabilizes the P-loop and C-helix portion of the kinase domain (He et al. Clin Cancer Res 2012). The fact that L884P was reported in two B-ALL patients lacking additional JAK2 mutations (Torra et al. Blood (ASH Annual Meeting Abstracts) 2010) raised the possibility it was also an activating mutation. We confirmed L884P is an activating allele, as Ba/F3 cells expressing CRLF2, IL7R, and JAK2 L884P proliferated in the absence of TSLP ligand. To improve CHZ868 efficacy, we tested for synergy with multiple chemotherapy agents currently used in B-ALL treatment. Dexamethasone was the most highly synergistic with CHZ868 in MHH-CALL4 cells. To assess the combination in vivo, we treated mice transplanted with CRLF2/JAK2 R683G/Pax5+/-/Ts1Rhr murine B-ALL with vehicle, CHZ868, dexamethasone, or CHZ868 + dexamethasone for 14 days post engraftment. CHZ868 treatment prolonged survival compared to vehicle (p Disclosures Hofmann: Novartis Institutes for BioMedical Research: Employment. Baffert:Novartis: Employment. Vangrevelinghe:Novartis Institutes for BioMedical Research: Employment. Gaul:Novartis: Employment. Radimerski:Novartis: Employment. Weinstock:Novartis: Consultancy, Research Funding.

Abstract 62 Mutation within the kinase domain of tyrosine kinases is a common mechanisms of resistance to enzymatic inhibitors. Inhibitors of janus kinase 2 (JAK2) are under evaluation in patients with myeloproliferative neoplasms (MPNs), B-cell acute lymphoblastic leukemia (B-ALL) with rearrangements of the cytokine receptor subunit CRLF2, and other tumors with constitutive JAK2 signaling. To identify resistance mutations in JAK2, we randomly mutagenized human JAK2 R683G, which is observed in approximately half of CRLF2-rearranged B-ALL. We transduced the mutagenized JAK2 cDNA library into murine Ba/F3 cells that express CRLF2. Expression of CRLF2 and JAK2 R683G confers IL3 independent growth in Ba/F3 cells. The transduced population was selected in the JAK2-selective inhibitor NVP-BVB808 in the absence of IL3. Multiple BVB808-resistant clones were recovered that harbored either E864K, Y931C or G935R mutations in JAK2. Alignment of homologous regions of the JAK2 kinase domain (JH1) with ABL1 demonstrated that the three mutations are located in regions homologous to imatinib resistance hotspots in ABL1. Codons Y931 and G935 are within the hinge region of the kinase domain. Based on structural modeling, Y931C is likely to inhibit substrate binding. E864K is located in the middle of b3 following the P-loop in the N-lobe and may modify the structure and flexibility of the preceding P-loop, thus destabilizing the conformation required for inhibitor binding. We expressed JAK2 V617F alleles harboring Y931C, G935R or E864K in Ba/F3-EPOR cells and exposed the cells to the JAK2 enzymatic inhibitors JAK inhibitor-1, NVP-BSK805, TG101348, tofacitinib (formerly tasocitnib), ruxolitinib (formerly INCB18424) and BVB808. All three mutations conferred 2- to >10-fold resistance against BVB808, NVP-BSK805, TG101348, ruxolitinib and JAK inhibitor-1. Y931C and E864K but not G935R conferred resistance to tofactinib. Modeling of G935R indicated that a 935R side-chain would occlude the hydrophobic channel of the ATP-binding pocket. As a consequence, this mutation would decrease the binding affinity of compounds occupying the hydrophobic channel like JAK inhibitor-1 or BSK805, but not affect the potency of tofactinib, which does not bind in this region. Mutation of G935 to arginine, histidine or glutamine reduced the inhibitory effects of JAK inhibitor-1, but not tofacitinib, on JAK2 kinase domain activity. None of the codon 935 mutations had significant effects on Km or Vmaxin vitro. BVB808 treatment partially reduced activation state-specific phosphorylation of STAT5 in Ba/F3-EPOR/JAK2 V617F cells but not in Ba/F3-EPOR/JAK2 V617F/G935R or G935H cells. JAK2 is a known client of HSP90, and HSP90 inhibitors promote the degradation of both wild-type and mutant JAK2. We hypothesized that resistance mutations within the JAK2 kinase domain would not affect JAK2 degradation induced by HSP90 inhibitors. We assayed the cytotoxicity of the resorcinylic isoxazole amide NVP-AUY922 and the benzoquinone ansamycin 17-AAG in Ba/F3 cells that express the erythropoietin receptor (EPOR) and JAK2 V617F, which is observed in more than half of MPNs. Mutation of JAK2 V617F to include E864K, Y931C or G935R did not affect sensitivity to either AUY922 or 17-AAG. In fact, AUY922 was more active against cells harboring G935R (GI50, 3.87 nM) or E864K (GI50, 6.14 nM) compared to cells with no resistance mutation (GI50, 14.7 nM; p Disclosures: Gaul: Novartis: Employment. Vangrevelinghe:Novartis: Employment. De Pover:Novartis: Employment. Regnier:Novartis: Employment. Erdmann:Novartis: Employment. Hofmann:Novartis: Employment. Eck:Novartis: Consultancy, Research Funding. Kung:Novartis Pharmaceuticals: Consultancy, Research Funding. Radimerski:Novartis Pharma AG: Employment. Weinstock:Novartis: Consultancy, Research Funding.