BCL-2 Family Of Genes Is a Key Regulator In The Pathogenesis Of SF3B1 Mutant and Wild Type MDS With Ring Sideroblasts and Represents a Novel Drug Target In This Disease
Authors: Visconte Valeria , Mahfouz Reda Z , Tabarroki Ali , Hasrouni Edy , Rogers Heesun J. , Saunthararajah Yogen , Hill Brian T. , Kalaycio Matt , Sekeres Mikkael A. , Barnard John , Tiu Ramon V. 
Published: October 2019

Abstract
Myelodysplastic syndromes (MDS) are a group of neoplastic diseases characterized by impairment in hematopoietic cell differentiation which leads to disease related consequences like cytopenias and cellular dysplasia. Mutations in splicing factor 3b subunit 1 (SF3B1) are the most frequently identified genetic abnormalities in refractory anemia with ring sideroblasts (RARS) and RARS with thrombocytosis (RARS-T). However, the mechanisms whereby cells acquire clonal dominance and evolution, cytopenias and impairment in differentiation remain unknown. To understand the role of SF3B1 mutations in the pathogenesis of RARS/-T, we analyzed the transcriptome of SF3B1 mutant (MUT) and wild type (WT) RARS/-T patients (pts). RNA was isolated from bone marrow of healthy subjects (n=3), SF3B1 MUT (n=3) and WT (n=3) RARS/-T, and other MDS (n=5). cDNA was made from RNA (1.5-3ug) and fragmented for library preparation. RNA-sequencing was performed on 20 million sequence reads on an Illumina HiSeq2000 and aligned to the human genome 19. RNA-splicing patterns were analyzed by a published bioinformatics algorithm at three levels (exon usage, gene expression, and pathway analysis) (Visconte V; Blood. 2012). Differential exon usage identified 271 and 71 genes with at least 1 exon alternatively spliced in SF3B1 MUT, WT, and healthy subjects (P<.05). SF3B1 preferentially affected the 3’-end regions. The chromosomes (chrs) with the highest number of genes displaying differential exon usage were chr1:30, chr17:25, chr7:22, and chrs19: 20, chr11:17, chrs:6 and 2:15 following by chrs:8, 5, and 16: 11. No significant differences in mRNA levels of genes relevant to MDS (U2AF1, TET2, DNMT3A, ASXL1, EZH2, TP53, RUNX1, ETV6, CBL, IDH1/2, and N/KRAS) were found leading us to think that SF3B1 affects alternative targets. Using GSEA/ MiSigDB v3.0 analysis we found a specific molecular signature: out of 27 mitochondrial gene sets, one gene set differed between SF3B1 MUT and WT RARS pts (P=.01). This gene set contains 11 genes involved in mitochondrial membrane/organization/biogenesis network. BCL-2 family genes were highly expressed: BCL-2 (mean: 22.8 vs 68.1 vs 15.2), BNIP3 (mean: 50.1 vs 55.3 vs 31.9) and BCL2-L1 (mean: 3221 vs 2905 vs 595) in SF3B1 MUT vs WT pts and vs healthy subjects. Changes in BCL2 mRNA levels were also confirmed by western blotting in MUT (n=2) vs WT (n=2). In myeloid cell lineage, the relationship between BCL-2 protein levels is inversely related to the maturation stage with myeloblasts and promyelocytes having higher BCL-2 while more differentiated cells like metamyelocytes and polymorphonuclear cells have less to no BCL-2 expression. Contrary to prevailing knowledge and consistent with what would be expected in explaining MDS persistence by development of clonal advantage, in our hands RARS/-T pts were not characterized by increased cleaved PARP and cellular stress as evidenced by no difference in reactive oxygen species between SF3B1 MUT and WT (93.83% ± 7.94 vs 83.28% ± 13.82; n=3 vs 4) further supported by the overexpression of an antiapoptotic gene BCL-2. We previously presented the biological rationale of the different clinical outcomes between SF3B1 MUT and WT (Visconte V; Blood (ASH Annual Meeting Abstracts), Nov 2012;120: 922). SF3B1 WT carries more high risk chromosomal defects and higher g-H2AX levels indicative of greater DNA damage. Higher g-H2AX and genomic instability are associated with BCL-2 over-expressing lymphoid malignancies (Laulier C; Cancer Res. 2011) which may explain the higher number of chromosomal defects and propensity for leukemia transformation in WT pts. SF3B1 WT tends to respond less to hypomethylating therapy (HMT) vs MUT (n=2 vs 2). Cells presenting an immature hematopoietic phenotype have lower response rate to HMT in-vitro. Indeed, differences in erythroid maturation were found in SF3B1 MUT vs WT RARS. SF3B1 MUT showed higher mRNA levels of GATA-1, a marker of hematopoietic maturation (FC: 4.0), and a down-regulation (FC: 0.48) of GATA-2, an early hematopoietic marker vs WT. In conclusion, BCL-2 is a key regulator in the pathogenesis of RARS/-T and may provide clonal advantage. Differences in BCL-2 expression can explain different survival outcomes in SF3B1 MUT vs WT. BCL-2 may be a novel pathway in the pathogenesis of RARS/-T opening the possibility that a BCL2- antagonist either with/without a spliceosome inhibitor will be an ideal therapeutic approach in RARS/-T.

Disclosures:
No relevant conflicts of interest to declare.