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Aberrant activation of β-catenin is a common event in AML and is an independent predictor of poor prognosis. Although increased β-catenin signaling in AML has been associated with oncogenic translocation products and activating mutations in the FLT3R, the mechanisms that activate β-catenin in AML more broadly are still unclear. Here, we describe a novel link between IL-3 signaling and the regulation of β-catenin in myeloid transformation and AML. In a murine model of HoxB8 and IL-3 cooperation, we show that β-catenin protein levels are modulated by IL-3 and that Cre-induced deletion of β-catenin abolishes IL-3-dependent growth and colony formation. In IL-3-dependent leukemic TF-1.8 cells, we observed increased β-catenin protein levels and nuclear localization in response to IL-3, and this correlated with transcriptional induction of β-catenin target genes. Furthermore, IL-3 promoted β-catenin accumulation in a subset of AML patient samples, and gene-expression profiling of these cells revealed induction of WNT/β-catenin and TCF4 gene signatures in an IL-3-dependent manner. This study is the first to link β-catenin activation to IL-3 and suggests that targeting IL-3 signaling may be an effective approach for the inhibition of β-catenin activity in some patients with AML., (© 2014 Society for Leukocyte Biology.)

p53 is critical in the normal response to a variety of cellular stresses including DNA damage and loss of p53 function is a common feature of many cancers. In hematological malignancies, p53 deletion is less common than in solid malignancies but is associated with poor prognosis and resistance to chemotherapy. Compared to their wild-type (WT) counterparts, hematopoietic progenitor cells lacking p53 have a greater propensity to survive cytokine loss, in part, due to the failure to transcribe Puma, a proapoptotic Bcl-2 family member. Using expression arrays, we have further characterized the differences that distinguish p53(-/-) cells from WT myeloid cells in the presence of Interleukin-3 (IL-3) to determine if such differences contribute to the increased clonogenicity and survival responses observed in p53(-/-) cells. We show that p53(-/-) cells have a deregulated intracellular signaling environment and display a more rapid and sustained response to IL-3. This was accompanied by an increase in active ERK1/2 and a dependence on an intact MAP kinase signaling pathway. Contrastingly, we find that p53(-/-) cells are independent on AKT for their survival. Thus, loss of p53 in myeloid cells results in an altered transcriptional and kinase signaling environment that favors enhanced cytokine signaling.

Loss of p53-dependent apoptosis contributes to the development of hematologic malignancies and failure to respond to treatment. Proapoptotic Bcl-2 family member Puma is essential for apoptosis in HoxB8-immortalized interleukin-3 (IL-3)-dependent myeloid cell lines (FDM cells) provoked by IL-3 deprivation. p53 and FoxO3a can transcriptionally regulate Puma. To investigate which transcriptional regulator is responsible for IL-3 deprivation-induced Puma expression and apoptosis, we generated wild-type (WT), p53(-/-), and FoxO3a(-/-) FDM cells and found that p53(-/-) but not FoxO3a(-/-) cells were protected against IL-3 withdrawal. Loss of p21(cip/waf), which is critical for p53-mediated cell-cycle arrest, afforded no protection against IL-3 deprivation. A survival advantage was also observed in untransformed p53(-/-) hematopoietic progenitor cells cultured in the presence or absence of cytokines. In response to IL-3 deprivation, increased Puma protein levels in p53(-/-) cells were substantially delayed compared with WT cells. Increased p53 transcriptional activity was detected after cytokine deprivation. This was substantially less than that induced by DNA damage and associated not with increased p53 protein levels but with loss of the p53 regulator, MDM2. Thus, we conclude that p53 protein is activated after IL-3 deprivation by loss of MDM2. Activated p53 transcriptionally up-regulates Puma, which initiates apoptosis.

Growth and survival of hematopoietic cells is regulated by growth factors and cytokines, such as interleukin 3 (IL-3). When cytokine is removed, cells dependent on IL-3 kill themselves by a mechanism that is inhibited by overexpression of Bcl-2 and is likely to be mediated by proapoptotic Bcl-2 family members. Bad and Bim are 2 such BH3-only Bcl-2 family members that have been implicated as key initiators in apoptosis following growth factor withdrawal, particularly in IL-3-dependent cells. To test the role of Bad, Bim, and other proapoptotic Bcl-2 family members in IL-3 withdrawal-induced apoptosis, we generated IL-3-dependent cell lines from mice lacking the genes for Bad, Bim, Puma, both Bad and Bim, and both Bax and Bak. Surprisingly, Bad was not required for cell death following IL-3 withdrawal, suggesting changes to phosphorylation of Bad play only a minor role in apoptosis in this system. Deletion of Bim also had no effect, but cells lacking Puma survived and formed colonies when IL-3 was restored. Inhibition of the PI3 kinase pathway promoted apoptosis in the presence or absence of IL-3 and did not require Bad, Bim, or Puma, suggesting IL-3 receptor survival signals and PI3 kinase survival signals are independent.

Granulocyte-macrophage colony-stimulating factor (GM-CSF) is a pluripotent cytokine produced by many cells in the body, which regulates normal and malignant hemopoiesis as well as innate and adaptive immunity. GM-CSF assembles and activates its heterodimeric receptor complex on the surface of myeloid cells, initiating multiple signaling pathways that control key functions such as cell survival, cell proliferation, and functional activation. Understanding the molecular composition of these pathways, the interaction of the various components as well as the kinetics and dose-dependent mechanics of receptor activation provides valuable insights into the function of GM-CSF as well as the related cytokines, interleukin-3 and interleukin-5. This knowledge provides opportunities for the development of new therapies to block the action of these cytokines in hematological malignancy and chronic inflammation. [ABSTRACT FROM AUTHOR]

Summary: Puma (p53 upregulated modulator of apoptosis) belongs to the BH3 (Bcl-2 homology 3)-only protein family of apoptotic regulators. Its expression is induced by various apoptotic stimuli, including irradiation and cytokine withdrawal. Using an inducible system to express Puma, we investigated the nature of Puma-induced apoptosis. In BaF3 cells, expression of Puma caused rapid caspase-mediated cleavage of ICAD (inhibitor of caspase-activated deoxyribonuclease) and Mcl-1 (myeloid cell leukemia 1), leading to complete loss of cell viability. Surprisingly, Puma protein levels peaked within 2 h of its induction and subsequently declined to basal levels. Maximal Puma abundance coincided with the onset of caspase activity. Subsequent loss of Puma was prevented by the inhibition of caspases, indicating that its degradation was caspase dependent. In cells expressing transfected Bcl-2, induced Puma reached significantly higher levels, but after a delay, caspases became active and cell death occurred. Puma co-immunoprecipitated endogenous Bcl-2 and Mcl-1 but not Bax and Bak, suggesting that Puma did not associate with either Bax or Bak in these cells to initiate cell death. In mouse embryonic fibroblasts (MEFs), the amount of Puma peaked within 4 h of its induction. In contrast, in bax/bak double-knockout MEFs, Puma was stably expressed following its induction and was unable to trigger apoptosis even at very high levels. Overexpression of Bcl-2 in wild-type MEFs, like in BaF3 cells, resulted in higher levels of Puma being reached but did not prevent cell death from occurring. These results demonstrate that the level of the Bcl-2 prosurvival family sets the threshold at which Puma is able to indirectly activate Bax or Bak, leading in turn to activation of caspases that not only cause cell death but also rapidly induce Puma degradation. [Copyright &y& Elsevier]