HSP90 Inhibition Targets JAK2 and Is Highly Effective in CRLF2-Rearranged Acute Lymphoblastic Leukemia
Authors: Weigert Oliver , Bird Liat , Kopp Nadja , Lane Andrew A. , Chapuy Bjoern , van Bodegom Diederik , Marubayashi Sachie , Yoda Akinori , Romanet Vincent , Murakami Masato , Sallan Stephen E. , Levine Ross L , Kung Andrew L. , Radimerski Thomas , Weinstock David M 
Published: October 2019

Abstract
Abstract 576
Enzymatic inhibitors of Janus kinase 2 (JAK2) are in clinical development for the treatment of B-cell acute lymphoblastic leukemia (B-ALL) with rearrangements of the cytokine receptor subunit CRLF2, myeloproliferative neoplasms, and other tumors with constitutive JAK2 signaling. In addition, JAK2 is a client of heat shock protein 90 (HSP90) and HSP90 inhibition results in JAK2 degradation. To explore the utility of blocking JAK2 in CRLF2-rearranged B-ALL, we exposed the MHH-CALL4 and MUTZ-5 cell lines, which both have CRLF2/IGH rearrangements and activating JAK2 mutations to a panel of JAK2 inhibitors (JAK inhibitor-1, INCB18424, tofacitinib, NVP-BSK805, NVP-BVB808, TG101348) and the HSP90 inhibitor NVP-AUY922. Both MUTZ-5 and MHH-CALL4 were highly sensitive to AUY922 (GI50, 25–26 nM), with 50- to >1,000-fold superior potency compared with the panel of JAK2 enzymatic inhibitors. AUY922 and the structurally divergent HSP90 inhibitors 17-AAG, PU-H71 and HSP990 were all potently active against a panel of Ba/F3 lines dependent on CRLF2 and JAK2 signaling (GI50, 1–11 nM). Treatment of MUTZ-5 and MHH-CALL4 cells with JAK inhibitor-1 reduced but did not eliminate phospho (P-) STAT5 and P-ERK1/2 in both lines but promoted increases in P-AKT in MUTZ-5 and P-JAK2 in MHH-CALL4. In contrast, AUY922 treatment more extensively reduced or eliminated phosphorylation of all the targets. The combination of AUY922+JAK inhibitor-1 had little or no additional effect on target phosphorylation compared with AUY922 alone and pairwise dose-response studies with isobologram analysis failed to identify synergistic effects. We performed transcriptional profiling on MUTZ-5 and MHH-CALL cells treated with either vehicle (DMSO), JAK inhibitor-1, AUY922 or JAK inhibitor-1+AUY922. Unsupervised hierarchical clustering distinguished samples treated with AUY922 (or combination) from those treated with JAK inhibitor-1 or vehicle. To formally assess whether AUY922 targets the same genes as JAK inhibitor-1, we defined a ‘JAK inhibitor signature' from the top/bottom 250 most differentially expressed genes following treatment with JAK inhibitor-1. Using gene set enrichment analysis (GSEA), the ‘JAK inhibitor signature' was highly enriched upon treatment with AUY922 (p=0.003). GSEA also demonstrated that STAT5A signatures were enriched upon treatment with JAK inhibitor-1, AUY922, or JAK inhibitor-1+AUY922. To identify additional targets of HSP90 inhibition beyond the inhibition of JAK2, we used the C3 database from the MsigDB compendium to perform a transcription factor binding site enrichment analysis on the most differentially expressed genes between JAK inhibitor-1 and AUY922. The top 5 hits were all heat-shock factors (HSF, FDR<0.05). GSEA revealed that an HSF1 signature was only enriched upon treatment with AUY922 or AUY922+JAKinh-1, but not after JAKinh-1 alone. To extend our findings to in vivo treatment of human B-ALL, we established primary B-ALL xenografts from CRLF2-rearranged, patient-derived bone marrow samples in NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ (NSG) mice. To stringently assay established disease in vivo, we waited until bone marrow leukemia burden exceeded 30% and then initiated treatment with BVB808 50mg/kg twice daily, AUY922 50mg/kg thrice weekly, BVB808+AUY922 or vehicle. After 5 days of treatment, spleens from mice treated with vehicle or BVB808 had nearly complete effacement by B-ALL, while AUY922 or BVB808+AUY922 treatment resulted in visible islands of hematopoiesis. Based on immunohistochemistry, mice receiving AUY922 or BVB808+AUY922, but not BVB808 or vehicle, had nearly complete loss of P-STAT5 as well as upregulation of the pharmacodynamic marker HSP70. Immunoblotting of spleens from treated mice demonstrated reductions in P-STAT5, P-JAK2, and total JAK2 in AUY922- or BVB808+AUY922-treated mice. In contrast, treatment with single-agent BVB808 only modestly suppressed P-STAT5. Treatment with either BVB808 or AUY922 prolonged overall survival compared to vehicle (p=0.01 for both xenografts). Treatment with AUY922 further prolonged overall survival compared to BVB808 (p<0.01 for both xenografts), while the combination of BVB808 and AUY922 had no additional benefit compared to AUY922 alone. In conclusion, HSP90 is a promising therapeutic target in CRLF2-rearranged B-ALL and merits clinical evaluation.

Disclosures:
Romanet: Novartis Pharma AG: Employment. Murakami:Novartis Pharma AG: Employment. Sallan:Enzon Pharmaceuticals: Honoraria. Kung:Novartis Pharmaceuticals: Consultancy, Research Funding. Radimerski:Novartis Pharma AG: Employment. Weinstock:Novartis: Consultancy, Research Funding.