Tifab, a Del(5q) MDS/AML Gene, Regulates USP15 Activity and p53

Interstitial deletion of a single copy of chromosome 5q is the most frequent cytogenetic alteration in Myelodysplastic Syndromes (MDS), which results in reduced dosage of numerous genes. TRAF-interacting protein with forkhead-associated domain B (TIFAB) resides within the proximal commonly-deleted region on band 5q31.1, and belongs to a family of forkhead-associated domain proteins. TIFAB is deleted in nearly all reported cases of del(5q) MDS and AML. As expected, TIFAB expression is significantly lower in CD34+ and BM mononuclear cells isolated from MDS patients with del(5q) as compared with cells from MDS patients diploid at chr 5q. Recently we have shown that hematopoietic-specific deletion of Tifab results in progressive BM and blood defects, including aberrant HSPC proportions, altered myeloid differentiation, and progressive cytopenia (Varney and Niederkorn et al., JEM 2015). Approximately 10% of mice transplanted with Tifab KO HSPCs develop a BM failure with neutrophil dysplasia and cytopenia. Gene expression analysis of Tifab KO lineage-Sca1+cKit+ (LSK) cells identified dysregulation of immune-related signatures, and hypersensitivity to Toll-like receptor stimulation. To investigate the molecular function of TIFAB, we performed a tandem-affinity tag purification and mass-spectrometry analysis of TIFAB complexes in a del(5q) AML cell line (HL60), and identified unique TIFAB-interacting proteins. The top interacting candidate was an ubiquitin-specific peptidase (USP), USP15. USPs play a major role in ubiquitin-dependent processes including DNA damage response signaling, protein degradation, and kinase activation. Specifically, USP15 has been shown to promote p53 degradation via deubiquitination and stabilization of its major negative regulator, MDM2. Through biochemical assays and a series of deletion mutants, we confirmed that TIFAB interacts with USP15. Moreover, we find that TIFAB enhances the deubiquitination of USP15 substrates, including MDM2 and histone 2B. To examine whether TIFAB directly regulates USP15 DUB activity, we performed in vitro deubiquitination assays in a cell-free system using fluorescent reporter di-ubiquitin substrates with purified USP15. We found that the addition of purified TIFAB increases the rate of USP15 catalytic activity on both lysine (K)48 and K63-linked di-ubiquitins in a dose-dependent manner. Collectively, these findings indicated that the USP15-TIFAB interaction leads to increased USP15 activity.

USP15 stabilizes MDM2 via its DUB function, and MDM2 is known to bind and inhibit p53. Moreover, p53 is implicated in the pathogenesis of del(5q) MDS: 1) BM cells from murine models and BM from del(5q) patients exhibit increased p53 activity, which is thought to contribute to ineffective hematopoiesis and anemia; and 2) del(5q) MDS patients often acquire concurrent TP53 mutations that result in rapid transformation to AML and poor treatment response. To examine the effects of TIFAB on p53 function, we examined p53 target genes in TIFAB-overexpressing and -deficient cells. Gene expression profiling and qRT-PCR analysis of Tifab KOHSPCs revealed significant upregulation of p53 regulatory genes. In contrast, overexpression of TIFAB in a p53-competent cell line reduced the expression of p53 target gene, p21. Collectively, our findings identify a novel role for TIFAB as an activating adapter of USP15 and mediator of p53 activity. These findings have important implications in the potential role of TIFAB and p53 signaling in the pathogenesis of del(5q) MDS and transformation to AML.