Chromatin state dynamics confers specific therapeutic strategies in enhancer subtypes of colorectal cancer.

OBJECTIVE: Enhancer aberrations are beginning to emerge as a key epigenetic feature of colorectal cancers (CRC), however, a comprehensive knowledge of chromatin state patterns in tumour progression, heterogeneity of these patterns and imparted therapeutic opportunities remain poorly described. DESIGN: We performed comprehensive epigenomic characterisation by mapping 222 chromatin profiles from 69 samples (33 colorectal adenocarcinomas, 4 adenomas, 21 matched normal tissues and 11 colon cancer cell lines) for six histone modification marks: H3K4me3 for Pol II-bound and CpG-rich promoters, H3K4me1 for poised enhancers, H3K27ac for enhancers and transcriptionally active promoters, H3K79me2 for transcribed regions, H3K27me3 for polycomb repressed regions and H3K9me3 for heterochromatin. RESULTS: We demonstrate that H3K27ac-marked active enhancer state could distinguish between different stages of CRC progression. By epigenomic editing, we present evidence that gains of tumour-specific enhancers for crucial oncogenes, such as ASCL2 and FZD10, was required for excessive proliferation. Consistently, combination of MEK plus bromodomain inhibition was found to have synergistic effects in CRC patient-derived xenograft models. Probing intertumour heterogeneity, we identified four distinct enhancer subtypes (EPIgenome-based Classification, EpiC), three of which correlate well with previously defined transcriptomic subtypes (consensus molecular subtypes, CMSs). Importantly, CMS2 can be divided into two EpiC subgroups with significant survival differences. Leveraging such correlation, we devised a combinatorial therapeutic strategy of enhancer-blocking bromodomain inhibitors with pathway-specific inhibitors (PARPi, EGFRi, TGFßi, mTORi and SRCi) for EpiC groups. CONCLUSION: Our data suggest that the dynamics of active enhancer underlies CRC progression and the patient-specific enhancer patterns can be leveraged for precision combination therapy.

Epigenetic Perspective of Immunotherapy for Cancers.

Immunotherapy has brought new hope for cancer patients in recent times. However, despite the promising success of immunotherapy, there is still a need to address major challenges including heterogeneity in response among patients, the reoccurrence of the disease, and iRAEs (immune-related adverse effects). The first critical step towards solving these issues is understanding the epigenomic events that play a significant role in the regulation of specific biomolecules in the context of the immune population present in the tumor immune microenvironment (TIME) during various treatments and responses. A prominent advantage of this step is that it would enable researchers to harness the reversibility of epigenetic modifications for their druggability. Therefore, we reviewed the crucial studies in which varying epigenomic events were captured with immuno-oncology set-ups. Finally, we discuss the therapeutic possibilities of their utilization for the betterment of immunotherapy in terms of diagnosis, progression, and cure for cancer patients.

Single-cell chromatin accessibility profiling of acute myeloid leukemia reveals heterogeneous lineage composition upon therapy-resistance.

Acute myeloid leukemia (AML) is a heterogeneous disease characterized by high rate of therapy resistance. Since the cell of origin can impact response to therapy, it is crucial to understand the lineage composition of AML cells at time of therapy resistance. Here we leverage single-cell chromatin accessibility profiling of 22 AML bone marrow aspirates from eight patients at time of therapy resistance and following subsequent therapy to characterize their lineage landscape. Our findings reveal a complex lineage architecture of therapy-resistant AML cells that are primed for stem and progenitor lineages and spanning quiescent, activated and late stem cell/progenitor states. Remarkably, therapy-resistant AML cells are also composed of cells primed for differentiated myeloid, erythroid and even lymphoid lineages. The heterogeneous lineage composition persists following subsequent therapy, with early progenitor-driven features marking unfavorable prognosis in The Cancer Genome Atlas AML cohort. Pseudotime analysis further confirms the vast degree of heterogeneity driven by the dynamic changes in chromatin accessibility. Our findings suggest that therapy-resistant AML cells are characterized not only by stem and progenitor states, but also by a continuum of differentiated cellular lineages. The heterogeneity in lineages likely contributes to their therapy resistance by harboring different degrees of lineage-specific susceptibilities to therapy.

Reprogramming of H3K9bhb at regulatory elements is a key feature of fasting in the small intestine.

ß-hydroxybutyrate (ß-OHB) is an essential metabolic energy source during fasting and functions as a chromatin regulator by lysine ß-hydroxybutyrylation (Kbhb) modification of the core histones H3 and H4. We report that Kbhb on histone H3 (H3K9bhb) is enriched at proximal promoters of critical gene subsets associated with lipolytic and ketogenic metabolic pathways in small intestine (SI) crypts during fasting. Similar Kbhb enrichment is observed in Lgr5+ stem cell-enriched epithelial spheroids treated with ß-OHB in vitro. Combinatorial chromatin state analysis reveals that H3K9bhb is associated with active chromatin states and that fasting enriches for an H3K9bhb-H3K27ac signature at active metabolic gene promoters and distal enhancer elements. Intestinal knockout of Hmgcs2 results in marked loss of H3K9bhb-associated loci, suggesting that local production of ß-OHB is responsible for chromatin reprogramming within the SI crypt. We conclude that modulation of H3K9bhb in SI crypts is a key gene regulatory event in response to fasting.

Reprogramming of bivalent chromatin states in NRAS mutant melanoma suggests PRC2 inhibition as a therapeutic strategy.

The dynamic evolution of chromatin state patterns during metastasis, their relationship with bona fide genetic drivers, and their therapeutic vulnerabilities are not completely understood. Combinatorial chromatin state profiling of 46 melanoma samples reveals an association of NRAS mutants with bivalent histone H3 lysine 27 trimethylation (H3K27me3) and Polycomb repressive complex 2. Reprogramming of bivalent domains during metastasis occurs on master transcription factors of a mesenchymal phenotype, including ZEB1, TWIST1, and CDH1. Resolution of bivalency using pharmacological inhibition of EZH2 decreases invasive capacity of melanoma cells and markedly reduces tumor burden in vivo, specifically in NRAS mutants. Coincident with bivalent reprogramming, the increased expression of pro-metastatic and melanocyte-specific cell-identity genes is associated with exceptionally wide H3K4me3 domains, suggesting a role for this epigenetic element. Overall, we demonstrate that reprogramming of bivalent and broad domains represents key epigenetic alterations in metastatic melanoma and that EZH2 plus MEK inhibition may provide a promising therapeutic strategy for NRAS mutant melanoma patients.

Single cell T cell landscape and T cell receptor repertoire profiling of AML in context of PD-1 blockade therapy.

In contrast to the curative effect of allogenic stem cell transplantation in acute myeloid leukemia via T cell activity, only modest responses are achieved with checkpoint-blockade therapy, which might be explained by T cell phenotypes and T cell receptor (TCR) repertoires. Here, we show by paired single-cell RNA analysis and TCR repertoire profiling of bone marrow cells in relapsed/refractory acute myeloid leukemia patients pre/post azacytidine+nivolumab treatment that the disease-related T cell subsets are highly heterogeneous, and their abundance changes following PD-1 blockade-based treatment. TCR repertoires expand and primarily emerge from CD8+ cells in patients responding to treatment or having a stable disease, while TCR repertoires contract in therapy-resistant patients. Trajectory analysis reveals a continuum of CD8+ T cell phenotypes, characterized by differential expression of granzyme B and a bone marrow-residing memory CD8+ T cell subset, in which a population with stem-like properties expressing granzyme K is enriched in responders. Chromosome 7/7q loss, on the other hand, is a cancer-intrinsic genomic marker of PD-1 blockade resistance in AML. In summary, our study reveals that adaptive T cell plasticity and genomic alterations determine responses to PD-1 blockade in acute myeloid leukemia.

EF-hand domain containing 2 (Efhc2) is crucial for distal segmentation of pronephros in zebrafish.

BACKGROUND: The blood filtering organ in zebrafish embryos is the pronephros, which consists of two functional nephrons. Segmentation of a nephron into different domains is essential for its function and is well conserved among vertebrates. Zebrafish has been extensively used as a model to understand nephron segmentation during development. Here, we have identified EF-hand domain containing 2 (Efhc2) as a novel component of genetic programme regulating nephron segmentation in zebrafish. Human EFHC2 is a protein with one predicted calcium-binding EF-hand motif and three DM10 domains, whose function is unknown. EFHC2 has been implicated in several brain-related genetic diseases like Turner syndrome and juvenile myoclonic epilepsy. However, there is limited information on its normal physiological function. RESULTS: efhc2 mRNA is primarily expressed in the pronephros of zebrafish embryos. Other sites of expression include olfactory placode, notochord, otic vesicle, epiphysis and neuromast cells. Morpholino antisense oligonucleotide-mediated knock-down of Efhc2 resulted in defects in pronephros development and function in zebrafish embryos. Efhc2 knock-down leads to expansion of distal early segment of pronephros, whereas, the corpuscle of stannius and distal late segments were reduced. The number of multi-ciliated cells (MCC) that are present in a salt-and-pepper fashion throughout the middle of each nephron and vital for fluid flow were also reduced. It is known that retinoic acid (RA) signaling regulates pronephros segmentation in vertebrates and we show that Efhc2 function is crucial for nephron segmentation in zebrafish. Our data suggests that RA and Efhc2 function independent of each other in pronephros segmentation. However, Efhc2 and RA synergistically regulate MCC development. CONCLUSION: In this study, we have identified Efhc2 as a regulator of segmentation of the distal part of nephron and pronephros function during zebrafish development.

Nucleolar protein 4-like has a complex expression pattern in zebrafish embryos.

The nucleolar protein 4-like (NOL4L) gene is present on chromosome 20 (20q11.21) in humans. Parts of this gene have been shown to fuse with RUNX1 and PAX5 in acute myeloid leukemia and acute lymphoblastic leukemia, respectively. The normal function of NOL4L in humans and other organisms is not well understood. The expression patterns and functions of NOL4L homologs during vertebrate development have not been reported. We sought to address these questions by studying the expression pattern of zebrafish nol4l during embryogenesis. Our data show that Znol4l mRNA is expressed in multiple organs in zebrafish embryos. The sites of expression include parts of the brain, spinal cord, pronephros, hematopoietic cells and gut.