Rashim Pal Singh

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Rashim Pal Singh


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EDUCATION

M.Sc. (Biochemistry) Post-Graduate Institute of Medical Education and Research, Chandigarh, India.
PhD (Biomedicine) Technische universität Dresden, Dresden International Graduate School for Biomedicine and Bioengineering, Dresden, Germany.
14

Scopus Publications

Scopus Publications

  • Iron- And erythropoietin-resistant anemia in a spontaneous breast cancer mouse model
    Nuria Fabregas Bregolat, Maja Ruetten, Milene Costa Da Silva, Mostafa A. Aboouf, Hyrije Ademi, Nadine von Büren, Julia Armbruster, Martina Stirn, Sandro Altamura, Oriana Marques, Josep M. Monné Rodriguez, Victor J. Samillan, Rashim Pal Singh, Ben Wielockx, Martina U. Muckenthaler, Max Gassmann, Markus Thiersch
    Haematologica, 2022
    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, whereby functional iron deficiency and iron-restricted erythropoiesis are induced by increased hepcidin levels in response to raised levels of interleukin-6. 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 levels of interleukin-6 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 not be prevented by either iron or erythropoietin treatment. Trp53floxWapCre mice are the first mouse model in which erythropoietin-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 who do not respond to erythropoietin.
  • The metabolic enzyme hexokinase 2 localizes to the nucleus in AML and normal haematopoietic stem and progenitor cells to maintain stemness
    Geethu Emily Thomas, Grace Egan, Laura García-Prat, Aaron Botham, Veronique Voisin, Parasvi S. Patel, Fieke W. Hoff, Jordan Chin, Boaz Nachmias, Kerstin B. Kaufmann, Dilshad H. Khan, Rose Hurren, Xiaoming Wang, Marcela Gronda, Neil MacLean, Cristiana O’Brien, Rashim P. Singh, Courtney L. Jones, Shane M. Harding, Brian Raught, Andrea Arruda, Mark D. Minden, Gary D. Bader, Razq Hakem, Steve Kornblau, John E. Dick, Aaron D. Schimmer
    Nature Cell Biology, 2022
    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.
  • Transduction of Primary AML Cells with Lentiviral Vector for In Vitro Study or In Vivo Engraftment
    Aaron D. Schimmer, Rashim Pal Singh, Ayesh K. Seneviratne, Geethu E. Thomas, Neil MacLean, Rose Hurren
    STAR Protocols, 2020
    We describe a method to silence genes in primary acute myeloid leukemia cells by transducing them with shRNA in lentiviral vectors. The transduction of primary non-adherent cells is particularly challenging. The protocol will aid in performing such experiments and is particularly helpful to prepare cells for in vivo engraftment studies. Use of a special medium supplemented with cytokines preserves the viability of the leukemic stem cells and their ability to engraft the marrow of immune-deficient mice. For complete details on the use and execution of this protocol, please refer to Singh et al. (2020).
  • Mitochondria regulate AML differentiation independent of oxidative phosphorylation and metabolism
    Rashim Pal Singh, Aaron D Schimmer
    Molecular and Cellular Oncology, 2020
    Acute myeloid leukemia (AML) is an aggressive hematologic malignancy originating from acquired mutations in stem and progenitor cells. While many patients achieve remission with upfront therapy, most eventually relapse and succumb to their disease. AML relapse is often due to the persistence of quiescent, chemoresistant leukemic stem cells (LSC). Hence, it is important to understand the biological vulnerabilities of LSCs as a step toward developing new therapies that target these cells. We and subsequently others previously showed that AML cells, including LSC, have unique mitochondrial features with increased reliance on mitochondrial metabolism and oxidative phosphorylation. Disrupting pathways such as mitochondrial protein import or protein degradation kills AML cells and LSC in vitro and in vivo. In our recent paper, “Disrupting Mitochondrial Copper Distribution Inhibits Leukemic Stem Cell Self-Renewal” we explored mitochondrial protein import in AML and LSC and specifically the impact of inhibiting the folding of proteins in the inner mitochondrial space. The majority of the proteins in the mitochondria are translated in the cytoplasm and imported into the mitochondria. Proteins enter the mitochondria through the Translocase of the Outer Membrane (TOM) and Translocase of the Inner Membrane (TIM) protein import machinery. A subset of cysteine-rich proteins is destined for the mitochondrial intermembrane space where they are oxidized and folded into mature forms by Mitochondrial Intermembrane Assembly (MIA) pathway and the oxidase Augmenter of Liver Regeneration (ALR). In our recent publication, we evaluated the impact of inhibiting the MIA pathway by targeting ALR. Genetic knockdown of ALR or inhibiting the target with the selective ALR inhibitor MitoBloCK-6 decreased the growth of AML cells and LSC in vitro and in vivo and induced differentiation. We identified the copper chaperone, COX17, as a primary downstream target of ALR. Inhibition of ALR led to decreased levels of mitochondrial COX17, presumably due to impaired folding of the protein in the intermembrane space and subsequent exit from the mitochondria. Loss of COX17 phenocopied ALR inhibition and decreased AML growth and increased differentiation. Decreased AML growth upon ALR or COX17 inhibition was not due to reduced oxidative phosphorylation. Therefore, we searched for other mechanisms to explain how inhibition of ALR and COX17 decreased AML stem cell function and increased differentiation. COX17 is a copper chaperone that shuttles copper from the outer membrane of the mitochondria to the terminal complex of respiratory chain, cytochrome c oxidase. Loss of COX17 resulted in the redistribution of copper to S-adenosyl homocysteine hydrolase (SAHH). SAAH is a key enzyme that regulates levels of the global methyl donor, SAM, and thereby controls protein and DNA methylation. SAAH is a copperregulated enzyme. Copper binds and inhibits the activity of SAAH. Loss of COX17 inhibited the activity of SAHH, decreased levels of S-adenosyl methionine (SAM), and reduced global DNA methylation. Chelation of copper with penicillamine restored SAHH activity, returned levels of SAM and DNA methylation to the baseline and rescued changes in gene expression after ALR inhibition. Finally, chelation of copper prevented AML cell death after inhibiting ALR. Thus, this work highlights new mechanisms by which mitochondrial pathways regulate epigenetic marks to influence AML stem cell function and differentiation. We also describe a new role for mitochondrial copper in regulating leukemia stem cells and differentiation. Future work will investigate the critical genes whose methylation is influenced by redistributing mitochondrial copper. We will also compare changes in DNA methylation and gene expression after ALR or COX17 inhibition with changes after treatment with hypomethylating agents, such as azacytidine. The data from this paper support the future development of more potent ALR inhibitors as leads for potential anti-leukemic agents. Finally, this work reveals how mitochondrial pathways can influence stem cell function and differentiation independent of the effects of mitochondrial metabolism and oxidative phosphorylation.
  • Disrupting Mitochondrial Copper Distribution Inhibits Leukemic Stem Cell Self-Renewal
    Rashim Pal Singh, Danny V. Jeyaraju, Veronique Voisin, Rose Hurren, Changjiang Xu, James R. Hawley, Samir H. Barghout, Dilshad H. Khan, Marcela Gronda, Xiaoming Wang, Yulia Jitkova, David Sharon, Sanduni Liyanagae, Neil MacLean, Ayesh K. Seneviratene, Sara Mirali, Adina Borenstein, Geethu E. Thomas, Joelle Soriano, Elias Orouji, Mark D. Minden, Andrea Arruda, Steven M. Chan, Gary D. Bader, Mathieu Lupien, Aaron D. Schimmer
    Cell Stem Cell, 2020
  • Hematopoietic Stem Cells but Not Multipotent Progenitors Drive Erythropoiesis during Chronic Erythroid Stress in EPO Transgenic Mice
    Rashim Pal Singh, Tatyana Grinenko, Beáta Ramasz, Kristin Franke, Mathias Lesche, Andreas Dahl, Max Gassmann, Triantafyllos Chavakis, Ian Henry, Ben Wielockx
    Stem Cell Reports, 2018
    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.
  • Endothelial Cell-Specific Overexpression of Del-1 Drives Expansion of Haematopoietic Progenitor Cells in the Bone Marrow
    Lan-Sun Chen, Ioannis Kourtzelis, Rashim Singh, Sylvia Grossklaus, Ben Wielockx, George Hajishengallis, Triantafyllos Chavakis, Ioannis Mitroulis
    Thrombosis and Haemostasis, 2018
    Hematopoietic stem cells (HSCs) are responsible for the maintenance of hematopoiesis under normal conditions and its adaptation to hematopoietic stress (1). HSCs are localised in the bone marrow (BM) in a micro-anatomic space designated as the HSC niche (2) comprising different cellular components. While osteoblasts predominantly contribute to the restoration of hematopoiesis after transplantation, endothelial and perivascular stromal cells form a perivascular niche, which promotes the self-renewal of HSCs and their multi-lineage differentiation, and, hence the maintenance of hematopoiesis (2). Cells of the perivascular niche not only provide the mechanical barrier between the BM microenvironment and circulation, but also regulate hematopoiesis, either through adhesive interactions with HSCs (3) or by the release of paracrine factors (4,5). The adhesive interactions of HSCs with endothelial and perivascular cells mediate both the trafficking of HSCs into and out of the BM (6) and their maintenance and proliferation potential (3). Developmental endothelial locus-1 (Del-1) is a secreted glycoprotein that interacts with integrins We have recently shown that Del-1 is expressed by important cellular components (endothelial, mesenchymal stromal and cells of the osteoblastic of the HSC niche, where it regulates hematopoietic progenitor function and their myeloid differentiation (10). This ability of Del-1 to act as a HSC niche factor was attributed to its interaction with β 3 integrin on hematopoietic progenitors (10). Here, we set out to precisely define the role of Del-1 as an endothelial cell-derived perivascular niche factor. To this end, we studied steady-state and regeneration hematopoiesis using an independent in vivo approach, namely mice with endothelial-specific overexpression of Del-1. We engaged transgenic mice with endothelial cell-specific overexpression of Del-1 (EC-Del1), generated by utilizing a tie2 promoter/enhancer construct (11,12). EC-Del1 mice
  • Secreted protein Del-1 regulates myelopoiesis in the hematopoietic stem cell niche
    Ioannis Mitroulis, Lan-Sun Chen, Rashim Pal Singh, Ioannis Kourtzelis, Matina Economopoulou, Tetsuhiro Kajikawa, Maria Troullinaki, Athanasios Ziogas, Klara Ruppova, Kavita Hosur, Tomoki Maekawa, Baomei Wang, Pallavi Subramanian, Torsten Tonn, Panayotis Verginis, Malte von Bonin, Manja Wobus, Martin Bornhäuser, Tatyana Grinenko, Marianna Di Scala, Andres Hidalgo, Ben Wielockx, George Hajishengallis, Triantafyllos Chavakis
    Journal of Clinical Investigation, 2017
    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.
  • Increased EPO Levels Are Associated With Bone Loss in Mice Lacking PHD2 in EPO-Producing Cells
    Martina Rauner, Kristin Franke, Marta Murray, Rashim Pal Singh, Sahar Hiram-Bab, Uwe Platzbecker, Max Gassmann, Merav Socolovsky, Drorit Neumann, Yankel Gabet, Triantafyllos Chavakis, Lorenz C Hofbauer, Ben Wielockx
    Journal of Bone and Mineral Research, 2016
    The main oxygen sensor hypoxia inducible factor (HIF) prolyl hydroxylase 2 (PHD2) is a critical regulator of tissue homeostasis during erythropoiesis, hematopoietic stem cell maintenance, and wound healing. Recent studies point toward a role for the PHD2‐erythropoietin (EPO) axis in the modulation of bone remodeling, even though the studies produced conflicting results. Here, we used a number of mouse strains deficient of PHD2 in different cell types to address the role of PHD2 and its downstream targets HIF‐1α and HIF‐2α in bone remodeling. Mice deficient for PHD2 in several cell lineages, including EPO‐producing cells, osteoblasts, and hematopoietic cells (CD68:cre‐PHD2f/f) displayed a severe reduction of bone density at the distal femur as well as the vertebral body due to impaired bone formation but not bone resorption. Importantly, using osteoblast‐specific (Osx:cre‐PHD2f/f) and osteoclast‐specific PHD2 knock‐out mice (Vav:cre‐ PHD2f/f), we show that this effect is independent of the loss of PHD2 in osteoblast and osteoclasts. Using different in vivo and in vitro approaches, we show here that this bone phenotype, including the suppression of bone formation, is directly linked to the stabilization of the α‐subunit of HIF‐2, and possibly to the subsequent moderate induction of serum EPO, which directly influenced the differentiation and mineralization of osteoblast progenitors resulting in lower bone density. Taken together, our data identify the PHD2:HIF‐2α:EPO axis as a so far unknown regulator of osteohematology by controlling bone homeostasis. Further, these data suggest that patients treated with PHD inhibitors or EPO should be monitored with respect to their bone status. © 2016 American Society for Bone and Mineral Research.
  • Loss of prolyl hydroxylase-2 in myeloid cells and T-lymphocytes impairs tumor development
    Soulafa Mamlouk, Joanna Kalucka, Rashim Pal Singh, Kristin Franke, Antje Muschter, Anika Langer, Christiane Jakob, Max Gassmann, Gustavo B. Baretton, Ben Wielockx
    International Journal of Cancer, 2014
    The tumor microenvironment plays a pivotal role during cancer development and progression. The balance between suppressive and cytotoxic responses of the tumor immune microenvironment has been shown to have a direct effect on the final outcome in various human and experimental tumors. Recently, we demonstrated that the oxygen sensor HIF‐prolyl hydroxylase‐2 (PHD2) plays a detrimental role in tumor cells, stimulating systemic growth and metastasis in mice. In our current study, we show that the conditional ablation of PHD2 in the hematopoietic system also leads to reduced tumor volume, intriguingly generated by an imbalance between enhanced cell death and improved proliferation of tumor cells. This effect seems to rely on the overall downregulation of protumoral as well as antitumoral cytokines. Using different genetic approaches, we were able to confine this complex phenotype to the crosstalk of PHD2‐deficient myeloid cells and T‐lymphocytes. Taken together, our findings reveal a multifaceted role for PHD2 in several hematopoietic lineages during tumor development and might have important implications for the development of tumor therapies in the future.
  • HIF prolyl hydroxylase 2 (PHD2) is a critical regulator of hematopoietic stem cell maintenance during steady-state and stress
    Rashim Pal Singh, Kristin Franke, Joanna Kalucka, Soulafa Mamlouk, Antje Muschter, Agnieszka Gembarska, Tatyana Grinenko, Carsten Willam, Ronald Naumann, Konstantinos Anastassiadis, A. Francis Stewart, Stefan Bornstein, Triantafyllos Chavakis, Georg Breier, Claudia Waskow, Ben Wielockx
    Blood, 2013
  • HIF-1α is a protective factor in conditional PHD2-βdeficient mice suffering from severe HIF-2-induced excessive erythropoiesis
    Kristin Franke, Joanna Kalucka, Soulafa Mamlouk, Rashim Pal Singh, Antje Muschter, Alexander Weidemann, Vasuprada Iyengar, Steffen Jahn, Kathrin Wieczorek, Kathrin Geiger, Michael Muders, Alex M. Sykes, David M. Poitz, Tatsiana Ripich, Teresa Otto, Sybille Bergmann, Georg Breier, Gustavo Baretton, Guo-Hua Fong, David R. Greaves, Stefan Bornstein, Triantafyllos Chavakis, Joachim Fandrey, Max Gassmann, Ben Wielockx
    Blood, 2013
  • Loss of epithelial hypoxia-inducible factor prolyl hydroxylase 2 accelerates skin wound healing in mice
    Joanna Kalucka, Andreas Ettinger, Kristin Franke, Soulafa Mamlouk, Rashim Pal Singh, Katja Farhat, Antje Muschter, Susanne Olbrich, Georg Breier, Dörthe M. Katschinski, Wieland Huttner, Alexander Weidemann, Ben Wielockx
    Molecular and Cellular Biology, 2013
  • Hypoxia-mediated regulation of stem cell fate
    Rashim Pal Singh, Kristin Franke, Ben Wielockx
    High Altitude Medicine and Biology, 2012