Complete Inhibition of STAT5 Phosphorylation Is Achieved By Combination of JAK1/2 and PI3K/mTOR Inhibitors in in Vitro and In Vivo MPN Models

Autonomous activation of JAK/STAT pathway by JAK2 V617F (VF) and similar mutations in myeloproliferative neoplasms (MPN) is associated with enhanced signaling of other downstream pathways including the PI3K/mTOR. Targeting the PI3K pathway resulted in inhibition of JAK2 VF cells ( J Cell Mol Med 2013;17:1385 ), and the Torc1 inhibitor Everolimus (RAD001) showed clinical benefits in a phase I/II trial ( Blood 2011;118:2069 ). In this study we evaluated the effects of BKM120, a pan-PI3K specific inhibitor, alone and in combination with the JAK1/JAK2 inhibitor Ruxolitinib (Ruxo) and Everolimus or PP242 (Torc1/2 inhibitor), in different MPN models and correlated efficacy with the extent of STAT5 phosphorylation inhibition. In vitro , we used BaF3 and BaF3-EPOR cells expressing wild type (WT) or VF mutated JAK2, the human VF mutated HEL and SET2 cell lines, and primary CD34 + cells from MPN patients. The combination index (CI) according to Chou and Talalay was used to evaluate drug combination; a CI in vivo studies we used two mouse models: (1) SCID mice receiving iv BaF3-EPOR VF-luciferase (luc) cells ( Mol Cancer Ther 2010;9:1945 ) were randomized on day 6 to different treatment groups based on baseline luminescence and animals were sacrificed when became moribund; (2) C57Bl6/J JAK2VF knock-in (KI) mice constitutively expressing JAK2VF protein in heterozygous status ( Blood 2013; 122:1464 ) were treated for 15 days before analysis. BKM120 preferentially inhibited BaF3VF and BaF3-EpoR-VF cells compared to the WT counterpart (IC50: 364±200nM and 1100±207nM vs 5300±800nM and 3122±1000nM respectively; P + cells at concentrations 2- to 8-fold lower than controls (P In vivo , BKM120 alone at a dose of 60mpk increased survival of BaF3VF-luc injected mice from 21d (controls) to 28d (P In vivo , triple combination treatment of KI mice with low drug doses (30mpk BKM120 + 30mpk Ruxo + 1.5mpk Everolimus) resulted in impressive improvement of splenomegaly: median spleen reduction compared to controls was -44.5%, -36.2%, -8%, for 60mpk BKM120, 60mpk Ruxo, 60mpk Everolimus as single agents and -69.5%, for triple treatment. Similarly greater activity was seen concerning reduction of leukocytosis and reticulocyte count. Compatible with findings in vitro , the level of p-4eBP1 and p-STAT5 in the spleen were significantly reduced in mice receiving combined treatment versus single agents. We explored the mechanisms by which PI3K/mTOR inhibitors resulted in reduced p-STAT5. We found that Ruxo treatment reduced tyrosine (Tyr) phosphorylation of the STAT5a isoform while leaving serines (Ser) unaffected, while BKM120 and Everolimus specifically resulted in dephosphorylation of STAT5b serine residues, in particular Ser193 and Ser731; accordingly, combined treatment resulted in complete STAT5 inactivation by dephosphorylation of both Ser and Tyr residues. Interestingly, the mRNA and protein levels of Cip2a, Cancerous Inhibitor of Phosphatase 2a (PP2a, that is normally involved in dephosphorylation of p-STAT residues), were strongly reduced by PI3K pathway inhibitors both in cell lines and in samples from patients responsive to Everolimus treatment. PP2a inhibition by Calyculin A resulted in enhanced STAT5b Ser193 and Ser731 phosphorylation while slightly altered STAT5a phosphoresidues suggesting a role of PP2a and its repressor Cip2a in PI3K/STAT5-dependent activation of JAK/STAT signaling. Concurrent inhibition of the JAK2 and PI3K pathways by Ruxo plus BKM120 and Everolimus results in enhanced activity in preclinical models of MPN and underlines the importance of complete STAT5 inactivation in order to magnify treatment efficacy, thereby providing a rationale for clinical trials. Disclosures Vannucchi: Novartis Pharmaceuticals Corporation: Membership on an entity9s Board of Directors or advisory committees, Research Funding, Speakers Bureau; Shire: Speakers Bureau; Baxalta: Membership on an entity9s Board of Directors or advisory committees.