Combined Inhibition of JAK2 and mTOR Signaling Results in Enhanced Efficacy in in-Vitro and Preclinical Mouse Models of JAK2V617F-Driven Myeloproliferative Disease

The JAK1/JAK2 inhibitor Ruxolitinib (ruxo) produced rapid and sustained responses in splenomegaly and symptomatic improvement in patients (pts) with myelofibrosis (MF), supporting the central role of dysregulated JAK2 signaling in myeloproliferative neoplasms (MPN). Splenomegaly and constitutional symptoms improved also after treatment with Everolimus (RAD001), a rapamycin-derivative inhibitor of the serine/threonine kinase mTOR, in a phase I/II study, pointing to mTOR pathway as a novel target for MPN therapy. BEZ235 is a dual phosphatidylinositol-3-kinase (PI3K) and mTOR inhibitor currently in clinical trials in solid cancer. Aim of the study was to investigate the efficacy of BEZ235, alone and in combination with ruxo, against MPN cells in vitro and in preclinical mouse models.

We used mouse (Ba/F3 and Ba/F3-EPOR expressing wild-type (WT) or V617F(VF) mutated JAK2) and human (VF HEL and SET2 or WT K562) cell lines and primary MPN CD34⁺ cells from pts with MF or polycythemia vera (PV); cell proliferation, colony formation, apoptosis, cell cycle and protein phosphorylation status were evaluated. Effects of drug combination were analyzed according to Chou and Talalay to calculate the combination index (CI); a CI <1.0 indicates synergistic activity. For in vivo studies, two mouse models were used. (1) SCID mice receiving iv Ba/F3-EPOR VF-luciferase (luc) cells (gift of T. Radimerski) were randomized on d6 to treatment groups (either drugs alone and in combination or vehicle (VE)) based on baseline luminescence. Bioluminescence measurement was done at week intervals until death. (2) A C57Bl6/J JAK2 V617F Knock-in mouse model was generated by the flex switch strategy with insertion of the reversed JAK2V617F exon 13 sequence; mating with Vav-Cre transgenic mice activates the VF allele producing a MPN phenotype in progenies from VF heterozygous expression. Mice were treated for 5d, then blood, spleen and bone marrow cells were analyzed.

We found that BEZ235 inhibited proliferation of Ba/F3 VF and Ba/F3-EPOR VF cells at concentrations significantly lower than the wt counterparts (IC50 was 64±10nM vs 10,000±500nM and 87±50nM vs 676±200nM; P<0.01); similar preferential inhibition was observed in HEL and SET2 cells compared to K562 cells (IC50, 387±90nM and 334±40nM vs 5,000±1,000nM; P<0.01). BEZ235 dose-dependently increased the percentage of cells in G0/G1 and induced apoptosis. Western blot analysis showed marked reduction of the mTOR target p4EBP1 as well as appreciable downregulation of pSTAT5 and pSTAT3 at 6 to 24h of treatment. BEZ235 impaired the proliferation of CD34⁺ cells from MF pts with an IC50= 43±20nM vs 780±150nM in healthy donors (P<0.01), and reduced colony formation of MF and PV hematopoietic progenitors at doses statistically lower (2 to 15-fold) than normal cells. The growth of EPO-independent colonies (EEC) in PV pts was potently inhibited (IC50=20±10nM). Co-treatment of BEZ235+ruxo resulted in synergistic inhibition of proliferation in SET2 (median CI=0.37) and BaF3-EPOR VF (CI=0.77) cells and increased apoptosis rate in SET2 (CI=0.25); drug combination was highly effective also in the EEC assay (CI=0.17). In the Ba/F3VF luc model, the median bioluminescence index (No. of pixel) on day 7 of treatment was 4.7×10⁶ in VE animals, 7.2×10⁶ in ruxo (P=ns), 1.1×10⁶ in BEZ235 (P=0.04) and 4.6×10⁵ in animals receiving the BEZ+ruxo combination (P<0.01). The median survival in mice treated with the combination of BEZ+ruxo was 30d, significantly longer than either ruxo (18.5d; P<0.01) or BEZ (24d; P<0.04) alone (15.0d in VE animals). In JAK2V617F KI mice treated for 5d, we found that drug combination was significantly more effective in reducing enlarged spleen (median spleen index (spleen weight/body weightx100): 38, 35, 27 and 7 for VE, BEZ, Ruxo and BEZ+Ruxo) and reticulocyte count (median No. per HPF: 48, 50, 44, and 3 for VE, BEZ, Ruxo and BEZ+Ruxo) than either drugs alone. The phosho levels of STAT5 and 4EBP1 in the spleen were significantly reduced in mice receiving BEZ+Ruxo as compared to single treatment.

Combined inhibition of PI3K/mTOR and JAK2 signaling resulted in enhanced activity in preclinical models of MPN compared with either treatment alone, providing a rationale for the development of combination clinical trials.

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