Impact of cell-type–specific circadian disruption in pediatric B-ALL on clock-based biomarkers with implications for chronotherapy.

Abstract

6541 Background: B-cell acute lymphoblastic leukaemia (B-ALL) is the most common paediatric malignancy. While cure rates exceed 90%, treatment remains intensive and is frequently associated with relapse, toxicity, and long-term adverse effects. The circadian clock regulates DNA repair, metabolism, immune function, and drug pharmacokinetics—processes central to cancer therapy. Although treatment timing has been shown to influence outcomes in childhood ALL, the molecular circadian landscape of malignant versus healthy hematopoietic cells in paediatric patients remains unexplored. Methods: We performed an integrated molecular and clinical circadian profiling study in paediatric ALL patients (N=7) and age-matched healthy controls (N=10). Peripheral blood, bone marrow, and saliva samples were collected at diagnosis. CD19⁺ (leukemic B cells) and paired CD19⁻ (non-B cells) populations were isolated and used to establish an internal, patient-specific circadian baseline. Expression of ~800 cancer-, immune-, and clock-related genes was quantified using NanoString SPRINT technology. Rhythmicity parameters (acrophase, amplitude, MESOR), differential expression, and machine-learning–based classification were integrated with longitudinal clinical and physiological data. Results: Malignant CD19⁺ cells exhibited marked circadian dysregulation compared with paired CD19⁻ cells from the same patients. Core clock architecture was altered specifically in leukemic cells, including disrupted BMAL1–PER2 phase relationships and reduced rhythmic amplitude. CD19 expression itself displayed cell-type–specific circadian modulation, with higher amplitude and phase variability in CD19⁺ cells. In contrast, CD19⁻ cells retained more coherent circadian organization, supporting their use as an internal normalization reference. Preliminary analyses suggest that the degree of circadian misalignment between CD19⁺ and CD19⁻ compartments correlates with treatment dynamics, including early response and therapy duration. Conclusions: Our findings reveal profound, cell-intrinsic circadian disruption in paediatric B-ALL and demonstrate that comparing malignant CD19⁺ cells against paired non-malignant CD19⁻ cells enables robust detection of clock dysregulation. These results provide a molecular framework for developing circadian biomarkers and support the rational design of chronotherapy strategies aimed at optimising efficacy while minimising toxicity in paediatric leukaemia.