Targeting Chromatin Research Articles

Abstract SY05-01: Targeting chromatin adaptor proteins in cancer

Abstract Menin interacts with oncogenic fusion proteins produced by MLL1 rearrangements, and small molecules capable of disrupting these interactions are currently under investigation in clinical trials for the treatment of leukemia. Our research, which combines chromatin-focused and genome-wide CRISPR screenings with a range of genetic, pharmacological, and biochemical strategies in both mouse and human models, has led to the discovery of a molecular mechanism that acts as a switch between the MLL1-Menin and MLL3/4-UTX chromatin modifying complexes, influencing the efficacy of Menin-MLL inhibitors. We demonstrate that the MLL1-Menin complex plays a critical role in maintaining leukemia cell survival by preventing the MLL3/4-UTX complex from binding to certain gene promoters. Interruption of the Menin-MLL1 interaction results in the UTX-dependent transcriptional activation of a tumor suppressor gene program, which significantly influences therapeutic outcomes in both murine and human leukemia models. Notably, the therapeutic activation of this gene program through CDK4/6 inhibitors can overcome resistance in leukemia cells that do not respond to Menin inhibitors. The identification of this molecular switch between MLL1-Menin and MLL3/4-UTX complexes on chromatin highlights the complex roles of these evolutionarily conserved epigenetic regulators and underscores their importance in understanding and addressing the molecular mechanisms underlying response and resistance in leukemia treatment trials. Citation Format: Yadira Soto-Feliciano. Targeting chromatin adaptor proteins in cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr SY05-01.

Abstract 2580: Targeting chromatin in ovarian cancer stem cells

Abstract The dynamic organization of chromatin, governed by epigenetic modifiers, critically regulates cell plasticity and determines cell fate in both normal and malignant stem cells. We hypothesized that chromatin architecture plays a role in regulating transcriptional heterogeneity in ovarian cancer stem cells (OCSCs) to maintain stemness and chemoresistance. PWS microscopy showed that ALDH+ OCSCs derived displayed higher nuclear fractal dimension D signals (P<0.05) than ALDH- non-OCSCs, indicating that chromatin in OCSCs possesses higher packing compared to non-OCSCs. Cut & TAG Sequencing showed that of the 3606-histone repressive H3K27m3 enriched regions (P<0.05, fold change >2), 3208 were detected in OCSCs while 398 peaks were present in non-OCSCs, consistent with findings supporting high D features in OCSCs. The active mark H3K4me3 was enriched at the promoter of stemness-associated genes, including SOX2, ALDH1A3, and FZD7 in OCSCs; and the repressive histone mark H3K27me3 was enriched at the promoter of the epithelial cell marker CDH1 in OCSCs. These results support that the chromatin of OCSCs contains open genomic regions, corresponding to stemness-associated genes and repressed regions associated with genes related to differentiation. RNA sequencing indicated that the coefficient of variance (COV) reflecting transcriptional heterogeneity in OCSCs was higher than in non-OCSCs at baseline. In response to platinum, genes with low baseline expression in OCSCs were most upregulated, supporting increased transcriptional heterogeneity in response to chemotherapy. Inhibitors of epigenetic modifiers (DNMTi, EZH2i, and Dot1Li) induced chromatin re-organization in OCSCs and promoted OCSCs transition toward differentiation, supporting that stemness-associated chromatin structure is targetable. We demonstrated that a Dot1L inhibitor (Dot1Li) targeted OCSCs by decreasing chromatin packing D, resulting in decreased transcriptional heterogeneity. In conclusion, OCSCs harbor highly packed chromatin, contributing to chemoresistance and cell plasticity. Epigenetic modifiers (such as Dot1Li) inhibit stemness by regulating chromatin organization and transcriptional malleability. Citation Format: Yinu Wang, Jane Frederick, Yaqi Zhang, Elizabeth Thomas Bartom, Greta Bauer, Edward Tanner, Vadim Backman, Daniela Matei. Targeting chromatin in ovarian cancer stem cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2580.

Mechanisms of action and resistance in histone methylation-targeted therapy

Epigenomes enable the rectification of disordered cancer gene expression, thereby providing new targets for pharmacological interventions. The clinical utility of targeting histone H3 lysine trimethylation (H3K27me3) as an epigenetic hallmark has been demonstrated1–7. However, in actual therapeutic settings, the mechanism by which H3K27me3-targeting therapies exert their effects and the response of tumour cells remain unclear. Here we show the potency and mechanisms of action and resistance of the EZH1–EZH2 dual inhibitor valemetostat in clinical trials of patients with adult T cell leukaemia/lymphoma. Administration of valemetostat reduced tumour size and demonstrated durable clinical response in aggressive lymphomas with multiple genetic mutations. Integrative single-cell analyses showed that valemetostat abolishes the highly condensed chromatin structure formed by the plastic H3K27me3 and neutralizes multiple gene loci, including tumour suppressor genes. Nevertheless, subsequent long-term treatment encounters the emergence of resistant clones with reconstructed aggregate chromatin that closely resemble the pre-dose state. Acquired mutations at the PRC2–compound interface result in the propagation of clones with increased H3K27me3 expression. In patients free of PRC2 mutations, TET2 mutation or elevated DNMT3A expression causes similar chromatin recondensation through de novo DNA methylation in the H3K27me3-associated regions. We identified subpopulations with distinct metabolic and gene translation characteristics implicated in primary susceptibility until the acquisition of the heritable (epi)mutations. Targeting epigenetic drivers and chromatin homeostasis may provide opportunities for further sustained epigenetic cancer therapies.

Biallelic Landscape of DNMT3A Mutant Myeloid Neoplasia

The structure of DNMT3A comprises an N-terminal regulatory part promoting nuclear localization and targeting chromatin and a C-terminal domain, mainly involved in DNA binding and methylation catalysis. 1DNMT3A mutations in myeloid neoplasia (MN) and clonal hematopoiesis are mostly heterozygous. Homozygous null Dnmt3amice are born runted and Dnmt3a-/- cells express a hypomethylation phenotype. 2 An invariant detection of heterozygous mutations without homozygous, hemizygous, or biallelic configurations suggests absence of acquisition of more malignant phenotypes by progressive inactivation of DNMT3A. Moreover, residual DNMT3A function is essential for cell survival and potentially suggests that synthetic lethality might be a viable therapeutic strategy whereby cases with two intact alleles would be more resistant allowing a therapeutic window. Consequently, identification of biallelic DNMT3A mutant cases may be extremely instructive for the development of therapeutics targeting DNMT3A. We analyzed targeted NGS sequencing results of 4,895 cases with MN enrolled at our institution from 2002 to 2021 and retrospectively reviewed the clinical characteristics of all patients with deleterious DNMT3A mutations. DNMT3A mutations were identified in 461 patients, of whom 39 had double and 1 triple hits. Most of the mutants had AML (n=236) including biphenotypic AML (n=5), followed by MDS (n=142), MDS/MPN overlap (n=46) and MPN (n=37). In addition, mutations were identified in 9% of CHIP/CCUS (n=117) and 4% of aplastic anemia (n=68). Multiple DNMT3A hits were more often encountered in AML than MDS and MDS/MPN overlap (62.5% vs. 32.5% vs. 2.5%; P = .045). Single hits were represented by missense (73%) within the hotspot R882 (35%), frameshifts (10%), nonsense (9%), splice sites (6%) and in-frame ins/del (2%). Double hits configuration had a constellation of missense/missense (40%), missense/frameshifts (25%), missense/nonsense (13%), and missense/splice sites (10%) ( Fig1). R882 accounted for 10% of the multi hit carriers. Of note is that biallelic involvement was not confined to non-R882. Patients harboring multi hits carriedmore likely IDH2 (27.5% vs. 10.5%; OR 2.73; P = .011) and ETV6 (7.5% vs. 1.7%; OR 4.32 P = .043) mutations compared to patients with single hits DNMT3A by multivariate logistic regression analysis. Since deletion or UPD of DNMT3A locus has not been found, one can assume that all cases with one hit harbor monoallelic rather than biallelic subclones. For carriers of multiple mutations, one can stipulate that when the sum of VAFs exceeds the theoretical 60%, the presence of a biallelic subclone might be suspected. Indeed, 24 cases fulfilled this criterion. However, in 40% of patients with double hits, arithmetic VAF did not allow for unequivocal resolution of the mutational configuration, with either smaller biallelic subclones vs. subclonal mosaicism being possible. Next, we compared clinical features and outcomes of patients with mono vs. biallelic cases. In MDS, compared with single hits DNMT3A, biallelic cases showed significantly higher WBC (median 7.49 vs. 3.15 k/µL; P = .0052), ANC (4.88 vs. 1.52 k/µL; P = .0056), and platelet count (264 vs. 83 k/µL; P = .0036). In high-risk MDS and AML, with a median follow-up of 12.5 months (0.2-201), patients with biallelic DNMT3Ashowed significantly poorer 1-year OS rate than patients with single hits (42.8% vs. 65.8%; P = .0095). Longitudinal molecular analysis of 8 cases (2 MDS at onset and upon progression to sAML; 4 AML at diagnosis and following relapse after conventional intensive chemotherapy-induced remission; 1 AML at diagnosis and CR post SCT; 1 AML and donor-derived AML post SCT) demonstrated an early origin of biallelic hits with leukemogenic potential. Indeed, biallelic clones persisted or expanded in 6 transformed cases. Our study describes the likelihood that biallelic configuration of DNMT3A mutations, while rare, is indeed compatible with clonal expansion and thus questions the applicability of a synthetic lethality strategy. We are currently confirming that, indeed, truly biallelic cases vs. mosaicism are permissive using DNASeq of single sorted progenitor cells. The results of this analysis will be available at the time of ASH meeting.

Abstract 3554: Targeting chromatin remodeling complex in pediatric cancers

Abstract Chromatin accessibility plays critical roles in maintaining normal gene regulatory programs in healthy tissue. Aberrant chromatin accessibility triggered by abnormal activity of a variety of chromatin remodeling complexes is known to activate and maintain tumor proliferation. The most widely known of these complexes is the SWI/SNF (also called BAF) complex. Studies have shown that BAF regulates chromatin accessibility in a more global manner and its long-term inhibition or functional loss may lead to non-specific downstream effects in both tumor and normal tissue. Thus, there is a need to further probe understudied complexes, such as the Imitation Switch (ISWI) family of chromatin remodelers, to find more cancer specific functions that can provide mechanistic insights that pave the way for novel therapeutic discoveries. We have examined the functions of these chromatin complex in different type of pediatric cancers to estsablish the pre-clinical rationale for potential novel pediatric cancer therapy. We discovered that fusion protein positive rhabdomyosarcoma (FP-RMS), an aggressive sub-type of RMS characterized by chromosomal translocations resulting in gene fusions between PAX3/7 and FOXO1 is highly dependent on chromatin accessibility. This aggressive subtype exhibits a significant vulnerability in its dependence on the oncogenic transcription factor PAX3-FOXO1, yet like many other TFs, PAX3-FOXO1 is currently not considered a viable therapeutic candidate. We have then carefully examined the effect of chemical inhibition and chemical degradation of BAF complex function in FP-RMS and discovered that BAF complex globally regulates chromatin accessibility, is not specifically regulating FP function. We next examined the function of other chromatin remodeling complexes that might regulate FP function specifically in FP-RMS. We identified that some of other chromatin remodeling complex, such as Nuclear Remodeling Factor (NURF) complex, a member of the ISWI chromatin remodeling complex family, interacts with a critical fusion protein, PAX3/7-FOXO1, in FP-RMS. There is limited understanding surrounding the functions of NURF complex, particularly in pediatric cancer. Here, we then studied the key functions of FP regulated by variety of chromatin remodeling complex utilizing combined chemistry and biology method to elaborate the essential functions of PAX3/7-FOXO1 fusion protein. We have developed variety of chemical strategies to target these complexes, and utilize these tool compounds to delineate the chromatin remodeling complex regulated FP functions. This study showcased our approach to advance cancer research by combining the power of chemistry, chemical biology, structural biology, computational biology and cellular biology to identify novel mechanistic understanding as well as novel therapeutic approach for pediatric cancers. Citation Format: Jun Qi. Targeting chromatin remodeling complex in pediatric cancers. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3554.

Targeting chromatin mediated cellular plasticity in vivo.

Advances in cancer treatment, including precision oncology, are significantly hindered by cancer cell plasticity. The ability to adapt to various external stressors is due to two attributes of highly plastic cells: transcriptional malleability, which is the ability of a cell to quickly upregulate survival pathways, and transcriptional heterogeneity, or the variance in the pathways utilized by cells to evade cell death. Both aspects of phenotypic plasticity have been directly linked to a measure of statistical chromatin organization, the chromatin packing scaling D, which facilitates changes in gene expression. To study the role of chromatin in plasticity-mediated survival, we performed time-course imaging of chemotherapy-treated cancer cells using label-free Partial Wave Spectroscopic (PWS) microscopy. The population distribution of D shifted over the course of treatment such that all surviving cells exhibited high D values. Accordingly, cell cluster tracking demonstrated a negative correlation between the initial D and the increase in D induced by treatment. We developed a computational model of cell fate based on changes in transcription due to D, which could recapitulate experimental results. We predicted that pharmacologically lowering D would reduce plasticity and improve chemotherapeutic efficacy. Specifically, we screened compounds that would lower D by facilitating chromatin-nucleoplasm interactions through either histone tail modifications or changes in the nuclear ionic environment. Adding a D-lowering agent to chemotherapy treatment increased the probability of cell death compared to chemotherapy alone, which matched model predictions. We next tested these treatments in vivo in a PDX model where our model predicted that although tumors would still be able to adapt to chemotherapy treatment, the combination treatment would exhibit a higher cell growth inhibition rate and stunt tumor adaptation. Significantly, our results demonstrated that lowering D improves treatment outcomes even in a conservative treatment with low-dose chemotherapy.

Durable Clinical Impacts and Mechanisms of Action and Resistance in EZH1/2-Targeting Epigenetic Therapy

Background: Epigenomes allow the rectification of disordered gene expressions, thereby providing new targets for pharmacological interventions in cancer therapy. The clinical utility of targeting histone H3 lysine tri-methylation (H3K27me3) as an epigenetic hallmark has been demonstrated. However, in actual therapeutic settings, the mechanism by which H3K27me3-targeting therapies exert their effects and the response of tumor cells remain unclear. Results and Discussion: We conducted intensive molecular monitoring of three patients enrolled in the valemetostat first-in-human phase 1 clinical trial (NCT02732275) for adult T-cell leukemia/lymphoma (ATL). Valemetostat was administered orally once daily (200 mg/day) until a sign of disease progression could be observed. The number of abnormal lymphocytes (Ably) in these three patients drastically decreased after one week of treatment. All patients could stay on valemetostat single agent for more than 2 years with acceptable safety and durable response. PBMC samples (n = 55) were collected from these three patients immediately before (Pre) and during treatment, as well as during the progressive disease (PD) stage. Flow cytometry-based histone methylation assay demonstrated that tumor H3K27me3 level was generally high in the Pre-samples, but the valemetostat treatment reduced it to a normal level in all patients. In addition, ChIP sequencing (ChIP-seq) could directly assess how valemetostat affects the tumor epigenome. The pre-treatment tumor cells showed an overall increasing trend in H3K27me3. For all H3K27me3 peaks (51,683 peaks), 85.9% of the peak signals decreased at the average fold-change of 0.742 after the valemetostat treatment. Focusing on the gene loci, an overall decrease could be observed around the TSS and across the gene body. The characteristic H3K27me3 clusters initially observed in the tumor cells were demethylated. We also conducted integrative multilayered single-cell analyses (scATAC-seq and scRNA-seq) and showed that valemetostat abolishes the highly condensed chromatin structure formed by the plastic H3K27me3 and neutralizes multiple gene loci, such as tumor suppressor genes (TSGs), in all patients. These results demonstrated that valemetostat sufficiently restored the epigenome of the tumor cells close to the healthy state, leading to clinical improvements. Although all tested cases showed a durable response for more than 2 years, these responses were eventually interrupted due to the recurrence of ATL. We identified characteristic somatic mutations of EZH2 during clonal repopulation at PD. The identified amino acid substitutions were all located at the interface between EZH2 and valemetostat. Direct evaluation of the H3K27me3 level in the PD sample harboring the acquired EZH2 mutation showed that tumor cells in a hypermethylated state were repopulating. The scATAC-seq analysis also showed strong chromatin compaction of the same gene set occurred in the rapidly propagating resistant clone, indicating that the clonally selected mutations should be responsible for resistance. In patients free of the EZH2 resistant mutations, we detected TET2 mutation or elevated DNMT3A expression in the propagated resistant clones. Genome-wide DNA methylation analysis revealed increased DNA methylation near the TSS in the resistant clones. The genes characterized by the H3K27me3-related chromatin condensation were restored by valemetostat treatment but were again strongly re-condensed by mCpG gain at PD. Consequently, the gene regulation of several important TSGs restored by valemetostat was repressed again. Furthermore, in the single-cell platform, we successfully identified subpopulations with distinct metabolic and gene translation characteristics implicated in primary susceptibility until the acquisition of the heritable (epi)mutations. Conclusion: This study illustrates, for the first time, the molecular and cellular dynamics in patients in response to a molecular-targeting inhibitor designed for histone methyltransferases. Eliminating H3K27me3 leads to the reprogramming of the cancer epigenome, thereby exerting a sustained benefit on tumors. Targeting chromatin homeostasis might provide opportunities for further sustained epigenetic therapies.

Abstract 15671: Targeting Chromatin Structure: The Combined Inhibition of Histone Deacetylases and Bromodomain Proteins in Normalizing Persistently Activated Pulmonary Hypertensive Fibroblasts

Rationale: The multifactorial nature of pulmonary hypertension (PH) suggests epigenetic changes as potential determinants of vascular remodeling through regulating gene expression (both up-regulated and down-regulated genes) in vascular cells. Changes in the chromatin state (especially histone acetylation) of specific genes can lead to their repression or activation. We and others have demonstrated histone deacetylases inhibitors (HDACi) and histone acetylation readers inhibitors (BRDi) can prevent and ameliorate PH changes by normalizing pathological gene expression. However, the non-histone protein targets and the narrow therapeutic window of currently available HDACi limits their clinical utility. In this study, we evaluated the effect of combination treatments using a new HDACi (OKI-005, US patent) with high potency and selectivity for HDACs (targeting decreased genes) together with BRDi (targeting increased genes), in low doses. Methods and Results: Cultured human pulmonary artery fibroblasts derived from patients with IPAH (PH-Fibs) or from control donors (CO-Fibs) were used as in vitro model system. PH-Fibs exhibit increased pro-inflammatory genes (e.g. CXCL12), proliferation markers (e.g. Mki67) and anti-apoptosis genes (e.g. BCL2) as well as decreased anti-inflammatory genes (e.g. HMOX1), anti-proliferation genes (e.g. P21) and pro-apoptotic genes (e.g. PERP). In PH-Fibs, the increased expressed CXCL2 gene exhibited more opened chromatin structure and BRD4 binding (ChIP-assay), and BRDi (JQ1) can decrease its expression in a dose dependent trend. At the low doses, JQ1 (100 nM) alone is sufficient to normalize most of increased genes, while the new HDACi (OKI-005) was more effective in normalizing decreased genes in PH- Fibs. The combination of low dose of JQ1 (100 nM) and OKI-005 (64 nM) was sufficient to normalize both increased genes and decreased genes thus inhibiting the persistently activated PH-phenotype without deleterious effects on CO-Fibs. Conclusion: Combination of HDACi and BRDi in low doses is effective in normalizing the persistent activation of PH-Fibs without deleterious effects on control cells, offering promise for epigenetic-targeted therapies in PH.

CircleBase: an integrated resource and analysis platform for human eccDNAs.

Rapid advances in high-throughput sequencing technologies have led to the discovery of thousands of extrachromosomal circular DNAs (eccDNAs) in the human genome. Loss-of-function experiments are difficult to conduct on circular and linear chromosomes, as they usually overlap. Hence, it is challenging to interpret the molecular functions of eccDNAs. Here, we present CircleBase (http://circlebase.maolab.org), an integrated resource and analysis platform used to curate and interpret eccDNAs in multiple cell types. CircleBase identifies putative functional eccDNAs by incorporating sequencing datasets, computational predictions, and manual annotations. It classifies them into six sections including targeting genes, epigenetic regulations, regulatory elements, chromatin accessibility, chromatin interactions, and genetic variants. The eccDNA targeting and regulatory networks are displayed by informative visualization tools and then prioritized. Functional enrichment analyses revealed that the top-ranked cancer cell eccDNAs were enriched in oncogenic pathways such as the Ras and PI3K-Akt signaling pathways. In contrast, eccDNAs from healthy individuals were not significantly enriched. CircleBase provides a user-friendly interface for searching, browsing, and analyzing eccDNAs in various cell/tissue types. Thus, it is useful to screen for potential functional eccDNAs and interpret their molecular mechanisms in human cancers and other diseases.

The lncRNAs in HBV-Related HCCs: Targeting Chromatin Dynamics and Beyond.

Simple SummaryHepatocellular carcinoma (HCC), a common and fast rising cause of cancer, is responsible for over 800,000 deaths/year. Chronic hepatitis B virus (HBV) infection accounts for >50% of the cases worldwide. Long non-coding RNAs (lncRNAs), untranslated transcripts longer than 200 nucleotides, by acting both in the nuclear and cytoplasmic compartments, regulate gene expression both at the transcriptional and post-transcriptional levels. The lncRNAs have been involved in the development and progression of many cancers, including HCC. In this review, we describe the role of lncRNAs in HBV infection and HBV-related liver carcinogenesis and discuss the potential of lncRNAs as predictive or diagnostic biomarkers.Hepatocellular carcinoma (HCC) represents the fourth leading and fastest rising cause of cancer death (841,000 new cases and 782,000 deaths annually), and hepatitis B (HBV), with 250 million people chronically infected at risk of developing HCC, accounts for >50% of the cases worldwide. Long non-coding RNAs (lncRNAs), untranslated transcripts longer than 200 nucleotides, are implicated in gene regulation at the transcriptional and post-transcriptional levels, exerting their activities both in the nuclear and cytoplasmic compartments. Thanks to high-throughput sequencing techniques, several lncRNAs have been shown to favor the establishment of chronic HBV infection, to change the host transcriptome to establish a pro-carcinogenic environment, and to directly participate in HCC development and progression. In this review, we summarize current knowledge on the role of lncRNAs in HBV infection and HBV-related liver carcinogenesis and discuss the potential of lncRNAs as predictive or diagnostic biomarkers.

Nanobody-mediated control of gene expression and epigenetic memory

Targeting chromatin regulators to specific genomic locations for gene control is emerging as a powerful method in basic research and synthetic biology. However, many chromatin regulators are large, making them difficult to deliver and combine in mammalian cells. Here, we develop a strategy for gene control using small nanobodies that bind and recruit endogenous chromatin regulators to a gene. We show that an antiGFP nanobody can be used to simultaneously visualize GFP-tagged chromatin regulators and control gene expression, and that nanobodies against HP1 and DNMT1 can silence a reporter gene. Moreover, combining nanobodies together or with other regulators, such as DNMT3A or KRAB, can enhance silencing speed and epigenetic memory. Finally, we use the slow silencing speed and high memory of antiDNMT1 to build a signal duration timer and recorder. These results set the basis for using nanobodies against chromatin regulators for controlling gene expression and epigenetic memory.

Targeting DNA Repair and Chromatin Crosstalk in Cancer Therapy.

Simple SummaryTargeting aberrant DNA repair in cancers in addition to transcription and replication is an area of interest for cancer researchers. Inhibition of DNA repair selectively in cancer cells leads to cytotoxic or cytostatic effects and overcomes survival advantages imparted by chromosomal translocations or mutations. In this review, we highlight the relevance of DNA repair-linked events in developmental diseases and cancers and also discuss mechanisms to overcome these events that participate in different cellular processes.Aberrant DNA repair pathways that underlie developmental diseases and cancers are potential targets for therapeutic intervention. Targeting DNA repair signal effectors, modulators and checkpoint proteins, and utilizing the synthetic lethality phenomena has led to seminal discoveries. Efforts to efficiently translate the basic findings to the clinic are currently underway. Chromatin modulation is an integral part of DNA repair cascades and an emerging field of investigation. Here, we discuss some of the key advancements made in DNA repair-based therapeutics and what is known regarding crosstalk between chromatin and repair pathways during various cellular processes, with an emphasis on cancer.

Targeting Chromatin Complexes in Myeloid Malignancies and Beyond: From Basic Mechanisms to Clinical Innovation.

The aberrant function of chromatin regulatory networks (epigenetics) is a hallmark of cancer promoting oncogenic gene expression. A growing body of evidence suggests that the disruption of specific chromatin-associated protein complexes has therapeutic potential in malignant conditions, particularly those that are driven by aberrant chromatin modifiers. Of note, a number of enzymatic inhibitors that block the catalytic function of histone modifying enzymes have been established and entered clinical trials. Unfortunately, many of these molecules do not have potent single-agent activity. One potential explanation for this phenomenon is the fact that those drugs do not profoundly disrupt the integrity of the aberrant network of multiprotein complexes on chromatin. Recent advances in drug development have led to the establishment of novel inhibitors of protein–protein interactions as well as targeted protein degraders that may provide inroads to longstanding effort to physically disrupt oncogenic multiprotein complexes on chromatin. In this review, we summarize some of the current concepts on the role epigenetic modifiers in malignant chromatin states with a specific focus on myeloid malignancies and recent advances in early-phase clinical trials.

Abstract 1058: Targeting chromatin remodeling-associated genetic vulnerabilities in cancer: PBRM1 defects are synthetic lethal with PARP and ATR inhibitors

Abstract Aim: Polybromo-1 (PBRM1), a specific subunit of the pBAF chromatin remodeling complex, is frequently inactivated in cancer. For example, 40% of clear cell Renal Cell Carcinoma (ccRCC) and 15% of cholangiocarcinoma present deleterious PBRM1 mutations. There is currently no precision medicine-based therapeutic approach that targets PBRM1 defects. To identify novel, targeted therapeutic strategies for PBRM1-defective cancers, we carried out high-throughput functional genomics and drug screenings followed by in vitro and in vivo validation studies. Methods: High-throughput siRNA-drug sensitization and drug sensitivity screens evaluating > 150 cancer-relevant small molecules in dose-response were performed in Pbrm1 siRNA-transfected mouse embryonic stem cells (mES) and isogenic PBRM1-KO or -WT HAP1 cells, respectively. After identification of PBRM1-selective small molecules, revalidation was carried out in a series of in-house-generated isogenic models of PBRM1 deficiency - including 786-O (ccRCC), A498 (ccRCC), U2OS (osteosarcoma) and H1299 (non-small cell lung cancer) human cancer cell lines - and non-isogenic ccRCC models, using multiple clinical compounds. Mechanistic dissection was performed using immunofluorescence, RT-qPCR, western blotting, DNA fiber assay, transcriptomics, proteomics and DRIP-sequencing to evaluate markers of DNA damage response (DDR), replication stress and cell-autonomous innate immune signaling. Preclinical data were integrated with TCGA tumor data. Results: Parallel high-throughput drug screens independently identified PARP inhibitors (PARPi) as being synthetic lethal with PBRM1 defects - a cell type-independent effect which was exacerbated by ATR inhibitors (ATRi) and which we revalidated in vitro in isogenic and non-isogenic systems and in vivo in a xenograft model. PBRM1 defects were associated with increased replication fork stress (higher γH2AX and RPA foci levels, decreased replication fork speed and increased ATM checkpoint activation), R-loop accumulation and enhanced genomic instability in vitro; these effects were exacerbated upon PARPi exposure. In patient tumor samples, we also found that PBRM1-mutant cancers possessed a higher mutational load. Finally, we found that ATRi selectively activated the cGAS/STING cytosolic DNA sensing pathway in PBRM1-deficient cells, resulting in increased expression of type I interferon genes. Conclusion: PBRM1-defective cancer cells present increased replication fork stress, R-loop formation, genome instability and are selectively sensitive to PARPi and ATRi through a synthetic lethal mechanism that is cell type-independent. Our data provide the pre-clinical rationale for assessing PARPi as a monotherapy or in combination with ATRi or immune-modulating agents in molecularly-selected patients with PBRM1-defective cancers. Citation Format: Roman Merial Chabanon, Daphné Morel, Léo Colmet-Daage, Thomas Eychenne, Nicolas Dorvault, Ilirjana Bajrami, Marlène Garrido, Suzanna Hopkins, Cornelia Meisenberg, Andrew Lamb, Theo Roumeliotis, Samuel Jouny, Clémence Astier, Asha Konde, Geneviève Almouzni, Jyoti Choudhary, Jean-Charles Soria, Jessica Downs, Christopher J. Lord, Sophie Postel-Vinay. Targeting chromatin remodeling-associated genetic vulnerabilities in cancer: PBRM1 defects are synthetic lethal with PARP and ATR inhibitors [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1058.

Revealing eukaryotic histone-modifying mechanisms through bacterial infection.

In the long co-evolution of host-pathogen interaction, bacteria have developed sophisticated strategies to manipulate host cell mechanisms and reprogram host transcription. Targeting chromatin, mainly through post-translational modification (PTM) of histone proteins, is one strategy that has been revealed over the last decade. Indeed, histone modifications play a crucial role in regulating transcription during cell type and stimulus specific responses, making them good targets during infection. Therefore, the study of host-pathogen interactions provides breakthroughs in understanding virulence mechanisms, but also in host cell mechanisms. Although chromatin is regulated by DNA methylation, noncoding RNAs, and post-translational modifications of histones, most studies have concentrated on bacteria-induced histone modifications, which will be the focus of this review. We will discuss the different mechanisms used by bacteria to induce histone PTMs, whether it is through direct targeting of pathogen effector enzymes, or indirectly through modulation of cellular signaling cascade. We will summarize the concepts we learned in cell biology from exploring bacteria-triggered histone modifications, by focusing on the signaling cascades modified by bacteria, bacterial mimics of eukaryotic enzymes, and the novel histone marks imposed upon infection.

Therapeutic Targeting of CDK12/CDK13 in Triple-Negative Breast Cancer

Epigenetic regulation enables tumors to respond to changing environments during tumor progression and metastases and facilitates treatment resistance. Targeting chromatin modifiers or catalytic effectors of transcription is an emerging anti-cancer strategy. The cyclin-dependent kinases (CDKs) 12 and 13 phosphorylate the C-terminal domain of RNA polymerase II, regulating transcription and co-transcriptional processes. Here we report the development of SR-4835, a highly selective dual inhibitor of CDK12 and CDK13, which disables triple-negative breast cancer (TNBC) cells. Mechanistically, inhibition or loss of CDK12/CDK13 triggers intronic polyadenylation site cleavage that suppresses the expression of core DNA damage response proteins. This provokes a "BRCAness" phenotype that results in deficiencies in DNA damage repair, promoting synergy with DNA-damaging chemotherapy and PARP inhibitors.

Targeting chromatin

Targeting chromatin

Targeting Chromatin Remodeling for Cancer Therapy.

Background:Epigenetic alterations comprise key regulatory events that dynamically alter gene expression and their deregulation is commonly linked to the pathogenesis of various diseases, including cancer. Unlike DNA mutations, epigenetic alterations involve modifications to proteins and nucleic acids that regulate chromatin structure without affecting the underlying DNA sequence, altering the accessibility of the transcriptional machinery to the DNA, thus modulating gene expression. In cancer cells, this often involves the silencing of tumor suppressor genes or the increased expression of genes involved in oncogenesis. Advances in laboratory medicine have made it possible to map critical epigenetic events, including histone modifications and DNA methylation, on a genome-wide scale. Like the identification of genetic mutations, mapping of changes to the epigenetic landscape has increased our understanding of cancer progression. However, in contrast to irreversible genetic mutations, epigenetic modifications are flexible and dynamic, thereby making them promising therapeutic targets. Ongoing studies are evaluating the use of epigenetic drugs in chemotherapy sensitization and immune system modulation. With the preclinical success of drugs that modify epigenetics, along with the FDA approval of epigenetic drugs including the DNA methyltransferase 1 (DNMT1) inhibitor 5-azacitidine and the histone deacetylase (HDAC) inhibitor vorinostat, there has been a rise in the number of drugs that target epigenetic modulators over recent years.Conclusion:We provide an overview of epigenetic modulations, particularly those involved in cancer, and discuss the recent advances in drug development that target these chromatin-modifying events, primarily focusing on novel strategies to regulate the epigenome.

A Histone Acetyltransferase Inhibitor with Antifungal Activity against CTG clade Candida Species.

Candida species represent one of the most frequent causes of hospital-acquired infections in immunocompromised patient cohorts. Due to a very limited set of antifungals available and an increasing prevalence of drug resistance, the discovery of novel antifungal targets is essential. Targeting chromatin modifiers as potential antifungal targets has gained attention recently, mainly due to their role in regulating virulence in Candida species. Here, we describe a novel activity for the histone acetyltransferase inhibitor Cyclopentylidene-[4-(4-chlorophenyl)thiazol-2-yl)hydrazone (CPTH2) as a specific inhibitor of CTG clade Candida species. Furthermore, we show that CPTH2 has fungicidal activity and protects macrophages from Candida-mediated death. Thus, this work could provide a starting point for the development of novel antifungals specific to CTG clade Candida species.

Targeting chromatin complexes in fusion protein-driven malignancies.

Recurrent chromosomal rearrangements leading to the generation of oncogenic fusion proteins are a common feature of many cancers. These aberrations are particularly prevalent in sarcomas and haematopoietic malignancies and frequently involve genes required for chromatin regulation and transcriptional control. In many cases, these fusion proteins are thought to be the primary driver of cancer development, altering chromatin dynamics to initiate oncogenic gene expression programmes. In recent years, mechanistic insights into the underlying molecular functions of a number of these oncogenic fusion proteins have been discovered. These insights have allowed the design of mechanistically anchored therapeutic approaches promising substantial treatment advances. In this Review, we discuss how our understanding of fusion protein function is informing therapeutic innovations and illuminating mechanisms of chromatin and transcriptional regulation in cancer and normal cells.