Characterization of Targets of Plitidepsin In JAK2V617F-Mutated Cells From Myeloproliferative Neoplasms

In a previous study (Verrucci M et al, J. Cell Physiol, 2010, Epub May10) we reported that the cyclic depsipeptide Aplidin®, a potent cytotoxic agent currently in phase II/III clinical trials for solid and hematologic neoplasia, displayed activity in a murine model of myelofibrosis, the GATA1-low mice. In fact, Aplidin improved the proliferation of Gata1-low hematopoietic cells, corrected abnormal microvessel density and reduced bone marrow fibrosis. These effects were largely attributed to improved maturation of megakaryocytes, as suggested by the increased platelet count in thrombocytopenic Gata1-low mice. It has been previously shown that a low expression of p27 in tumor cells might correlate with their response to Aplidin; of interest, GATA1-low megakaryocytes expressed reduced levels of p27. Overall, these results suggested that Aplidin could have the potential to alter the course of myelofibrosis-like disease in Gata1-low mice and could be useful for the treatment of myelofibrosis.

to evaluate activity and targets of Aplidin in cellular models of myeloproliferative neoplasms (MPN).

We measured the effect of Aplidin on the proliferation of JAK2V617F-mutated cell lines (UKE-1, HEL, SET2) and of primary cells from MPN patients in liquid cultures and semisolid medium, the rate of apoptosis using Annexin V flow cytometry analysis, and the cell cycle by propidium iodide staining. Expression of mRNA was quantified by real-time PCR, while protein level was measured by western blotting. Gene expression profiling was accomplished with Agilent Whole Human Genome Oligo Microarrays (44K).

Aplidin reduced the proliferation of all human V617F-mutated cell lines in the low nanomolar range, with IC50 from 0.5+/−0.03nM for UKE-1 to 1.5+/−0.05nM for HEL cells. Aplidin increased the proportion of cells in the G1/G0 phase of cell cycle, up to a mean of 80+/−5% from 60+/−3% in control cells (P<0.01). Also the proportion of apoptotic cells dose-dependently increased from 20+/−3% in control cultures to 50+/−6% at 10 nM in UKE-1 cells. After 24 h of incubation with Aplidin, the level of p27 mRNA was dose-dependently increased in all cell lines evaluated, while the BCR/ABL mutated K562 cells, whose proliferative rate was inhibited at higher Aplidin concentration, did not show significant changes of p27 mRNA. By western blotting analysis, level of p27 was overtly increased in UKE-1 cells treated with 5 and 10 nM Aplidin for 24h, and at the highest concentration we also observed reduction of phosphorylated STAT5. On the other hand, there was no change in JAK2, pJAK2, STAT5, STAT3, pSTAT3, Akt, pAkt. We then measured the level of p27 mRNA in peripheral blood cells obtained from patients with polycythemia vera (PV) or primary myelofibrosis (PMF). We found that PMF patients had significantly reduced p27 mRNA and protein level compared to controls and PV patients (P<.001 for both). After incubation with 1nM Aplidin there was a 2.5+/−2.0-fold increase in the p27 mRNA level and protein in cells of PMF patients while it was substantially unaffected in controls or PV patients. Gene expression analysis was performed in SET2 and UKE-1 cells that had been treated with 3nM of Aplidin for 24 hr. Aplidin treatment of SET2 cell line resulted in the up-regulation (UR) of 268 probsets and down-regulation (DR) of 247 (in total, about 1.5% of 40,961 genes called as “present”); corresponding figures in UKE-1 cells were 261 for UR (0.6%) and 364 (0.9%) for DR genes. MAPK signalling, TGF-beta signaling pathway, Cytokine-cytokine receptor interaction, cell adhesion molecules (CAMs), neuroactive ligand-receptor interaction, T cell receptor signaling, calcium signaling pathway, and regulation of actin cytoskeleton were the most involved pathways.

Current results suggest that Aplidin has growth inhibitory activity and induces apoptosis in MPN cells. Response to Aplidin involves increasing p27 level, similar to findings in myelofibrotic GATA1-low mice; furthermore, we have identified low p27 expression as a characteristic of PMF cells compared to normal and PV cells. Finally, gene expression analysis allowed to identify a set of genes and involved pathways undergoing differential regulation in response to Aplidin. These information could be of value also for analyzing the response to Aplidin of PMF patients enrolled in an ongoing Phase II clinical trial.

Aracil:PharmaMar: Employment. Vannucchi:PharmaMar: Research Funding.