br Results br Discussion These

Results
Discussion These data identify a transmembrane molecule that negatively regulates AKT signaling via modulation of mTORC2 (Figure 4E). It joins the small number of mTORC2 upstream interactors, such as ribosome components (Zinzalla et al., 2011). Its transmembrane, putative cell-surface localization suggests that it may be a means by which exogenous cues can dampen mTORC2 activation. We identified this molecule by virtue of its amplification in the context of a microenvironmentally induced AML (Raaijmakers et al., 2010), in line with AKT playing a tumor-suppressive role in a substantial proportion of patients with this disease and in maintenance of leukemia stem Decitabine in an animal model (Sykes et al., 2011). However, we cannot draw a direct link between UT2 amplification and aberrant signals from the modified stroma in that model. Of note, UT2 had modest effects on normal hematopoietic function (Figure S4). We did observe that enforced UT2-expressing donor cells contributed to lower levels of blood cells (Figure S4B), the frequency of Lin−SCA1+c-KIT+ (L−S+K+) cells (Figure S4C), and colony formation as compared with control cells (Figure S4D). Moreover, hematopoietic cells from the BM of animals with the altered microenvironment showed evidence of decreased pAKTS473 (Figure S1I) consistent with a role for UT2, but whether that was then selected for in the emergence of the leukemia cannot be discerned by our studies to date. In T-ALL, where mTORC2/AKT plays a clearer oncogenic role, we were able to demonstrate that UT2 could be a therapeutic target to modulate that disease. Collectively, our work points to the value of models that can be used to examine niche contributions to oncogenesis and reveals a previously unrecognized transmembrane modulator of a critical pathway with therapeutic implications for cancer.
Experimental Procedures
Author Contributions
Acknowledgments
Introduction The IKAROS transcription factor is essential for normal mouse lymphopoiesis, and its suppression by dominant-negative isoforms produces T cell tumors (Payne and Dovat, 2011). In human cells, the IK6 dominant-negative isoform has been reported to inhibit erythroid and B cell production (Dijon et al., 2008; Tonnelle et al., 2001; Tonnelle et al., 2009) and to produce an acute leukemia in cord blood cells cotransduced with a virus encoding BCR-ABL1 (Theocharides et al., 2014). A greater understanding of the role of IKAROS in the human blood system is of particular interest given the high frequency of inactivating mutations in IKZF1 (encoding IKAROS) in human B cell leukemias as well as occasional myeloid malignancies (Grossmann et al., 2011; Jäger et al., 2010; Mullighan et al., 2008; Nacheva et al., 2013; Nakayama et al., 1999).
Results
Discussion Growing evidence suggests that the mechanisms by which signaling networks regulate normal human HSC self-renewal and lineage restriction are highly complex processes that involve many genes implicated in human leukemogenesis. Nevertheless, the role in normal human hematopoiesis played by many of these leukemia-associated genes remains poorly understood. Much has been learned from studies of genetically manipulated mouse cells, in which the expression of genes from an endogenous promoter can be controlled both temporally and in a lineage-specific manner. However, the conclusions derived from mouse models are necessarily limited by species-specific differences (Payne and Crooks, 2007; Rangarajan and Weinberg, 2003). Fortunately, recent technical advances are making it increasingly possible to assess the consequences of direct gene manipulation in human hematopoietic cells. These include the development of lentiviral vectors that can efficiently transduce primitive human hematopoietic cell populations, improved multiparameter flow cytometry, array-based techniques applicable to small numbers of cells, and the generation of highly immunodeficient long-lived strains of mice that can reproducibly support sustained, high-level reconstitution with human hematopoietic cells (Billerbeck et al., 2011; Doulatov et al., 2012; Miller et al., 2013). Here, we exploited such methods to examine and compare the cellular and molecular consequences of IK6 in primitive normal human and mouse hematopoietic cells. In addition, we made use of an experimental design in which matched numbers of IK6- and control-transduced cells were cotransplanted into the same recipients or analyzed in the same cultures to enable a precise and accurate comparison of absolute (rather than relative) lineage contributions not necessarily otherwise possible.