Cumulative reports reveal the protean nature of NOTCH signaling in the maintenance of normal and malignant hematopoiesis 
. While Notch2 signaling regulates regeneration of mouse long-term HSC, ligand-driven NOTCH1 activation induces human hematopoietic progenitor expansion and differentiation 
. Ligand binding to the NOTCH1 extracellular domain activates ADAM family metalloprotease and γ-secretase complex-mediated cleavage and intracellular release of the NOTCH1 intracellular domain (ICN1). Subsequently, nuclear translocation of ICN1 followed by engagement of transcriptional activators such as CBF1/Su(H)/Lag2 (CSL) and mastermind-like (MAML) sets the stage for NOTCH1 target gene transcription. Conversely, activation of NOTCH1 signaling through gain-of-function mutations in NOTCH1
, first described in T-ALL 
, or loss-of-function mutations in NOTCH1
regulators, such as FBXW7
, has been linked to therapeutic recalcitrance of hematologic malignancies 
. Chronic antagonism of both NOTCH1 and NOTCH2 processing with small molecule inhibitors of the γ-secretase complex has been associated with loss of intestinal crypt progenitor cells, thereby providing the impetus for development of selective NOTCH1 inhibitors 
. Recent pre-clinical studies demonstrate that inhibition of NOTCH1 using synthetic stapled peptides or monoclonal antibody-mediated strategies effectively decreases T-ALL cell line growth 
. However, the consequences of selective NOTCH1 inhibition for normal hematopoietic progenitor and patient-derived T-ALL LIC survival and self-renewal have been unclear.
In this study, CD34+ cells from 6 of 12 T-ALL samples harbored NOTCH1 activating mutations. In these patients, NOTCH1Mutated CD34+ LIC had greater engraftment and serial transplantation potential than their CD34− counterparts. Conversely, both CD34+ and CD34− subpopulations from NOTCH1WT samples harbored roughly equivalent bioluminescent engraftment potential, albeit at lower levels than NOTCH1Mutated LIC and with lower serial transplantation capacity. With the exception of one sample (patient 02) that harbored high NOTCH1 transcript levels in the absence of identifiable NOTCH1 mutations, bioluminescent imaging and FACS analyses of leukemic engraftment suggest that phenotypic markers other than CD34 will be needed to identify LIC in the NOTCH1WT samples. In contrast to experiments with NOTCH1WT and normal cord blood CD34+ samples, NOTCH1Mutated LIC survival was significantly impaired by selective hN1 mAb-mediated inhibition, concomitant with reductions in ICN1 and NOTCH1 mRNA expression and protein levels. Furthermore, serial transplantation potential was also reduced by hN1 mAb treatment of mice transplanted with NOTCH1-activated T-ALL samples. Thus, NOTCH1Mutated CD34+ cells from these pediatric T-ALL patients constitute the apex of a leukemic hierarchy.
Notably, patient samples with NOTCH1 activation, conferred either by mutation or elevated WT NOTCH1 expression levels, show enrichment of a subset of the CD34+
human progenitor cell population distinguished by co-expression of CD2 and CD7. Seminal studies reveal that CD7 expression enriches for a therapeutically recalcitrant LIC population 
. Our analyses of the serial transplantation capacity of the CD34+
population reveal that this population is maintained over multiple generations of T-ALL LIC transplantation, and these cells harbor robust leukemic initiating potential in medullary and extramedullary reservoirs of resistance. In experiments aimed at elucidating the fate of these cells in mice treated with hN1 mAb, we observed a significant reduction in this population compared to animals that received control IgG1 antibody. Taken together, these data further refine the markers that identify LIC in NOTCH1Mutated
T-ALL samples, and demonstrate that the CD34+
population is sensitive to and depleted following hN1 mAb treatment. While in the present studies, our analyses of the refined LIC marker were focused on the NOTCH1Mutated
samples, additional markers, or activation of other receptor-mediated signaling pathways such as insulin-like growth factor 1 receptor 
, may also be informative to determine the leukemic potential of LIC in non-NOTCH1Mutated
While mutations in tumor suppressor genes co-exist in some samples, NOTCH1Mutated
T-ALL LIC appear to be oncogenically addicted to NOTCH1 activation, rendering them uniquely susceptible to inhibition with a NOTCH1-targeted mAb, hN1. In contrast, hN1 mAb treatment did not significantly impair the survival of normal human hematopoietic progenitor cells. This favorable therapeutic index may be explained, at least in part, by mouse models of hematopoiesis, which demonstrate that Notch2, rather than Notch1, regulates mouse HSC regeneration 
. In summary, characterization of LIC based on functional molecular drivers provides a useful paradigm for identification and selective elimination of malignant stem cells. Moreover, these findings provide a compelling rationale for clinical evaluation of hN1 mAb therapy in clinical trials aimed at eliminating self-renewing LIC that promote therapeutic resistance and relapse in T-ALL and potentially in other NOTCH1-driven malignancies.