Studies on Myelodysplastic syndromes and Leukemia
   

Myelodysplastic syndrome (MDS) are hematologic malignancies that are characterized by ineffective hematopoiesis. Even though these are preleukemic disorders that can transform to Acute Myeloid Leukemia in a third of cases, most of the morbidity is due to low blood counts. Our lab if focused in studying the molecular pathogenesis of myelodysplastic syndromes and identifying targetable pathways in this disease.

Role of p38 MAP kinase in pathogenesis of MDS:


Figure 1
Our laboratory identified that the p38 MAPK is constitutively activated in low grade MDS, mediates progenitor apoptosis in MDS and provides an excellent therapeutic target to stimulate MDS hematopoiesis. We demonstrated that p38 MAPK activation is required for interactions between the marrow stroma and hematopoietic cells that lead to production of myelosuppressive cytokines by the stroma. These studies provided the preclinical rationale for the use of p38 MAPK inhibitors in MDS and was the basis for a multicenter clinical trial with the p38 inhibitor, SCIO-469, in low-grade MDS, that was recently completed (A Randomized, Multicenter, Open-Label, Modified Dose-Ascension, Parallel Study of the Safety, Tolerability, and Efficacy of Oral SCIO-469 in Patients With Myelodysplastic Syndromes ClinicalTrials.gov Identifier: NCT00113893) This trial showed that SCIO-469 was generally well tolerated and was active as monotherapy in low/int-1 MDS at dose levels of 30-120 mg po TID. Responses in all three blood lineages were seen. Additional trials with another p38 MAPK inhibitor, ARRY-614, have now been initiated and are ongoing, underscoring the high translational potential of this work in MDS therapeutics (ARRY-614 in Patients with Low or Intermediate-1 Risk MDS ClinicalTrials.gov Identifier: NCT00916227). Our lab is presently studying the efficacy of ARRY-614 in preclinical models. 

    Navas T, Mohindru M, Estes M, Ma JY, Sokol L, Pahanish P, Parmar S, Haghnazari E, Zhou L, Collins RC, Kerr I, Nguyen A, Xu Y, Platanias LC, List AA, Higgins LS, Verma A. Inhibition of overactivated p38 MAPK can restore hematopoiesis in myelodysplastic syndrome progenitors Blood 2006, 15;108(13):4170-7 PMID: 16940419

    Verma A, Sassano A, Deb D, Wickrema, A., VanBesien K., and Platanias L. “Blockade of p38 MAP kinase reverses cytokine mediated inhibition in a plastic anemia.” Journal of Immunology 2002, 168 (12) (Cutting edge section) PMID: 12055203

    Verma A, Deb D.K., Sassano A., Uddine S., Wickrema A., Varga and Platanias LC “Activation of the p38 mitogen activated protein kinase mediates suppressive effects of Type I interferons and transforming growth factor- on normal hematopoiesis.” Journal of Biological Chemistry 2002, 277(10):7726-35 PMID: 11773065

    Bachegowda L, Gligich O, Mantzaris I, Schinke C, Wyville D, Carrillo T, Braunschweig I, Steidl U, Verma A. Signal transduction inhibitors in treatment of myelodysplastic syndromes. J Hematol Oncol. 2013 Jul 10;6:50. doi: 10.1186/1756-8722-6-50. Review. PMID: 23841999

Role of TGF-β in the pathogenesis of MDS


Figure 2
Our lab provided the first comprehensive evidence for TGF-β signaling in MDS and defined the functional role of TGF-β receptor I (TBRI) kinase-Smad2 pathway in hematopoietic suppression in MDS. This work demonstrated that (i) a TGF-β secreting transgenic mouse can serve as a novel model of cytokine-induced bone marrow failure, and (ii) that inhibition of the TGF-β signaling pathway by genetic and pharmacologic means can stimulate in vitro hematopoiesis in MDS. These findings strongly suggest that novel TBRI kinase inhibitors may be clinically useful in the treatment of MDS. Recently we showed that Smad7, an important negative regulator of TBRI kinase, is markedly downregulated in MDS marrow progenitors leading to increased TBRI signaling. They also found that the novel TBRI inhibitor, LY-2157299, shows activity in mouse models of MDS. More recently, we have shown that increased levels of miR-21 are seen in MDS and target SMAD7 in hematopoietic stem and progenitor cells. This work has shown that inhibiting miR-21 can also reverse TGF-β signaling and stimulate hematopoiesis in MDS.

    Bhagat T, Zhou L, Sokol L, Caceres G, Gundabolu K, Gordon S, Mantzaris I, Gligich O, Yu Y, Bhattacharyya S, Jing X, Polineni R, Tamari R, Bhatia K, Pellagatti A , Boultwood J, Kambhampati S, Steidl U, Stein C, Ju W, Liu G, Kenny P, List A, Bitzer M, Verma A. miR-21 mediates hematopoietic suppression in MDS by activating TGF- signaling Blood 2013, (In Press) PMID: 23390194

    L Zhou, C McMahon, T Bhagat, C Alencar, Y Yu, M Fazzari, D Sohal, C Heuck, K Gundabolu, C Ng, Y Mo, W Shen, A Wickrema, G Kong, E Friedman, L Sokol, G Mantzaris, A Pellagatti , J Boultwood, LC. Platanias. U Steidl, L Yan, JM Yingling, MM Lahn, A List, M Bitzer and A Verma Reduced SMAD7 leads to overactivation of TGF-β signaling in MDS that can be reversed by a specific inhibitor of TGF-β receptor I kinase. Cancer Research 2011 Feb 1;71(3):955-63.PMID: 21189329

    Zhou L, Nguyen AN, Sohal D, Ma JY, Pahanish P, Gundabolu K, Hayman J, Chubak A, Mo Y, Bhagat T, Das B, Haghnazari E, Navas T, Parmar S, Kambhampati S, Pellagati A, Braunchweig I, Zhang YE, Wickrema A, Boultwood J, Platanias LC, Higgins LS, List A, Bitzer M, Verma A. Inhibition of Transforming Growth Factor beta receptor I kinase can stimulate hematopoiesis in MDS Blood 2008 Oct 15;112(8):3434-43 PMID: 18474728

Identifying Stem cell and progenitor alterations in MDS and acute myeloid leukemia


Figure 3
In collaboration among the the Steidl lab we have identified the involvement of precisely defined stem and progenitor compartments in patients with MDS and AML. Primary bone marrow samples were sorted and analyzed by multi-parameter fluorescence-activated cell sorting, genome-wide DNA cytosine methylation, and transcriptional analysis, in combination with cytogenetic analysis. These studies indicate that in most patients cytogenetic aberrations are already detectable in the earliest stem cells, and that those cells have characteristic changes in DNA methylation and transcriptional profiles. Most importantly, the abnormal stem cells are not eliminated by treatment with approved agents such as 5-azacytidine, and can expand before clinical relapses occur. Current studies are validating several promising targets, such as STAT3, in functional analyses. One important objective of these studies is the identification of (i) markers for leukemia/MDS-initiating cells for the detection of minimal residual (stem cell) disease, and (ii) novel targets for the development of therapies specific for leukemia/MDS-initiating cells. The sorting strategy used in these studies will be utilized in identifying leukemic stem cells in an investigator initiated study of Lenalidomide maintenance in AML at AECC.

    Will B, Zhou L, Vogler TO, Ben-Neriah S, Schinke C,Tamari R, Yu Y, Bhagat T, Bhattacharya S, Barreyro L, Heuck C, Mo Y, Parekh S, McMahon C, Pellagatti A, Boultwood J, Montagna C, Silverman L, Maciejewski J, Greally JM, Ye BH, List AF, Steidl C, Steidl U, Verma A Stem and progenitor cells in myelodysplastic syndromes show aberrant stage specific expansion and harbor genetic and epigenetic alterations, Blood 2012; 120(10):2076-86. PMID: 22753872

    Elias HK, Schinke C, Bhattacharyya S, Will B, Verma A, Steidl U.
    Stem cell origin of myelodysplastic syndromes.
    Oncogene. 2013 Dec 16. (review) PMID: 24336326

Role of DNA methylation in normal and malignant hematopoiesis.

Even though epigenetic alterations have been seen in MDS marrow progenitors, very little is known about the molecular alterations in dysplastic peripheral blood cells. We analyzed the methylome of MDS leucocytes by the HpaII tiny fragment Enrichment by Ligation-mediated PCR (HELP) assay and determined that it was globally distinct from age-matched controls and was characterized by numerous, novel, aberrant hypermethylated marks that were located mainly outside of CpG islands. The marks preferentially affected GTPase regulators and other cancer-related pathways, and were enriched for binding sites for SP1 and other transcription factors. Additionally, array CGH revealed that novel as well as previously-characterized deletions and amplifications could also be visualized in peripheral blood leucocytes, thus potentially reducing the need for bone marrow samples for future studies.

We have also conducted high resolution analysis of DNA methylation during normal erythropoesis. We demonstrated that loss of methylation is the predominant epigenetic change during this process and affects regulatory regions of the genome. Furthermore, integrative analysis enabled us to identify novel, potential regulatory areas of the genome as evident by epigenetic changes in a predicted PU.1 binding site in intron 1 of the GATA1 gene. This intronic site was found to be conserved across species and was validated to be a novel PU.1 binding site by quantitative ChIP in erythroid cells. Altogether, our study provides a comprehensive analysis of methylomic and transcriptomic changes during erythroid differentiation and demonstrates that human terminal erythropoiesis is surprisingly associated with hypomethylation of the genome.

    Yu Y, Mo Y, Liu H, Bhattacharyya S, Ebenezer D, Sundaravel S, Caces B, Ulaszek J, Artz A, Nischal S, Bhagat T, Bathon , Maqbool S, Suzuki M, Steidl U, Godley L, Skoultchi A, Greally J, Wickrema A, Verma A 

    High resolution methylome analysis reveals widespread hypomethylation during human erythroid differentiation JBC (In Press) PMID: 23306203

    Zhou L, Opalinska J, Sohal D, Yu Y, Mo Y, Bhagat T, Abdel-Wahab O, Fazzari M, Figueroa M, Alencar C, Zhang J, Kanbhampati S, Parmar S, Nischal S, Heuck C, Suzuki M, Friedman E, Pellagatti A, Boultwood J, Steidl U, Sauthararajah Y, Yajnik V, Gore SD, Platanias LC, Levine R, Melnick A, Greally JM, Verma A. Aberrant epigenetic and genetic marks are seen in myelodysplastic leucocytes and reveal DOCK4 as a candidate pathogenic gene on chr7q. J Biol Chem. 2011 Apr 30. PMID: 21532034

Development of Gene expression profiles from MDS CD34+ cells:

In collaboration with Prof Jackie Boutlwood and Dr Andrea Pellagatti (Oxford) we have maintained a database of gene expression of MDS stem cells that have been used in numerous studies.

    Pellagatti A, Benner A, Mills K, Cazzola M, Giagounidis AAN, Perry J, Malcovati L, Giovanni M, Porta D, Jädersten M, Verma A, McDonald E, Killick S, Hellstrom-Lindberg E, Bullinger L, Wainscoat JS, Boultwood J. Identification of gene expression based prognostic markers in the hematopoietic stem cells of patients with myelodysplastic syndromes JCO 2013 (In Press)

    Pellagatti A, Cazzola M, Giagounidis A, Perry J, Malcovati L, Della Porta MG, Jädersten M, Verma A, Norbury C Hellström-Lindberg E, Wainscoat JS, Boultwood J Deregulated gene expression pathways in myelodysplastic syndrome hematopoietic stem cells. Leukemia. 2010 Apr;24(4):756-64. PMID: 20220779

    Sohal D, Yeatts A, Ye K, Pellagatti A, Zhou L, Pahanish P, Mo Y, Bhagat T, Mariadason J, Boultwood J, Melnick A, Greally JM, Verma A. Meta-analysis of microarray studies reveals a novel hematopoietic progenitor cell signature and demonstrates feasibility of inter-platform data integration. PLOS One 2008 Aug 13;3(8):e2965 PMID: 18698424

 
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