Transcriptome Sequencing Reveals Alternative Splicing Patterns and an Increased Sensitivity to Spliceosome Inhibition Associated MYD88 L265P Mutation in Chronic Lymphocytic Leukemia

Background: Chronic lymphocytic leukemia (CLL) is the most common leukemia in adults in the Western countries but is relatively rare in East Asia. Mutations in myeloid differentiation primary response gene 88 (MYD88) occur in 2.0-4.4% of Caucasian patients with CLL. However, Asian subjects showed a higher MYD88 mutation frequency of 8% and MYD88 mutations were associated with unfavorable prognosis in Asian CLL patients with mutated IGHV gene. To further explore the molecular and pathological mechanisms of the MYD88 L265P mutation in CLL, we performed a pilot study to investigate the transcriptome-wide differential expression between CLL patients with and without MYD88 L265P mutation.

Methods: mRNA sequencing was performed on a cohort of 15 Chinese CLL patients with MYD88 L265P mutation (n=5) or wild-type MYD88 gene (n=10). Differential expression and pathway enrichment analyses were performed based on FPKM values after processing the raw sequencing reads. The alternative splicing events were analyzed using JuncBASE and the differences in splicing patterns were compared between the two groups. The MYD88 L265P and wild-type genes were overexpressed in the MEC-1 cell line using lentiviral vectors, and stable cell lines were established after puromycin selection. Real time quantitative PCR was used to determine the mRNA expression of spliceosome pathway genes. The protein expression of TLR pathway was assessed with Western Blot. The PrestoBlue™ assay was used for determining IC-50 of spliceosome inhibitor-treated CLL cell lines, and flow cytometry was utilized for apoptosis and cell death analyses.

Results: mRNA sequencing analysis revealed that 4231 genes showed significant differences in mRNA expression between MYD88 mutant and wild-type CLL patients. Of these genes, 3033 were up regulated and 1198 were down regulated in patients with MYD88 L265P mutation. Pathway analysis demonstrated that tumor necrosis factor (TNF) signaling, cell cycle, spliceosome, ribosome, p53 signaling, and NF-κB signaling pathways were significantly enriched among differentially expressed genes. Interestingly, about two thirds (19 out of 30) of differentially expressed genes in the spliceosome pathway were down regulated in CLL samples with MYD88 mutation. Quantitative RT-PCR confirmed the down regulation of spliceosome genes including SF3A1, SF3A2, SF3B4, SF3B5 and PUF60 in MYD88 mutated samples in an independent CLL patient cohort. Analysis of alternative splicing events revealed a total of 180 genes with inter-group splicing differences, among which differential splicing events were most common in the genes involved in immune system regulation, i. e. CD22, CD79B, CD200 and IKZF1 etc. The MEC1 cells stably overexpressing MYD88 L265P demonstrated decreases in RelA and TRAF6 expression as compared to wild type MYD88-overexpressing cells, which was in agreement with RNA-seq analysis results in CLL patient samples. In addition, we found that overexpression of MYD88 L265P mutant resulted in decreased mRNA expression of several spliceosome genes such as SF3A1, SF3A2, SF3B4, SF3B5 and PUF60 in the stable cell line. Furthermore, MYD88 L265P increased the sensitivity of MEC1 cells to spliceosome inhibitor sudemycin D6 (SD6) by 3 fold, while reducing their sensitivity to Ibrutinib by two fold.

Conclusion: We discovered the differential mRNA expression landscapes related to MYD88 mutation status in CLL and their contributions to the progression of CLL through multiple pathways. Of particular interest was the down regulation of spliceosome pathway genes. In a CLL cell line model, we found that, compared with the MYD88 wild-type CLL cell line, CLL cells overexpressing the mutant MYD88 were more sensitive to spliceosome inhibitor SD6, suggesting that inhibiting spliceosome function may be a potential therapeutic target for CLL patients with mutant MYD88 gene. In summary, our findings provide novel insights for further understanding of the molecular and pathological mechanisms of MYD88 mutation in CLL.