Retention of CD19 intron 2 contributes to CART-19 resistance in leukemias with subclonal frameshift mutations in CD19

Chimeric antigen receptor T cell (CAR-T) targeting the CD19 antigen represents an innovative therapeutic approach to improve the outcome of relapsed or refractory B-cell acute lymphoblastic leukemia (B-ALL). Yet, despite a high initial remission rate, CAR-T therapy ultimately fails for some patients. Notably, around half of relapsing patients develop CD19 negative (CD19neg) B-ALL allowing leukemic cells to evade CD19-targeted therapy. Herein, we investigate leukemic cells of a relapsing B-ALL patient, at two-time points: before (T1) and after (T2) anti-CD19 CAR-T treatment. We show that at T2, the B-ALL relapse is CD19 negative due to the expression of a non-functional CD19 transcript retaining intron 2. Then, using single-cell RNA sequencing (scRNAseq) approach, we demonstrate that CD19neg leukemic cells were present before CAR-T cell therapy and thus that the relapse results from the selection of these rare CD19neg B-ALL clones. In conclusion, our study shows that scRNAseq profiling can reveal pre-existing CD19neg subclones, raising the possibility to assess the risk of targeted therapy failure.

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“…1e ). We observed that both samples express a previously described 18 nonfunctional CD19 transcript retaining intron 2 (Supplementary Fig. 4c ).…”

Section: Resultssupporting

confidence: 60%

Single-cell profiling identifies pre-existing CD19-negative subclones in a B-ALL patient with CD19-negative relapse after CAR-T therapy

et al. 2021

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“…Moreover, the dysregulation of some of these splicing factors could control the expression of at least a subset of circRNAs that would then entail important functional consequences that could contribute to neoplastic transformation of B-cells. This has already been shown by us in the context of aberrant splicing of SRSF3-dependent mRNA transcript encoding CD19 (Asnani, 2019;Black et al, 2018;Sotillo et al, 2015), but here we provide increasing evidence that gene dysregulation could also occur via circRNAs.…”

Section: Introductionsupporting

confidence: 78%

The role of SRSF3 splicing factor in generating circular RNAs

Asnani

Black

et al. 2019

Preprint

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Circular RNAs (circRNAs) represent a novel class of non-coding RNAs that are emerging as potentially important regulators of gene expression. circRNAs are typically generated from host gene transcripts through a non-canonical back-splicing mechanism, whose regulation is still not well understood. To explore regulation of circRNAs in cancer, we generated sequence data from RNase R-resistant transcripts in human p493-6 B-lymphoid cells and identified thousands of novel as well as previously identified circRNAs. Approximately 40% of expressed genes generated a circRNA, with half of them generating multiple isoforms, suggesting the involvement of alternative back-splicing and regulatory RNA-binding proteins (RBPs). We observed that genes generating circRNAs with back-spliced exonic junctions were enriched for RBP recognition motifs, including multiple splicing factors, most notably SRSF3, a splicing factor known to promote exon inclusion. To test the role of SRSF3 role in circRNA production, we performed traditional RNA-seq in p493-6 B-lymphoid cells with and without SRSF3 knockdown, and identified 926 mRNA transcripts, whose canonical splicing was affected by SRSF3. We found that a subset (205) of these SRSF3 targets served as host transcripts for circRNA, suggesting that SRSF3 may regulate exon circularization. Since this splicing factor is deregulated in hematologic malignancies, we hypothesize that SRSF3-dependent circRNAs, similar to their mRNA counterparts, might contribute to the pathogenesis of lymphomas and leukemias.

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“…LR-seq of human and mouse cell and tissue transcriptomes has revealed a rich diversity of spliced isoforms (Tilgner et al, 2015;Lagarde et al, 2017;Byrne et al, 2017;Tilgner et al, 2018;Workman et al, 2019;Sheynkman et al, 2020). In cancer research, the use of LR-seq to identify primary tumorassociated spliced isoforms remains underexploited and has been limited to the study of human leukemia samples (Asnani et al, 2020;Tang et al, 2020). Importantly, the ability to aquire the depth of coverage needed to accurately quantitate transcripts using LR data is prohibitively expensive.…”

Section: Introductionmentioning

confidence: 99%

Long-read isoform sequencing reveals survival-associated splicing in breast cancer

Nesta

Zhao

et al. 2020

Preprint

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SummaryTumors display widespread transcriptome alterations, but the full repertoire of isoform-level alternative splicing in cancer is not known. We developed a long-read RNA sequencing and analytical platform that identifies and annotates full-length isoforms, and infers tumor-specific splicing events. Application of this platform to breast cancer samples vastly expands the known isoform landscape of breast cancer, identifying thousands of previously unannotated isoforms of which ~30% impact protein coding exons and are predicted to alter protein localization and function, including of the breast cancer-associated genes ESR1 and ERBB2. We performed extensive cross-validation with -omics data sets to support transcription and translation of novel isoforms. We identified 3,059 breast tumor-specific splicing events, including 35 that are significantly associated with patient survival. Together, our results demonstrate the complexity, cancer subtype-specificity, and clinical relevance of novel isoforms in breast cancer that are only annotatable by LR-seq, and provide a rich resource of immuno-oncology therapeutic targets.

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Retention of CD19 intron 2 contributes to CART-19 resistance in leukemias with subclonal frameshift mutations in CD19

Cited by 70 publications

References 22 publications

Single-cell profiling identifies pre-existing CD19-negative subclones in a B-ALL patient with CD19-negative relapse after CAR-T therapy

Single-cell profiling identifies pre-existing CD19-negative subclones in a B-ALL patient with CD19-negative relapse after CAR-T therapy

The role of SRSF3 splicing factor in generating circular RNAs

Long-read isoform sequencing reveals survival-associated splicing in breast cancer

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