The HDAC INHIBITOR ITF2357 MODULATES KEY HEMATOPOIETIC GENES in JAK2^V617F CELLS From MYELOPROLIFERATIVE Neoplasm PATIENTS

Abstract 797

We have previously shown that the pan-HDAC inhibitor ITF2357 has strong cytotoxic activity against cells from patients with myeloproliferative neoplasms (MPN) bearing JAK2 mutation at position 617. Indeed ITF2357 inhibited colony growth of JAK2^V617F positive cells at doses 5–10 fold lower than those required to block JAK2 wild type cells. We have therefore investigated here the molecular mechanism of this effect. Three cell lines homozygotes (HEL, UKE1) or heterozygotes (SET2) for the JAK2^V617F mutation were used along with cell lines bearing JAK2 wild type (K562 and KG1). We confirmed the higher sensitivity of mutated with respect to unmutated cell lines in colony formation assay (mean IC₅₀ 42 nM versus 179 nM) and alamar blue assay (mean IC₅₀ 84 nM vs 325 nM, respectively). In proliferation assays measuring number of live and dead cells at different time points, we observed that 100 nM ITF2357 blocked the proliferation of both JAK2 mutated and unmutated cell lines to a similar extent, with mean inhibition of 31–69% at 72 hours, but induced apoptosis more efficiently in JAK2 mutated (mean 34%) versus unmutated cells (mean 2%). By cell cycle analysis we could show a block in G1 phase of cell cycle in JAK2^V617F cells treated with 100 nM drug. In order to unravel the mechanism of specific inhibition of JAK2 mutated cells by ITF2357, we first investigated expression of HDAC isoforms in the different cell lines. We could detect HDAC1, HDAC2 and HDAC3 proteins in Western blots but these were not differentially expressed in a panel of 3 JAK2 mutated and 3 wild type cell lines. We then set out to analyse the molecular mechanism of action of ITF2357 by global gene expression analysis. Using the Rank Product method with a false positive prediction (pfp) of 0.05 and a 2 fold change cut off parameters, we observed 716 and 863 genes modulated at 6 hours by 250 nM ITF2357 in HEL and UKE-1 cell lines, respectively; 293 of these, (179 up- and 114 down-regulated), were common between both cell lines and 10 were subsequently validated by Q-RT-PCR. Among differentially expressed genes, a number are known to play an important role in the control of proliferation and /or apoptosis, most notably APAF1, BCL2L11, CCNG2, NFKB2, MXD1 and TP53INP1, while additional 6 genes (C-MYB, A-MYB, TAL1, NFE2, MLF1, NOTCH2) are involved in the control of hematopoietic differentiation. Of particular interest is NFE2, which was down modulated 2.7 fold by ITF2357 at 6 hours at the RNA level and by about 2 fold at 24 hours at the protein level. NFE2 has been reported to be hyperexpressed in JAK2^V617 MPN patients. We also showed that ITF2357 downmodulated NFE2 expression 2 fold also in CD34⁺ cells purified from these patients. Given the accepted role of NFE2 in the control of erythroid progenitor cell proliferation and differentiation, and its enhanced expression in MPN patients, our data suggest that NFE2 down-regulation by ITF2357 may at least partially explain the drug effect on growth of MPN progenitor cells. The regulation of NFE2 expression and that of other hematopoietic transcription factors and regulatory proteins in response to ITF2357 is under investigation in our laboratory and data will be presented.