Chromatin dynamics and gene regulation in normal and malignant hematopoiesis
Acute myeloid leukemia (AML) and several hematological malignancies arise from the 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 areas are:
1. Small molecule-mediated targeted therapy in inv(16) AML
The core binding factor (CBF), composed of CBFβ and RUNX subunits, plays a critical role in most hematopoietic lineages and is deregulated in acute myeloid leukemia (AML). The fusion oncogene CBFβ-SMMHC, expressed in AML with the chromosome inversion inv(16)(p13q22), acts as a driver oncogene in hematopoietic stem cells and induces AML. Although the survival of patients with inv(16) AML is higher than that of patients with other AML subtypes, they still experience relapses and often die from the disease, highlighting the need for improved treatment strategies. CBFβ-SMMHC directly interacts with RUNX1 and sequesters it away from DNA binding, thereby inducing AML. We have developed AI-10-49, a small molecule inhibitor that specifically disrupts the interaction between RUNX1 and CBFβ-SMMHC. AI-10-49 selectively induces apoptosis in inv(16) AML cells and extends the survival of mice with inv(16) leukemia. AI-10-49 is currently being developed as an anti-leukemic drug. In follow-up studies, we found that MYC and N-MYC are downstream targets of AI-10-49 in inv(16) AML. We also identified several novel enhancers essential for MYC and N-MYC transcription, and demonstrated that RUNX1 represses MYC and N-MYC during AI-10-49 treatment by displacing the SWI/SNF complex from these enhancers via the RUNX1-PRC1 complex. We are examining the following questions: (1) What are the mechanisms of AI-10-49 resistance in inv(16) AML cells? (2) Can inhibiting eIF4G1 be combined with AI-10-49 for inv(16) AML treatment? (3) Could targeting enhancer RNAs (eRNAs) serve as an effective therapy for AML?
References:
Peramangalam PS, Surapally S, Veltri AJ, Zheng S, Burns R, Zhu N, Rao S, Muller-Tidow C, Bushweller JH and Pulikkan JA. N-MYC regulates Cell Survival via eIF4G1 in inv(16) Acute Myeloid Leukemia. Science Advances 2024 Mar;10(9):eadh8493.
Surapally S, Tenen DG and Pulikkan JA. Emerging therapies for inv(16) Acute Myeloid Leukemia. Blood 2021May 13;137(19):2579-2584.
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.
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. Elucidate the mechanisms of CEBPA mutant proteins in AML
The transcription factor C/EBPα is a master regulator of granulopoiesis. Mutations in the gene encoding C/EBPα have been reported in AML patients. CEBPA N-terminal mutations are linked to poorer survival. These mutations lead to a 30 kDa truncated protein (C/EBPα-p30). The pathways regulated by C/EBPα-p30 in AML are not well understood. Therefore, further investigation of this pathway is urgently needed to develop better treatments targeting CEBPA N-terminal mutations in AML. To model AML with CEBPA N-terminal mutation, our lab recently created the first conditional knock-in mouse model for C/ebpa-p30 (manuscript in preparation). Our data suggest that chromatin deregulation by C/EBPα-p30 is the primary mechanism through which this oncogene drives leukemia. We are exploring these questions: (1) Which genes regulated by C/EBPα-p30 are crucial for self-renewal, survival, and proliferation? (2) Can we target chromatin deregulation in AML cells therapeutically without affecting normal hematopoietic stem/progenitor cells? (3) What are the preleukemic molecular events in CEBPA mutations that define the pattern for the acquisition of secondary mutations?
References:
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. Develop better models for studying human hematopoiesis and AML.
Accurate modeling of human hematopoiesis is essential for developing treatments for various blood diseases, including AML. Ex vivo culture of hematopoietic stem and progenitor cells (HSPCs) is less effective due to their limited self-renewal capacity, sensitivity to ex vivo conditions, and rapid differentiation, which hinder long-term maintenance and expansion. Although animal models, including genetic mouse models, have been developed as alternative systems, these models do not fully replicate the complexity and species-specific biology of human hematopoiesis—highlighting the need for improved human-relevant systems that better predict disease mechanisms and treatment responses.
Patient-derived xenograft (PDX) models are important tools for studying disease biology, clonal evolution, and pre-clinical drug testing. Although significant progress has been made in modeling human AML with PDX models like NSGS, NSGW41, and NOG-EXL, many primary AML samples still fail to engraft across different models. We are developing new PDX models to investigate human hematopoiesis and AML, and examining how altering the recipient mouse's bone marrow cytokine milieu to match the primary AML HSPC niche can improve the engraftment of human primary AML cells.
Organoids have become a more effective option for studying various human diseases, including blood disorders. Published human bone marrow organoids depend on induced pluripotent stem cells (iPSCs). However, iPSC-based bone marrow organoids have several limitations in modeling human hematopoiesis. We are developing a new hydrogel-based organoid platform that directly uses human primary HSPCs, mesenchymal stem cells, and endothelial cells.
References:
Peramangalam PS, Surapally S, Veltri AJ, Zheng S, Burns R, Zhu N, Rao S, Muller-Tidow C, Bushweller JH and Pulikkan JA. N-MYC regulates Cell Survival via eIF4G1 in inv(16) Acute Myeloid Leukemia. Science Advances 2024 Mar;10(9):eadh8493.