PF654 EPIGENETIC ALTERATION OF CSK BINDING PROTEIN(CBP) IN MYELOPROLIFERATIVE NEOPLASMS COMPLEMENTS THE JAK2V617F MUTATION

Abstract

Background:Csk binding protein (CBP), a ubiquitously expressed transmembrane protein, functions as a suppressor of Src‐mediated tumor progression by promoting the inactivation of Src. CBP is widely reported to act as a scaffold in the Csk‐mediated negative regulationof Src family kinases (SFKs). First, CBP is phosphorylated by SFKs, then it associates withC‐terminal Src kinase (Csk) through a specific site (Tyr‐317 in humans) and brings it into proximity with membrane‐associated SFKs. After that, Csk phosphorylates the C‐terminal negative regulatory tyrosine residue of SFKs, which suppresses their activation. SFKs are membrane‐associated non‐receptor protein tyrosine kinases that play pivotal roles in regulating various cellular processes including proliferation, differentiation, adhesion, migration, and survival. Thus, CBP plays the opposite role in various Csk‐mediated cellular processes and might be a significant target for Csk‐mediated tumors.Aims:To explore the role of CBP in myeloproliferative neoplasms complements the JAK2V617F mutation.Methods:Bone marrow of 78 JAK2/V617F mutation positive MPN patients were collected, including 18 of 78 idiopathic myelofibrosis, 32 of 78 polycythaemia vera and 28 of 78 essential thrombocythaemia. The mononucleated cells separated, RNA extracted and the CBP expression level detected using real‐time quantitative PCR and MSP. SPSS 23.0 used for data analysis.Results:We identified CBP as a functional tumor suppressor gene frequently methylated in myeloproliferative neoplasms complements the JAK2V617F mutation. We further uncovered CBP as one of the up regulated genes in JAK2/V617F mutation positive cell lines after pharmacologic demethylation with 5 aza 2’ deoxycytidine (Aza). Methylation of CBP was detected in several JAK2/V617F mutation positive cells. Aberrant methylation was further detected in 60% of various types of JAK2/V617F mutation positive MPN, but not in any normal PBMC sample, and is thus tumor specific. The levels of CBP mRNA were significantly lower in hypermethylated samples than unmethylated samples, suggesting CBP may be epigenetically inactivated in MPN patients. Aza treatment led to CBP promoter demethylation and transcriptional reactivation in silenced cell lines, indicating a methylation mediated silencing. Meanwhile, phosphorylated JAK2 and STAT3 were progressively reduced.Summary/Conclusion:Our results indicate that epigenetic inactivation of CBP contributes to the constitutiveactivation of JAK2/STAT signaling. CBP methylation is a frequent event in myeloproliferative neoplasms complements the JAK2V617F mutation. Restoration of CBP expression by HMA may contribute to clinical treatment for MPN patients.