Milica Vukovic

Death-associated protein kinase (DAPK) and signal transduction: regulation in cancer

Death‐associated protein kinase (DAPK) is a pro‐apoptotic serine/threonine protein kinase that is dysregulated in a wide variety of cancers. The mechanism by which this occurs has largely been attributed to promoter hypermethylation, which results in gene silencing. However, recent studies indicate that DAPK expression can be detected in some cancers, but its function is still repressed, suggesting that DAPK activity can be subverted at a post‐translational level in cancer cells. This review will focus on recent data describing potential mechanisms that may alter the expression, regulation or function of DAPK.

Megakaryocytes assemble podosomes that degrade matrix and protrude through basement membrane

Megakaryocytes give rise to platelets via extension of proplatelet arms, which are released through the vascular sinusoids into the bloodstream. Megakaryocytes and their precursors undergo varying interactions with the extracellular environment in the bone marrow during their maturation and positioning in the vascular niche. We demonstrate that podosomes are abundant in primary murine megakaryocytes adherent on multiple extracellular matrix substrates, including native basement membrane. Megakaryocyte podosome lifetime and density, but not podosome size, are dependent on the type of matrix, with podosome lifetime dramatically increased on collagen fibers compared with fibrinogen. Podosome stability and dynamics depend on actin cytoskeletal dynamics but not matrix metalloproteases. However, podosomes degrade matrix and appear to be important for megakaryocytes to extend protrusions across a native basement membrane. We thus demonstrate for the first time a fundamental requirement for podosomes in megakaryocyte process extension across a basement membrane, and our results suggest that podosomes may have a role in proplatelet arm extension or penetration of basement membrane.

Hif-2α is not essential for cell-autonomous hematopoietic stem cell maintenance.

Local hypoxia in hematopoietic stem cell (HSC) niches is thought to regulate HSC functions. Hypoxia-inducible factor-1 (Hif-1) and Hif-2 are key mediators of cellular responses to hypoxia. Although oxygen-regulated α-subunits of Hifs, namely Hif-1α and Hif-2α, are closely related, they play overlapping and also distinct functions in nonhematopoietic tissues. Although Hif-1α-deficient HSCs lose their activity on serial transplantation, the role for Hif-2α in cell-autonomous HSC maintenance remains unknown. Here, we demonstrate that constitutive or inducible hematopoiesis-specific Hif-2α deletion does not affect HSC numbers and steady-state hematopoiesis. Furthermore, using serial transplantations and 5-fluorouracil treatment, we demonstrate that HSCs do not require Hif-2α to self-renew and recover after hematopoietic injury. Finally, we show that Hif-1α deletion has no major impact on steady-state maintenance of Hif-2α-deficient HSCs and their ability to repopulate primary recipients, indicating that Hif-1α expression does not account for normal behavior of Hif-2α-deficient HSCs.

Hif-1α and Hif-2α synergize to suppress AML development but are dispensable for disease maintenance

Vukovic et al. report that Hif-1α and Hif-2α are not required for leukemia stem cell maintenance and AML propagation, but they act synergistically to suppress leukemia development in mice. Furthermore, knockout of HIF-2α or pharmacological inhibition of the HIF pathway in human AML cells has no impact on their survival and proliferation under hypoxic conditions.

Concise Review: Genetic Dissection of Hypoxia Signaling Pathways in Normal and Leukemic Stem Cells

Adult hematopoiesis depends on rare multipotent hematopoietic stem cells (HSCs) that self‐renew and give rise to progenitor cells, which differentiate to all blood lineages. The strict regulation of the fine balance between self‐renewal and differentiation is essential for normal hematopoiesis and suppression of leukemia development. HSCs and progenitor cells are commonly assumed to reside within the hypoxic BM microenvironment, however, there is no direct evidence supporting this notion. Nevertheless, HSCs and progenitors do exhibit a hypoxic profile and strongly express Hif‐1α. Although hypoxia signaling pathways are thought to play important roles in adult HSC maintenance and leukemogenesis, the precise function of Hif‐dependent signaling in HSCs remains to be uncovered. Here we discuss recent gain‐of‐function and loss‐of‐function studies that shed light on the complex roles of hypoxia‐signaling pathways in HSCs and their niches in normal and malignant hematopoiesis. Importantly, we comment on the current and often contrasting interpretations of the role of Hif‐dependent signaling in stem cell functions. Stem Cells 2014;32:1390–1397

Genetic dissection of hypoxia signalling pathways in normal and leukaemic stem cells

Adult hematopoiesis depends on rare multipotent hematopoietic stem cells (HSCs) that self‐renew and give rise to progenitor cells, which differentiate to all blood lineages. The strict regulation of the fine balance between self‐renewal and differentiation is essential for normal hematopoiesis and suppression of leukemia development. HSCs and progenitor cells are commonly assumed to reside within the hypoxic BM microenvironment, however, there is no direct evidence supporting this notion. Nevertheless, HSCs and progenitors do exhibit a hypoxic profile and strongly express Hif‐1α. Although hypoxia signaling pathways are thought to play important roles in adult HSC maintenance and leukemogenesis, the precise function of Hif‐dependent signaling in HSCs remains to be uncovered. Here we discuss recent gain‐of‐function and loss‐of‐function studies that shed light on the complex roles of hypoxia‐signaling pathways in HSCs and their niches in normal and malignant hematopoiesis. Importantly, we comment on the current and often contrasting interpretations of the role of Hif‐dependent signaling in stem cell functions. Stem Cells 2014;32:1390–1397

Adult hematopoietic stem cells lacking Hif-1α self-renew normally.

The hematopoietic stem cell (HSC) pool is maintained under hypoxic conditions within the bone marrow microenvironment. Cellular responses to hypoxia are largely mediated by the hypoxia-inducible factors, Hif-1 and Hif-2. The oxygen-regulated α subunits of Hif-1 and Hif-2 (namely, Hif-1α and Hif-2α) form dimers with their stably expressed β subunits and control the transcription of downstream hypoxia-responsive genes to facilitate adaptation to low oxygen tension. An initial study concluded that Hif-1α is essential for HSC maintenance, whereby Hif-1α-deficient HSCs lost their ability to self-renew in serial transplantation assays. In another study, we demonstrated that Hif-2α is dispensable for cell-autonomous HSC maintenance, both under steady-state conditions and following transplantation. Given these unexpected findings, we set out to revisit the role of Hif-1α in cell-autonomous HSC functions. Here we demonstrate that inducible acute deletion of Hif-1α has no impact on HSC survival. Notably, unstressed HSCs lacking Hif-1α efficiently self-renew and sustain long-term multilineage hematopoiesis upon serial transplantation. Finally, Hif-1α-deficient HSCs recover normally after hematopoietic injury induced by serial administration of 5-fluorouracil. We therefore conclude that despite the hypoxic nature of the bone marrow microenvironment, Hif-1α is dispensable for cell-autonomous HSC maintenance.

Fumarate hydratase is a critical metabolic regulator of hematopoietic stem cell functions

Strict regulation of stem cell metabolism is essential for tissue functions and tumor suppression. In this study, we investigated the role of fumarate hydratase (Fh1), a key component of the mitochondrial tricarboxylic acid (TCA) cycle and cytosolic fumarate metabolism, in normal and leukemic hematopoiesis. Hematopoiesis-specific Fh1 deletion (resulting in endogenous fumarate accumulation and a genetic TCA cycle block reflected by decreased maximal mitochondrial respiration) caused lethal fetal liver hematopoietic defects and hematopoietic stem cell (HSC) failure. Reexpression of extramitochondrial Fh1 (which normalized fumarate levels but not maximal mitochondrial respiration) rescued these phenotypes, indicating the causal role of cellular fumarate accumulation. However, HSCs lacking mitochondrial Fh1 (which had normal fumarate levels but defective maximal mitochondrial respiration) failed to self-renew and displayed lymphoid differentiation defects. In contrast, leukemia-initiating cells lacking mitochondrial Fh1 efficiently propagated Meis1/Hoxa9-driven leukemia. Thus, we identify novel roles for fumarate metabolism in HSC maintenance and hematopoietic differentiation and reveal a differential requirement for mitochondrial Fh1 in normal hematopoiesis and leukemia propagation.

Modulation of PKC-α promotes lineage reprogramming of committed B lymphocytes.

During hematopoietic lineage development, hematopoietic stem cells sequentially commit toward myeloid or lymphoid lineages in a tightly regulated manner, which under normal circumstances is irreversible. However, studies have established that targeted deletion of the B‐lineage specific transcription factor, paired box gene 5 (Pax5), enables B cells to differentiate toward other hematopoietic lineages, in addition to generating progenitor B‐cell lymphomas. Our previous studies showed that subversion of protein kinase C (PKC)‐α in developing B cells transformed B‐lineage cells. Here, we demonstrate that PKC‐α modulation in committed CD19+ B lymphocytes also promoted lineage conversion toward myeloid, NK‐, and T‐cell lineages upon Notch ligation. This occurred via a reduction in Pax5 expression resulting from a downregulation of E47, a product of the E2A gene. T‐cell lineage commitment was indicated by the expression of T‐cell associated genes Ptcra, Cd3e, and gene rearrangement at the Tcrb gene locus. Importantly, the lineage‐converted T cells carried Igh gene rearrangements reminiscent of their B‐cell origin. Our findings suggest that modulation of PKC‐α induces hematopoietic‐lineage plasticity in committed B‐lineage cells by perturbing expression of critical B‐lineage transcription factors, and deregulation of PKC‐α activity/expression represents a potential mechanism for lineage trans‐differentiation during malignancies.

Generation of a poor prognostic chronic lymphocytic leukemia-like disease model: PKCα subversion induces up-regulation of PKCβII expression in B lymphocytes.

Overwhelming evidence identifies the microenvironment as a critical factor in the development and progression of chronic lymphocytic leukemia, underlining the importance of developing suitable translational models to study the pathogenesis of the disease. We previously established that stable expression of kinase dead protein kinase C alpha in hematopoietic progenitor cells resulted in the development of a chronic lymphocytic leukemia-like disease in mice. Here we demonstrate that this chronic lymphocytic leukemia model resembles the more aggressive subset of chronic lymphocytic leukemia, expressing predominantly unmutated immunoglobulin heavy chain genes, with upregulated tyrosine kinase ZAP-70 expression and elevated ERK-MAPK-mTor signaling, resulting in enhanced proliferation and increased tumor load in lymphoid organs. Reduced function of PKCα leads to an up-regulation of PKCβII expression, which is also associated with a poor prognostic subset of human chronic lymphocytic leukemia samples. Treatment of chronic lymphocytic leukemia-like cells with the selective PKCβ inhibitor enzastaurin caused cell cycle arrest and apoptosis both in vitro and in vivo, and a reduction in the leukemic burden in vivo. These results demonstrate the importance of PKCβII in chronic lymphocytic leukemia-like disease progression and suggest a role for PKCα subversion in creating permissive conditions for leukemogenesis.