PO-445 E7107 treatment results in aberrantly spliced transcripts and protein products of P53 pathway genes

Abstract

IntroductionRecent studies indicate that E7107, a spliceosome inhibitor, causes altered splicing of key genes in CLL. We evaluated the effect of E7107 on cell viability and key proteins in the p53 pathway.Material and methodsEight leukaemia/lymphoma cell lines and eight primary CLL samples (6 wild-type and 2 mutant for SF3B1) were exposed to E7107 (H3 Biomedicine) for 72 and 48 hours. Cell viability was assessed by XTT assay. To understand the effect of splicing modulation on key proteins in the p53 pathway, including p21 and MDM2, five B-cell lines were treated with E7107 for 24 hours.Results and discussionsE7107 decreased cell viability at low nanomolar concentrations in all CLL samples (mean LC50=10.5±2.0 nM; but >300 nM in two healthy PBMC controls). No correlation between drug sensitivity and SF3B1 status was observed in CLL samples (p=0.5). Six out of eight cell lines were sensitive to E7107 (mean GI50=6±1.8 nM). The GI50 values were 60.2 and 203.5 nM for the resistant HEL and HAL-01 cells, respectively. The most frequently mutated regions of the SF3B1 gene, exons 14, 15 and 16, had no mutations detected by Sanger sequencing. There was no correlation between drug sensitivity and TP53 status.Western immunoblot revealed a marked decrease of MDM2 protein level in all cell lines, accompanied by a reciprocal concentration-dependent increase in p53. The normal molecular weight p21 disappeared at higher doses of E7107, with concomitant appearance of a high molecular weight p21 isoform (~30 kDa) in TP53 wild-type cells. In addition, a lower molecular weight p53 isoform was seen in OCI-Ly3 cells treated with 50 nM E7107, likely due to alternative splicing. RT-PCR of p21 mRNA, spanning exons 3 to 4, revealed an additional longer transcript in OCI-Ly3 cells treated with E7107(50 nM, 24 hours), which sequencing confirmed was due to intron retention, predicted to result in a high molecular weight protein with an alternative c-terminal sequence. RT-PCR of MDM2 mRNA spanning exons 1 to 11 detected an altered MDM2 transcript with exons 3–10 spliced out.ConclusionE7107 treatment resulted in decreased viability of B-cell lines and primary CLL samples independently of SF3B1 status. This was associated with the production of an aberrant high molecular weight isoform of p21 due to intron retention, and a short isoform of MDM2 missing exons 3–10. Loss of normal MDM2 was accompanied by increased p53. Further investigation is needed to understand the contribution of abnormal isoforms to cell fate.