We have recently reported that MDM2 levels are increased in PV CD34+ cells as a consequence of JAK2617F, while the p53 levels are reduced in CD34+ cells from both patients with PV and PMF. These observations led us to explore therapeutic approaches by which to up-regulate p53 as a strategy to treat MPN patients. We previously reported that combination treatment with low doses of Peg IFNα 2a and Nutlin-3, an antagonist of MDM2, induced PV CD34+ apoptosis and inhibited PV colony formation significantly. The combination of these agents also decreased the proportion of JAK2V617F-positive HPCs. We demonstrated that these two drugs affect p53 through different pathways with Peg IFNα 2a activating p38 MAP kinase and STAT1 leading to increased p53 transcription while nutlin-3 prevents the degradation of p53 (Lu et al, Blood, 2012; In Press).
RG7112 is an orally bioactivity small molecule inhibitor of p53-MDM2 binding which activates the p53 pathway. RG7112 is currently being evaluated in several phase 1 cancer trials. Based on our previous data, we hypothesized that RG7112 would be an effective drug to combine with low doses of IFN alpha to treat MPN patients. In order to evaluate the therapeutic effects of RG7112 alone or in combination with Peg IFNα 2a on hematopoietic progenitor cells from MPN patients, CD34+ cells were isolated from patients with PV and PMF patients and were treated with varying of doses of RG7112 alone or/and in combination with a low dose of Peg IFNα 2a and its effects on PV CD34+ cell proliferation, apoptosis and colony formation was evaluated. The effects of this drug on MPN CD34+ cells was compared to its effects on cord blood CD34+ cells After treatment with RG7112 at doses ranging from 100 nM to 5000 nM for 4 days, the numbers of PV CD34+ cells were reduced to a far greater degree (about 60%) than that observed with cord blood CD34+ cells (90% and 80%) at the doses between 200 and 500 nM indicating that low doses of RG7112 selectively affects MPN HPCs. 200nM of RG7112 and 200 U/ml of Peg-IFNα 2a alone induced cell cycle arrest of PV CD34+ cells, and combination treatment significantly promoted apoptosis of MPN CD34+ cells (2.5 folds). This observation leads us to examine the possibility that low doses of RG7112 might be used in combination with Peg-IFNα 2a to treat MPN patients. Treatment with 200 nM of RG7112 alone decreased PV CFU-GM and BFU-E-derived colony formation by 20% while combination treatment with 200 U/ml of Peg-IFNα 2a decreased PV CFU-GM and BFU-E-derived colony formation by 60% and 70%, respectively. Similarly, treatment with 200 nM of RG7112 alone decreased PMF CFU-GM and BFU-E-derived colony formation by 20% and 40%, respectively, while combination treatment with 200 U/ml of Peg-IFNα 2a decreased PMF CFU-GM and BFU-E-derived colony formation by 60% and 80%, respectively. We also found that treatment with both agents in combination also decreased the size of the hematopoietic colonies. By contrast, treatment with the same low doses of RG7112 and Peg-IFNα 2a alone or in combination did not significantly affect the ability of normal CD34+ cells to generate CFU-GM and BFU-E-derived colonies. Combined treatment with low doses of RG7112 and Peg-IFNα 2a decreased the total number of JAKV617F-positive PV HPC by 9% - 25% (p=0.014) leading to an increased proportion of colonies with wild type JAK2. In conclusion, these data demonstrate that IFNα plus RG7112 specifically target malignant MPN HPC by activating p53 in vitro, which we propose can be exploited therapeutically to eliminate JAK2V617F positive HPC in both PV and PMF patients. These results strongly suggest that combinations of low doses of IFNα and RG7112 might serve as a novel therapeutic strategy for the long term treatment of MPN patients.
No relevant conflicts of interest to declare.
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