AG Rawat

  • Investigating the role of ParaHox  and Vent-like homeobox genes  in normal and malignant hematopoiesis

  • Epigenetics, retroviral gene transfer, stem cells function and non-coding RNA

Hematopoietic stem cells (HSCs) beside the property of self-renewal have the potential to proliferate rapidly, lack senescence, and have the unique capability to produce all specialized differentiated blood cells over the whole lifespan of an organism. Studies suggest that the methylation machinery in HSCs regulate their self-renewal property, thereby controlling their lineage–specific differentiation and proliferation. Recent evidence indicates that deregulation of DNA methylation plays an important pathogenic role in leukemogenesis. The cause for this tremendous deregulation of methylation pattern in leukemia remains unknown. Although the mechanism of DNA methylation has been well-studied and rigorously established, active DNA demethylation in vertebrates has remained enigmatic. Only recently, it was observed that DNA demethylation is critical steps in the generation of induce pluripotent stem cells when reprogramming the somatic nuclei to a pluripotent stage. Therefore, this finding strongly supports the hypothesis that DNA methylation is a reversible process.
Our research concentrates on following topics.

  1. Identification and characterization of the role of demethylation enzyme in normal and malignant hematopoiesis.
    Recently a demethylation pathway of 5mC was unraveled; this cascade is crucially initiated by the so called ten eleven translocation (TET1-3) oxidases, which demethylate 5-methylcytosine (5mC) into 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC), and 5-carboxylcytosine (5caC). An embryonic study clearly indicates the family members of TET genes is playing a role in ES cell maintenance and inner cell mass (ICM) cell specification and was demonstrated to be involved in hematological malignancies. Recently a correlation between low genomic 5hmC and TET2 mutation status was shown in patients with myeloid malignancies. At our lab, we are interested in identifying and characterizing the function of TET genes in normal and malignant hematopoiesis.
    Funding – Juniorprofessorenprogramm, University of Ulm
    Collaborator- Prof. C. Buske (Germany), Prof. K. Döhner (Germany), Prof. K. Akashi (Japan)
  2. Role of ParaHox genes in normal and malignant hematopoiesis.
    The identification of novel proteins which regulate propagation of human leukemic cells and do not either express in normal HSC and early progenitors or do not impact their functions is one of the major goals for experimental and clinical hematology. In this regard we have shown that ParaHox gene - caudal type homeobox gene 2 (CDX2), is a highly conserved transcription factor frequently ectopically expressed proto-oncogenes in human AML as well as acute lymphoblastic leukemia, but is silenced in postnatal hematopoiesis. Importantly, levels of CDX2 were closely correlated with deregulated gene expression of developmentally important HOX genes, substantiating the leukemogenic potential of Cdx2. Ectopic expression of Cdx2 in murine hematopoietic progenitors induces AML in murine model and causes perturbation of leukemogenic Hox genes. Deletion of its N-terminal domain abolished the leukemogenic potential of Cdx2. We want to test the hypothesis that CDX2 is an essential driving factor for leukemia propagation and can act as novel drug target in the treatment of acute leukemia
    Co-investigator –  Prof. C. Buske
    Collaborator-  Prof. H. Döhner, Prof. K. Döhner, Prof. Dr. R. Schlenk all from Germany

  3. VENTX: a novel regulator in human leukemias
    Previously, we have shown that human Vent-like homeobox gene VENTX, a putative homologue of the early developmental Xenopus xvent2 gene, is a novel regulatory hematopoietic gene. We could document that enforced VENTX expression promotes myeloid development while paralleled by downregulation of lymphoid development genes. VENTX is aberrantly expressed in CD34+ leukemic stem cell (LSC) candidates in human AML, predominantly in t(8;21) positive and normal karyotype (NK) AML.  Lentiviral mediated knock-down of VENTX in human AML cell lines significantly impaired leukemic cell growth, indicating that VENTX is a key protein for human leukemic growth. Based on these findings we want to evaluate the regulatory function of VENTX by analyzing upstream regulators and downstream targets of VENTX in leukemic hematopoiesis. We want to identify factors which contribute to the aberrant expression of VENTX in LSC and evaluate the functional relevance. Furthermore, we want to analyze a potential collaboration of VENTX with AML1-ETO in perturbing hematopoiesis and evaluate the role of the known VENTX pseudogenes in leukemic transformation.  Finally, we want to search for potential therapeutic strategies to inhibit aberrant VENTX expression in CD34+ LSC´s.
    Funding –  DFG
    Co-investigator –  PD. Dr. M. Feuring-Buske
    Collaborator-  Prof. C. Buske, Prof. H. Döhner, Prof. K. Döhner, Dr. F.  Kuchenbauer all from Germany

  4. Characterization of the role of piRNA-related proteins, PIWI in human and murine hematopoietic stem cell development and leukemogenesis.
    To carry out their biological functions, small non coding RNA such as miRNA require a unique class of highly conserved proteins called Argonautes. A sub family of Argonaute proteins known as the Piwi proteins is expressed mainly in germ cells and germ stem cells, and is known to bind with a class of small non coding RNA called piwi interacting RNA or piRNA.  Piwi proteins are known to be essential for germ- line- stem -cell self renewal and together with piRNA, have been implicated in transposon methylation (silencing) in mammalian germ cells and germ stem cells. Piwil gene promotes euchromatic histone modifications and piRNA transcription in subtelomeric heterochromatin in Drosophila. Piwil genes have shown to be expressed in humancCD34+ BM cells and silenced in leukemic cell lines. Our focus lies in the area of Piwi like proteins, piRNA’s and their possible effect on self- renewal capability and transposon silencing in hematopoietic stem cells.
    Funding –  DFG
    Co-investigator –  Prof. C. Buske
    Collaborator-  Prof. Dr. C. Plass (DKFZ, Heidelberg), Prof. Dr. Klaus Förstemann (Gene Center Munich, LMU, Department of Chemistry and Pharmaceutics, Munich), Dr. M. Feuring-Buske ( Ulm, Germany)

  5. Role of none coding RNA in normal and malignant hematopoiesis
    Homeobox gene expression in normal cells is highly regulated by complex gene regulatory networks. Disruption of these networks lead to hematological malignancy. Several global gene expression profiles revealed that homeobox genes are highly over-expressed in majority of human leukemia’s. Recently it has been shown that miRNAs (mir-196a and b and mir-10a and b) encoded at three paralogous locations in the A, B, and C mammalian HOX clusters, has extensive evolutionarily conserved complementarities to HOX genes, thereby regulating their expression. This opens a possibility to modulate homeobox gene expression by miRNAs. Importantly, mir-196 and mir-10 are highly expressed in murine normal hematopoietic stem cells and there expression goes down during differentiation. However, recently over expression of these miRNA has been reported in certain subtype of human leukemia, indicating that may be deregulated expression could lead to the hematological malignancy. We want to identification and characterization the role of homeobox genes associated miRNA in normal and malignant hematopoiesis in vivo.