Plitidepsin Inhibits the Growth of Cells Harboring JAK2V617F Mutation.

Abstract 3907

Poster Board III-843

Plitidepsin (Aplidin^®) is a novel cyclic depsipeptide derived from the marine tunicate Aplidium albicans, currently obtained by chemical synthesis, that is under Phase II clinical development. Plitidepsin is effective against a large panel of tumor cells, and although precise action mechanisms have still to be ascertained the drug induces an oxidative stress, activation of Rac1 GTPase and inhibition of protein phosphatases, overall leading to sustained activation of JNK and p38MAPK. In a previous report (Verrucci M et al, ASH 2008, 2787A) we evaluated Plitidepsin activity in the GATA-1low murine model of myelofibrosis. Plitidepsin corrected thrombocytopenia of myelofibrotic mice, reduced the frequency of megakaryocytes (Mk) and normalized angiogenesis in the bone marrow, and prevented extramedullary hematopoiesis. In the present study, we assessed the effects of Plitidepsin on cell lines harboring homozygous (HEL and UKE-1, a gift of W. Fiedler) or heterozygous (SET2) JAK2V617F mutation and on cells from patients (pts) with myeloproliferative neoplasms (MPN). In a short-term (3 days) proliferation assay we found that Plitidepsin prevented cell growth with IC50 values of 1.0±0.3 nM for HEL, 0.5±0.03 nM for UKE-1, and 0.8±0.02 nM for SET2, that were all lower than 1.5±0.1 nM for the BCR/ABL mutated K562 cell line (P<.001 in case of UKE-1 cells). Also Ba/F3 cells transduced with the V617F allele (a gift of R. Skoda) were found more sensitive to Plitidepsin (IC50= 0.03±0.01 nM) than the wild-type counterpart (IC50= 0.4±0.03 nM; P<0.02). Similar results were obtained using a 14-day clonogenic assay in agar cultures. These data indicated that Plitidepsin was active at very low nanomolar concentrations against cell lines harboring JAK2V617F mutation. We then evaluated the effects of Plitidepsin on the growth of BFU-E, CFU-GM and CFU-Mk from MPN pts; all five Polycythemia Vera (PV) and 4/5 Primary Myelofibrosis (PMF) pts analyzed were JAK2V617F mutated. As shown in the Table, PMF pts presented significantly lower IC50 value than controls (Ctrl; P<.002) for all type of clonogenic progenitors; cells from PMF pts resulted also significantly more sensitive to Plitidepsin than those from PV patients (P<.02), while the difference between PV and Ctrl did not reach the significance level. To evaluate whether Plitidepsin also affected the latest stages of differentiation and maturation of MKs, that is the most overtly affected cell lineage in PMF, we added Plitidepsin on day +7 of a two-stage liquid culture system initiated with CD34+ cells purified from the PB of PMF patients; the generation of CD61+ Mks was measured 5 days later by FACS analysis. However, we found that the number of CD61+ cells was no different between cultures containing or not Plitidepsin, overall suggesting that the drug mainly affected early proliferation of Mk progenitors rather than influencing their differentiation. We then performed single colony genotyping to quantify the proportion of hematopoietic colonies harboring the JAK2V617F mutation which grew in growth factor-supplemented methylcellulose cultures initiated with purified CD34+ cells from PMF patients in the presence of 1 nM Plitidepsin. Initial data in 3 pts were available; in one, the proportion of JAK2-mutated BFU-E colonies decreased from 51% to 27% while no changes were observed in the other two pts. Finally, since a correlation between levels of p27(Kip1) and the response of tumor cells to Plitidepsin has been described, we measured p27 levels in different cell lines after exposure to Plitidepsin. We observed that p27 mRNA levels increased 15-fold and 30-fold in UKE1 and HEL cells, respectively, compared to K562 cells after 24 hr with 1nM Plitidepsin; such an increase was mirrored by a protein content 1.9- to 3.5-fold greater than baseline in UKE-1 cells at 1 and 10 nM Plitidepsin, suggesting that JAK2V617F mutated cells responded to the drug by modulating their p27 levels. Collectively, we provided evidence that Plitidepsin has in-vitro activity against MPN cells, particularly from PMF pts. These results, as well as those which were previously described in the GATA1low murine model, provided the rationale for a clinical trial in patients with myelofibrosis that is being developed within the Myeloproliferative Disorders Research Consortium (MPD-MRC).