Treatment with Nutlin-3, a Small-Molecule Antagonist of MDM2, in Combination with Low Doses of Pegylated Interferon Alpha 2a Targets Hematopoietic Progenitor Cells From Polycythemia Vera (PV) Patients

Abstract 796^FN2

The chronic myeloproliferative neoplasms (MPN) originate at the level of the hematopoietic stem cell (HSC). The prolonged and frequently unpredictable natural history of the MPNs has limited the use of many therapeutic approaches some of which are associated with unacceptable toxicities. IFNα therapy reverses morphological marrow abnormalities, eliminates cytogenetic abnormalities, reduces the JAK2V617F allele burden and re-establishes polyclonal hematopoiesis in patients with PV. Prolonged IFNα therapy is, however also frequently difficult due to drug related toxicity. We hypothesized that the addition of a non-genotoxic therapeutic agent to lower doses of IFNα might be better tolerated and be effective in eliminating the PV clone. The tumor suppressor, p53 gene plays an important role in the control of DNA repair, cell cycling, and apoptosis and is frequently inactivated in many cancers. The cellular level of p53 is controlled by a master regulator, MDM2. Nutlin-3, a small-molecule antagonist of MDM2 which activates the p53 pathway in cancer cells with wild-type p53 and is currently being evaluated in clinical trials. In MPN, p53 mutations and heterozygous deletions have been identified in a limited number of patients undergoing transformation to acute leukemia. We have previously reported that IFNα acts through p38 MAP kinase (Lu M, et al. Exp Hematol. 2010;38:472-80) while others have shown that activating this pathway leads to increased p53 (Sancéau J, Oncogene. 2000;19:3372-83). We hypothesized that targeting alternative pathways which can up-regulate p53 might serve as a novel therapeutic strategy for treating MPN patients. In this study, the therapeutic effects of nutlin-3 combined with Pegylated interferon alpha 2a on PV and normal CD34⁺ cells were investigated. FACS and western blot analysis demonstrated that the p53 protein levels in PV CD34⁺ cells were lower than normal CD34⁺ cells. CD34⁺ cells isolated from 8 PV patients and 7 normal donors were exposed to low doses of Peg IFNa 2a (200 U/ml) and/or nutlin-3 (200 nM) for 4 days and the number of hematopoietic progenitor cells (HPC) assayed. A dose dependent anti-proliferative effect of nutlin-3 on PV CD34⁺ cells was documented. PV CD34⁺ cells treated with nutlin-3 at doses ranging from 100 nM to 1000 nM, led to a progressively greater inhibition of colony formation from 10% to 80%. Treatment of normal CD34⁺ cells with 200 nM of nutlin-3 did not affect BFU-E or CFU-GM numbers, but inhibited PV CFU-GM by 20% and BFU-E by 50%. Simultaneous treatment with low doses of both nutlin-3 and Peg IFNa 2a inhibited PV CFU-GM by 55% and BFU-E by 90%, while normal cord blood CFU-GM and BFU-E were inhibited by 18% and 22%, respectively. This inhibition of PV HPC was far greater than that observed following the treatment with either agent alone. The combination of drugs also further decreased the proportion of JAK2V617F positive HPCs, as compared to CD34⁺ cells treated with Peg IFNa 2a alone (between 12.5% and 30% greater reduction). In addition, in a case of JAK2V617F negative myelofibrosis with a deletion of the long arm of chromosome 20 del(20) (q11–q12), Peg IFNα 2a and Nutlin-3 treatment of CD34⁺ cells cultured in the presence of SCF, TPO, IL-3 and FLT3 ligand reduced the number of cytogenetically abnormal total cells to 1% as compared to 15% observed following Peg IFNa 2a treatment alone. These data suggest that Peg IFNα 2a combined with Nutlin-3 treatment can substantially reduce the number of malignant MPN HPCs. The mechanism by which nutlin-3 and Peg IFNa 2a act on PV HPC was explored by FACS and Western Blot analysis using a JAK2V617F positive erythroleukemia cell line (HEL cells) and K562 cells. Peg IFNa 2a and nutlin-3 treatment each individually increased the p53 protein level and nuclear translocation in HEL but not in K562 cells. A further increase in p53 was observed when HEL cells were exposed to the combination of Peg IFNa 2a and nutlin-3. These data suggest that Peg IFNa 2a leads to increased p53 production while nutlin-3 results in reduced p53 degradation thereby providing a means of up regulating wild type p53 in PV cells. It is anticipated that the completion of these studies will provide a rationale for a phase I clinical trial evaluating the effects of nutlin3 alone or in combination with low doses of IFNα for the treatment of MPN patients.