SRSF2 is a RNA-binding protein frequently mutated in CMML tasked with marking exon-intron boundaries during alternative splicing. SRSF2 contains a domain consisting of serine-arginine repeats that are dynamically phosphorylated and comprise a major regulatory mechanism in RNA capture. It has been previously demonstrated that EGFR signaling can elicit the phosphorylation of SRSF2 in an AKT dependent manner, resulting in functional alterations in exon usage of SRSF2 target genes. We previously reported that GM-CSF hypersensitivity is a reproducible feature of CMML. Because AKT is a known signaling intermediate of GM-CSF, we hypothesize that aberrant GM-CSF signaling could induce mutation-independent splicing abnormalities in CMML via phosphorylation of SRSF2. To test this hypothesis, we selected CMML samples that displayed GM-CSF hypersensitivity but lacked mutations by Sanger sequencing in 5 splicing-related genes commonly mutated in myeloid malignancies (SRSF2, SF3B1,U2AF1, ZRSF2,and PRPF40B) and labeled this group as CMML ‘splicing WT'. A second sample set included CMML specimens with isolated SRSF2 mutation (SRSF2 ‘splicing MT'). Bone marrow mononuclear cells from the SRSF2 ‘splicing WT' group (n=4), CMML ‘splicing MT' group (n=4), and normal controls (n=4) were lymphodepleted (CD3 and CD19) to enrich for the clonal myeloid population. RNA was extracted and whole transcriptome RNA sequencing and exon arrays were performed to compare the relative global exon usage of CMML ‘splicing WT', CMML ‘splicing MT', and normal controls. Briefly, the human exon 1.0 ST array was used for exon array analysis and splice index was calculated with the Exon/Gene Array Analyzer Web Tool (Gellert Bioinformatics 2009). RNA sequencing data was generated and aligned with TopHat2 (Kim Genome Bio 2013) while read counts and exon usage assignments were made with DexSeq (Anders Genome Res 2012). Splice indices determined by exon array demonstrated statistically significant exon usage differences between CMML ‘splicing MT' and normal controls consistent with previously published results (Figure 1a). Interestingly, when CMML ‘splicing WT' was compared to normal controls, a statistically significant difference in exon usage was also identified (Figure 1b). Further, when ‘splicing MT' and ‘splicing WT' were compared by splice index, differences in exon usage differences were dramatically reduced indicating similar splicing abnormalities in the CMML 'splicing WT' group (1c). Euclidian hierarchical clustering at the gene- and exon-level separated normal controls from CMML groups but failed to separate ‘splicing MT' from ‘splicing WT', supporting the notion that mutation-independent splicing aberrancy may characterize CMML (1d). Global patterns in exon usage were also tested by whole transcriptome RNA-sequencing and were similar to those described above identified by exon array splice index. [Display omitted] We first attempted to determine if the transcript levels of core splicing components in CMML ‘splicing WT' were altered but were unable to identify differences in expression when compared to CMML 'splicing MT' and controls. Next we explored the possibility that GM-CSF signaling, known to be upregulated in CMML, could post-translationally modify SRSF2. The THP-1 monocytic leukemia cell line was stimulated with 10ng/ml of GM-CSF for 15 minutes. Cell lysates were immediately prepared and western blot analysis was used to assess SRSF2 phosphorylation after stimulation with GM-CSF, demonstrating induction of pSRSF2 at 15 minutes as accompanied by induction of the known GM-CSF signaling intermediates pJAK2, pAKT, and pERK. Pharmacologic inhibition of these intermediates abrogated GM-CSF-induced pSRSF2, whereas only the JAK 1/2 inhibitor ruxolitinib was able to inhibit pSTAT5, pAKT, and pERK, suggesting that JAK2 is a proximal kinase in the signaling cascade. Our results indicate that splicing abnormalities are present in CMML specimens lacking mutations in splicing-related genes. GM-CSF induces phosphorylation of SRSF2 for post-translational regulation of the RS domain. Low dose GM-CSF dependent pSRSF2 and functional splicing analyses are currently underway in primary CMML ‘SRSF2 WT' samples to directly determine whether GM-CSF hypersensitivity is responsible for mutation-independent splicing aberrancies. Disclosures:List: Celgene: Membership on an entity's Board of Directors or advisory committees.
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Jul 24, 2014
Brandon E Aubol + 7
Open Access
