Iron- and erythropoietin-resistant anemia in a spontaneous breast cancer mouse model

Anemia of cancer (AoC) with its multifactorial etiology and complex pathology is a poor prognostic indicator for cancer patients. One of the main causes of AoC is cancer-associated inflammation that activates mechanisms, commonly observed in anemia of inflammation, where functional iron deficiency and iron-restricted erythropoiesis is induced by increased hepcidin levels in response to IL-6 elevation. So far only a few AoC mouse models have been described, and most of them did not fully recapitulate the interplay of anemia, increased hepcidin levels and functional iron deficiency in human patients. To test if the selection and the complexity of AoC mouse models dictates the pathology or if AoC in mice per se develops independently of iron deficiency, we characterized AoC in Trp53floxWapCre mice that spontaneously develop breast cancer. These mice developed AoC associated with high IL-6 levels and iron deficiency. However, hepcidin levels were not increased and hypoferremia coincided with anemia rather than causing it. Instead, an early shift in the commitment of common myeloid progenitors from the erythroid to the myeloid lineage resulted in increased myelopoiesis and in the excessive production of neutrophils that accumulate in necrotic tumor regions. This process could neither be prevented by iron nor erythropoietin (EPO) treatment. Trp53floxWapCre mice are the first mouse model where EPO-resistant anemia is described and may serve as a disease model to test therapeutic approaches for a subpopulation of human cancer patients with normal or corrected iron levels that do not respond to EPO

Secreted protein Del-1 regulates myelopoiesis in the hematopoietic stem cell niche

Hematopoietic stem cells (HSCs) remain mostly quiescent under steady-state conditions but switch to a proliferative state following hematopoietic stress, e.g., bone marrow (BM) injury, transplantation, or systemic infection and inflammation. The homeostatic balance between quiescence, self-renewal, and differentiation of HSCs is strongly dependent on their interactions with cells that constitute a specialized microanatomical environment in the BM known as the HSC niche. Here, we identified the secreted extracellular matrix protein Del-1 as a component and regulator of the HSC niche. Specifically, we found that Del-1 was expressed by several cellular components of the HSC niche, including arteriolar endothelial cells, CXCL12-abundant reticular (CAR) cells, and cells of the osteoblastic lineage. Del-1 promoted critical functions of the HSC niche, as it regulated long-term HSC (LT-HSC) proliferation and differentiation toward the myeloid lineage. Del-1 deficiency in mice resulted in reduced LT-HSC proliferation and infringed preferentially upon myelopoiesis under both steady-state and stressful conditions, such as hematopoietic cell transplantation and G-CSF- or inflammation-induced stress myelopoiesis. Del-1-induced HSC proliferation and myeloid lineage commitment were mediated by β3 integrin on hematopoietic progenitors. This hitherto unknown Del-1 function in the HSC niche represents a juxtacrine homeostatic adaptation of the hematopoietic system in stress myelopoiesis

The metabolic enzyme hexokinase 2 localizes to the nucleus in AML and normal haematopoietic stem and progenitor cells to maintain stemness

Thomas, Egan et al. report that hexokinase 2 localizes to the nucleus of leukaemic and normal haematopoietic cells to maintain stemness by interacting with nuclear proteins and modulating chromatin accessibility independently of its kinase activity. Mitochondrial metabolites regulate leukaemic and normal stem cells by affecting epigenetic marks. How mitochondrial enzymes localize to the nucleus to control stem cell function is less understood. We discovered that the mitochondrial metabolic enzyme hexokinase 2 (HK2) localizes to the nucleus in leukaemic and normal haematopoietic stem cells. Overexpression of nuclear HK2 increases leukaemic stem cell properties and decreases differentiation, whereas selective nuclear HK2 knockdown promotes differentiation and decreases stem cell function. Nuclear HK2 localization is phosphorylation-dependent, requires active import and export, and regulates differentiation independently of its enzymatic activity. HK2 interacts with nuclear proteins regulating chromatin openness, increasing chromatin accessibilities at leukaemic stem cell-positive signature and DNA-repair sites. Nuclear HK2 overexpression decreases double-strand breaks and confers chemoresistance, which may contribute to the mechanism by which leukaemic stem cells resist DNA-damaging agents. Thus, we describe a non-canonical mechanism by which mitochondrial enzymes influence stem cell function independently of their metabolic function

Relationship Between Flavonoid Structure And Phase-II Metabolism

Objective: The overall objective is to develop structure-metabolism relationships (SMRs) between UGTs and flavonoids for predicting glucuronidation of flavonoids. The goals of this research project were to: 1) identify the major UGT isoform(s) contributing to the glucuronidation of flavonoids and predicting the major organ of metabolism; 2) establish the substrate-selectivity and regiospecificity of these major UGT isoform(s); 3) develop the in silico prediction models for UGT 1A8 and UGT1A9 using pharmacophore and 2-D/3-D QSAR modeling techniques; 4) study the effect of change in backbone on the disposition of flavonoids in Caco-2 cells; 5) study the rate-limiting role of efflux transporters in the disposition of flavonols in Caco-2 cells; and 6) study the regiospecific disposition of flavones in Caco-2 cells. Method: For objectives 1 and 2, in vitro recombinant human UGT isoforms glucuronidation model was used. For objective 3, in silico pharmacophore and 2-D/3-D QSAR modeling was used along with in vitro glucuronidation intrinsic clearance values in recombinant human UGT isoforms. For objectives 4, 5 and 6, intact Caco-2 cell monolayers was used as the transport model, and Caco-2 cell lysate was used for measuring glucuronide formation rates. Results: 1) To identify the major UGT isoform(s) contributing to the glucuronidation of flavonoids and predicting the major organ of metabolism, we found that flavonoids were mainly glucuronidated by UGT1A1, 1A8 and 1A9 at the substrate concentration of 2.5, 10 and 35μM. 2) To establish the substrate-selectivity and regiospecificity of these major UGT isoform, we found that UGT1A1 showed no regiospecificity for glucuronidating any position, whereas, UGT1A8 and UGT1A9 showed either dominant, preferred or weak regiospecificity for 3-O or 7-O position, depending on the structure of the compound. In general, the addition of hydroxyl group at C-4' reduced, whereas the addition of hydroxyl group at C-5 and/or C-7 improved the rates of glucuronidation of flavonoids by UGT1A8 and 1A9. On the other hand, the rates of glucuronidation by UGT1A1 reduced as number of hydroxyl group in the structure increased. 3) To develop the in silico prediction models for UGT1A8 and UGT1A9 using pharmacophore and 2-D/3-D QSAR modeling techniques, we found that pharmacophore-based semi-quantitative SMR models for UGT1A9 with >75% predictive ability could be developed. But neither semi-quantitative SMR models for UGT1A8 nor the quantitative SMR models for UGT1A8 and UGT1A9 could be successfully developed. 4) To study the effect of change in backbone on the disposition of flavonoids in Caco-2 cells, we found that the change in backbone impacts the excretion of flavonoid sulfates more significantly than the excretion of their glucuronides except for genistein. 5) To study the rate-limiting role of efflux transporters in the disposition of flavonols in Caco-2 cells, we found that excretion of flavonol glucuronides in Caco-2 cells were not limited by efflux transporters and glucuronides of flavonols showed basolateral preference in their excretion. 6) To study the regiospecific conjugation of flavones in Caco-2 cells, we found that both glucuronidation and sulfation of flavones mainly happened at hydroxyl group at C-7 position. Conclusion: UGT1A9, UGT1A8 and UGT1A1 are the most important isoforms that can glucuronidate vast majority of tested flavonoids. Based on published UGT isoform expression pattern in human liver and intestine, they should serve as the major first-pass metabolism organs for flavonoids. UGT1A8 and UGT1A9 showed regiospecificity for 3-O or 7-O position, depending on the structure of the compound, whereas UGT1A1 showed no regiospecificity. Also, the addition of hydroxyl group at C-4' reduced, whereas the addition of hydroxyl group at C-5 and/or C-7 improved the rates of glucuronidation of flavonoids by UGT1A8 and 1A9, with rare exceptions. In contrast, the rates of glucuronidation by UGT1A1 reduced as number of hydroxyl group in the structure increased. Isoform-specific semi-quantitative Pharmacophore-based 3-D SMR prediction models could be developed for UGT1A9 with the predictive ability of more than 75%, but more efforts are needed to develop better quantitative models of prediction. We also probed the SMR experimentally using the Caco-2 model, and the results showed that the excretion of glucuronides was impacted more by the change in number and position of hydroxyl group in the flavonoid structure than changes in backbone. The excretion of glucuronides of flavones but not flavonols is rate-limited by efflux transporters. Future SMR research will incorporate more experimentally derived information to develop better models to predict glucuronidation of flavonoids in humans.Pharmacological and Pharmaceutical Sciences, Department o

Low velocity penetrating head injury with impacted foreign bodies in situ

Penetrating head injury is a potentially life-threatening condition. Penetrating head injuries with impacted object (weapon) are rare. The mechanism of low velocity injury is different from high velocity missile injury. Impacted object (weapon) in situ poses some technical difficulties in the investigation and management of the victims, and if the anticipated problems are not managed properly, they may give rise to serious consequences. The management practice of eight patients with impacted object in situ in context of earlier reported similar cases in literature is presented

Three-Dimensional Quantitative Structure-Activity Relationship Studies on UGT1A9-Mediated 3-O-Glucuronidation of Natural Flavonols Using a Pharmacophore-Based Comparative Molecular Field Analysis ModelS⃞

Glucuronidation is often recognized as one of the rate-determining factors that limit the bioavailability of flavonols. Hence, design and synthesis of more bioavailable flavonols would benefit from the establishment of predictive models of glucuronidation using kinetic parameters [e.g., Km, Vmax, intrinsic clearance (CLint) = Vmax/Km] derived for flavonols. This article aims to construct position (3-OH)-specific comparative molecular field analysis (CoMFA) models to describe UDP-glucuronosyltransferase (UGT) 1A9-mediated glucuronidation of flavonols, which can be used to design poor UGT1A9 substrates. The kinetics of recombinant UGT1A9-mediated 3-O-glucuronidation of 30 flavonols was characterized, and kinetic parameters (Km, Vmax, CLint) were obtained. The observed Km, Vmax, and CLint values of 3-O-glucuronidation ranged from 0.04 to 0.68 μM, 0.04 to 12.95 nmol/mg/min, and 0.06 to 109.60 ml/mg/min, respectively. To model UGT1A9-mediated glucuronidation, 30 flavonols were split into the training (23 compounds) and test (7 compounds) sets. These flavonols were then aligned by mapping the flavonols to specific common feature pharmacophores, which were used to construct CoMFA models of Vmax and CLint, respectively. The derived CoMFA models possessed good internal and external consistency and showed statistical significance and substantive predictive abilities (Vmax model: q2 = 0.738, r2 = 0.976, rpred2 = 0.735; CLint model: q2 = 0.561, r2 = 0.938, rpred2 = 0.630). The contour maps derived from CoMFA modeling clearly indicate structural characteristics associated with rapid or slow 3-O-glucuronidation. In conclusion, the approach of coupling CoMFA analysis with a pharmacophore-based structural alignment is viable for constructing a predictive model for regiospecific glucuronidation rates of flavonols by UGT1A9

Hematopoietic stem cells but not multipotent progenitors drive erythropoiesis during chronic erythroid stress in EPO transgenic mice

The hematopoietic stem cell (HSC) compartment consists of a small pool of cells capable of replenishing all blood cells. Although it is established that the hematopoietic system is assembled as a hierarchical organization under steady-state conditions, emerging evidence suggests that distinct differentiation pathways may exist in response to acute stress. However, it remains unclear how different hematopoietic stem and progenitor cell subpopulations behave under sustained chronic stress. Here, by using adult transgenic mice overexpressing erythropoietin (EPO; Tg6) and a combination of in vivo, in vitro, and deep-sequencing approaches, we found that HSCs respond differentially to chronic erythroid stress compared with their closely related multipotent progenitors (MPPs). Specifically, HSCs exhibit a vastly committed erythroid progenitor profile with enhanced cell division, while MPPs display erythroid and myeloid cell signatures and an accumulation of uncommitted cells. Thus, our results identify HSCs as master regulators of chronic stress erythropoiesis, potentially circumventing the hierarchical differentiation-detour