Notch Expression Expands the Pre-Malignant Pool of T-Cell Acute Lymphoblastic Leukemia Clones without Affecting Self-Renewal

Abstract 3161

60% of human T-cell acute lymphoblastic leukemias (T-ALL) harbor NOTCH1 activating mutations, making it the most commonly mutated oncogene in T-ALL. Notch signaling is critical for T cell development, and activating Notch mutations are found in all subtypes of T-ALL, suggesting that Notch deregulation may be a dominant initiating event in human disease. In human and rodent models of T-ALL, Notch directly induces cMyc expression. However, cMyc over expression cannot completely rescue Notch inhibition, suggesting that Notch may have other important roles in T-ALL progression. Classic viral insertion screens in mice have indentified that insertional activation of Notch1 is common in Myc induced T-cell malignancies, suggesting that Notch imparts a distinct advantage to leukemic clones independent of cMyc.

Notch-induced transgenic zebrafish models of T-ALLs are unique in that Notch signaling does not induce cMyc expression, allowing new opportunities to determine the function of Notch which are independent of cMyc. As with rodent models, the co-expression of Notch and cMyc in zebrafish T cells significantly enhanced T-ALL progression compared to cMyc or Notch alone (p<0.001). However, Notch co-expression with Myc did not enhance proliferation, alter cell cycle kinetics, or modify apoptosis in leukemic cells when compared to Myc alone expressing T-ALLs. Moreover, clonality assays using RT-PCR analyses for T-cell receptor-beta rearrangements indicate that Notch collaborates with Myc to increase the number of T-ALL clones contained within the primary tumor by 2–4 fold when compared to single transgenic animals that express only cMyc. Following serial transplantation, a large portion of T-ALL clones present in primary Notch/Myc leukemias are not found in transplanted animals. This starkly contrasts to results seen in Myc-alone expressing T-ALLs where all primary clones are capable of engraftment and reinitiation of leukemia. Paradoxically, transplant animals developing T-ALL from leukemias that coexpress Notch and Myc have similar numbers of clones as found in primary Myc-induced leukemias. Primary Myc-induced T-ALLs express high levels of endogenous scl and lmo2, recapitulating the most common and treatment resistant subtype in human T-ALL. By contrast, T-ALLs that co-express Notch and Myc fail to upregulate any of the T-ALL oncogenes; however, following transplantation into recipient animals, double transgenic Notch/Myc leukemias now express high levels of scl and lmo2. Finally, large scale limiting dilution cell transplantation analyses using syngeneic zebrafish demonstrated that Notch does not collaborate with Myc to increase self-renewal of leukemia initiating cells (LICs). Primary T-ALLs expressing both Notch and Myc have 10-fold less leukemia-initiating frequency when compared to T-ALLs that express only cMyc; however, following serial passaging, these Notch/Myc leukemias exhibit similar leukemia-initiating frequency as Myc-induced T-ALLs. Taken together, our data supports a model where Notch expands a pool of pre-malignant T-ALL clones within the primary tumor, a subset of which acquire additional mutations to confer a fully transformed phenotype. By contrast, Myc alone is insufficient to increase the overall pool of pre-malignant clones but confers a fully-transformed phenotype to leukemic cells accounting for the longer latency likely reflecting acquisition of additional genetic changes in clones. Our data may explain why a subset of relapse human T-ALLs develop disease from an underrepresented clone found in the primary leukemia. Primary human T-ALLs likely have a large pool of premalignant clones resulting from NOTCH-pathway activation that are unable to self-renew and thus, cannot give rise to relapsed T-ALL.