Pietro Zafferani

T-ALL relapses are characterized by chemotherapy resistance, cellular diversity and dismal outcome. To gain a deeper understanding of the mechanisms underlying relapses, we conduct single-cell RNA sequencing on 13 matched pediatric T-ALL patient-derived samples at diagnosis and relapse, along with samples derived from 5 non-relapsing patients collected at diagnosis. This comprehensive longitudinal single-cell study in T-ALL reveals significant transcriptomic diversity. Notably, 11 out of 18 samples exhibit a subpopulation of T-ALL cells with stem-like features characterized by a common set of active regulons, expression patterns and splice isoforms. This subpopulation, accounting for a small proportion of leukemia cells at diagnosis, expands substantially at relapse, indicating resistance to therapy. Strikingly, increased stemness at diagnosis is associated with higher risk of treatment induction failure. Chemotherapy resistance is validated through in-vitro and in-vivo drug testing. Thus, we report the discovery of treatment-resistant stem-like cells in T-ALL, underscoring the potential for devising future therapeutic strategies targeting stemness-related pathways.

T cell acute lymphoblastic leukemia (T-ALL) is an aggressive leukemia predominantly affecting adolescents and young adults. T-ALL relapses are characterized by chemotherapy resistance, cellular heterogeneity and dismal outcome. To gain a deeper understanding of the cellular heterogeneity and mechanisms driving relapse, we conducted single-cell full-length RNA sequencing of 13 matched pediatric T-ALL patient-derived xenografts (PDX) samples obtained at initial diagnosis and relapse, generating the to date most comprehensive longitudinal single cell study in paired T-ALL samples, along with 5 non-relapsing PDX samples collected at initial diagnosis. This dataset identifies considerable transcriptomic diversity among individual T-ALL cell populations. Notably however, 11 of the 18 patients exhibit a small T-ALL cell subpopulation with a shared set of gene regulatory networks characterized by a common set of active regulons, expression patterns and splice isoforms. This profile involves the upregulation of a stem-like cell signature with enrichment of cell adhesion, and inhibition of the cell cycle and metabolic activity. Comprehensive investigations of these networks identify transcripts enforcing drug resistance through NF-κB expression and TGF-β signaling and of anti-apoptotic T cell signaling. Longitudinal monitoring of these stem-like cells demonstrates this subpopulation to account for only a small proportion of leukemia cells initially with a substantial expansion at relapse suggesting resistance to first line therapy. Chemotherapy resistance is functionally corroborated through in vitro and in vivo drug testing. We thus report the discovery of inherently treatment-resistant stem-like T-ALL cell populations underscoring the potential for devising future therapeutic strategies aimed at targeting stemness pathways in pediatric T-ALL. Key Points: Single-cell full-length RNA sequencing reveals dormant, treatment-resistant cells expanding upon T cell acute lymphoblastic leukemia relapse. Stem-like T-ALL cells exhibit a shared gene regulatory network conferring chemotherapy resistance and relapse.

Long-lived species suppress cancer despite accumulating somatic mutations throughout life, but how this is achieved in renewing tissues remains unclear. Here, we show that naked mole-rat skin uncouples high cellular turnover from cancer risk through coordinated epithelial and stromal mechanisms that constrain clonal outgrowth and suppress tumor-promoting inflammation. Despite elevated epidermal proliferation and mutational burden, naked mole-rat skin maintains tissue integrity through an expanded pool of early committed progenitor (hybrid) cells and replication-coupled genome maintenance pathways. Under chronic carcinogenic stress, undifferentiated basal cells replenish the hybrid progenitor pool. This dilutes initiated clones and permits only limited selective expansion of rare cancer gene–mutant clones. Depletion of the hybrid compartment shifts this protective state toward clonal expansion and inflammatory activation. These expanding clones are further constrained by a highly tumor-suppressive stromal microenvironment, driven by fibroblasts that adopt a metabolically restricted, non-inflammatory program. Together, our data uncover a multi-layered tumor-suppressive strategy that couples turnover-driven regeneration with an anti-permissive stromal niche to prevent malignant progression under mutagenic stress.