The Landscape of Circular RNAs in Drug Resistant TCF3-Rearranged Childhood Leukemia

Treatment resistance is associated with poor prognosis in acute lymphoblastic leukemia (ALL). The underlying causes are largely unknown and specific risk markers and treatment options are consequently lacking. Efforts need to be made to identify new therapeutic targets in drug resistant patients who are left without other treatment options. This applies in particular to ALL patients bearing the t(17;19)(q22;p13) translocation that leads to the expression of the oncogenic chimeric transcription factor TCF3-HLF . Due to treatment resistance these patients typically suffer from relapse and die within two years from diagnosis.

Circular RNAs may present such novel targets. Similar to linear RNAs, circRNAs are derived from parental genes. They are circularized by backsplicing and are remarkably stable. CircRNAs may regulate the expression of their parental gene, e.g. by sequestration of miRNAs or RNA binding proteins. CircRNAs have been shown to play a role in cancer by regulating microRNAs with tumor suppressive or oncogenic function (including let-7 and miR-7) and dysregulated circRNA expression has been observed in a variety of human tumors.

Therefore we analyzed the landscape of circular RNAs in the drug resistant TCF3-HLF + cell line HAL01 and compared our findings to RNA expression data of a cohort of TCF3-HLF + patients (n=5) at diagnosis and remission. As controls, we used a cell line (697) and data derived from matched diagnosis/remission samples of patients (n=5) with the drug sensitive ALL subtype TCF3-PBX1 .

Cell lines underwent whole-exome, -transcriptome and -circRNome sequencing. Single nucleotide variation, indel and copy number analysis revealed only few and not previously in TCF3-HLF+ ALL reported somatic mutations (MSH6, EP300) in HAL01. Transcriptome sequencing confirmed the expression of a TCF3-HLF fusion transcript joining exon 16 of TCF3 to exon 4 of HLF. 54 inserted nontemplate nucleotides indicated terminal deoxynucleotide transferase activity characteristic of an early B cell stage. Similar to leukemic TCF3-HLF+ patient samples, expression of the B cell transcription factor PAX5 was halved, which may account for the block in B cell development. Gene expression analysis restricted to genes specifically expressed in HAL01 and not expressed in 697 revealed a functional cluster comprised of GO terms implicated in drug resistance as most highly enriched.

We used a bioinformatic approach to identify circRNAs in RNAseq data of TCF3-HLF+ and TCF3-PBX1+ patient samples. We detected 556 putative circRNAs. 249 genes expressed specific circRNAs only in leukemic and not in remission samples. Of those, 109 circRNAs were exclusively expressed in drug resistant TCF3-HLF -positive leukemia and 9 derived from parental genes such as the lysine demethylase KDM1A involved in p53 mediated DNA damage signaling were recurrently detected.

Enrichment of circRNAs by RNase R digest and subsequent sequencing led to detection of 14,311 circular RNAs in HAL01 and 697. 7,918 of those were described in the circBase database, 6,393 were novel. The circRNAs were derived from 4,652 parental genes. 1 to 39 circRNAs were transcribed per gene. The most highly, but ubiquitiously expressed circRNA was derived from the gene NRIP1, a corepressor that modulates a variety of transcription factors. Several circRNAs derived from genes known to be involved in ALL pathogenesis (PAX5, IKZF1 and RUNX1) were detected. CircRNAs derived from the drug transporter gene Multidrug resistance-associated protein 1 (MRP1) were expressed in drug resistant but not in sensitive cells. Two of those were also detected in a leukemic TCF3-HLF+ patient sample. MRP1 detoxifies chemotherapeutic drugs by export of these substances out of the cells. Clinically relevant substrates of MRP1 are anticancer drugs, including vincristine, etoposide, topotecan and methotrexate. Drug screening of TCF3-HLF+ patient derived xenografts previously demonstrated explicit resistance against these drugs. Overexpression of MRP1 and similar transporters is considered a major cause of multidrug resistant tumor phenotypes and may be due to promoter hypomethylation or gene amplification. Upregulation of these transporters up to 10 fold has been observed in TCF3-HLF+ leukemia. Our results may add another layer of regulation and may indicate circular RNAs as novel biomarkers or therapeutic targets for patients with therapy resistant, fatal disease.