Leukemia cell clones that rebound after treatment have differential levels of therapeutic tolerance.

  • Major Finding: Leukemia cell clones that rebound after treatment have differential levels of therapeutic tolerance.

  • Approach: ClonMapper enables clonal fitness measurements, clonally resolved single-cell RNA sequencing, and live clonal isolation.

  • Impact: This technique links transcriptomic, proteomic, and genetic data with cell fate to provide insight into chemoresistance.

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Characterizing clonal dynamics is critical to understanding cancer biology, including treatment resistance and relapse. Many existing techniques have enabled dissection of many key aspects of clonal dynamics, but they do not allow further analysis of live cells. To address this challenge, Gutierrez, Al'Khafaji, Brenner, and colleagues developed a novel lineage-tracing system, dubbed ClonMapper, which leverages multifunctional barcodes to enable high-resolution tracking of clonal fitness, clonally resolved single-cell RNA sequencing, and live clonal isolation. Applying this method to chronic lymphocytic leukemia (CLL) and mantle cell lymphoma (MCL) cell lines revealed that fludarabine–mafosfamide or mafosfamide–doxorubicin–vincristine treatment, representing standard-of-care first-line chemotherapy regimens for CLL and MCL, respectively, led to the consistent selection of a specific set of clones. The authors then leveraged the single-cell capabilities of ClonMapper to characterize the transcriptomic profiles of surviving clones, identifying two clonally distinct subpopulations with differing survivorship trajectories. The more drug-tolerant subpopulation, termed “high-survivorship” (HS), was noted to already be present prior to treatment and exhibited marked differences from their “low-survivorship” (LS) counterpart that preceded the initiation of treatment and persisted following treatment. For example, gene-expression analyses revealed that HS clones exhibited upregulation of genes involved in WNT signaling, Notch signaling, and chromatin remodeling pathways, whereas LS clones displayed upregulation of focal adhesion and MHC class II antigen presentation pathways. Importantly, transcriptomic data from blood samples collected from a cohort of patients with CLL before and after treatment with fludarabine-based chemotherapy or the BTK inhibitor ibrutinib concorded with the in vitro findings, suggesting that the presence of preexisting HS clones is a defining feature of chemotherapy resistance and relapse in CLL. To further dissect the nature of clonal identity, the authors applied supervised machine learning to transcriptomic data from isolated subclones and identified transcriptional features that were heritable over the course of therapeutic stimuli and accruing genetic alterations. This work provides a road map for systematically parsing heterogeneous cell populations and linking these insights with clonal fitness.

Gutierrez C, Al'Khafaji AM, Brenner E, Johnson KE, Gohil SH, Lin Z, et al. Multifunctional barcoding with ClonMapper enables high-resolution study of clonal dynamics during tumor evolution and treatment. Nat Cancer 2021;2:758–72.

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