Year: 2026
Type: article
Study design: Observational Study
Abstract: Alternative splicing has emerged as a key mechanism contributing to cancer biology. In Chronic Myeloid Leukemia (CML), dysregulation of splicing regulators, including U2AF1, HNRNPA1, CELF2, MYC and the epigenetic regulator EZH2, has been reported. The spliceosome pathway is also enriched in CML, especially in blast crisis patients. Although CML is primarily driven by the BCR::ABL1 fusion gene, increasing evidence indicates supplementary co-mutational patterns and abnormalities in splicing. Recurrent splicing variants, including exon 7 skipping (Δ7) and a 35-bp insertion (INS35), have been detected in 25–30% of CML cases by Sanger sequencing (SS) and in up to 70% of cases by next-generation sequencing. These findings suggest that alternative splicing may provide an additional layer of molecular regulation in CML pathogenesis. Our previous findings associated these variants with higher BCR::ABL1 transcript levels, supporting the hypothesis that aberrant splicing contributes to CML biology rather than being an isolated transcriptional event. In this study, we investigated the expression of splicing-related genes (EZH2, CELF2 and MYC) associated with changes in Δ7 and INS35 frequencies in CML samples and their correlation with BCR::ABL1 transcript levels. Δ7 and INS35 were evaluated by SS or fragment analysis in 882 CML samples collected between 2020 and 2026, including 263 diagnostic and 639 follow-up samples, of which 267 were longitudinal samples from 31 patients, as well as 53 samples from healthy donors. The study was conducted with Ethics Committee approval (CAAE: 96613326.3.0000.5274). Relative expression of EZH2, CELF2, and MYC was quantified by RT-qPCR and normalized to HPRT1 and GAPDH (GeNorm) in samples arranged according to splicing variants identification. In parallel, splicing-related genes in public RNA-seq data (GSE100026; GSE144119) was also analyzed. The analysis proceeds using STAR for alignment, followed by a dedicated splicing filter with rMATS. Differential expression analysis proceeded using Limma. Δ7 VAF positively correlated with leukemic burden, as measured by %BCR::ABL1IS (p = 0.03), and significantly discriminated patients achieving deep molecular response (MR 4.0-4.5) using a VAF cut-off of 16.88% (p = 0.009). In contrast, INS35 showed no association with %BCR::ABL1IS or molecular response. Longitudinal analyses demonstrated that variants fluctuate across patients' follow-up with no prognostic value; and significantly higher Δ7 VAFs were detected at diagnosis rather than at relapse (11% vs. 6%; p=0.02). Although both splicing variants were detected at lower frequencies in healthy donors, both variants were significantly enriched in BCR::ABL1-positive samples. Preliminary qPCR analyses showed increased EZH2 expression in splicing variant-positive samples (log2FC +3.7/+3.0 vs. controls; HPRT/GAPDH normalized) in comparison to either wild-type BCR::ABL1 CML patients and donors, whereas CELF2 showed no significant gene expression alteration. RNA-seq analysis of the GSE100026 dataset identified EZH2, U2AF1, and PRPF8 as the most differentially expressed genes in CML samples vs donors. In contrast, MYC and CELF2 exhibited no significant gene expression alteration between CML patients and healthy control samples. Overall, these findings identify EZH2 as a candidate molecular correlate of splicing variant-positive CML and support further investigation of aberrant splicing as a novel aspect of CML biology.
Language: English
Subfield: Hematology
Field: Medicine
Domain: Health Sciences
Open Access status: diamond
OpenAlex ID: W7223159307