The mTOR Inhibitor, RAD001, Inhibits the Growth of Cells From Patients with Myeloproliferative Neoplasms.

Abstract 2914

Poster Board II-890

The JAK2 and MPL somatic mutations in chronic myeloproliferative neoplasms (MPN) result in constitutive activation of JAK2/STAT pathway; in-vitro JAK2 inhibitors reduced the proliferation of JAK2V617F mutant cells while in early clinical trials in patients (pts) with myelofibrosis resulted in clinical improvement, although the effects on the burden of JAK2-mutant clone were less impressive than anticipated. On the other hand, little is known about other signalling pathways, such as PI3K/Akt and ERK, that also resulted activated in cells harboring the JAK2V617F mutation. In this study we focused on the mammalian target of rapamycin (mTOR), a downstream target of key signalling molecules including Akt, PI3K, GTPase Ras and Rheb; mTOR is often abnormally activated in cancer cells and represents an attractive target for anticancer therapy, as shown by the effects of mTOR inhibitors in solid tumors and lymphomas. First, we documented constitutive phosphorylation of mTOR and of the eukaryotic initiation factor 4E-binding protein 1 (4EBP1), a downstream target of mTOR, in Ba/F3 cells transduced with JAK2 V617F (kindly provided by R. Skoda); conversely, phosphorylation of both mTOR and 4EBP1 in wild-type (wt) Ba/F3 cells was largely dependent on exposure to IL3. Then, to assess the effects of mTOR inhibition on the growth of JAK2V617F mutant cells we used the specific mTOR inhibitor RAD001. We observed that day-14 clonogenic growth of JAK2V617F mutant cells HEL, UKE-1 (a gift of W. Fiedler) and SET2 was potently inhibited by RAD001 compared to BCR-ABL-pos K562 cells: IC50 values were 85±30 nM, 60±20 nM, 330±50nM and 12,000±340 nM, respectively (all P<.001). Also the IC50 of JAK2V617Fpos Ba/F3 cells (2.0±3.0 nM) was significantly lower than that of wt Ba/F3 cells (1,990±500 nM). RAD001 caused an accumulation of HEL cells in the G0 phase of the cell cycle from 60.5±11.5% to 71.8±10.3%, and reduced cells in the S-phase from 19.1±5.9% to 10.7±1.6%. The proportion of apoptotic HEL cells increased from 8.9±1.8% to 40.8±8.3% after 48 hr with 100 nM RAD001. We then evaluated whether inhibition of mTOR could affect the growth of hematopoietic progenitors from MPN pts by using serial dilutions of the drug added to cytokine-supplemented clonogenic cultures. Results shown in the Table demonstrated that RAD001 efficiently prevented the growth of clonogenic progenitors from Polycythemia Vera (PV) or Primary Myelofibrosis (PMF) pts compared to controls (Ctrl) (P<.01 for all). The proportion of wt and JAK2V617F-mutant colonies was measured in cytokine supplemented cultures of 5 pts by single-colony genotyping ; we found that JAK2V617Fpos colonies decreased of a median value of 30% (range 10-50%) in the presence of RAD001. Finally, we found that the sensitivity to RAD001 of erythropoietin-independent erythroid progenitors from PV pts was significantly greater for JAK2V617F homozygous (IC50= 3.0±2.0 nM) than heterozygous (33.0±5.0 nM; P<.01) progenitors. In HEL and UKE1 cells, and in JAK2V617Fpos Ba/F3, exposed to RAD001 the phosphorylation of mTOR and 4EBP1 was efficiently prevented, as expected; however, we also found that RAD001 caused a significant reduction of STAT5 phosphorylation (Western Blotting and FACS analysis). To confirm that reduction of pSTAT5 was actually dependent on mTOR inhibition, we transduced (Amaxa) HEL cells with siRNA against mTOR; we observed a parallel reduction of 4EBP1 and STAT5 phosphorylation at 48-72 hr that followed a >50% decrease of mTOR mRNA and protein content. In conclusion, we observed constitutive activation of mTOR pathway associated with V617F mutation and provided evidence of a significant sensitivity to RAD001 of JAK2V617F mutated cell lines (HEL, Ba/F3) and hematopoietic progenitors from MPN patients. These effects may be largely related to a previously uncharacterized effect on STAT5 phosphorylation by member(s) of the mTOR pathway, that is inhibited by RAD001. Overall, these data suggest an involvement of mTOR in MPN pathogenesis, and anticipate a possible relevance for treatment. A phase I/II trial with RAD001 in pts with myelofibrosis is currently ongoing in Italian Institutions (AM Vannucchi et al., ASH 2009, abstract submitted).