Inhibition of AKT Signaling Potently Inhibits Growth of MPL-Mutant Cells in the Myeloproliferative Neoplasms.

Abstract 1896

Poster Board I-919

Somatic mutations in JAK2 and MPL are associated with BCR-ABL negative myeloproliferative neoplasms (MPNs) including polycythemia vera (PV), essential thrombocythemia (ET), and primary myelofibrosis (PMF). Mutations in these genes have also been identified in rare cases of acute megakaryoblastic leukemia (AMKL). To investigate the role of activated JAK2 and MPL signaling in aberrant megakaryopoiesis associated with MPN and AMKL, we overexpressed JAK2 V617F or MPL W515L in primary murine hematopoietic progenitor cells. We found that expression of mutant MPL, but not JAK2, led to a dramatic increase in the size and number of megakaryocyte colonies. Overexpression of constitutively activated alleles of STAT3 or STAT5 did not, however, enhance megakaryocyte colony formation. Moreover, knock-down of STAT3 or STAT5 failed to inhibit the growth of JAK2 V617F or MPL W515L transduced clones of the G1ME megakaryocytic cell line. Together, these observations argue against the simple model of JAK2 and MPL mutants conferring cytokine independent growth and excessive proliferation solely by inducing STAT signaling. Since TPO is known to activate additional signaling pathways, including MAPK/ERK and PI3K/AKT, we hypothesized that alterations in pathways other than JAK/STAT contribute to MPNs. To determine which of these signaling pathways are involved, we cultured JAK2 V617F and MPL W515L expressing clones of the G1ME and 6133 megakaryocytic cell lines with a panel of small molecule kinase inhibitors. We discovered that inhibition of the PI3K/AKT and MAPK/JNK signaling pathways with triciribine and SP600125 respectively, dramatically suppressed growth of these cells by inducing G1 arrest and apoptosis. Of note, the inhibitory effect of these two small molecules was more pronounced than that caused by JAK2 inhibitor II. Inhibitors of Ras, ERK, Src family tyrosine kinase, and PKC all failed to significantly inhibit proliferation of the JAK2 or MPL mutant expressing cells. Since triciribine showed the strongest activity against the mutant cell lines, we next assayed its activity on a mouse model of MPL-induced AMKL, on primary murine cells expressing MPL W515L and on human MPN specimens. Twice daily injection with 0.1mg/kg of triciribine significantly delayed AMKL development in mice transplanted with 6133/MPL mutant cells. Triciribine also repressed CFU-Mk formation from MPL W515L transduced mouse bone marrow as well as BFU-E formation from human PV patient samples. These findings demonstrate that AKT is a potential target for therapy of human myeloproliferative neoplasms.