Triple Negative (JAK2 exon 12 /14 and MPL wild type) Myelofibrosis Have Higher Expression Of CDC25A and Greater Sensitivity To CDC25A Inhibition Compared To JAK2 Mutant Cases

Pharmacologic therapies that target the JAK-STAT pathway are clinically used to alleviate splenomegaly and disease-related constitutional symptoms in MF. However, it is clear that some patients develop intolerance or resistant to this therapy. Furthermore, there are MF related complications especially cytopenias that are not alleviated by these therapies. Therefore, alternative and complementarytherapies are warranted in the management of MF. We hypothesized that other pathways downstream of the JAK-STAT signaling pathway can play a role in the pathophysiology of MF. We used whole exome (WES) and RNA sequencing technologies to interrogate new molecular markers and pathways which can serve as novel targets for this disease. In 4 MF patients [JAK2 mutant (MUT) =2, and wild type (WT) =2], WES was performed using the Illumina platform. All of the variants were filtered based on PHRED score (>=30) with coverage was set at 30X. Analysis of data in JAK2/MPL WT patients demonstrated the presence of 263 candidate genes. After clarifying the status of tumor nucleotide variants in each gene compared to germline (CD3+) fraction, 7 genes (RBL1, ADSS, ZNF717,MUC4, TUBB4Q and CDC25A) were selected for further somatic confirmation by direct sequencing. Among these genes, only alteration in CDC25A, a regulator of cyclinE/cdk2 (cyclin-dependent kinase-2) and cyclinA/cdk2 kinase, was confirmed to be somatic. This genetic change was previously reported as somatic by WES in lung cancer although not confirmed by direct sequencing (Bartkova et al, Nature, 2005, Apr 14; 434 (7035):864-70). Based on these observations and since CDC25A acts as a downstream effector of JAK-STAT signaling, we hypothesized that, CDC25A phosphatase, may be a driver in MF pathogenesis. The transcriptome of two patients, one MUT and one WT for JAK2 was then analyzed. RNA was isolated from bone marrow (BM) cells of healthy individuals (HI) (N=3). cDNA was made from 1.5-3 ug of RNA and fragmented for library preparation. RNA-sequencing was performed on 20 million sequence reads. Paired-end 90 base pair reads were generated on an Illumina HiSeq2000 sequencer and aligned to the human genome 19. RNA-splicing patterns were analyzed by a bioinformatics algorithm and gene expression analysis was carried out using GSEA (Visconte V; Blood. 2012). By using FDR<0.2, 11,460 genes were expressed. Further analysis demonstrated, CDC25A was over-expressed in both cases compared to HI but interestingly more highly expressed in JAK2 WT cases [fold change (FC): 0.39].This finding was validated by performing Western blotting and immunohistochemistry (IHC). To evaluate the protein expression of CDC25A in 10 MF (JAK2 MUT=5, JAK WT=5) patients and 5 HI, western blotting was performed; and higher expression in WT and MUT compared to HI were observed. Furthermore, its expression was also higher in WT compared to MUT cases. This is in contrast to a previous report by Gautier EF et al (Blood, 2012 Feb 2;119 (5):1190-9) where CDC25A expression was less in JAK2 WT cases compared to MUT cases. IHC was performed to confirm the difference of expression level of CDC25A in JAK2 MUT and WT bone marrow samples (N=8). IHC showed that JAK2 WT samples had many positive megakaryocytes stained with CDC25A antibody (>80%) while JAK2 MUT samples had only a few positive megakaryocytes (<20%). To test the feasibility of targeting this pathway in patients with MF and to assess for differential response between JAK2 MUT and WT cases, a potent cell permeable 7-substituted quinolinedione derivedCDC25 phosphatase inhibitor (NSC663284) was tested in JAK2 MUT (N=2) vs WT (N=1). Cell proliferation was determined by Trypan Blue and MTT assay after cell exposure to different concentrations of the inhibitor [3, 5, 7, 10 and 30uM] in 24 hours observation. NSC663284 induced higher dose-dependent cell growth inhibition in JAK2 WT compared to MUT cases (% of viable cells in WT vs MUT using previously mentioned concentrations, 3 uM= 98% vs 86%, 5 uM= 93% VS 77%, 7 uM= 88% vs 65%, 10 uM= 71% vs 43% and 30 uM= 25% vs 61%; p=0.01).In conclusion, CDC25A is more highly expressed in patients with wild type JAK2 compared to the mutant counterpart and primary cells from WT JAK2 patients demonstrate higher sensitivity to CDC25A inhibition, warranting further clinical testing of this therapeutic strategy.