Chromatin dynamics and gene regulation in normal and malignant hematopoiesis

Acute myeloid leukemia (AML) and several hematological malignancies arise from acquisition of multiple stepwise genetic and epigenetic changes in hematopoietic stem and progenitor cells (HSPCs). Understanding the regulatory pathways that are deregulated in HSPCs is important to better understand the development of leukemia and to design novel therapeutic strategies for the treatment of leukemia. Our lab applies genetic, epigenetic and biochemical approaches in genetically modified mouse models, humanized mouse models and human primary leukemic cells. Our research focus on three areas:

 

  1. Interplay between transcription factors and chromatin dynamics in normal and malignant hematopoiesis

  2. Transcriptional deregulation in AML

  3. Non-coding RNA function in hematopoiesis and leukemia

1. Interplay between transcription factors and chromatin dynamics in normal and malignant hematopoiesis

Epigenetic gene regulation is mediated by DNA methylation, histone modification and chromatin modeling which in turn modify chromatin structure there-by giving chromatin access to transcription factors and other associated regulators (Figure 1). A major question in gene regulation is how is the transmission between transcription factors and chromatin modulated. Understanding the mechanisms behind this mutual interplay between transcription factors and chromatin dynamics will provide novel insights into our understanding of gene regulation in physiology and diseases. We are exploring the following questions: (1) What are the mechanisms through which transcription factor binding at a distal enhancer can act as a bridge between the enhancer and its target promoter in hematopoiesis? (2) Having both activation and repression potential, how do transcription factors decide between which genes should be activated versus which ones should be repressed? (3) What are the implications of chromosomal rearrangements observed in hematological malignancies in topologically associated domain (TAD) architecture and gene expression? (4) What is the functional significance of enhancer RNAs (eRNAs) in hematopoiesis and how they are deregulated in leukemia?

Figure 1: Interplay between transcription factors and chromatin dynamics in gene regulation

References:

Pulikkan JA, Hegde M, Ahmed H, Belaghzal H, Illendula A, Yu J, O’Hagen K, Ou J, Muller-Tidow C, Wolfe SA, Zhu LJ, Dekker J, Bushweller JH, Castilla LH. CBFβ-SMMHC inhibition triggers apoptosis by disrupting MYC chromatin dynamics in acute myeloid leukemia. Cell 2018 Jun 28;174(1):172-186.

Illendula A*, Pulikkan JA*, Zong H, Grembecka J, Xue L, Sen S, Zhou Y, Boulton A, Kuntimaddi A, Gao Y, Rajewski RA, Guzman ML, Castilla LH, Bushweller JH. A small-molecule inhibitor of the aberrant transcription factor CBFβ-SMMHC delays leukemia in mice. Science 2015 Feb 13;347(6223):779-84.

Choi A, Illendula A, Pullikkan JA, Roderick JE, Tesell JT, Yu J, Hermance N, Zhu L, Castilla LH,  Bushweller JH and Kelliher MA. RUNX1 is required for oncogenic Myb and Myc enhancer activity in T cell acute lymphoblastic leukemia. Blood 2017 Oct 12;130(15):1722-1733.

2. Transcriptional deregulation in AML

Mutations in transcription factors have long been shown to be central in tumorigenesis. Our lab is interested in understanding transcriptional regulation of myeloid differentiation and how this is altered in AML. In particular, we are studying deregulation of transcription factors C/EBPα and core-binding factors, CBFs (consisting of RUNX and CBFβ proteins) in AML. Genetic research based on conditional knock-in adult mouse models for mutated C/EBPα and chromosomally rearranged RUNX1/ CBFβ showed a distinct pattern of differentiation block compared to conditional knockout adult mouse models for C/EBPα and RUNX1/ CBFβ, respectively (Figure 2). These results suggest that when mutated these transcription factors induce an altered transcriptional program rather than a loss of function phenotype. We are asking the following questions: (1) What are the genes regulated by C/EBPα-p30 that play key roles in self-renewal, survival and proliferation? (2) What are the preleukemic molecular events in CEBPA mutations and CBF leukemia that define the pattern for acquisition of secondary mutations? (3) Can we specifically target preleukemic HSPCs without affecting normal HSPCs?

Figure 2: Distinct stages of differentiation blocks observed during conditional deletions and AML associated mutations for RUNX1, CBFβ and C/EBPα. [LT-HSC, Long-term hematopoietic stem cells; ST-HSC, short-term hematopoietic stem cells, MPP, multi-potential progenitors; CMP, common myeloid progenitors; GMP, granulocyte/macrophage progenitors; cMP, committed myeloid progenitors].

References:

Pulikkan JA, Hegde M, Ahmed H, Belaghzal H, Illendula A, Yu J, O’Hagen K, Ou J, Muller-Tidow C, Wolfe SA, Zhu LJ, Dekker J, Bushweller JH, Castilla LH. CBFβ-SMMHC inhibition triggers apoptosis by disrupting MYC chromatin dynamics in acute myeloid leukemia. Cell 2018 Jun 28;174(1):172-186.

Illendula A*, Pulikkan JA*, Zong H, Grembecka J, Xue L, Sen S, Zhou Y, Boulton A, Kuntimaddi A, Gao Y, Rajewski RA, Guzman ML, Castilla LH, Bushweller JH. A small-molecule inhibitor of the aberrant transcription factor CBFβ-SMMHC delays leukemia in mice. Science 2015 Feb 13;347(6223):779-84.

Pulikkan JA*, Madera D*, Xue L, Bradley P, Landrette SF, Kuo YH, Abbas S, Zhu LJ, Valk P, Castilla LH. Thrombopoietin/MPL participates in initiating and maintaining RUNX1-ETO acute myeloid leukemia via PI3K/AKT signaling. Blood 2012 Jul 26;120(4):868-79

Pulikkan JA and Castilla LH. Pre-leukemia in inv(16) acute myeloid leukemia development. Frontiers in Oncology 2018 Apr  26: 8(129): 1-7.

Pulikkan JA, Peramangalam PS, Dengler V, Müller-Tidow C, Bohlander SK, Preudhomme C, Tenen DG, Behre G. C/EBPα regulated microRNA-34a targets E2F3 during granulopoiesis and is downregulated in AML with CEBPA mutations. Blood 2010 Dec 16;116(25):5638-49.

 

Pulikkan JA, Dengler V, Peer Zada AA, Kawasaki A, Geletu MH, Pasalic Z, Bohlander SK, Ryo A, Tenen DG, Behre G. Elevated PIN1 expression by C/EBPα-p30 blocks C/EBPα induced granulocytic differentiation via c-Jun in AML. Leukemia 2010 May;24(5):914-23.

 

Pulikkan JA*, Dengler  V*, Peramangalam PS, Peer Zada AA, Müller-Tidow C, Bohlander SK, Tenen DG, Behre G. Cell cycle regulator E2F1 and microRNA-223 comprise an autoregulatory negative feedback loop in acute myeloid leukemia. Blood 2010 Mar 4;115(9):1768-78

Pulikkan JA, Tenen DG, Behre G. C/EBPα deregulation as a paradigm for leukemogenesis. Leukemia 2017 Nov;31(11):2279-2285.

3. Non-coding RNA function in hematopoiesis and leukemia

The Encyclopedia of DNA Elements (ENCODE) project revealed that about 76% of the human genome is transcribed into RNA. However only 2% of the genome is translated to proteins. Recent findings in RNA biology made a paradigm shift in our understanding of RNA function beyond acting as templates for protein synthesis. An emerging theme in gene regulation is the central role played by long non-coding RNAs (lncRNAs), which have been characterized as epigenetic factors, protein scaffolds, antisense transcripts and miRNA sponges (Figure 3). lncRNA function in AML is relatively an unexplored research path. We are interested in exploring the following questions: (1) What are the lncRNAs regulated by CBFs and C/EBPα and what are the mechanisms of action of these lncRNAs? (2) What is the functional relevance of deregulated lncRNAs in AML with altered CBF and C/EBPα activity? (3) What factors regulate the interaction of lncRNAs with DNA, RNA and protein targets? (4) What is the therapeutic relevance of using antisense oligonucleotides (ASOs) targeting lncRNAs in AML treatment?

Figure 3: lncRNAs and gene regulation

References:

Pulikkan JA, Madera D, Illendula A, Zhu LJ, Bushweller JH, Castilla LH. The microRNAs miR-17/20a mediate inv(16) acute myeloid leukemia initiation and maintenance (submitted).

Pulikkan JA, Peramangalam PS, Dengler V, Müller-Tidow C, Bohlander SK, Preudhomme C, Tenen DG, Behre G. C/EBPα regulated microRNA-34a targets E2F3 during granulopoiesis and is downregulated in AML with CEBPA mutations. Blood 2010 Dec 16;116(25):5638-49.

 

Pulikkan JA*, Dengler  V*, Peramangalam PS, Peer Zada AA, Müller-Tidow C, Bohlander SK, Tenen DG, Behre G. Cell cycle regulator E2F1 and microRNA-223 comprise an autoregulatory negative feedback loop in acute myeloid leukemia. Blood 2010 Mar 4;115(9):1768-78

2279-2285.

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