Patients in organ failure of vascular origin have increased circulating hematopoietic stem cells and
progenitors (HSC/P). Plasma levels of angiotensin II (Ang-II), are commonly increased in
vasculopathies. Hyperangiotensinemia results in activation of a very distinct Ang-II receptor set,
Rho-family GTPase members, and actin in bone marrow endothelial cells (BMEC) and HSC/P,
which results in decreased membrane integrin activation in both BMEC and HSC/P, and in HSC/P
de-adhesion and mobilization. The Ang-II effect can be reversed pharmacologically and
genetically by inhibiting Ang-II production or signaling through BMEC AT2R, HSCP AT1R/
AT2R or HSC/P RhoA, but not by interfering with other vascular tone mediators.
Hyperangiotensinemia and high counts of circulating HSC/P seen in sickle cell disease (SCD) as a
result of vascular damage, is significantly decreased by Ang-II inhibitors. Our data define for the
first time the role of Ang-II HSC/P traffic regulation and redefine the hematopoietic consequences
of anti-angiotensin therapy in SCD.
We have designed ROS-activated cytotoxic agents that are active against AML cancer cells. In this study the mechanism and synergistic effects against cells co-expressing the AML oncogenes MLL-AF9 fusion and FLT3-ITD was investigated. The agent had an IC50 value of 1.8±0.3 μM with a selectivity of 9-fold compared to untransformed cells. Treatment induced DNA strand breaks, apoptosis, and cell cycle arrest. Proteomics and transcriptomics revealed enhanced expression of the pentose phosphate pathway, DNA repair, and pathways common to cell stress. Western blotting confirmed repair by homologous recombination. Importantly, RAC1 treatment was synergistic in combination with multiple pathway targeting therapies in AML cells but less so in untransformed cells. Taken together, these results demonstrate that RAC1 can selectively target poor prognosis AML and do so by creating DNA double strand breaks that require homologous recombination.
The primary structure of the major surface glycoconjugate of Leishmania donovani parasites, a lipophosphoglycan, has been further characterized. The repeating PO4-6Galp beta 1-4Man disaccharide units, which are a salient feature of the molecule, are shown to terminate with one of several neutral structures, the most abundant of which is the branched trisaccharide Galp beta 1-4(Manp alpha 1-2)Man. The phosphosaccharide core of lipophosphoglycan, which links the disaccharide repeats to a lipid anchor, contains 2 phosphate residues. One of the core phosphates has previously been localized on O-6 of the galactosyl residue distal to the lipid anchor; the second phosphate is now shown to be on O-6 of the mannosyl residue distal to the anchor and to bear an alpha-linked glucopyranosyl residue. Also, the anomeric configuration of the unusual 3-substituted Galf residue in the phosphosaccharide core is established as beta. The complete structure of the core is thus PO4-6Galp alpha 1-6Galp alpha 1-3Galf beta 1-3[Glcp alpha 1-PO4-6]Manp alpha 1-3Manp alpha 1-4GlcN alpha 1-. This further clarification of the structure of lipophosphoglycan may prove beneficial in determining the structure-function relationships of this highly unusual glycoconjugate.
An extracellular phosphoglycan (exPG), present in the culturem edium of the promastigote form L oefi shmania donovani, was purified and structurally characterized. The purification scheme included ethanol precipitation of the culture medium, anion exchange chromatography, hydrophobic chromatography on phenyl-Sepharose, and preparative polyacrylamgeild e electrophoresis. Structural analysis by ‘H-’H NMR, methylation linkage analysis, and glycosidase digestion revealed that the exPG consisted of thfoel lowing structure: (CAP)+[P04-6Galp@1-4Manpal]lo-11-POr6GalpB1-4Man. The capw as found to be ones eovf eral small, neutral oligosaccharides, the most abundant of which was the trisaccharide Galp@l-4(Manpal-2)Man. The results indicated structural analogy to the cellular-derived lipophosphoglycan (LPG) from L. donovani. The important exceptions are a lacko f the lipid anchor, the entire phosphosaccharide core, and several of the repeating disaccharide units. Although the function of exPGis presently unknowni,t may play a protective role for the promastigote in the insect vector or during infection of a mammalian host
PM-18. EGFR-STAT3 ACTIVATES b-CATENIN SIGNALING TO
DRIVE NEUROFIBROMA INITIATION IN NF1, AND PLAYS A
ROLE IN TUMOR MAINTENANCE
Nancy Ratner1, Vincent Keng2, Deanna M. Patmore1, Jed K. Kendall1,
Edwin Jousma1, Kwangmin Choi1, Danhua Fan2, Eric B. Schwartz2, James
R. Fuchs2, Yuanshu Zou2, Mi-Ok Kim1, Eva Dombi5, David E. Levy6, Jose
A. Cancelas1, Anat Stemmer-Rachamimov4, Robert J. Spinner3, and David
Cincinnati Children’s, Cincinnati, OH, USA; 2
Minnesota, Minneapolis, MN, USA; 3
Mayo Clinic, Rochester, MN, USA; 4
Massachusetts General Hospital, Boston, MA, USA; 5
Institute Pediatric Branch, Bethesda, MD, USA; 6
New York University School
of Medicine, New York, NY, USA
To identify genes and signaling pathways that drive peripheral nerve tumor
initiationand growth beyond the Ras-MAPK pathwaywe used unbiased insertional
mutagenesis screening. We identified Stat3 as a potential driver of
Neurofibromatosis type 1 neurofibroma. Targeted genetic deletion of Stat3
in Schwann cell precursors (SCPs) and Schwann cells (SCs) largely prevented
neurofibroma formation, and self-renewal of tumor initiating cells. Genetic
gain- and loss-of-function identified EGFR as the major upstream regulator
of P-Stat3 in mouse and human neurofibroma SCP and in neurofibroma initiation;
IL-6 reinforced EGFR/Jak/Stat signaling. Preclinical tests of a Jak2/
Stat3 inhibitor reduced established neurofibroma growth, supporting an additional
role for Stat3 in benign nerve tumor maintenance. Unexpectedly, downstream
of Stat3, we identified b-catenin, and b-catenin expression rescued
phenotypic effects of Stat3 loss in SCPs. Phosphorylated STAT3 (Y705) and
b-catenin were strongly correlated in NF1 human plexiform neurofibromas.
The data support testing of JAK/STAT inhibition and Wnt/ b-catenin
pathway inhibition in neurofibroma therapeutic trials. Supported by: NIH
R01 NS28840 to N.R. and NIH P50 NS057531 to N.R. and D.L.), a
DAMD New Investigator Award (W81XWH-11-1-0259) and an Ohio State
University Comprehensive Cancer Center Pelotonia Idea Grant (to J.W.).
The American Cancer Society (IRG-67-003-44) supported J.R.F.
Severe congenital neutropenia (SCN) is often associated with inherited heterozygous point mutations in ELANE, which
encodes neutrophil elastase (NE). However, a lack of appropriate models to recapitulate SCN has substantially hampered
the understanding of the genetic etiology and pathobiology of this disease. To this end, we generated both normal and SCN
patient–derived induced pluripotent stem cells (iPSCs), and performed genome editing and differentiation protocols that
recapitulate the major features of granulopoiesis. Pathogenesis of ELANE point mutations was the result of promyelocyte
death and differentiation arrest, and was associated with NE mislocalization and activation of the unfolded protein
response/ER stress (UPR/ER stress). Similarly, high-dose G-CSF (or downstream signaling through AKT/BCL2) rescues
the dysgranulopoietic defect in SCN patient–derived iPSCs through C/EBPβ-dependent emergency granulopoiesis. In
contrast, sivelestat, an NE-specific small-molecule inhibitor, corrected dysgranulopoiesis by restoring normal intracellular
NE localization in primary granules; ameliorating UPR/ER stress; increasing expression of CEBPA, but not CEBPB; and
promoting promyelocyte survival and differentiation. Together, these data suggest that SCN disease pathogenesis includes
NE mislocalization, which in turn triggers dysfunctional survival signaling and UPR/ER stress. This paradigm has the
potential to be clinically exploited to achieve therapeutic responses using lower doses of G-CSF combined with targeting to
correct NE mislocalization.