Somatic Mutations in the Wiskott-Aldrich Syndrome Protein Family Member 3 (WASF3) Are Associated with Poor Prognosis in Myeloid Malignancies

Myelodysplastic syndromes (MDS) encompass a clinically and pathologically heterogeneous group of blood cancers characterized by dysplasia, cytopenias, and a significant risk for transformation to acute myeloid leukemia (AML). The understanding of MDS biology is the key to deciphering disease pathogenesis and offers the ability to identify specific pathways that may be important for diagnosis, prognosis, and most importantly for designing new targets amenable to therapies. Genetic factors specifically molecular mutations in genes involved in many important cellular processes such as epigenetics, signaling, and RNA splicing have been identified in MDS. These mutations affect disease pathogenesis and are prognostic. Whole exome sequencing (WES) technologies have advanced genomic discoveries in MDS. We applied WES on 2 ug of DNA of 14 patients (pts) (MDS=11 and MF=3). Non clonal CD3⁺ cells served as germline control. 20 million reads were run on an Illumina HiSeq2000 sequencer. A stringent bio-informatic algorithm filtered all variants based on a variation score (>=30) and a coverage (30X). Tumor nucleotide variation analysis was performed for each pair (tumor vs. germline), where only variants unique to the tumor were retained after the exclusion of SNPs. Using this tool, we discovered the first recurrent somatic mutation (R178H) in a Wiskott-Aldrich syndrome protein (WASP) family of genes specifically Wiskott-Aldrich syndrome protein family member 3 (WASF3) also known as WAVE-3 in a pt with MDS. Further analysis by Sanger sequencing on a larger cohort of pts with myeloid neoplasms [n=241; (MDS= RARS/RCMD: n=30; RAEB1/2: n=49); primary AML (n=42), CMML1/2 (n=30), MPN (MF, ET, PV, CML: n=90)] showed WASF3 mutations in 5/49 (10%) higher risk MDS and 2/25 (8%) JAK2^WT PV pts. MDS pts with WASF3 mutations (WASF3^MUT) had higher peripheral (3.7% vs. 0.5%, P = .002) and bone marrow blasts % (4.7% vs. 1.4%, P = .005), and lower PLT count (71 vs. 191 k/uL, P = .005) compared to WASF3^WT pts. Molecular analysis for a panel of genes known to be mutated in MDS and MPN like ASXL1, U2AF1, SRSF2, SF3B1 and IDH1/2 showed that 2/7 WASF3^MUT carried also SRSF2 mutations (P95R/L). WASF3 mutations had a negative impact on the survival outcomes. Indeed WASF3^MUT pts had a shorter overall survival (OS^Mut 10.5 mos vs. OS^WT 29.1 mos, P=.01) and progression free survival (PFS^MUT 5.1 mos vs. 15.9 PFS^WT mos, P = .03) compared to WASF3^WT pts. WASF3^MUT MDS pts also carried worse OS compared to pts with MDS and normal karyotype (14 mos vs. 41.8 mos, P =.019) and SF3B1^MUT which are known to carry a good prognostic outcome in MDS (14 mos vs. 54.1 mos, P =.003). Western blotting showed increased WASF3 protein expression in the index case compared to WASF3^WT pts (n=2). We performed microarray analysis on 6 higher risk MDS and 6 MPN pts, with one pt harboring the WASF3 mutation. Illumina Human HT-12 v4 expression beadchips were used to estimate mRNA expression levels. The WASF3^R178H mutant pt had a higher WASF3 mRNA expression compared to 2 matched WT pts (Fold Change [FC] =1.44, P=.16). Further, protein complex pathways, specifically, Arp2/3, which has been shown to interact with WASF3 and other WASF family members also showed similar patterns of expression. ARP2 (ACTR2) and ARP3 (ACTR3) were both elevated in WASF3^MUT vs. WT (ACTR2: FC=1.79, P=.029; ACTR3: FC=1.60, P=.144). We also found that WAS, which encodes the WASP protein, was expressed in our MDS samples and shows elevated expression in WASF3^MUT vs. WT (FC=1.58, P = .05). This suggests that other members of the WASP family of proteins may be involved in MDS pathogenesis. WASP was originally found to be mutated in Wiskott Aldrich syndrome (WAS), an X-linked recessive immunodeficiency disorder. Mutations in this gene are important in actin polymerization, a process essential for cytokinesis, chromosome segregation, endocytosis, migration, and formation of filopodia and lamellipodia. In prostate and breast cancers, overexpression of WASF3 has been associated with increased disease metastasis and tumor invasion and treatment resistance. In conclusion, WASF3 somatic mutations are found in MDS and MPN pts and associated with increased mRNA/ protein expression. The presence of WASF3 mutations is associated with poor survival in myeloid malignancies. Understanding the mechanism by which WASF3 gene act may provide new targeted therapeutic options for myeloid cancers which harbor this mutation.