Mutations in the Splicosome Lead to Alternative Splicing of CSF3R in Promoting Myelodysplastic Syndromes

Despite advances into the genetic basis of myelodysplastic syndromes (MDS), the mechanisms of their pathophysiology remain poorly understood, and frequently terminates in acute myeloid leukemia. Mutually exclusive mutations have been identified in splicing factors U2AF1, SF3B1, SRSF2, SF3A1, ZRSR2 and LUcL7 in 85% of MDS cases. We and others have identified aberrant forms of Granulocyte Colony Stimulating Factor Receptor (CSF3R) in patients with myeloid malignancies and partially characterized their phenotypic and signal transduction effects. Two of the seven isoforms of CSF3R have clinical relevance: Class I (full-length form) and Class IV (alternatively spliced form). Compared to Class I, Class IV isoform is differentiation defective and displays enhanced proliferation. Increased expression of the Class IV isoform has been found in MDS/AML and results in enhanced cell proliferation and impaired differentiation of granulocytes (Mehta et al, Leukemia 2014). The Class IV CSF3R transcript is derived by intron excision, whereas Class I results from retention of that intron. We hypothesize aberrant splicing of the CSF3R transcript promoted by mutations in splicing factors drives abnormal granulopoiesis in MDS due to increased expression of the differentiation defective Class IV CSF3R.

We identified U2AF1, SRSF2, and SAM68 sites within the nucleotide sequence of the spliced intron. To study the effect of splicing factor mutations on CSF3R splicing, we constructed a minigene consisting of 5' exon of the CAT gene with an ATG site and a partial intron fused to intron region of G-CSFR upstream of Exon 17. The retained intron is present in the exon 17 of CSF3R. The minigene consists of 2 introns and 3 exons. We transduced 293FT cells with the minigene ± wild-type U2AF1 or U2AF1 S34F mutant, as well as the minigene ± wild-type SF3B1 or SF3B1 K300E. The cells were then treated with either 1µM sodium orthovanadate (Na₃VO₄) or 10 nM phorbol myristic acetate (PMA). The former inhibits tyrosine phosphatases, promoting tyrosine protein phosphorylation, whereas the latter promotes activation of the protein kinase C pathway, which eventually activates ERK1/2 an upstream activator of SAM68. Protein phosphorylation of spliceosome proteins is fundamental to regulation of splicing. The effect of PMA and Na₃VO₄ were compared with each other and those treated with control diluent. Qualitative and semi-quantitative evaluations were performed. We identified expression with SF3B1 K700E enhances CSF3R splicing resulting in increased Class IV, however the U2AF1 S34F inhibits splicing resulting in reduced Class IV. The increase and decrease were in relation to the wild type. Further exploration of how cellular protein phosphorylation influences splicing using Na₃VO₄ (identified that cells transduced with SF3B1, U2AF1 wt or mutant shown decreased splicing resulting in Class IV transcript. PMA however had no effect on either class expression. These data suggest that CSF3R transcript processing is sensitive to diverse tyrosine kinase signaling, resulting in increased intron retention, suggesting that phosphorylation events inhibit Class IV transcript formation. Additionally the mutations in splicing factors which are recurrently found in MDS are associated with altered CSF3R transcripts and may affect CSF3R signaling and cell phenotype.

(This work is supported by DOD and NIH R01R01HL128173.)