Upregulation of ZEB1-Derived Circular RNAs Is Associated with SF3B1-Mutated Myelodysplastic Neoplasms

Myelodysplastic neoplasms (MDS) are a heterogeneous group of bone marrow (BM) disorders characterized by ineffective hematopoiesis, peripheral cytopenia, and increased tendency to leukemic transformation. Mutations of splicing factor gene SF3B1 are frequently found in MDS, resulting in aberrant splicing of multiple genes such as those involved in mitochondrial metabolism. Splicing process results not only in a variety of linear mRNAs but it can also produce covalently enclosed RNAs, circular RNAs (circRNAs). CircRNAs regulate biological processes through various molecular mechanisms, e.g. miRNA sponging. CircRNAs are deregulated in many types of cancer, including MDS. Based on our previous data (Dostalova Merkerova M, et al., Cancer Genomics Proteomics. 2022; 19:205-228), we hypothesized that there is an association between MDS and the level of circRNAs derived from ZEB1 gene. ZEB1 is an essential hematopoietic transcription factor governing blood lineage commitment. Here, we studied expression and function of these circRNAs in SF3B1-mutated MDS.

The study included 85 patients and 13 healthy controls. We examined somatic mutations (Illumina Myeloid Panel) and gene expression (RNA-seq). Further, isogenic CRISPR/Cas9-edited NALM-6 cell line with or without SF3B1 mutation was used for in vitro testing. RT-qPCR method was applied for analysis of expression of individual genes or circRNAs. Cellular bioenergetics was measured using a Seahorse platform.

Initially, we examined ZEB1 gene expression, its alternative splicing, and ZEB1-circRNA expression using RNA-seq data from CD34+ BM cells. Although expression of ZEB1 gene as a whole remained unchanged, we observed substantial changes on the level of transcripts, including upregulation of ZEB1 circularization in patients with a SF3B1 mutation in comparison to controls and non-mutated patients. Within the data, we detected expression of ten ZEB1-circRNAs. Of these, increased levels of seven neighboring circRNAs from the central part of ZEB1 gene were detected and their levels were significantly coregulated. More specifically, four alternatively spliced exons of ZEB1 were present in the upregulated circRNAs, suggesting that the minimal region essential for this alteration occurs in chr10:31,661,947-31,676,195 (hg19).

Hsa_circ_0000228 showed more than five-fold higher expression than other ZEB1-circRNAs. Thus, we particularly focused on this circRNA in subsequent experiments. Initially, we confirmed the sequence of the backsplicing junction site of hsa_circ_0000228 by Sanger sequencing. Treatment of isolated RNA with RNase R exonuclease, which specifically cleaves linear RNAs and leaves circRNAs intact, was used to prove that hsa_circ_0000228 is of circular nature. Further, NALM-6 cell line was treated with actinomycin D, which blocks RNA transcription, showing that hsa_circ_0000228 is more stable that linear ZEB1-derived mRNAs.

To better describe the relation between ZEB1-circRNAs and MDS pathogenesis, we studied correlation of hsa_circ_0000228 levels with various mutational and clinical variables. Importantly, we observed that the circRNA upregulation is exclusive to SF3B1 mutations and not for mutations in other splicing factors (SRSF2, U2AF1, and ZRSR2) or other recurrently mutated genes. Further, the circRNA upregulation was specifically associated with SF3B1 mutations and not with any other tested clinical variables. Upregulation of ZEB1-circRNAs expression in NALM-6 cells with SF3B1 K700E mutation (compared to wildtype) proved that this mutation directly leads to increased circularization.

To study functions of hsa_circ_0000228 in MDS cells, we performed siRNA-mediated knock-down of this circRNA in NALM-6 cells with SF3B1 mutation. Subsequent RNA-seq and pathway analysis suggested that hsa_circ_0000228 inhibition positively regulates mitochondrial complex I. Effect of hsa_circ_0000228 on function of complex I was proved by the Seahorse analysis, which showed that the siRNA-transfection led to metabolic switch - induction of oxidative phosphorylation and reduction of glycolysis.

Overall, we demonstrated that SF3B1 mutation in MDS leads to upregulation of ZEB1-circRNAs and this deregulation contributes to changes in mitochondrial metabolism which have been associated with SF3B1-mutated MDS.

Supported by GA CR N20-19162S, AZV NU21-03-00565, and MH CZ-DRO UHKT, 00023736.

No relevant conflicts of interest to declare.