Notch pathway is regulated by leukemia microenvironmental cues and positively modulates IL-7 signaling

C295 Notch signals are critical in modulating normal and malignant T-cell development. Aberrant Notch activation by activating Notch1 mutations or deregulation of Notch degradation is an important genetic determinant in T-cell acute lymphoblastic leukemia (T-ALL). However, the mechanisms by which dysregulated Notch mediate proliferation and survival of leukemia T-cells is poorly understood. Increasing evidence shows that microenvironment cues crosstalk with genetic lesions to sustain tumor cells. IL-7, a pro-leukemogenic cytokine present in the leukemic microenvironment, plays a fundamental role in T-ALL biology, triggering multiple signaling pathways critical for cell expansion, survival and metabolic activity. Here, we investigated the potential interplay between Notch and IL-7 signaling in the biology of T-ALL. All primary T-ALL cases studied express Notch receptors and exhibit constitutive Notch activation, with the presence of truncated Notch1 (Notch1 IC ) and HES1 transcripts. Importantly, most T-ALL patients are responsive to Notch stimulation by recombinant Dll4, with more than 50% increase on IL-7-mediated cell proliferation. These observations indicate that primary leukemia T-cells, irrespective of the presence of Notch mutations, are responsive to Notch activation by extrinsic signals. Since primary leukemia T cells and cells in the leukemic microenvironment express Notch ligands, both homotypic and heterotypic signals may be operative in T-ALL. Accordingly, Notch activation was increased by cell-cell interactions among leukemia cells and between leukemia and BM endothelial cells. Since IL-7 is a main mediator of T-ALL cell survival and proliferation in the leukemic microenvironment, we evaluated whether Notch activation affects IL-7-mediated signals. The IL-7-dependent T-ALL line TAIL7 was transduced with constitutive active forms of Notch (TAIL7-ICN; TAIL7-ΔE). Overexpression of activated Notch per se failed to confer factor-independence to TAIL7 cells. However, TAIL7-ICN and TAIL7-ΔE showed marked proliferative and survival advantage over parental cells, an effect that required the presence of IL-7. Analysis of CD127/IL-7Rα expression showed that TAIL7-ICN and TAIL7-ΔE express higher levels of this receptor than control wild-type cells. Interestingly, in response to cellular stress, Notch-transduced cells were more efficient in inducing the transcription and surface expression of CD127; these effects were abrogated by Notch blockade with γ-secretase inhibitors (GSI). Notch blockade markedly inhibited IL-7-triggered activation of Akt, STAT5 and STAT3 signaling in leukemia T-cells. Finally, we observed that Notch blockade by GSI synergized with the targeted disruption of PI3K/Akt, mTOR, and Jak/STAT to inhibit IL-7-induced proliferation of T-ALL cells. In this work, we present evidence that Notch signaling in T-ALL is sustained by a partnership of cell-autonomous oncogenic mechanisms and microenvironmental signals. We show that by modulating CD127 expression, Notch signaling sensitizes leukemia T-cells to signals triggered by IL-7. These observations suggest that therapeutic targeting of signaling pathways in T-ALL should account for the critical role of microenvironmental signals in leukemogenesis and tumor progression.