Shestova, O. | bioRxiv Science

bioRxiv2025

scDNA: Single Cell DNA analysis software toolkit for subclonality discovery and assessment.

Advances in single cell multi-omics technologies have allowed for investigation into genotype-immunophenotype relationships and dynamic clonal changes in cancer patient samples. These technologies provide rich insights into genetic profiling, drivers of disease progression, and subclonal dynamics. Increased adoption and throughput of these technologies, necessitate accessible computational tools for processing and analysis. Toward developing easy to use computational tools, we introduce the Single Cell DNA (scDNA) package that allows for rapid analysis of single-cell molecular profiling. Our platform aims to provide a diagnostic summary tool for sample quality control, representative subclonal architecture within a sample, demultiplexing functionality for multi-sample processing, copy number variation, and mutation trajectory analysis to identify order of subclonal mutation acquisition. We showcase a series of vignettes on hematopoiesis datasets for each aim that reflects recently deployed uses of scDNA. Additionally, we show scDNA provides a modular, user-friendly framework that readily feeds into other standard software pipelines.

genomics↗

bioRxiv2019

Impaired tumor death receptor signaling drives resistance to CAR T cell therapy

Cellular immunotherapy using T cells engineered to express chimeric antigen receptors targeting CD19 (CART19) leads to long-term remission in patients with B-cell malignancies1-5. Unfortunately, a significant fraction of patients demonstrate primary resistance to CART19 or experience relapse after achieving remission. Beyond loss of target antigen, the molecular pathways governing CART19 failure are unknown. Here we demonstrate that death receptors, cell surface signaling molecules that induce target cell apoptosis, are key mediators of leukemic resistance and CART19 failure. Using a functional CRISPR/Cas9-based genome-wide knockout screen6 in B-cell acute lymphoblastic leukemia (ALL), we identified the death receptor signaling pathway as a central regulator of sensitivity to CART19-induced cell death. In the absence of the pro-apoptotic death receptor signaling molecules BID or FADD, ALL cells were resistant to CART19 cytotoxicity, resulting in rapid disease progression in mice. We found that this initial resistance to cytotoxicity led to persistence of tumor cells, which drove the development of T cell dysfunction that further compromised anti-tumor immunity and permitted tumor outgrowth. We validated these findings using clinical samples collected from patients with ALL treated with CD19-targeted CAR T cells and found that expression of pro-apoptotic death receptor pathway genes in pre-treatment tumor samples correlated with CAR T cell expansion and persistence, as well as patient response and overall survival. Our findings indicate that tumor-intrinsic death receptor signaling directly contributes to CAR T cell failure.

immunology↗