STAT3 Is Required for Notch-Induced Leukemogenesis, and Functions As a Critical Survival Effector in T-Cell Leukemia

Abstract 920

The disruption of main signaling mediators of critical leukemia cell functions represents the most promising approach for the development of more effective targeted therapies for acute lymphoblastic leukemia (ALL). Increasing evidence suggests an important combinatorial role for cell autonomous, epigenetic and extrinsic factors in tumorigenesis and tumor progression. However, little is known on how oncogenic and microenvironmental signals are integrated within leukemia T-cells and how they influence T-cell leukemogenesis. Our studies in patients' leukemia T-cells showed that oncogenic Notch enhances their fitness by regulating the transcription of IL-7Rα, thus increasing the sensitivity of leukemia cells to IL-7, which triggers survival and mitogenic pathways. Interestingly, analysis of the Notch1/CBF-1 complex regulating IL7-Rα transcription show that it contains STAT3, but not STAT5, the main STAT molecule implicated in lymphoid cell signaling. Further analyses showed that STAT3 signaling is active in patients' T-ALL specimens and in leukemia cells from mice with Notch-induced leukemia. We observed that IL-7 signaling triggers STAT3 phosphorylation and transcriptional activity, regulating the expression of the STAT3 target genes Bcl-xL, survivin, Cyclin D3 and miR-21.

We then performed studies using a genetic model of conditional STAT3 deletion in the hematopoietic system. We observed that STAT3 loss-of-function markedly decreased the response of T-lineage cells (including thymocytes) to IL-7 stimulation, even if these cells exhibit functional STAT5 signaling. Importantly, in comparison to wild-type animals, STAT3-null mice did not exhibit defects in common lymphoid progenitors (CLP) in the bone marrow (BM) or in T-cell development, namely on DN thymocyte subsets. Also, no changes were observed in STAT3-null mice in the surface expression or transcriptional levels of IL-7Rα in CLP or thymocytes. To investigate the putative role of STAT3 in leukemia development, we performed the Notch-induced tumor model using BM primitive hematopoietic cells from STAT3-null mice, which could be effectively transduced with the constitutively-active Notch (ICN) construct. We observed that STAT3 deletion significantly inhibited the development of T-cell leukemia, with animals surviving for long periods without evidence of leukemia. Importantly, this was not due to defects in homing or in early engraftment of transplanted cells, which was further documented using intravital microscopy.

Finally, pre-clinical studies were performed to evaluate the therapeutic potential of STAT3 pharmacological targeting on leukemia T-cells. STAT3 blockade by the selective inhibitors STATTIC or S3I-201 or the JAK2 inhibitor WP-1066 abrogated STAT3 phosphorylation, inhibiting the transcription of Bcl-xL and Survivin. This inhibition resulted in marked inhibition of leukemia T-cells, which was observed in primary T-ALL cells, in a IL-7-dependent T-ALL line and in leukemia cells from mice carrying ICN-induced leukemia. Mechanistically, we observed that STAT3 blockade results in and massive leukemia cell apoptosis, which is associated with activation of the pro-apoptotic p53(Ser46), and PARP cleavage. Also, STAT3 silencing by shRNA prevented leukemia cell expansion. Strong anti-leukemia synergism was observed between low doses of STATTIC and the frontline therapeutic drug Dexamethasone, or selective PI3K or mTOR inhibitors.

In summary, our studies indicate that STAT3 signaling plays an important role in the integration of IL-7 and Notch signals in T-cell leukemia, and unveiled an unexpected role for STAT3 in T-cell leukemogenesis. Finally, our work suggests that the targeted disruption of STAT3 represents a valid therapeutic approach in this cancer.