ALBERT

Description: The class IA subgroup of phosphoinositide 3-kinase (PI3K) is activated downstream of antigen receptors, costimulatory molecules, and cytokine receptors on lymphocytes. Targeted deletion of individual genes for class IA regulatory subunits severely impairs the development and function of B cells but not T cells. Here we analyze conditional mutant mice in which thymocytes and T cells lack the major class IA regulatory subunits p85α, p55α, p50α, and p85β. These cells exhibit nearly complete loss of PI3K signaling downstream of the T-cell receptor (TCR) and CD28. Nevertheless, T-cell development is largely unperturbed, and peripheral T cells show only partial impairments in proliferation and cytokine production in vitro. Both genetic and pharmacologic experiments suggest that class IA PI3K signaling plays a limited role in T-cell proliferation driven by TCR/CD28 clustering. In vivo, class IA–deficient T cells provide reduced help to B cells but show normal ability to mediate antiviral immunity. Together these findings provide definitive evidence that class IA PI3K regulatory subunits are essential for a subset of T-cell functions while challenging the notion that this signaling mechanism is a critical mediator of costimulatory signals downstream of CD28.

Description: Recent studies have suggested not only that stem cells have different metabolic requirements than terminally differentiated cells, but also that metabolic intermediates may play a role in the maintenance of stem cells. It has long been clear that changes in acetylation and methylation of histones, as well as methylation of DNA play critical roles in deciding cell fate. The availability of critical intermediates in metabolism, especially S-adenosylmethionine (SAM), acetyl-CoA, nicotinamide adenine dinucleotide (NAD) and a-ketoglutarate play critical roles in modulating acetylation and methylation of histones and methylation of DNA. In the course of evaluating an unusual requirement of threonine (Thr) for the growth of murine embryonic stem cells, we found that metabolism of Thr to glycine (Gly) and the subsequent use of the methyl group of Gly for converting homocysteine to methionine is critical for maintaining high levels of SAM and low levels of S-adenosyl-homocysteine. Importantly, depletion of Thr from the media resulted in decreased tri-methylation of histone H3 lysine-4 (H3K4me3), leading to slowed growth and increased differentiation. Thus, abundance of SAM appears to influence H3K4me3, providing a possible mechanism by which modulation of a metabolic pathway might influence stem cell fate. Demethylation of histones and DNA can also be controlled by metabolic intermediates. Mutated forms of isocitrate dehydrogenase 1 (IDH1) and IDH2 that drive acute myeloid leukemia (AML) and other cancers, produce an oncometabolite (2-hydrogyglutarate) that can compete with the a-ketoglutarate requirement for enzymes involved in hydroxy-methylation and subsequent demethylation of DNA and histones. Recent studies indicate that 2-hydroxyglutarate plays a role in blocking differentiation of cancer cells. These and other observations linking intermediates in metabolism to stem cell maintenance will be discussed. Disclosures Cantley: Agios Pharmaceuticals: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties.

Description: How glucose is metabolized can influence cell function, but whether differences in glucose metabolism reflect, or dictate, cell state is not clear and is of particular interest given the association of cancer with aerobic glycolysis. Studies on cancer cell lines have indicated that increased glucose uptake with lactate production regardless of oxygen concentration, a phenomenon also known as the Warburg effect, is promoted in part by expression of the M2 isoform of pyruvate kinase (PKM2) and the muscle form of lactate dehydrogenase A (LDHA). Normal somatic cells thought to also preferentially use glycolytic metabolism are tissue stem cells, particularly the self-renewing hematopoietic stem cells (HSC) resident in the hypoxic microenvironment of the bone marrow. It remains to be defined, however whether proliferating hematopoietic progenitor cells rely on aerobic glycolysis and whether malignant and normal hematopoietic cells are dependent on the same metabolic regulation. We observed that PKM2 and LDHA are the predominant isoforms expressed by all BM hematopoietic cells. To further understand the role of glycolytic metabolism in hematopoiesis and hematological malignancy, we utilized a mouse strain that allows conditional deletion of the PKM2 specific exon 10. Deletion of PKM2 in hematopoetic cells leads to expression of PKM1, accompanied with partial inhibition of lactate production and decreased glycolytic intermediates in the hematopoietic stem/progenitor cell (HSPC) population. Loss of PKM2 compromises the long-term repopulation capacity of HSPCs as revealed by serial transplantation assay. Interestingly, the repopulating defects resulting from PKM2 depletion appear to involve progenitors, perhaps due to inadequate biomass generation necessary for robust cell proliferation. To confirm that the effect of PKM2 deletion on HSPC function is due to metabolic changes rather than other putative PKM2 functions, we engineered a mouse strain that allowed conditional knockout of LDHA to more potently impair aerobic glycolysis. LDHA deletion completely inhibited lactate production, enhanced ROS levels in hematopoietic cells and impaired long-term BM repopulating activity. In contrast to PKM2 deletion that affects progenitor but not stem cells, LDHA depletion impacts both stem cell maintenance and progenitor cell proliferation. Deletion of either PKM2 or LDHA markedly suppressed leukemia initiation by either putative stem cell (BCR-ABL) or progenitor (MLL-AF9) transforming alleles. Therefore, modulating aeroblic glycolysis has effects on normal hematopoietic cells that depend upon cell state and negatively impacts leukemic growth regardless of cell state. The differential sensitivity of normal and malignant cells to modulation of aerobic glycolysis suggests a potential therapeutic opportunity for leukemia intervention. Disclosures: No relevant conflicts of interest to declare.

Description: To produce blood platelets, megakaryocytes elaborate proplatelets, accompanied by expansion of membrane surface area and dramatic cytoskeletal rearrangements. The invaginated demarcation membrane system (DMS), a hallmark of mature cells, has been proposed as the source of proplatelet membranes. By direct visualization of labeled DMS, we demonstrate that this is indeed the case. Late in megakaryocyte ontogeny, the DMS gets loaded with PI-4,5-P2, a phospholipid that is confined to plasma membranes in other cells. Appearance of PI-4,5-P2 in the DMS occurs in proximity to PI-5-P-4-kinase α (PIP4Kα), and short hairpin (sh) RNA-mediated loss of PIP4Kα impairs both DMS development and expansion of megakaryocyte size. Thus, PI-4,5-P2 is a marker and possibly essential component of internal membranes. PI-4,5-P2 is known to promote actin polymerization by activating Rho-like GTPases and Wiskott-Aldrich syndrome (WASp) family proteins. Indeed, PI-4,5-P2 in the megakaryocyte DMS associates with filamentous actin. Expression of a dominant-negative N-WASp fragment or pharmacologic inhibition of actin polymerization causes similar arrests in proplatelet formation, acting at a step beyond expansion of the DMS and cell mass. These observations collectively suggest a signaling pathway wherein PI-4,5-P2 might facilitate DMS development and local assembly of actin fibers in preparation for platelet biogenesis.

Description: To produce blood platelets, the megakaryocyte (MK) cytoplasm elaborates proplatelets, accompanied by expansion of membrane surface area and dramatic cytoskeletal rearrangements. Invaginated demarcation membranes (DMS) are thought to be the source for the proplatelet and platelet membranes, however, they have THUS far BEEN INSUFFICIENTLY characterized. We first used a mouse model where the cDNA encoding enhanced yellow fluorescence protein (EYFP) with a C-terminally introduced myristoyl acceptor site has been introduced into the GPIIb locus. Heterozygous knock-in mice reveal yellow fluroescent MKs with an internal staining pattern that resembles the reticiulated pattern of the DMS as found in micrographs. Proplatelet-forming MKs reveal contiguous membrane connection between the internally stained membranes and the outlines of the proplatelet shaft resulting in production of fluorescent platelets. We next sought to characterize the internal membranes biochemically and retrovirally infected MKs to express the green fluorescence protein (EGFP) tagged with the pleckstrin homology domain of phospholipase Cδ1 (PLCδ1) which binds with high specificity to phosphatidylinositol(4,5)P2 (PIP2). Young MKs stain the cell periphery as described for most other cell types. Mature MKs, however, stain the internal membranes, whereas the plasma membrane becomes PIP2-negative as shown by co-staining with CD41. Proplatelet membranes emanate from these internal PIP2-positive membranes, proving that the DMS is indeed the membrane reservoir during platelet biogenesis. Appearance of PI-4,5-P2 in the DMS occurs in proximity to PI-5-P-4-kinaseα (PI4Kα), a protein highly expressed in MKs and platelets, as shown by overexpressing EGFP-tagged kinase in primary MKs. In addition, shRNA-mediated loss of PIP4Kα or depletion of its presumptive substrate block DMS development and expansion of MK size. Thus, PI-4,5-P2 is a marker and essential component of internal membranes and is most likely introduced about the non-canonical pathway using PI5P as the substrate. PI-4,5-P2 promotes actin polymerization by activating small GTPases from the Rac/Rho superfamily as well as Wiskott-Aldrich Syndrome (WASp) family proteins. Indeed, PIP2 is associated with filamentous actin when MKs are co-stained with phalloidin. Expression of a dominant-negative N-WASp C-terminal fragment (CA-domain) that inactivats all WASp/WAVE family members leads to Arp3 binding without assembling the complete Arp2/3 complex, thus inhibiting actin filament nucleation. F-Actin staining in the infected MKs reveals a pattern similar to that of MKs treated with pharmacologic dosage of actin polymerization-antagonists like cytochalasin D, which disrupts actin filaments and inhibits proplatelet formation when administered early in MK culture. Dominant-negative WASp impairs proplatelet elaboration similarly, acting at a step past expansion of the cell volume. These observations implicate a signaling pathway wherein PI-4,5-P2 facilitates DMS development and suggests a pathway that links a DMS lipid marker with local assembly of actin fibers as a requirement for platelet biogenesis.