Thymocyte Reprogramming by the Scl, Lmo1 and Notch1 Oncogenes

Abstract 207

Normal thymic progenitors are devoid of self-renewal capacity, which is a distinctive stem cell property. These thymic progenitors progress into the thymus through several stages of differentiation (DN1, DN2-4, DP) before giving rise to CD4⁺ or CD8⁺ immunocompetent cells that are released into the periphery. Therfore, thymic output requires continuous seeding from stem cell-derived progenitors. T cell acute lymphoblastic leukemia (T-ALL) is a common cancer in children. Almost 25% of childhood T-ALL involve the SCL transcription factor and/or its nuclear partners LMO1/2 and more than 50% harbour gain of function mutations of NOTCH1. Using a transgenic mouse model that reproduces the human disease, we previously showed that activation of SCL, LMO1 and Notch1 in the thymus is sufficient to transform thymocyte progenitors and induce T-ALL (Tremblay M et al., 2010). Here, we explore the mechanism of transformation by these three oncogenes in primary thymocytes during the pre-leukemic stage. Our results indicate that the SCL and LMO1 oncogenes collaborate to confer an aberrant self-renewal potential to a subset of pre-leukemic thymocytes, via induction of a stem cell gene signature that also distinguishes primary T-ALL patient samples in which LMO2 is expressed. Furthermore, our clonality and functional analyses indicate that only a few clones of preleukemic thymocytes from Scl-Lmo1 mice are able to colonize the thymus of recipient mice in transplantation assays and produce mature T-cells during the pre-leukemic stage. Precisely, we show that self-renewal activity is enriched in DN3 thymocytes which eventually acquire Notch1 gain of function mutations and leukemia initiating activity. On the other hand, the Notch1 oncogene by itself does not confer self-renewal properties to thymocytes. Rather, we show that the Notch1 oncogene enhances the activity of the SCL-LMO1 oncogene to increase the frequency of pre-Leukemic Stem Cells (pre-LSCs) without modifying the clonal expansion of individual pre-LSC when transplanted at limiting dilutions. These results indicate that the Notch1 oncogene modifies the self-renewal activity enforced by the SCL^tgLMO1^tg oncogenes into a self-renewal of expansion, typical of a transformed state. Furthermore, NOTCH1 confers an invasive potential to SCL^tgLMO1^tg thymocytes that become thymus-independent and acquire the capacity to develop in peripheral organs. Therefore, our observations are consistent with the view that the SCL-LMO1 oncogenic transcription factors reprogram DN3 thymocytes to acquire self-renewal potential, thereby establishing a pre-leukemic state. Finally, NOTCH1 activation provides a strong signal that collaborates with the SCL-LMO1 oncogenes to induce T-ALL by favoring self-renewal divisions in pre-LSC together with an invasive capacity.