Jennifer Chase

Ash1l controls quiescence and self-renewal potential in hematopoietic stem cells

Rapidly cycling fetal and neonatal hematopoietic stem cells (HSCs) generate a pool of quiescent adult HSCs after establishing hematopoiesis in the bone marrow. We report an essential role for the trithorax group gene absent, small, or homeotic 1-like (Ash1l) at this developmental transition. Emergence and expansion of Ash1l-deficient fetal/neonatal HSCs were preserved; however, in young adult animals, HSCs were profoundly depleted. Ash1l-deficient adult HSCs had markedly decreased quiescence and reduced cyclin-dependent kinase inhibitor 1b/c (Cdkn1b/1c) expression and failed to establish long-term trilineage bone marrow hematopoiesis after transplantation to irradiated recipients. Wild-type HSCs could efficiently engraft when transferred to unirradiated, Ash1l-deficient recipients, indicating increased availability of functional HSC niches in these mice. Ash1l deficiency also decreased expression of multiple Hox genes in hematopoietic progenitors. Ash1l cooperated functionally with mixed-lineage leukemia 1 (Mll1), as combined loss of Ash1l and Mll1, but not isolated Ash1l or Mll1 deficiency, induced overt hematopoietic failure. Our results uncover a trithorax group gene network that controls quiescence, niche occupancy, and self-renewal potential in adult HSCs.

Absence of a Red Blood Cell Phenotype in Mice with Hematopoietic Deficiency of SEC23B

ABSTRACT Congenital dyserythropoietic anemia type II (CDAII) is an autosomal recessive disease of ineffective erythropoiesis characterized by increased bi/multinucleated erythroid precursors in the bone marrow. CDAII results from mutations in SEC23B. The SEC23 protein is a core component of coat protein complex II-coated vesicles, which transport secretory proteins from the endoplasmic reticulum to the Golgi apparatus. Though the genetic defect underlying CDAII has been identified, the pathophysiology of this disease remains unknown. We previously reported that SEC23B-deficient mice die perinatally, exhibiting massive pancreatic degeneration, with this early mortality limiting evaluation of the adult hematopoietic compartment. We now report that mice with SEC23B deficiency restricted to the hematopoietic compartment survive normally and do not exhibit anemia or other CDAII characteristics. We also demonstrate that SEC23B-deficient hematopoietic stem cells (HSC) do not exhibit a disadvantage at reconstituting hematopoiesis when compared directly to wild-type HSC in a competitive repopulation assay. Secondary bone marrow transplants demonstrated continued equivalence of SEC23B-deficient and WT HSC in their hematopoietic reconstitution potential. The surprising discordance in phenotypes between SEC23B-deficient mice and humans may reflect an evolutionary shift in SEC23 paralog function and/or expression, or a change in a specific COPII cargo critical for erythropoiesis.

Loss of mTORC1 signaling alters pancreatic α cell mass and impairs glucagon secretion

Glucagon plays a major role in the regulation of glucose homeostasis during fed and fasting states. However, the mechanisms responsible for the regulation of pancreatic &agr; cell mass and function are not completely understood. In the current study, we identified mTOR complex 1 (mTORC1) as a major regulator of &agr; cell mass and glucagon secretion. Using mice with tissue-specific deletion of the mTORC1 regulator Raptor in &agr; cells (&agr;RaptorKO), we showed that mTORC1 signaling is dispensable for &agr; cell development, but essential for &agr; cell maturation during the transition from a milk-based diet to a chow-based diet after weaning. Moreover, inhibition of mTORC1 signaling in &agr;RaptorKO mice and in WT animals exposed to chronic rapamycin administration decreased glucagon content and glucagon secretion. In &agr;RaptorKO mice, impaired glucagon secretion occurred in response to different secretagogues and was mediated by alterations in KATP channel subunit expression and activity. Additionally, our data identify the mTORC1/FoxA2 axis as a link between mTORC1 and transcriptional regulation of key genes responsible for &agr; cell function. Thus, our results reveal a potential function of mTORC1 in nutrient-dependent regulation of glucagon secretion and identify a role for mTORC1 in controlling &agr; cell–mass maintenance.

Pancreatic SEC23B deficiency is sufficient to explain the perinatal lethality of germline SEC23B deficiency in mice.

In humans, loss of function mutations in SEC23B result in Congenital Dyserythropoietic Anemia type II (CDAII), a disease limited to defective erythroid development. Patients with two nonsense SEC23B mutations have not been reported, suggesting that complete SEC23B deficiency might be lethal. We previously reported that SEC23B-deficient mice die perinatally, exhibiting massive pancreatic degeneration and that mice with hematopoietic SEC23B deficiency do not exhibit CDAII. We now show that SEC23B deficiency restricted to the pancreas is sufficient to explain the lethality observed in mice with global SEC23B-deficiency. Immunohistochemical stains demonstrate an acinar cell defect but normal islet cells. Mammalian genomes contain two Sec23 paralogs, Sec23A and Sec23B. The encoded proteins share ~85% amino acid sequence identity. We generate mice with pancreatic SEC23A deficiency and demonstrate that these mice survive normally, exhibiting normal pancreatic weights and histology. Taken together, these data demonstrate that SEC23B but not SEC23A is essential for murine pancreatic development. We also demonstrate that two BAC transgenes spanning Sec23b rescue the lethality of mice homozygous for a Sec23b gene trap allele, excluding a passenger gene mutation as the cause of the pancreatic lethality, and indicating that the regulatory elements critical for Sec23b pancreatic function reside within the BAC transgenes.

High efficacy vasopermeability drug candidates identified by screening in an ex ovo chorioallantoic membrane model

The use of rodent models to evaluate efficacy during testing is accompanied by significant economic and regulatory hurdles which compound the costs of screening for promising drug candidates. Vasopermeation Enhancement Agents (VEAs) are a new class of biologics that are designed to increase the uptake of cancer therapeutics at the tumor site by modifying vascular permeability in the tumor to increase the therapeutic index of co-administered drugs. To evaluate the efficacy of a panel of VEA clinical candidates, we compared the rodent Miles assay to an equivalent assay in the ex ovo chicken embryo model. Both model systems identified the same candidate (PVL 10) as the most active promoter of vasopermeation in non-tumor tissues. An ex ovo chicken embryo system was utilized to test each candidate VEA in two human tumor models at a range of concentrations. Vasopermeation activity due to VEA was dependent on tumor type, with HEp3 tumors displaying higher levels of vasopermeation than MDA-MB-435. One candidate (PVL 10) proved optimal for HEp3 tumors and another (PVL 2) for MDA-MB-435. The use of the ex ovo chicken embryo model provides a rapid and less costly alternative to the use of rodent models for preclinical screening of drug candidates.

Abstract 3803: Development of genetic and chemical tools for understanding the recruitment of DOT1L in MLL-fusion driven leukemia and normal hematopoiesis

Leukemias harboring rearrangements of mixed-lineage leukemia gene (MLL1) are associated with poor clinical outcomes, and new therapeutic approaches are needed. Rearrangements of the MLL1 gene generate fusion oncoproteins which drive the high expression of the clustered homeobox (HOX) genes and induce leukemic transformation. Genome wide histone methylation studies have revealed that the abnormal expression of MLL1 fusion target genes is associated with high levels of histone H3 lysine 79 (H3K79) methylation. Recruitment of DOT1L (disruptor of telomeric silencing 1-like), a unique histone methyltransferase that catalyzes methylation of H3K79, proved to be essential for the transforming activity of multiple MLL fusion proteins. To gain insights into the unique functions of DOT1L in MLL-driven leukemia, we elucidated the mechanisms of DOT1L recruitment to the MLL fusion partners. The binding site was mapped to a short segment of 10 amino acids in DOT1L and peptides derived from this region disrupted the interaction between DOT1L and MLL-AF9. DOT1L mutants lacking these 10 residues did not support transformation by MLL-AF9. This discovery has established a foundation for disease-specific therapies that target chromatin modifications in highly malignant leukemias. Applying high throughput screening approach several different chemical classes of small molecules that disrupt the protein-protein interactions between DOT1L and oncogenic MLL-fusion proteins were identified and validated. To evaluate if the AF9-binding domain of DOT1L is critical for its functions in normal hematopoietic stem cells as opposed to leukemias driven by MLL fusion proteins, genetic tools were developed to functionally investigate the importance of the DOT1L AF9-binding domain in MLL-AF9-driven leukemia and its role in the physiological functions of DOT1L in normal hematopoiesis. Our findings demonstrate that pharmacological inhibition of the DOT1L complex through disrupting the AF9-DOT1L interactions may provide therapeutic benefits in an array of malignancies with abnormal HOXA gene expression. Citation Format: Sierrah Grigsby, Jennifer Chase, James Ropa, Justin Serio, Chenxi Shen, Martha Larsen, Preston Donover, Melvin Reichman, Andrew Muntean, Ivan Maillard, Zaneta Nikolovska-Coleska. Development of genetic and chemical tools for understanding the recruitment of DOT1L in MLL-fusion driven leukemia and normal hematopoiesis. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3803.