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Development of normal blood cells is often suppressed in juvenile myelomonocytic leukemia (JMML), a myeloproliferative neoplasm (MPN) of childhood, causing complications and impacting therapeutic outcomes. However, the mechanism underlying this phenomenon remains uncharacterized. To address this question, we induced the most common mutation identified in JMML (Ptpn11E76K) specifically in the myeloid lineage with hematopoietic stem cells (HSCs) spared. These mice uniformly developed a JMML-like MPN. Importantly, HSCs in the same bone marrow (BM) microenvironment were aberrantly activated and differentiated at the expense of self-renewal. As a result, HSCs lost quiescence and became exhausted. A similar result was observed in wild-type (WT) donor HSCs when co-transplanted with Ptpn11E76K/+ BM cells into WT mice. Co-culture testing demonstrated that JMML/MPN cells robustly accelerated differentiation in mouse and human normal hematopoietic stem/progenitor cells. Cytokine profiling revealed that Ptpn11E76K/+ MPN cells produced excessive IL-1β, but not IL-6, T NF-α, IFN-γ, IL-1α, or other inflammatory cytokines. Depletion of the IL-1β receptor effectively restored HSC quiescence, normalized their pool size, and rescued them from exhaustion in Ptpn11E76K/+/IL-1R-/- double mutant mice. These findings suggest IL-1β signaling as a potential therapeutic target for preserving normal hematopoietic development in JMML.

Aberrant cytokine signaling initiated from mutant receptor tyrosine kinases (RTKs) provides critical growth and survival signals in high risk acute myeloid leukemia (AML). Inhibitors to FLT3 have already been tested in clinical trials, however, drug resistance limits clinical efficacy. Mutant receptor tyrosine kinases are mislocalized in the endoplasmic reticulum (ER) of AML and play an important role in the non-canonical activation of signal transducer and activator of transcription 5 (STAT5). Here, we have tested a potent new drug called imipramine blue (IB), which is a chimeric molecule with a dual mechanism of action. At 200-300 nM concentrations, IB is a potent inhibitor of STAT5 through liberation of endogenous phosphatase activity following NADPH oxidase (NOX) inhibition. However, at 75-150 nM concentrations, IB was highly effective at killing mutant FLT3-driven AML cells through a similar mechanism as thapsigargin (TG), involving increased cytosolic calcium. IB also potently inhibited survival of primary human FLT3/ITD+ AML cells compared to FLT3/ITDneg cells and spared normal umbilical cord blood cells. Therefore, IB functions through a mechanism involving vulnerability to dysregulated calcium metabolism and the combination of fusing a lipophilic amine to a NOX inhibiting dye shows promise for further pre-clinical development for targeting high risk AML.

Inflammatory Bowel Disease (IBD) is a multi-factorial chronic inflammation of the gastrointestinal tract prognostically linked to CD8+ T-cells, but little is known about their mechanism of activation during initiation of colitis. Here, Grb2-associated binding 2/3 adaptor protein double knockout mice (Gab2/3−/−) were generated. Gab2/3−/− mice, but not single knockout mice, developed spontaneous colitis. To analyze the cellular mechanism, reciprocal bone marrow (BM) transplantation demonstrated a Gab2/3−/− hematopoietic disease-initiating process. Adoptive transfer showed individual roles for macrophages and T-cells in promoting colitis development in vivo. In spontaneous disease, intestinal intraepithelial CD8+ but much fewer CD4+, T-cells from Gab2/3−/− mice with rectal prolapse were more proliferative. To analyze the molecular mechanism, reduced PI3-kinase/Akt/mTORC1 was observed in macrophages and T-cells, with interleukin (IL)-2 stimulated T-cells showing increased pSTAT5. These results illustrate the importance of Gab2/3 collectively in signaling responses required to control macrophage and CD8+ T-cell activation and suppress chronic colitis. [ABSTRACT FROM AUTHOR]

Reactive oxygen species (ROS) are now recognized as important second messengers with roles in many aspects of signaling during leukemogenesis. They serve as critical cell signaling molecules that regulate the activity of various enzymes including tyrosine phosphatases. ROS can induce inactivation of tyrosine phosphatases, which counteract the effects of tyrosine kinases. ROS increase phosphorylation of many proteins including signal transducer and activator of transcription-5 (STAT5) via Janus kinases (JAKs). STAT5 is aberrantly activated through phosphorylation in many types of cancer and this constitutive activation is associated with cell survival, proliferation, and self-renewal. Such leukemic activation of STAT5 is rarely caused by mutation of the STAT5 gene itself but instead by overactive mutant receptors with tyrosine kinase activity as well as JAK, SRC family protein tyrosine kinases (SFKs), and Abelson murine leukemia viral oncogene homolog (ABL) kinases. Interestingly, STAT5 suppresses transcription of several genes encoding antioxidant enzymes while simultaneously enhancing transcription of NADPH oxidase. By doing so, STAT5 activation promotes an overall elevation of ROS level, which acts as a feed-forward loop, especially in high risk Fms-related tyrosine kinase 3 (FLT3) mutant leukemia. Therefore, efforts have been made recently to target ROS in cancer cells. Drugs that are able to either quench ROS production or inversely augment ROS-related signaling pathways both have potential as cancer therapies and may afford some selectivity by activating feedback inhibition of the ROS-STAT5 kinome. This review summarizes the cooperative relationship between ROS and STAT5 and explores the pros and cons of emerging ROS-targeting therapies that are selective for leukemia characterized by persistent STAT5 phosphorylation. [ABSTRACT FROM AUTHOR]

The difficulty in maintaining the reconstituting capabilities of hematopoietic stem cells (HSCs) in culture outside of the bone marrow microenvironment has severely limited their utilization for clinical therapy. This hurdle is largely due to the differentiation of long-term stem cells. Emerging evidence suggests that energy metabolism plays an important role in coordinating HSC self-renewal and differentiation. Here, we show that treatment with alexidine dihydrochloride, an antibiotic and a selective inhibitor of the mitochondrial phosphatase Ptpmt1, which is crucial for the differentiation of HSCs, reprogrammed cellular metabolism from mitochondrial aerobic metabolism to glycolysis, resulting in a remarkable preservation of long-term HSCs ex vivo in part through hyperactivation of adenosine 5'-monophosphate-activated protein kinase (AMPK). In addition, inhibition of mitochondrial metabolism and activation of AMPK by metformin, a diabetes drug, also decreased differentiation and helped maintain stem cells in culture. Thus, manipulating metabolic pathways represents an effective new strategy for ex vivo maintenance of HSCs. [ABSTRACT FROM AUTHOR]

Inflammatory Bowel Disease (IBD) is a multi-factorial chronic inflammation of the gastrointestinal tract prognostically linked to CD8 + T-cells, but little is known about their mechanism of activation during initiation of colitis. Here, Grb2-associated binding 2/3 adaptor protein double knockout mice (Gab2/3 -/- ) were generated. Gab2/3 -/- mice, but not single knockout mice, developed spontaneous colitis. To analyze the cellular mechanism, reciprocal bone marrow (BM) transplantation demonstrated a Gab2/3 -/- hematopoietic disease-initiating process. Adoptive transfer showed individual roles for macrophages and T-cells in promoting colitis development in vivo . In spontaneous disease, intestinal intraepithelial CD8 + but much fewer CD4 + , T-cells from Gab2/3 -/- mice with rectal prolapse were more proliferative. To analyze the molecular mechanism, reduced PI3-kinase/Akt/mTORC1 was observed in macrophages and T-cells, with interleukin (IL)-2 stimulated T-cells showing increased pSTAT5. These results illustrate the importance of Gab2/3 collectively in signaling responses required to control macrophage and CD8 + T-cell activation and suppress chronic colitis.

STAT5 transcription factors are frequently activated in hematopoietic neoplasms and are targets of various tyrosine kinase oncogenes. Evidences for a crosstalk between STAT5 and reactive oxygen species (ROS) metabolism have recently emerged but mechanisms involved in STAT5-mediated regulation of ROS still remain elusive. We demonstrate that sustained activation of STAT5 induced by Bcr-Abl in chronic myeloid leukemia (CML) cells promotes ROS production by repressing expression of two antioxidant enzymes, catalase and glutaredoxin-1(Glrx1). Downregulation of catalase and Glrx1 expression was also observed in primary cells from CML patients. Catalase was shown not only to reduce ROS levels but also, to induce quiescence in Bcr-Abl-positive leukemia cells. Furthermore, reduction of STAT5 phosphorylation and upregulation of catalase and Glrx1 were also evidenced in leukemia cells co-cultured with bone marrow stromal cells to mimic a leukemic niche. This caused downregulation of ROS levels and enhancement of leukemic cell quiescence. These data support a role of persistent STAT5 signaling in the regulation of ROS production in myeloid leukemias and highlight the repression of antioxidant defenses as an important regulatory mechanism.

A significant role of the microenvironment in leukemogenesis is beginning to emerge. The leukemia cell microenvironment consists of not only the stromal and endothelial cell components but also the normal hematopoietic cells. Signal transducer and activator of transcription 5 (STAT5) is a latent transcription factor that is normally transiently activated by phosphorylation in response to microenvironmental signals. In hematopoietic cells, persistently activated STAT5 via aberrant receptor signaling, Janus kinases (JAKs), or intracellular tyrosine kinases is a bona fide driver of leukemogenesis. However, active IL-7/STAT5 signaling also protects the early B-cell genome by suppressing error-prone recombination and vulnerability to transformation. Along these lines, we have reported that lymphocyte development from transplanted STAT5-deficient fetal liver cells was blocked at the pre-pro-B-cell stage but when combined with transgenic Myc and Bcl-2 promoted faster initiation of B-ALL. Furthermore, inflammatory responses may also be involved in leukemia initiation in both pediatric and adult patients which are associated with decreased phosphorylation of STAT5. Likewise, additional targeted agents continue to be developed for precision medicine that prominently suppress signaling pathways. A common theme of all of these perturbations is potential risk for dysregulating hematopoiesis through general transcriptional modulation. Here we discuss the potential for STAT5 inhibition as a double edged sword in certain hematologic disorders, such as early B-cell lymphoblastic leukemias. Considering the rapid pace of understanding of the pre-leukemic decrease in poly-clonality that precedes leukemia, the functional changes associated with microenvironmental influences are thus of potential clinical significance.