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Notch plays critical roles in both cell fate decisions and tumorigenesis. Notch receptor engagement initiates signaling cascades that include a phosphatidylinositol 3-kinase/target of rapamycin (TOR) pathway. Mammalian TOR (mTOR) participates in two distinct biochemical complexes, mTORC1 and mTORC2, and the relationship between mTORC2 and physiological outcomes dependent on Notch signaling is unknown. In this study, we report contributions of mTORC2 to thymic T-cell acute lymphoblastic leukemia (T-ALL) driven by Notch. Conditional deletion of Rictor, an essential component of mTORC2, impaired Notch-driven proliferation and differentiation of pre-T cells. Furthermore, NF-κB activity depended on the integrity of mTORC2 in thymocytes. Active Akt restored NF-κB activation, a normal rate of proliferation, and differentiation of Rictor-deficient pre-T cells. Strikingly, mTORC2 depletion lowered CCR7 expression in thymocytes and leukemic cells, accompanied by decreased tissue invasion and delayed mortality in T-ALL driven by Notch. Collectively, these findings reveal roles for mTORC2 in promoting thymic T cell development and T-ALL and indicate that mTORC2 is crucial for Notch signaling to regulate Akt and NF-κB.

LMO2 is a target of chromosomal translocations in T-cell tumors and was activated by retroviral vector insertions in T-cell tumors from X-SCID patients in gene therapy trials. To better understand the cooperating genetic events in LMO2-associated T-cell acute lymphoblastic leukemia (T-ALL), we investigated the roles of Arftumor suppressor loss and Notch activation in murine models of transplantation. Lmo2 overexpression enhanced the expansion of primitive DN2 thymocytes, eventually facilitating the stochastic induction of clonal CD4+/CD8+malignancies. Inactivation of the Arftumor suppressor further increased the self-renewal capacity of the primitive, preleukemic thymocyte pool and accelerated the development of aggressive, Lmo2-induced T-cell lympholeukemias. Notch mutations were frequently detected in these Lmo2-induced tumors. The Arfpromoter was not directly engaged by Lmo2 or mutant Notch, and use of a mouse model in which activation of a mutant Notch allele depends on previous engagement of the Arfpromoter revealed that Notch activation could occur as a subsequent event in T-cell tumorigenesis. Therefore, Lmo2 cooperates with Arfloss to enhance self-renewal in primitive thymocytes. Notch mutation and Arfinactivation appear to independently cooperate in no requisite order with Lmo2 overexpression in inducing T-ALL, and all 3 events remained insufficient to guarantee immediate tumor development.

Frequent hallmarks of T-cell acute lymphoblastic leukemia (T-ALL) include aberrant NOTCH signaling and deletion of the CDKN2Alocus, which contains 2 closely linked tumor suppressor genes (INK4Aand ARF). When bone marrow cells or thymocytes transduced with a vector encoding the constitutively activated intracellular domain of Notch1 (ICN1) are expanded ex vivo under conditions that support T-cell development, cultured progenitors rapidly induce CD4+/CD8+T-ALLs after infusion into healthy syngeneic mice. Under these conditions, enforced ICN1 expression also drives formation of T-ALLs in unconditioned CD-1 nude mice, bypassing any requirements for thymic maturation. Retention of Arfhad relatively modest activity in suppressing the formation of T-ALLs arising from bone marrow–derived ICN1+progenitors in which the locus is epigenetically silenced, and all resulting Arf+/+tumors failed to express the p19Arfprotein. In striking contrast, retention of Arfin thymocyte-derived ICN1+donor cells significantly delayed disease onset and suppressed the penetrance of T-ALL. Use of cultured thymocyte-derived donor cells expressing a functionally null Arf-GFPknock-in allele confirmed that ICN1 signaling can induce Arfexpression in vivo. Arfactivation by ICN1 in T cells thereby provides stage-specific tumor suppression but also a strong selective pressure for deletion of the locus in T-ALL.

Comment on: Volanakis EJ, et al. Blood 2009; 114:4451-9.

Abstract 143