Altered thymic niche synergistically drives the massive proliferation of malignant thymocytes

ABSTRACT The discovery of genetic alterations in patient samples over the last decades has reinforced a cell-autonomous view of proliferative expansion during T- cell acute lymphoblastic leukemia (T-ALL) development in the thymus. However, the potential contribution of non-cell-autonomous factors, particularly the impact of thymic epithelial cells (TECs) within the thymic niche during the initiation phase, remains unexplored. In this study, we combine a cell-based computational model of the thymus with complementary in vivo experiments in medaka (Oryzias latipes) to systematically analyze the impact of 12 cell-autonomous and non- autonomous factors, individually and in combination, on the proliferation of normal and malignant thymocytes carrying interleukin-7 receptor (IL7R) gain-of-function mutations or elevated IL7R levels, as observed in T-ALL patients. By simulating over 1500 scenarios, we show that while a dense TEC network favored the proliferation of normal thymocytes, it inhibited the proliferation of malignant lineages, which achieved their maximal proliferative capacity when TECs were sparsely distributed. Our in silico model further predicts that specific mutations could accelerate proliferative expansion within a few days. This prediction was experimentally validated, revealing the rapid onset of thymic lymphoma and systemic infiltration of malignant T-cells within just 8 days of embryonic development. These findings demonstrate that synergistic interaction between oncogenic alterations and modifications in the thymic niche can significantly accelerate disease progression. Our results also suggest that negative feedback from the proliferative state suppresses thymocyte differentiation. Overall, this multidisciplinary work reveals the critical role of TEC-thymocyte interactions in both the initiation and progression of T-ALL, highlighting the importance of the thymic microenvironment in early leukemogenesis. Competing Interest Statement The authors have declared no competing interest. Footnotes We wrote an extensive Appendix to better explain the model implementation. The Abstract was rewritten to improve clarity on what was done and to highlight the main findings. Subheadings to paragraphs were rewritten to better emphasize the main findings. Font sizes in Figure 2J and Figure 4E were increased to improve readability. The spacing of graphical elements in the legend of Figure 4E was improved. An error in Figure 5B was corrected (the legend labels had been accidentally swapped). A new supplementary figure to Figure 4 shows the sensitivity of clone size in control simulations for a subset of the tested parameter combinations. The Conclusion section was rewritten to better highlight limitations of the study and Improve the summary of the main findings. Minor wording improvements were done throughout the text to improve readability.