Enhancing Hematopoietic Stem Cell Transplantation Efficacy by Mitigating Oxygen Shock

Hematopoietic stem cells (HSCs) reside in hypoxic niches within bone marrow and cord blood. Yet, essentially all HSC studies have been performed with cells isolated and processed in non-physiologic ambient air. By collecting and manipulating bone marrow and cord blood in native conditions of hypoxia, we demonstrate that brief exposure to ambient oxygen decreases recovery of long-term repopulating HSCs and increases progenitor cells, a phenomenon we term extraphysiologic oxygen shock/stress (EPHOSS). Thus, true numbers of HSCs in the bone marrow and cord blood are routinely underestimated. We linked ROS production and induction of the mitochondrial permeability transition pore (MPTP) via cyclophilin D and p53 as mechanisms of EPHOSS. The MPTP inhibitor cyclosporin A protects mouse bone marrow and human cord blood HSCs from EPHOSS during collection in air, resulting in increased recovery of transplantable HSCs. Mitigating EPHOSS during cell collection and processing by pharmacological means may be clinically advantageous for transplantation

Netrin-1 acts as a survival factor for aggressive neuroblastoma

Neuroblastoma (NB), the most frequent solid tumor of early childhood, is diagnosed as a disseminated disease in >60% of cases, and several lines of evidence support the resistance to apoptosis as a prerequisite for NB progression. We show that autocrine production of netrin-1, a multifunctional laminin-related molecule, conveys a selective advantage in tumor growth and dissemination in aggressive NB, as it blocks the proapoptotic activity of the UNC5H netrin-1 dependence receptors. We show that such netrin-1 up-regulation is a potential marker for poor prognosis in stage 4S and, more generally, in NB stage 4 diagnosed infants. Moreover, we propose that interference with the netrin-1 autocrine loop in malignant neuroblasts could represent an alternative therapeutic strategy, as disruption of this loop triggers in vitro NB cell death and inhibits NB metastasis in avian and mouse models

Mutant IDH inhibits HNF-4α to block hepatocyte differentiation and promote biliary cancer

Mutations in isocitrate dehydrogenase 1 (IDH1) and IDH2 are among the most common genetic alterations in intrahepatic cholangiocarcinoma (IHCC), a deadly liver cancer1, 2, 3, 4, 5. Mutant IDH proteins in IHCC and other malignancies acquire an abnormal enzymatic activity allowing them to convert α-ketoglutarate (αKG) to 2-hydroxyglutarate (2HG), which inhibits the activity of multiple αKG-dependent dioxygenases, and results in alterations in cell differentiation, survival, and extracellular matrix maturation6, 7, 8, 9, 10. However, the molecular pathways by which IDH mutations lead to tumour formation remain unclear. Here we show that mutant IDH blocks liver progenitor cells from undergoing hepatocyte differentiation through the production of 2HG and suppression of HNF-4α, a master regulator of hepatocyte identity and quiescence. Correspondingly, genetically engineered mouse models expressing mutant IDH in the adult liver show an aberrant response to hepatic injury, characterized by HNF-4α silencing, impaired hepatocyte differentiation, and markedly elevated levels of cell proliferation. Moreover, IDH and Kras mutations, genetic alterations that co-exist in a subset of human IHCCs4, 5, cooperate to drive the expansion of liver progenitor cells, development of premalignant biliary lesions, and progression to metastatic IHCC. These studies provide a functional link between IDH mutations, hepatic cell fate, and IHCC pathogenesis, and present a novel genetically engineered mouse model of IDH-driven malignancy

Molecular mechanisms of cell death: recommendations of the Nomenclature Committee on Cell Death 2018.

Over the past decade, the Nomenclature Committee on Cell Death (NCCD) has formulated guidelines for the definition and interpretation of cell death from morphological, biochemical, and functional perspectives. Since the field continues to expand and novel mechanisms that orchestrate multiple cell death pathways are unveiled, we propose an updated classification of cell death subroutines focusing on mechanistic and essential (as opposed to correlative and dispensable) aspects of the process. As we provide molecularly oriented definitions of terms including intrinsic apoptosis, extrinsic apoptosis, mitochondrial permeability transition (MPT)-driven necrosis, necroptosis, ferroptosis, pyroptosis, parthanatos, entotic cell death, NETotic cell death, lysosome-dependent cell death, autophagy-dependent cell death, immunogenic cell death, cellular senescence, and mitotic catastrophe, we discuss the utility of neologisms that refer to highly specialized instances of these processes. The mission of the NCCD is to provide a widely accepted nomenclature on cell death in support of the continued development of the field

Protein kinases of the Hippo pathway: Regulation and substrates

The "Hippo" signaling pathway has emerged as a major regulator of cell proliferation and survival in metazoans. The pathway, as delineated by genetic and biochemical studies in Drosophila, consists of a kinase cascade regulated by cell-cell contact and cell polarity that inhibits the transcriptional coactivator Yorkie and its proliferative, anti-differentiation, antiapoptotic transcriptional program. The core pathway components are the GC kinase Hippo, which phosphorylates the noncatalytic polypeptide Mats/Mob1 and, with the assistance of the scaffold protein Salvador, phosphorylates the ndr-family kinase Lats. In turn phospho-Lats, after binding to phospho-Mats, autoactivates and phosphorylates Yorkie, resulting in its nuclear exit. Hippo also uses the scaffold protein Furry and a different Mob protein to control another ndr-like kinase, the morphogenetic regulator Tricornered. Architecturally homologous kinase cascades consisting of a GC kinase, a Mob protein, a scaffolding polypeptide and an ndr-like kinase are well described in yeast; in Saccharomyces cerevisiae, e. g., the MEN pathway promotes mitotic exit whereas the RAM network, using a different GC kinase, Mob protein, scaffold and ndr-like kinase, regulates cell polarity and morphogenesis. In mammals, the Hippo orthologs Mst1 and Mst2 utilize the Salvador ortholog WW45/Sav1 and other scaffolds to regulate the kinases Lats1/Lats2 and ndr1/ndr2. As in Drosophila, murine Mst1/Mst2, in a redundant manner, negatively regulate the Yorkie ortholog YAP in the epithelial cells of the liver and gut; loss of both Mst1 and Mst2 results in hyperproliferation and tumorigenesis that can be largely negated by reduction or elimination of YAP. Despite this conservation, considerable diversification in pathway composition and regulation is already evident; in skin, e. g., YAP phosphorylation is independent of Mst1Mst2 and Lats1Lats2. Moreover, in lymphoid cells, Mst1/Mst2, under the control of the Rap1 GTPase and independent of YAP, promotes integrin clustering, actin remodeling and motility while restraining the proliferation of naive T cells. This review will summarize current knowledge of the structure and regulation of the kinases Hippo/Mst1&2, their noncatalytic binding partners, Salvador and the Rassf polypeptides, and their major substrates Warts/Lats1&2, Trc/ndr1&2, Mats/Mob1 and FOXO. (C) 2012 Elsevier Ltd. All rights reserved.NIH [DK17776, CA136567]; Sidney Kimmel Foundation for Cancer Research; Linda J. Verville Cancer Research Foundation; 111 Project of Education of China [B06016]; Fundamental Research Funds for the Central Universities of China [2010111079]; National Natural Science Foundation of China [81101503]; Natural Science Foundation of Fujian [2011J05096

Cellular Metabolism Regulates Hippo–YAP Signaling through AMPK-mediated Phosphorylation and Stabilization of Angiomotin Like-1 Protein

Hippo signaling is a tumor-suppressor pathway involved in organ size control and tumorigenesis through the inhibition of YAP and TAZ. Here, we show that energy stress induces YAP cytoplasmic retention and S127 phosphorylation and inhibits YAP transcriptional activity and YAP-dependent transformation. These effects require the central metabolic sensor AMP-activated protein kinase (AMPK) and the upstream Hippo pathway components Lats1/Lats2 and angiomotin-like 1 (AMOTL1). Furthermore, we show that AMPK directly phosphorylates S793 of AMOTL1. AMPK activation stabilizes and increases AMOTL1 steady-state protein levels, contributing to YAP inhibition. The phosphorylation-deficient S793Ala mutant of AMOTL1 showed a shorter half-life and conferred resistance to energy-stress-induced YAP inhibition. Our findings link energy sensing to the Hippo-YAP pathway and suggest that YAP may integrate spatial (contact inhibition), mechanical, and metabolic signals to control cellular proliferation and survival

Energy Stress Regulates Hippo-YAP Signaling Involving AMPK-Mediated Regulation of Angiomotin-like 1 Protein

SummaryHippo signaling is a tumor-suppressor pathway involved in organ size control and tumorigenesis through the inhibition of YAP and TAZ. Here, we show that energy stress induces YAP cytoplasmic retention and S127 phosphorylation and inhibits YAP transcriptional activity and YAP-dependent transformation. These effects require the central metabolic sensor AMP-activated protein kinase (AMPK) and the upstream Hippo pathway components Lats1/Lats2 and angiomotin-like 1 (AMOTL1). Furthermore, we show that AMPK directly phosphorylates S793 of AMOTL1. AMPK activation stabilizes and increases AMOTL1 steady-state protein levels, contributing to YAP inhibition. The phosphorylation-deficient S793Ala mutant of AMOTL1 showed a shorter half-life and conferred resistance to energy-stress-induced YAP inhibition. Our findings link energy sensing to the Hippo-YAP pathway and suggest that YAP may integrate spatial (contact inhibition), mechanical, and metabolic signals to control cellular proliferation and survival

Superenhancer reprogramming drives a B-cell-epithelial transition and high-risk leukemia.

IKAROS is required for the differentiation of highly proliferative pre-B-cell precursors, and loss of IKAROS function indicates poor prognosis in precursor B-cell acute lymphoblastic leukemia (B-ALL). Here we show that IKAROS regulates this developmental stage by positive and negative regulation of superenhancers with distinct lineage affiliations. IKAROS defines superenhancers at pre-B-cell differentiation genes together with B-cell master regulators such as PAX5, EBF1, and IRF4 but is required for a highly permissive chromatin environment, a function that cannot be compensated for by the other transcription factors. IKAROS is also highly enriched at inactive enhancers of genes normally expressed in stem-epithelial cells. Upon IKAROS loss, expression of pre-B-cell differentiation genes is attenuated, while a group of extralineage transcription factors that are directly repressed by IKAROS and depend on EBF1 relocalization at their enhancers for expression is induced. LHX2, LMO2, and TEAD-YAP1, normally kept separate from native B-cell transcription regulators by IKAROS, now cooperate directly with them in a de novo superenhancer network with its own feed-forward transcriptional reinforcement. Induction of de novo superenhancers antagonizes Polycomb repression and superimposes aberrant stem-epithelial cell properties in a B-cell precursor. This dual mechanism of IKAROS regulation promotes differentiation while safeguarding against a hybrid stem-epithelial-B-cell phenotype that underlies high-risk B-ALL

Superenhancer reprogramming drives a B-cell-epithelial transition and high-risk leukemia

IKAROS is required for the differentiation of highly proliferative pre-B-cell precursors, and loss of IKAROS function indicates poor prognosis in precursor B-cell acute lymphoblastic leukemia (B-ALL). Here we show that IKAROS regulates this developmental stage by positive and negative regulation of superenhancers with distinct lineage affiliations. IKAROS defines superenhancers at pre-B-cell differentiation genes together with B-cell master regulators such as PAX5, EBF1, and IRF4 but is required for a highly permissive chromatin environment, a function that cannot be compensated for by the other transcription factors. IKAROS is also highly enriched at inactive enhancers of genes normally expressed in stem-epithelial cells. Upon IKAROS loss, expression of pre-B-cell differentiation genes is attenuated, while a group of extralineage transcription factors that are directly repressed by IKAROS and depend on EBF1 relocalization at their enhancers for expression is induced. LHX2, LMO2, and TEAD-YAP1, normally kept separate from native B-cell transcription regulators by IKAROS, now cooperate directly with them in a de novo superenhancer network with its own feed-forward transcriptional reinforcement. Induction of de novo superenhancers antagonizes Polycomb repression and superimposes aberrant stem epithelial cell properties in a B-cell precursor. This dual mechanism of IKAROS regulation promotes differentiation while safeguarding against a hybrid stem epithelial B-cell phenotype that underlies high-risk B-ALL.Funding Agencies|National Institutes of Health [R01CA162092, R21AI124326, R01CA090576, R01CA190964]</p