HDAC8 suppresses the epithelial phenotype and promotes EMT in chemotherapy-treated basal-like breast cancer

Background Basal-like breast cancer (BLBC) is one of the most aggressive malignant diseases in women with an increased metastatic behavior and poor prognosis compared to other molecular subtypes of breast cancer. Resistance to chemotherapy is the main cause of treatment failure in BLBC. Therefore, novel therapeutic strategies counteracting the gain of aggressiveness underlying therapy resistance are urgently needed. The epithelial-to-mesenchymal transition (EMT) has been established as one central process stimulating cancer cell migratory capacity but also acquisition of chemotherapy-resistant properties. In this study, we aimed to uncover epigenetic factors involved in the EMT-transcriptional program occurring in BLBC cells surviving conventional chemotherapy. Results Using whole transcriptome data from a murine mammary carcinoma cell line (pG-2), we identified upregulation of Hdac4, 7 and 8 in tumor cells surviving conventional chemotherapy. Subsequent analyses of human BLBC patient datasets and cell lines established HDAC8 as the most promising factor sustaining tumor cell viability. ChIP-sequencing data analysis identified a pronounced loss of H3K27ac at regulatory regions of master transcription factors (TFs) of epithelial phenotype like Gata3, Elf5, Rora and Grhl2 upon chemotherapy. Interestingly, impairment of HDAC8 activity reverted epithelial-TFs levels. Furthermore, loss of HDAC8 activity sensitized tumor cells to chemotherapeutic treatments, even at low doses. Conclusion The current study reveals a previously unknown transcriptional repressive function of HDAC8 exerted on a panel of transcription factors involved in the maintenance of epithelial cell phenotype, thereby supporting BLBC cell survival to conventional chemotherapy. Our data establish HDAC8 as an attractive therapeutically targetable epigenetic factor to increase the efficiency of chemotherapeutics. Graphical abstract

PRDM16 Is a Compact Myocardium-Enriched Transcription Factor Required to Maintain Compact Myocardial Cardiomyocyte Identity in Left Ventricle

Background: Left ventricular noncompaction cardiomyopathy (LVNC) was discovered half a century ago as a cardiomyopathy with excessive trabeculation and a thin ventricular wall. In the decades since, numerous studies have demonstrated that LVNC primarily impacts left ventricles (LVs), and is often associated with LV dilation and dysfunction. However, owing in part to the lack of suitable mouse models that faithfully mirror the selective LV vulnerability in patients, mechanisms underlying susceptibility of LV to dilation and dysfunction in LVNC remain unknown. Genetic studies have revealed that deletions and mutations in PRDM16 cause LVNC, but previous conditional Prdm16 knockout mouse models do not mirror the LVNC phenotype in patients, and importantly, the underlying molecular mechanisms by which PRDM16 deficiency causes LVNC are still unclear. Methods: Prdm16 cardiomyocyte (CM)-specific knockout ( Prdm16 cKO ) mice were generated and analyzed for cardiac phenotypes. RNA sequencing and ChIP sequencing were performed to identify direct transcriptional targets of PRDM16 in CMs. Single cell RNA sequencing in combination with Spatial Transcriptomics were employed to determine CM identity at single cell level. Results: CM-specific ablation of Prdm16 in mice caused LV-specific dilation and dysfunction, as well as biventricular noncompaction, which fully recapitulated LVNC in patients. Mechanistically, PRDM16 functioned as a compact myocardium-enriched transcription factor, which activated compact myocardial genes while repressing trabecular myocardial genes in LV compact myocardium. Consequently, Prdm16 cKO LV compact myocardial CMs shifted from their normal transcriptomic identity to a transcriptional signature resembling trabecular myocardial CMs and/or neurons. Chamber-specific transcriptional regulation by PRDM16 was in part due to its cooperation with LV-enriched transcription factors Tbx5 and Hand1. Conclusions: These results demonstrate that disruption of proper specification of compact CM may play a key role in the pathogenesis of LVNC. They also shed light on underlying mechanisms of LV-restricted transcriptional program governing LV chamber growth and maturation, providing a tangible explanation for the susceptibility of LV in a subset of LVNC cardiomyopathies.

Effects of ABCB1 DNA methylation in donors on tacrolimus blood concentrations in recipients following liver transplantation.

Aims: To investigate the effects of ABCB1 DNA methylation in donors on individual differences in tacrolimus blood concentrations following liver transplantation. Methods: Twenty-three donor liver samples carrying the CYP3A5*3/*3 genotype were classified into two groups based on the initial tacrolimus concentration/dose (C0/D) ratio following liver transplantation. ABCB1 mRNA levels in liver tissues and HepG2 cells were determined by qRT-PCR. DNA methylation status in liver tissues and HepG2 cells was determined using Illumina 850 methylation chip sequencing technology and pyrosequencing. 5-Aza-2dC was used to reverse methylation in HepG2 cells. Intracellular tacrolimus concentrations were determined by liquid mass spectrometry. Results: Genome-wide methylation sequencing and pyrosequencing analyses showed that the methylation levels of three ABCB1 CpG sites (cg12501229, cg00634941, and cg05496710) were significantly different between groups with different tacrolimus C0/D ratios. ABCB1 mRNA expression in donor livers was found to be positively correlated with tacrolimus C0/D ratio (r = 0.458, P < 0.05). After treatment with 5-Aza-2-Dc, the methylation levels of the ABCB1 CpG sites in HepG2 cells significantly decreased, and this was confirmed by pyrosequencing; there was also a significant increase in ABCB1 transcription, and this most likely induced a decrease in intracellular tacrolimus concentrations. Conclusions: ABCB1 CpG site methylation affects tacrolimus metabolism in humans by regulating ABCB1 expression. Therefore, ABCB1 DNA methylation in donor livers might be an important epigenetic factor that affects tacrolimus blood concentrations following liver transplantation.

Decreased PRC2 activity supports the survival of basal-like breast cancer cells to cytotoxic treatments

Breast cancer (BC) is the most common cancer occurring in women but also rarely develops in men. Recent advances in early diagnosis and development of targeted therapies have greatly improved the survival rate of BC patients. However, the basal-like BC subtype (BLBC), largely overlapping with the triple-negative BC subtype (TNBC), lacks such drug targets and conventional cytotoxic chemotherapies often remain the only treatment option. Thus, the development of resistance to cytotoxic therapies has fatal consequences. To assess the involvement of epigenetic mechanisms and their therapeutic potential increasing cytotoxic drug efficiency, we combined high-throughput RNA- and ChIP-sequencing analyses in BLBC cells. Tumor cells surviving chemotherapy upregulated transcriptional programs of epithelial-to-mesenchymal transition (EMT) and stemness. To our surprise, the same cells showed a pronounced reduction of polycomb repressive complex 2 (PRC2) activity via downregulation of its subunits Ezh2, Suz12, Rbbp7 and Mtf2. Mechanistically, loss of PRC2 activity leads to the de-repression of a set of genes through an epigenetic switch from repressive H3K27me3 to activating H3K27ac mark at regulatory regions. We identified Nfatc1 as an upregulated gene upon loss of PRC2 activity and directly implicated in the transcriptional changes happening upon survival to chemotherapy. Blocking NFATc1 activation reduced epithelial-to-mesenchymal transition, aggressiveness, and therapy resistance of BLBC cells. Our data demonstrate a previously unknown function of PRC2 maintaining low Nfatc1 expression levels and thereby repressing aggressiveness and therapy resistance in BLBC.

Different SP1 binding dynamics at individual genomic loci in human cells

Using a tamoxifen-inducible time-course ChIP-sequencing (ChIP-seq) approach, we show that the ubiquitous transcription factor SP1 has different binding dynamics at its target sites in the human genome. SP1 very rapidly reaches maximal binding levels at some sites, but binding kinetics at other sites is biphasic, with rapid half-maximal binding followed by a considerably slower increase to maximal binding. While ∼70% of SP1 binding sites are located at promoter regions, loci with slow SP1 binding kinetics are enriched in enhancer and Polycomb-repressed regions. Unexpectedly, SP1 sites with fast binding kinetics tend to have higher quality and more copies of the SP1 sequence motif. Different cobinding factors associate near SP1 binding sites depending on their binding kinetics and on their location at promoters or enhancers. For example, NFY and FOS are preferentially associated near promoter-bound SP1 sites with fast binding kinetics, whereas DNA motifs of ETS and homeodomain proteins are preferentially observed at sites with slow binding kinetics. At promoters but not enhancers, proteins involved in sumoylation and PML bodies associate more strongly with slow SP1 binding sites than with the fast binding sites. The speed of SP1 binding is not associated with nucleosome occupancy, and it is not necessarily coupled to higher transcriptional activity. These results with SP1 are in contrast to those of human TBP, indicating that there is no common mechanism affecting transcription factor binding kinetics. The biphasic kinetics at some SP1 target sites suggest the existence of distinct chromatin states at these loci in different cells within the overall population.

Nuclear receptor subfamily 4A signaling as a key disease pathway of CD1c+ dendritic cell dysregulation in systemic sclerosis

Objectives: To identify key disease pathways driving conventional dendritic cell (cDC) alterations in Systemic Sclerosis (SSc). Methods: Transcriptomic profiling was performed on peripheral blood CD1c+ cDCs (cDC2s) isolated from 12 healthy donors and 48 SSc patients with all major disease subtypes. Differential expression analysis comparing the different SSc subtypes and healthy donors was performed to uncover genes dysregulated in SSc. To identify biologically relevant pathways, a gene co-expression network was built using Weighted Gene Correlation Network Analysis. We validated the role of key transcriptional regulators using ChIP-sequencing and in vitro functional assays. Results: We identified 17 modules of co-expressed genes in cDCs that correlated with SSc subtypes and key clinical traits including auto-antibodies, skin score, and occurrence of interstitial lung disease. A module of immune regulatory genes was markedly down regulated in patients with the diffuse SSc subtype characterized by severe fibrosis. Transcriptional regulatory network analysis performed on this module predicted NR4A (nuclear receptor 4A) subfamily (NR4A1, NR4A2, NR4A3) genes as the key transcriptional mediators of inflammation. Indeed, ChIP-sequencing analysis supported that these NR4A members target numerous differentially expressed genes in SSc cDC2s. Inclusion of NR4A receptor agonists in culture-based experiments provided functional proof that dysregulation of NR4As affects cytokine production by cDC2s and modulates downstream T-cell activation. Conclusions: NR4A1, NR4A2 and NR4A3 are important regulators of immunosuppressive and fibrosis-associated pathways in SSc cDCs. Thus, the NR4A family represent novel potential targets to restore cDC homeostasis in SSc.

Nup93 and CTCF modulate spatiotemporal dynamics and function of the HOXA gene locus during differentiation

Nucleoporins regulate nuclear transport and are also involved in DNA damage, repair, cell cycle, chromatin organization, and gene expression. Here, we studied the role of nucleoporin Nup93 and the chromatin organizer CTCF in regulating HOXA expression during differentiation. ChIP sequencing revealed a significant overlap between Nup93 and CTCF peaks. Interestingly, Nup93 and CTCF are associated with the 3' and 5′HOXA genes respectively. Depletions of Nup93 and CTCF antagonistically modulate expression levels of 3′and 5′HOXA genes in undifferentiated NT2/D1 cells. Nup93 also regulates the localization of the HOXA gene locus, which disengages from the nuclear periphery upon Nup93 but not CTCF depletion, consistent with its upregulation. The dynamic association of Nup93 and CTCF with the HOXA locus during differentiation correlates with its spatial positioning and expression. While Nup93 tethers the HOXA locus to the nuclear periphery, CTCF potentially regulates looping of the HOXA gene cluster in a temporal manner. In summary, Nup93 and CTCF complement one another in modulating the spatiotemporal dynamics and function of the HOXA gene locus during differentiation.

EZH1/2 Dual Inhibitor Valemetostat Tosylate (DS-3201b) Acts Differently from EZH2 Selective Inhibitor on Epigenetic Landscape to Exert Greater Anti-Tumor Effect Against Diffuse Large B-Cell Lymphoma

Enhancer of zeste homologous (EZH) 2 and its close homolog EZH1 are catalytic subunits of polycomb repressive complex (PRC) 2 protein complex, and play redundant and crucial role for the maintenance of transcriptional repression by tri-methylating histone H3 lysine 27 (H3K27). Hyper trimethylation of H3K27 has been associated with malignant lymphoma and myeloma progression, thus several small molecules suppressing PRC2 complex activity has been developed for hematological malignancy therapy. We have developed valemetostat tosylate (DS-3201b, also known as valematostat), a potent dual inhibitor of EZH1/2, and demonstrated its superior anti-proliferative effect against DLBCL cells to tazemetostat (EPZ-6438, E7438) a selective EZH2 inhibitor currently in clinic. In addition, valemetostat synergized with wide variety of 1st and 2nd line drugs used in DLBCL therapy both in vitro and in vivo proposing its potential combination opportunities. However, it is still elusive how valemetostat modulates epigenetic landscape and represses malignant B-cell proliferation more potently than selective EZH2 inhibitors. Therefore, impact on epigenetic landscape between valemetostat and tazemetostat was analyzed by RNA/ChIP-sequencing. Though these two inhibitors significantly reduced cellular global H3K27me3 level, we observed ectopic EZH1/2 accumulation in several tumor suppressor gene loci after tazemetostat treatment resulting in partial reduction in H3K27me3 and de-repression of silenced gene expression. Meanwhile valemetostat treatment evidently triggered gene expression by depleting H3K27me3 and enhancing H3K27Ac mark without inducing ectopic enrichment of EZH1/2, suggesting that valemetostat has a distinct effect on genome wide distribution of EZH1/2 from tazemetostat. In conclusion, these results suggest that valemetostat has a capacity of averting ectopic relocation of EZH1/2 on tumor suppressor genes mainly induced by EZH2 specific inhibition and thereby exerts greater anti-B cell tumor effect than EZH2 preferential inhibitor. A phase 2 clinical study of valemetostat is now ongoing for patients with Relapse/Refractory B-cell Lymphoma [ClinicalTrials.gov Identifier: NCT04842877] Disclosures Banjo: Daiichi Sankyo Co., Ltd.: Current Employment. Hama: Daiichi Sankyo Co., Ltd.: Current Employment. Nosaka: Daiichi Sankyo Co., Ltd.: Current Employment. Takata: Daiichi Sankyo Co., Ltd.: Current Employment. Honma: Daiichi Sankyo Co., Ltd.: Current Employment. Kitagawa: Daiichi Sankyo Co., Ltd.: Current Employment. Yamamoto: Daiichi Sankyo RD Novare Co., Ltd.: Current Employment. Wada: Daiichi Sankyo RD Novare Co., Ltd.: Current Employment. Yoshida: Daiichi Sankyo RD Novare Co., Ltd.: Current Employment. Lim: Daiichi Sankyo RD Novare Co., Ltd.: Current Employment. Okamoto: Daiichi Sankyo RD Novare Co., Ltd.: Current Employment. Sato: Daiichi Sankyo RD Novare Co., Ltd.: Current Employment. Katayama: Daiichi Sankyo RD Novare Co., Ltd.: Current Employment. Sato: Daiichi Sankyo RD Novare Co., Ltd.: Current Employment. Goto: Daiichi Sankyo RD Novare Co., Ltd.: Current Employment. Abe: Daiichi Sankyo Co., Ltd.: Current Employment.

Super-Enhancer Driven Regulation of CKS1B in Multiple Myeloma: Implications in Mediating Response to BET Inhibitor and Celmod Agent Combination

Introduction: The Myeloma Genome Project (MGP) characterized the genomic landscape of patients with newly diagnosed multiple myeloma (NDMM) (Walker BA, et al. Blood 2018; 132[6]:587-597). Using a multi-omics unsupervised clustering approach, 12 molecularly-defined disease segments were identified (Ortiz M, et al. Blood 2018; 132[suppl 1]:3165). Here, we performed experimental validation of CDC28 Protein Kinase Regulatory Subunit 1B (CSK1B) that was identified as a putative target from the disease segment with poorest clinical outcome. CKS1B was selected for in-depth validation due to their role in cell cycle pathways associated with high-risk disease, biological mechanisms of chromosome 1q amplification and druggability. Methods: Association of CKS1B with outcomes was analyzed in NDMM patients, across relapses and with clinical outcome datasets from MGP and Mayo clinic. Inducible shRNAs of CKS1B and bromodomain containing protein 4 (BRD4, a member of the BET [bromodomain and extra terminal domain] family) were generated in MM cell lines. BRD4 and Aiolos ChIP-seq datasets were analyzed for binding on CKS1B gene. BRD4 inhibitors JQ1 and CC-90010 were utilized for inhibition studies in MM cell lines. Results: Higher expression of CKS1B was associated with significantly poorer PFS, OS, disease severity and relapse. Knock-down of CKS1B in MM cells led to a significant decrease in proliferation (P&lt;0.001) and enhanced apoptosis in MM cell lines. BRD4-ChIP sequencing studies revealed that the expression of CKS1B was regulated by super-enhancer (SE) associated elements. As expected, two BRD4 inhibitors, JQ1 and CC-90010 and inducible BRD4 shRNAs downregulated the expression of CKS1B resulting in decreased proliferation, cell cycle arrest and apoptosis in MM cell lines. Furthermore, MM cell lines harboring chromosome 1q gain/amp showed higher sensitivity to BRD4 inhibition compared to cell lines with normal 1q copy number. Mechanistic studies revealed that BRD4inh and BRD4 shRNAs downregulated the expression of Aiolos and Ikaros in MM cell lines. Interestingly, Aiolos ChIP-sequencing studies demonstrated the binding of Aiolos at the transcriptional start sites of CKS1B with the transcriptional activation mark. The immunomodulatory agent (IMiD ®) pomalidomide (Pom) transcriptionally downregulated CKS1B in Pom-sensitive cells downstream of Aiolos, Ikaros degradation. Based on these mechanisms, IMiD agents, lenalidomide, Pom and the novel Cereblon E3 ligase modulating degrader (CELMoD ®) agent CC-92480 in combination with BRD4inh promoted a synergistic decrease in proliferation, cell cycle arrest and increase in apoptosis in both Pom-sensitive and -resistant cell lines. The combination of IMiD or novel CELMoD agent with BRD4inh also promoted deeper downregulation of CKS1B, Aiolos, Ikaros, c-Myc and survivin proteins with enhanced levels of apoptotic marker cleaved Caspase 3 as compared to single agents alone. Conclusions: In summary, we have identified CKS1B as a key target associated with poor outcome in MM patients. Translational studies suggest a profound downregulation of CKS1B and key pro-survival effector proteins following combination treatment with BRD4inh and IMiD agents/novel CELMoD agents resulting in synergistic anti-tumor effects. These data provide rationale for testing these agents in the clinic for high-risk and IMiD-relapsed patients. Figure: Changes in cell proliferation and protein levels of key signaling mediators were studied in K12PE cell line treated with increasing doses of Lenalidomide, Pomalidomide and CC-92480 in combination with JQ1. Figure 1 Figure 1. Disclosures Ahsan: BMS: Current Employment, Current equity holder in publicly-traded company. Polonskaia: Bristol Myers Squibb: Current Employment, Current equity holder in publicly-traded company. Hsu: Bristol Myers Squibb: Current Employment, Current equity holder in publicly-traded company. Bjorklund: BMS: Current Employment, Current equity holder in publicly-traded company. Ortiz Estevez: Bristol Myers Squibb: Current Employment, Current equity holder in publicly-traded company. Towfic: Bristol Myers Squibb: Current Employment, Current equity holder in publicly-traded company. Bahlis: Takeda: Consultancy, Honoraria; Abbvie: Consultancy, Honoraria; GlaxoSmithKline: Consultancy, Honoraria; Sanofi: Consultancy, Honoraria; Amgen: Consultancy, Honoraria; Genentech: Consultancy; Pfizer: Consultancy, Honoraria; BMS/Celgene: Consultancy, Honoraria; Janssen: Consultancy, Honoraria; Karyopharm: Consultancy, Honoraria. Pourdehnad: Bristol Myers Squibb: Current Employment, Current equity holder in publicly-traded company, Patents & Royalties: No royalty. Flynt: BMS: Current Employment, Current equity holder in publicly-traded company. Ahsan: Bristol Myers Squibb: Current Employment, Current equity holder in publicly-traded company. Thakurta: Bristol Myers Squibb: Current Employment, Current equity holder in publicly-traded company, Patents & Royalties.

Bivalent Histone Modifications By Reiibp Leads to Transcriptional Reprogramming and TLR7-BTK Driven IL-6 Pro-Inflammatory Response in t(4;14) Myelomagenesis

Multiple Myeloma (MM) is characterized by the uncontrolled proliferation of malignant plasma cells that are incapable of producing functional antibodies. Current treatment regime involves novel drug classes such as proteasome inhibitors, immunomodulatory drugs and monoclonal antibodies, which have significantly improved survival outcomes in patients. Patients with t(4;14) translocation represents ~15% of MM cases, and displays a dysregulation of the MMSET locus. It has one of the worst prognosis when compared to other subgroups, but represents an intermediate-risk group given its response towards bortezomib. REIIBP is a t(4;14)-deregulated isoform arising from alternative promoter usage within the MMSET locus. Despite sharing identical sequence with the C-terminus of MMSET II, we found that REIIBP displayed mutually exclusive expression with the full-length MMSET II arising from MB4-1 breakpoint. Additionally, the expression of REIIBP can be regulated through microRNAs by another histone methyltransferase, EZH2 in a Dicer-dependent manner. We generated a stable cell line that overexpresses REIIBP in the t(4;14)-negative human myeloma cells, RPMI8226, and revealed REIIBP as an epigenetic regulator of repressive H3K27me3 and active H3K4me3 modifications. Transcriptome profiling and ChIP-sequencing identified an upregulation of Toll-like receptor 7 (TLR7) by REIIBP through the enrichment of H3K4me3 on its promoter coupled with decrease in intragenic H3K27me3. This led to a BCR-independent activation of Bruton's tyrosine kinase (BTK) and NF-ĸB signaling in t(4;14) myeloma cells. Using Cancer Cell Line Encyclopedia and DepMap portal (Broad Institute) to compare the expression of BTK and BTK dependency scores across lineages revealed that BTK is an important target in MM, and REIIBP expression correlated with BTK in the CoMMpass dataset. Activation of TLR7-BTK by REIIBP conferred bortezomib resistance through the dysregulation of pro-inflammatory cytokine expression such as IL-6. Importantly, cells with REIIBP overexpression displayed enhanced lethality towards BTK inhibitor Ibrutinib, and combination with Bortezomib potentiated inhibition in myeloma cell lines and mice engrafted with RPMI8226-REIIBP tumors. Altogether, our results indicated that blockade of REIIBP in t(4;14) cells through combining proteasome and BTK inhibitors is a therapeutic strategy in the clinic for further evaluation. Disclosures Chng: Novartis: Honoraria, Research Funding; Antengene: Honoraria; Pfizer: Honoraria; Sanofi: Honoraria; AbbVie: Honoraria; Amgen: Honoraria; BMS/Celgene: Honoraria, Research Funding; Johnson & Johnson: Honoraria, Research Funding; Takeda: Honoraria; Aslan: Research Funding.