Hypermethylation of SH2-Containing Phosphatase-1 (SHP-1) and Suppressor of Cytokine Signaling-1 and -3 (SOCS-1 and -3) in Philadelphia Negative Chronic Myeloproliferative Disorders (Ph-CMPD).

A fraction of Ph- CMPD is characterized by JAK2F617F mutation leading to constitutive JAK-STAT activation. The negative regulators of cytokine signaling SHP-1, SOCS-1 and SOCS-3 have a crucial function in the negative regulation of JAK2 activation/phosphorylation in response to EPO, G-CSF and a subset of cytokines. SHP-1, SOCS-1 and SOCS-3 may be silenced by aberrant DNA methylation in human malignancies. Here we tested chronic phase Ph- CMPD and acute myeloid leukaemia (AML) from Ph- CMPD for aberrant DNA methylation of SHP-1, SOCS-1 and SOCS-3. The study was based on: i) 85 Ph- CMPD, including 35 essential thrombocythemia (ET), 20 polycythemia vera (PV), 15 idiopathic myelofibrosis (IMF), 6 chronic myelomonocytic leukemia (CMML) and 9 Ph- chronic myeloid leukemia (Ph- CML); and on ii) 19 AML from Ph- CMPD, including 4 AML from PV, 10 AML from ET and 5 AML from IMF. Cases were analysed for SHP-1, SOCS-1 and SOCS-3 aberrant methylation by methylation-specific PCR and for JAK2V617F mutation by allele specific PCR. For comparison, 10 samples of normal bone marrow hematopoietic cells were also investigated. Among Ph- CMPD, methylation of SHP-1 occurred in 4/20 (20%) PV and 4/35 (11%) ET, while it was absent in IMF (0/15), CMML (0/6) and Ph- CML (0/9). Methylation of SOCS-1 occurred in 5/20 (25%) PV, 5/35 (14%) ET, 2/15 (13%) IMF and in 1/9 (11%) Ph- CML while it was absent in CMML (0/6). Methylation of SOCS-3 occurred in 11/20 (55%) PV, 13/35 (37%) ET, 4/15 (26%) IMF, 3/6 (50%) CMML and 3/9 (33%) Ph- CML. JAK2V617F mutation was detected in 47/85 (55%) Ph-CMPD, including 17/20 (85%) PV, 18/35 (51%) ET, 12/15 (80%) IMF, 0/6 CMML and 0/9 Ph- CML. SHP-1, SOCS-1 and SOCS-3 methylation was analysed according to JAK2 mutation status in PV, ET and IMF. In this group of patients, SHP-1, SOCS-1 and SOCS-3 methylation occurred in both JAK2 mutated cases (6/47, 13% for SHP-1; 10/47, 21% for SOCS-1 and 18/47, 38% for SOCS-3) and germline cases (2/38, 5% for SHP-1; 2/38, 5% for SOCS-1 and 10/38, 26% for SOCS-3). By combining the results of SHP-1, SOCS-1 and SOCS-3 methylation status, 21/47 (45%) JAK2 mutated cases carried SHP-1 and/or SOCS-1 and/or SOCS-3 methylation as opposed to 12/38 (31%) germline cases. This pattern of SHP-1 and SOCS-1 methylation was conserved also when the analysis was restricted to PV, ET and IMF each as a single group and after stratification for JAK2V617F mutation. Among AML from Ph- CMPD, methylation of SHP-1 occurred in 1/10 (10%) AML from ET, while it was absent in AML from PV and AML from IMF. Methylation of SOCS-1 occurred in 1/4 (25%) AML from PV and 1/10 (10%) AML from ET. One ET patient acquired SHP-1 methylation at transformation to AML. All normal bone marrow samples (n=10) scored negative for SHP-1, SOCS-1 and SOCS-3 methylation. The implications of these results are threefold. First, inactivation by aberrant methylation of SHP-1, SOCS-1 and SOCS-3 is involved in the pathogenesis of Ph- CMPD and is selectively associated with neoplastic hemopiesis. Second, among PV, ET and IMF, methylation of SHP-1, SOCS-1 and SOCS-3 may occur in both JAK2V617F positive and negative cases. Third, the low prevalence of SHP-1 and SOCS-1 methylation in AML from Ph- CMPD suggests that SHP-1 and SOCS-1 silencing is not involved in leukemic transformation.