Tifab, a Novel Candidate Gene in Deletion Chromosome 5q, Contributes to Deregulation of NF-κB Signaling in MDS/AML.
Authors: Varney Melinda , Jerez Andres , Fang Jing , Miller David , Bolanos Lyndsey , Karsan Aly , Maciejewski Jaroslaw P. , Starczynowski Daniel T 
Published: October 2019

Abstract 2812

Myelodysplastic syndromes (MDS) are hematologic disorders defined by blood cytopenias due to ineffective hematopoiesis, altered cytogenetics, and predisposition to acute myeloid leukemia (AML). The most common cytogenetic alteration in de novo and treatment-related MDS is deletion of chromosome 5q (del(5q)). There are two commonly deleted regions (CDR) mapped to chr 5q, however the gene(s) in these regions responsible for the manifestation of del(5q) MDS are not clearly defined. A search of annotated genes revealed that TRAF-interacting protein with forkhead-associated domain B (TIFAB), a known inhibitor of TRAF6 and a novel gene identified by an in silico search for TIFA-related genes, resides within the proximal CDR on band 5q31.1. We first determined whether TIFAB is expressed in normal hematopoietic stem/progenitor cell (HSPC) by qRT-PCR. We find that expression of TIFAB is enriched in human CD34+/CD38+ and mouse lineage-/cKit+ progenitors as compared to more differentiated populations, suggesting that it plays a role in normal HSPC function. To determine whether TIFAB is implicated in del(5q) MDS, we measured TIFAB expression in del(5q) MDS patients. According to a microarray analysis, TIFAB mRNA was significantly lower in CD34+cells isolated from MDS patients with del(5q) as compared with cells from MDS patients diploid at chr 5q (Pellagatti, et al., 2006). In an independent subset of patients, we confirmed that TIFAB expression was lower in marrow cells isolated from del(5q) MDS patients. Therefore, we hypothesize that TIFAB loss results in hematopoietic defects contributing to del(5q) MDS. To determine whether deletion of TIFAB affects hematopoiesis, we used lentiviral shRNAs to knockdown TIFAB mRNA in human cord blood CD34+ cells. To mimic haploinsufficiency of TIFAB in del(5q) MDS, we selected shRNAs that result in ∼50% knockdown of TIFAB mRNA and protein. Knockdown of TIFAB in human CD34+ cells results in increased survival, a competitive growth advantage, and altered hematopoietic progenitor function. Conversely, overexpression of TIFAB in human leukemia cell lines (THP1 and HL60) results in increased basal apoptosis, delayed G1/S-phase cell cycle progression, and impaired leukemic progenitor function in methylcellulose. Since TIFAB is predicted to regulate TRAF6, we examined the role of TIFAB on TRAF6 signaling. TIFAB suppressed TRAF6 lysine (K)-63 autoubiquitination (a measure of TRAF6 activity), and decreased total TRAF6 protein levels, suggesting that TIFAB may simultaneously inhibit TRAF6 function and protein expression. Consistent with this finding, TIFAB suppressed lipopolysaccharide-induced (TRAF6-dependent) NF-kB activation, but not TNF-induced (TRAF6-independent) NF-kB activation. TIFAB-mediated inhibition of TRAF6 also coincided with reduced phospho-IKK-beta (a measure of NF-kB activation) in leukemic cells. In summary, we have identified TIFAB as a novel del(5q) MDS/AML gene involved in regulating HSPC survival, progenitor function, and cell cycle. We propose that haploinsufficiency of TIFAB results in malignant clonal cell expansion and may contribute to the MDS/AML phenotype as a consequence of increased TRAF6-mediated activation of NF-kB.

Disclosures:

Maciejewski: NIH: Research Funding; Aplastic Anemia&MDS International Foundation: Research Funding.