Dysregulated JAK/STAT signaling, occurring mainly but not exclusively in cells harboring mutations in JAK2 or other proteins involved in JAK/STAT pathway such as MPL, CBL, or Lnk, represents a pathogenetic event in chronic myeloproliferative neoplasms (MPN). However, activation of other downstream pathways such as the ERK and PI3K/Akt/mTOR pathway has been also documented in JAK2V617F-mutated cells. In this study we explored in-vitro the potential relevance of targeting PI3K/Akt/mTor pathway with specific inhibitors, alone or in combination with JAK2 inhibitor. Indeed, clinical trials have recently documented the effectiveness of JAK1/2 inhibitors (Verstovsek S, NEJM, 2010;363:117; Pardanani A, JCO 2011; 29:789) and RAD001, an mTOR inhibitor (Guglielmelli 2001; Blood, in press), in patients with MPN, mainly with myelofibrosis. The following drugs were used: an allosteric (RAD001) and an ATP (PP242) mTOR competitor; a dual PI3K/mTOR inhibitor (BEZ235); the JAK1/2 kinase ATP competitor AZD1480 and INCB018424.
In the BA/F3/EPOR JAK2V617F-mutated cells, cell proliferation was prevented by lower doses of RAD001 (615±50nM, measured as IC50), PP242 (98±5nM) and BEZ235 (87±50nM) compared to BA/F3/EPOR JAK2wild-type (wt) cells (>10,000nM; 5,931±1,000nM; 676±200nM, respectively). In case of JAK inhibitors, IC50 was 313±23nM for AZD1480 and 51±2nM for INCB018424 as compared to 752±30nM and 457±15nM in wt cells, respectively. mTOR inhibitors induced cell cycle arrest in Go but were very poorly inducers of apoptosis (less than 15–20% at maximum); conversely JAK1/2 inhibitors induced dose-dependent increase of Annexin-V +ve cells up to >60% and BEZ235 induced 30–40% apoptosis at highest concentrations. All above drugs were able to prevent short-term cell proliferation and colony formation also in JAK2V617F-mutated HEL and SET2 cells. Western blot analysis demonstrated that, in addition to the expected inhibition of phosphorylation of specific drug targets (mTOR, 4EBP1), all three PI3K/mTOR inhibitors also reduced the degree of phophoSTAT5. siRNA-induced down-regulation of mTOR in SET2, HEL and BA/F3/EPOR JAK2V617F cells resulted in reduced phosphoSTAT5, indicating a direct mTOR-dependent phopshoSTAT5 regulation. Then, the activity of RAD001, BEZ235 and AZD1480 was analyzed in primary cells from MPN patients. All three drugs reduced clonogenic growth of MPN erythroid, myeloid and megakaryocytic progenitors at doses significantly lower (from 5 to 10-fold) than in normal cells, and prevented erythropoietin-independent colony (EEC) formation in the low nM range. Single colony genotyping in JAK2V617F mutated patients showed a median of 30±20% (range, 5–57%) reduction of V617F mutated colonies in favor of wt colonies Overall, these data indicated that inhibitors of PI3K/mTOR prevent proliferation and clonogenic capacity of MPN cells mainly through a cytostatic rather apoptotic effect (as JAK1/2 inhibitors do).
To exploit whether simultaneous treatment with PI3K/AKt and JAK1/2 inhibitor displayed synergism we treated SET2 cells with different drug doses and measured their proliferation and apoptotic rate. Synergism was calculated as the combination index (CI) according to Chou and Talalay. Evidence of synergism was obtained for AZD1480 and INCB018424 with RAD001 (CI: 0.13 and 0.20, respectively), PP242 (CI: 0.13 and 0.20, respectively) and BEZ235 (CI: 0.77 and 0.37, respectively). Synergism was similarly demonstrated in BA/F3/EPOR JAK2V617F-mutated cells. Activity of RAD001 with AZD1480 and INCB018242 was also assessed in a EEC assay. We found that addition of RAD001 (5nM) or BEZ235 (50nM) to very low doses of JAK1/2 inhibitors (in the range of 5 to 50 nM) resulted in significant synergism and almost completely prevented EEC formation.
In summary, these in vitro data indicate that PI3K/mTOR inhibitors are active against MPN cells and their combination with JAK1/2 inhibitors produced synergism, allowing to use lower doses of each drug; studies in murine models are ongoing to confirm these results in vivo. Thus, concurrent targeting of PI3K/mTOR and JAK/STAT pathway might represent a new therapeutic strategy to optimize efficacy and reduce toxicity in patients with MPN.
Vannucchi:Italfarmaco: Consultancy; Novartis: Honoraria.
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