Expression and Prognostic Impact of Candidate Suppressor Sequence-Specific Single-Stranded DNA Binding Protein (SSBP2) in AML Using Reverse Phase Proteins Arrays (RPPA).

Abstract

Sequence-specific single-stranded DNA binding protein 2 (SSBP2) is a candidate tumor suppressor gene, located at chromosome 5q13.3, that is thought to regulate hematopoietic growth and differentiation. Loss of SSPB2 expression is associated with deletions of chromosome 5 and replacement in cell lines induces partial differentiation and loss of clonogencity. Using a highly specific antibody, we studied the level of expression and prognostic relevance of SSBP2 protein expression (along with 51 other total and phospho proteins as described in ASH abstract #107, 2006) in leukemia-enriched samples from 423 patients (258 newly diagnosed, 47 primary refractory, 118 relapse 1, 2 or refractory) using high-throughput reverse phase protein array technology (RPPA). Levels of SSBP2 were lower, equal and higher than that of normal CD34+ cells in 2.7%, 58.7% and 11.6% of cases respectively. All 7 cases with low levels were female and 21/30 with high levels were male. The expression of SSBP2 was not equally distributed in the different FAB subtypes with the lowest median levels in M5 and the highest in M0, M1 and M2. Levels were significantly higher in favorable cytogenetics (t(8;21) and Inv (16) compared to intermediate (p=0.05) or unfavorable (p=0.008) cytogenetics. Median levels were lowest in cases with cytogenetic abnormalities on chromosome 5 or with an 11q23 abnormality. Levels were significantly higher in primary refractory and relapse samples compared to diagnostic samples. The levels of SSBP2 expression was strongly positively correlated with expression levels of anti-apoptotic proteins BCL2, Bcl-XL, caspase inhibitors SMAC, Survivin and XIAP, and inversely correlated with inactivated Bad-p112. Expression also was positively correlated with proliferation regulating proteins β-Catenin, Cyclin D, GSK3, Myc, P27, as well as VEGF receptor neuropilin and P53. Levels were strongly negatively correlated with activated signal transduction proteins (STP) including phosphorylated AKT (phosphorylated on threonine 308 or serine 473), pMEK, pERK2, pP38, pPKCα and as well as activated P70S6K. For outcome analysis martingale residuals were utilized to define an optimal cutpoint for dichotomization into groups with higher (n=130, 111 treated) vs. lower (n=128, 106 treated) SSBP2. Complete remission rates (58% vs. 63%, p=0.39), relapse rates (53% vs. 49%, P=0.59, and median remission duration (37 vs. 39 weeks, p=0.37) did not differ between patients with lower vs. higher SSBP2. However patients with higher SSPB2 levels had significantly longer median survival (40 vs. 31 weeks, p=0.015) compared to patients with lower SSBP2 levels. In summary, SSBP2 expression is heterogeneous in AML ranging from low in the poor and mixed prognosis subsets and high in the good prognosis t(8;21). Consistent with its demonstrated role in hematopoietic differentiation, the levels correlate negatively with expression of proliferation enhancing and STP. Nonetheless, the significance of positive correlation with antiapoptotic proteins is unclear at present. But the association with favorable longer overall survival suggests that therapeutic agents targeting the SSBP2 pathway function might have efficacy in AML.