Cancer Functional Epigenomics

Acute Myeloid Leukemia (AML) is a hematopoietic malignancy characterized by pathologic expansion of myeloid cells without terminal differentiation. It is the most common acute leukemia in adult and is highly aggressive, with ~70% of patients succumbing to the disease. AML is considered a paradigm of transcriptional dysregulation in cancer, as recurrent mutations associated with AML were found to commonly hit transcription factors (TFs), epigenetic modifiers and genome structure proteins (Figure 1). These mutations could alter the transcriptome through remodelling epigenetic environment and three-dimensional (3D) chromatin structure, converging on the dysregulation of gene networks to trigger malignant hematopoiesis.

Our initial research effort involved the identification of genetic aberrations with prognostic impact in myeloid malignancies, as well as the investigation into molecular mechanisms underlying the induction or maintenance of myeloid malignancies caused by gene alterations. Our work identified the dysregulated TFs including MN1 (Heuser et al. 2011) and IRF8 (Sharma et al. 2015), as well as the epigenetic regulator MLL5 (Yun et al. 2014) in the transcriptional control of AML-related gene network. We further contributed to the characterization of engineered mouse or cell models with deficiency in chromatin remodellers such as CBP (Horton et al. 2017), UTX/UTY (Gozdecka et al. 2018), EZH2 (Basheer et al. 2019) and cohesin complex members (Sasca et al. 2019), which are all commonly mutated in hematopoietic malignancies. Interestingly, loss of any of these epigenetic players could rewrite transcription programs through modulating enhancer functions, highlighting the crucial role of enhancer dysregulations in leukemogenesis.

Meanwhile, we applied an integrated multi-omics approach to study the epigenetic changes at 3D genome levels in the hematopoietic stem and progenitor (HSPC) compartment in a leukemia evolution model, utilizing a murine allelic series carrying the two most common mutations in AML, the Flt3-ITD and Npm1c (Yun et al. 2021; Figure 2a). We identified clusters of cis-regulatory elements exhibiting dynamic enhancer or promoter activity induced by mutations (Figure 2b,c). This analysis facilitated the capture of novel distal regulatory events responsible for the dysregulation of leukemia critical genes. An exemplar was the identification of a novel superenhancer sitting ~1Mb upstream of Hoxa genes cluster, regulating the transcription of Hoxa genes through chromatin interaction in Npm1c-mutated HSPCs (Figure. 3a). CRISPR-Cas9-mediated genetic disruption of this superenhancer resulted in downregulation of Hoxa genes and impairment of leukemia cell propagation (Figure 3b-d). Taken together, these data demonstrated that enhancer dysregulation symbolized epigenetic changes at higher-order chromatin level in leukemogenesis, and manipulation of altered enhancers represented a novel approach to target leukemia.

At Robert Bosch Center for Tumor Diseases, we will continue our dedication to the translational cancer research in the field of hematology and aim to expand our knowledge in understanding the multi-layered gene regulatory networks in hematological malignancies.

Previously we identified extensive bidirectional transcriptional signatures within a subset of leukemia-activated enhancers. The production of enhancer RNAs (eRNAs) is intricately linked to altered enhancer accessibility and modification, enhancer-promoter looping and targe gene dysregulation. These observations suggest that enhancer transcription might be a novel layer of epigenetic regulation through 3D enhancer remodelling. We therefore hypothesised that dysregulated eRNAs are essential to maintain leukemia propagation, through modulating chromatin at 3D level, and might be targetable for the eradication of leukemia cells. We will employ multiple genome-wide approaches to identify dysregulated eRNAs that are critical for leukemia maintenance, and investigate the underlying molecular mechanisms (Figure 4). Eventually we would evaluate the functional importance and clinical relevance of our candidate eRNAs in human AML samples, aiming to translating our finding into clinical practice.

Besides, we will endeavour to explore the epigenetic mechanisms controlling the dysregulation of key oncogenes associated with malignant hematopoiesis. These oncogenes include FLT3 and HOXA9, which are frequently mutated or overexpressed in AML, respectively. We will apply high-throughput profiling and screening methods on AML cell lines, primary samples and leukemia cell differentiation models, with expectation to capture bona fide TFs and cis-regulatory elements driving oncogene overexpression in leukemia cells. The results will assist the discovery of new targets for treating these specific types of leukemia.

Our other initiative will focus on characterizing and targeting leukemia-associated
oncoproteins for tumor eradication. In this part, we would dissect the role of mutant NPM1 (NPM1c) in chromatin remodelling. Using engineered fluorescence labelled NPM1c cell model, we will investigate how NPM1c triggers nuclear chromatin redistribution, and identify epigenetic regulators participating in this process. We will examine the possibilities of targeting these regulatory factors in NPM1c leukemia mouse models and patient samples. In addition, we will perform loss-of-function screens to find out gene networks responsible for the depletion of NPM1c in AML cells.