NOTCH1 is a prevalent signaling pathway in T cell acute lymphoblastic leukemia (T-ALL), but crucial NOTCH1 downstream signals and target genes contributing to T-ALL pathogenesis cannot be retrospectively analyzed in patients and thus remain ill defined. This information is clinically relevant, as initiating lesions that lead to cell transformation and leukemia-initiating cell (LIC) activity are promising therapeutic targets against the major hurdle of T-ALL relapse. Here, we describe the generation in vivo of a human T cell leukemia that recapitulates T-ALL in patients, which arises de novo in immunodeficient mice reconstituted with human hematopoietic progenitors ectopically expressing active NOTCH1. This T-ALL model allowed us to identify CD44 as a direct NOTCH1 transcriptional target and to recognize CD44 overexpression as an early hallmark of preleukemic cells that engraft the BM and finally develop a clonal transplantable T-ALL that infiltrates lymphoid organs and brain. Notably, CD44 is shown to support crucial BM niche interactions necessary for LIC activity of human T-ALL xenografts and disease progression, highlighting the importance of the NOTCH1/CD44 axis in T-ALL pathogenesis. The observed therapeutic benefit of anti-CD44 antibody administration in xenotransplanted mice holds great promise for therapeutic purposes against T-ALL relapse.

Mitochondrial calcium (Ca 2ϩ ) dyshomeostasis constitutes a critical step in the metabolic crossroads leading to cell death. Therefore, we have studied here whether 7-chloro-5-(2-chlorophenyl)-1,5-dihydro-4,1-benzothiazepin-2(3H)-one (CGP37157; CGP), a blocker of the mitochondrial Na . This drastic cytoprotective effect of CGP could be explained in part through its regulatory actions on the mNCX.In general, it is accepted that a dysregulation of the mechanism that fine tunes the transient or more sustained levels of the cytosolic Ca 2ϩ concentrations ([Ca 2ϩ ] c ), leads to excitotoxic neuronal death (Schanne et al., 1979) and to neurodegeneration (Mattson, 2007). However, Ca 2ϩ may behave as both a cell survival supporter and a cell death inducer. For instance, cell depolarization and subsequent Ca 2ϩ entry into the cytosol helps to sustain the survival of cerebellar granule cells (Gallo et al., 1987) and bovine chromaffin cells (Orozco et al., 2006). However, chronic elevation of [Ca 2ϩ ] c by ionophores induces apoptosis (Martikainen et al., 1991). The opposite is also true, i.e., Ca 2ϩ antagonists that reduce [Ca 2ϩ ] c also cause neuronal death (Koh and Cotman, 1992) and chromaffin cell death (Novalbos et al., 1999). These apparent contradictory findings may be explained in the frame of the hypothesis suggesting that the [Ca 2ϩ ] c changes occurring during cell activation must move within a critical set point; beyond this point a cytoprotective signal might turn into a cytotoxic one (Koike et al., 1989). In this context, the suggestion of Nicholls (1985) and White and Reynolds (1995) that Ca 2ϩ accumulation into mitochondria could play a neuropro- Article, publication date, and citation information can be found at http://jpet.aspetjournals.org. doi:10.1124/jpet.109.154765.ABBREVIATIONS: mNCX, mitochondrial Na ϩ /Ca 2ϩ -exchanger; DMSO, dimethyl sulfoxide; FPL64176, FPL, 2,5-dimethyl-4-[2-(phenylmethyl)benzoyl]-1H-pyrrole-3-carboxylic acid methyl ester; 30 K ϩ /FPL, 30 mM K ϩ /0.3 M FPL; MTT formazan, 1-(4,5-dimethylthiazol-2-yl)-3,5-diphenylformazan, thiazolyl blue formazan; CGP37157, 7-chloro-5-(2-chlorophenyl)-1,5-dihydro-4,1-benzothiazepin-2(3H)-one; TTX, tetrodotoxin citrate, octahydro-12-(hydroxymethyl)-2-imino-5,9:7,10a-dimethan o-10aH- [1,3] dioxocino [6,5-d]pyrimidine

Key Points IL-7R expression is a functional biomarker of T-ALL cells with leukemia-initiating potential and plays a crucial role in T-ALL pathogenesis. Targeting IL-7R–mediated signaling hampers leukemia-initiating activity and progression of human T-ALL.