Search

The reprogramming of human acute myeloid leukemia (AML) cells into induced pluripotent stem cell (iPSC) lines could provide new faithful genetic models of AML, but is currently hindered by low success rates and uncertainty about whether iPSC-derived cells resemble their primary counterparts. Here we developed a reprogramming method tailored to cancer cells, with which we generated iPSCs from 15 patients representing all major genetic groups of AML. These AML-iPSCs retain genetic fidelity and produce transplantable hematopoietic cells with hallmark phenotypic leukemic features. Critically, single-cell transcriptomics reveal that, upon xenotransplantation, iPSC-derived leukemias faithfully mimic the primary patient-matched xenografts. Transplantation of iPSC-derived leukemias capturing a clone and subclone from the same patient allowed us to isolate the contribution of a FLT3-ITD mutation to the AML phenotype. The results and resources reported here can transform basic and preclinical cancer research of AML and other human cancers. Significance: We report the generation of patient-derived iPSC models of all major genetic groups of human AML. These exhibit phenotypic hallmarks of AML in vitro and in vivo, inform the clonal hierarchy and clonal dynamics of human AML, and exhibit striking similarity to patient-matched primary leukemias upon xenotransplantation. See related commentary by Doulatov.

Supplemental Figure 1. Generation of a panel of iPSCs from patients with AML. Supplemental Figure 2. Reprogramming aids reconstruction of the evolutionary history and clonal composition of AML. Supplemental Figure 3. Transplantation of AML-iPSCs into immunodeficient mice. Supplemental Figure 4. Developmental block in a subset of AML-iPSC lines. Supplemental Figure 5. Transplantation of primary AML cells and patient-matched AMLiPSC lines. Supplemental Figure 6. Single-cell RNA-sequencing analyses of matched primary and iPSC-derived leukemia cells from patient AML-47. Supplemental Figure 7. Cell cycle and pseudotime analyses. Supplemental Figure 8. Comparison of scRNA-Seq data integration and clustering methods and pseudobulk differential gene expression analyses.

Table S1. Patient characteristics. AML: acute myeloid leukemia; MDS: myelodysplastic syndrome; MPN: myeloproliferative neoplasm; ET: essential thrombocythemia; PBMCs: peripheral blood mononuclear cells; BMMCs: bone marrow mononuclear cells; PDX: patient-derived xenografts Table S2. All patient samples used in this study with genetic characterization and reprogramming outcomes. Blue font denotes partially reprogrammed (as opposed to bona fide iPSC) colonies and clones. Table S3. All AML-iPSC lines phenotypically characterized. Table S4. Top 50 upregulated genes (highest log2 fold change) in each cluster. Table S5. Primers used for genotyping. Table S6. Primers used for qRT-PCR analyses.

The reprogramming of human acute myeloid leukemia (AML) cells into induced pluripotent stem cell (iPSC) lines could provide new faithful genetic models of AML, but is currently hindered by low success rates and uncertainty about whether iPSC-derived cells resemble their primary counterparts. Here we developed a reprogramming method tailored to cancer cells, with which we generated iPSCs from 15 patients representing all major genetic groups of AML. These AML-iPSCs retain genetic fidelity and produce transplantable hematopoietic cells with hallmark phenotypic leukemic features. Critically, single-cell transcriptomics reveal that, upon xenotransplantation, iPSC-derived leukemias faithfully mimic the primary patient-matched xenografts. Transplantation of iPSC-derived leukemias capturing a clone and subclone from the same patient allowed us to isolate the contribution of a FLT3-ITD mutation to the AML phenotype. The results and resources reported here can transform basic and preclinical cancer research of AML and other human cancers.Significance:We report the generation of patient-derived iPSC models of all major genetic groups of human AML. These exhibit phenotypic hallmarks of AML in vitro and in vivo, inform the clonal hierarchy and clonal dynamics of human AML, and exhibit striking similarity to patient-matched primary leukemias upon xenotransplantation.See related commentary by Doulatov.

Supplementary Data from Loss of PBRM1 Alters Promoter Histone Modifications and Activates ALDH1A1 to Drive Renal Cell Carcinoma

Supplementary Figure from Loss of PBRM1 Alters Promoter Histone Modifications and Activates ALDH1A1 to Drive Renal Cell Carcinoma

MacroH2A variants have been linked to inhibition of metastasis through incompletely understood mechanisms. Here, we reveal that solitary dormant disseminated cancer cells (DCCs) display increased levels of macroH2A variants in head and neck squamous cell carcinoma PDX in vivo models and patient samples compared to proliferating primary or metastatic lesions. We demonstrate that dormancy-inducing transforming growth factor–β2 and p38α/β pathways up-regulate macroH2A expression and that macroH2A variant overexpression is sufficient to induce DCC dormancy and suppress metastasis in vivo. Notably, inducible expression of the macroH2A2 variant in vivo suppresses metastasis via a reversible growth arrest of DCCs. This state does not require the dormancy-regulating transcription factors DEC2 and NR2F1; instead, transcriptomic analysis reveals that macroH2A2 overexpression inhibits cell cycle and oncogenic signaling programs, while up-regulating dormancy and senescence-associated inflammatory cytokines. We conclude that the macroH2A2-enforced dormant phenotype results from tapping into transcriptional programs of both quiescence and senescence to limit metastatic outgrowth.

The reprogramming of malignant cells to pluripotency has presented considerable challenges, hindering the application of induced pluripotent stem cell (iPSC) modeling technology to human cancers. In addition, how well iPSC-derived cells resemble their primary counterparts is currently largely unknown. We developed a reprogramming method tailored to human malignancies, “Complete Capture of Mutational Burden” (CCoMB), that combines comprehensive genetic characterization of the starting sample and inference of its clonal architecture with large-scale screening of clonally reprogrammed colonies. Using this method we were able to generate a panel of iPSC lines representing all major genetic groups of acute myeloid leukemia (AML). Specifically, we derived iPSC lines from 15 patients representing all major genetic groups of AML - PML-RARA; chromatin-spliceosome; TP53-mutated/aneuploidy; AML1-ETO, MLL-rearranged; NPM1-mutated and others - collectively capturing 21 distinct genotypes and 24 driver genetic lesions (mutations, translocations, deletions). Matched normal iPSCs were derived from 7 of these patients. Reprogramming to iPSCs captured both preleukemic (CH/initiating mutation only) and fully leukemic clones (baring the full set of patient mutations). In almost all cases, reprogramming informed reconstruction of the evolutionary hierarchy of the AML, with unexpected hierarchies unveiled in 4 of the cases. These AML-iPSCs retain genetic fidelity and, upon in vitro hematopoietic differentiation, produce hematopoietic cells with hallmark phenotypic leukemic features, including serial engraftment of a lethal myeloid leukemia into immunocompromised mice and extended self-renewal in vitro. Transplantation of cells derived from iPSCs representing two distinct AML clones from each of 3 patients revealed that these mimic the clonal dynamics in the patients, with more advanced clones showing increased representation in the xenografts, compared to the earlier clones. To compare the iPSC-derived to the primary leukemias, we performed single-cell transcriptomics analyses in patient-matched iPSC-derived and primary leukemic cells from 3 patients, both ex vivo/in vitro and after transplantation into NSGS mice. Clustering analyses identified cell types corresponding to primitive hematopoietic stem cell (HSC)/multipotent progenitor (MPP), hematopoietic progenitor cells (HPCs) and more mature myelomonocytic lineage cells in all samples from all patients, at varying frequencies. Leukemias derived through in vitro differentiation from iPSC lines exhibited both similarities, as well as differences, in their cellular composition and transcriptome, compared to the patient-matched ex vivo leukemias. However, upon transplantation of the same leukemias into NSGS mice, iPSC-derived xenografts were strikingly similar to the patient-derived xenografts. iPSC-derived leukemic cells exhibited a more stem/progenitor cell phenotype in vitro, with progressive maturation along the myeloid lineage upon primary and, even more, upon secondary transplantation, mimicking primary xenografts. In summary, our results reveal very few true biological barriers to the reprogramming of AML cells and show that AML-iPSC-derived leukemias faithfully mimic the primary patient leukemias upon xenotransplantation. Citation Format: Andriana Kotini, Saul Carcamo, Nataly Cruz-Rodriguez, Malgorzata Olszewska, Dan Hasson, Eirini Papapetrou. Patient-derived iPSCs faithfully represent the genetic diversity and cellular architecture of human acute myeloid leukemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr LB363.

Background: Small cell lung cancer (SCLC) is an exceptionally aggressive disease with limited treatment options that typically result in transient responses. SCLC is responsible for approximately 250,000 deaths globally per year. Major hurdles to improving SCLC treatment include the development of rapid chemo-resistance and limited second-line therapies. Lurbinectedin is FDA approved as a second-line treatment for SCLC but shows a response in a subset of patients. Therefore, improved mechanistic understanding and identifying predictive biomarkers of lurbinectedin treatment is a major unmet clinical need. Methods: We treated SCLC cell lines and patient-derived xenograft (PDX) models from all major SCLC subtypes with lurbinectedin. Lurbinectedin-mediated changes in signaling pathways were studied by bulk RNA sequencing, western blot, and flow cytometry. Anti-tumor efficacy and toxicity studies were performed in vivo. Result: All human and PDX-derived SCLC cell lines showed sensitivity to lurbinectedin at a nano-molar concentration ranging from 1.905 to 30 nM. Bulk RNA-seq analysis showed lurbinectedin induced changes in neuroendocrine phenotype, DNA damage response and tumor progression markers in vitro. Single agent treatment of lurbinectedin showed remarkable anti-tumor efficacy in an ASCL1-driven PDX model. RNA sequencing analysis identified modulation of genes in multiple signaling pathways including PI3K-AKT, apoptosis and EMT to be significantly associated with the lurbinectedin response in PDX models. Conclusion: There is an immediate need to understand the subsets of SCLC that would be most sensitive to lurbinectedin and identify predictive biomarkers. We demonstrate MYC as a predictive biomarker for lurbinectedin response. We are the first to show that single agent lurbinectedin shows remarkable anti-tumor efficacy in a ASCL1-driven PDX models of SCLC. Furthermore, our pre and post-lurbinectedin treatment transcriptomic analysis identify the pathways that may contribute to primary or acquired resistance to lurbinectedin in SCLC. Finally, we identity candidate targets that would guide the design of future combination clinical trials with lurbinectedin in SCLC. Citation Format: Subhamoy Chakraborty, Charles Coleman, Deniz Demircioglu, Dan Hasson, Triparna Sen. Preclinical analysis identifies predictive biomarker and potential pathways of resistance to lurbinectedin treatment in small cell lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr LB233.

Tumors exhibit widespread enhancer landscape reprogramming compared to normal tissue. The etiology is believed to be largely cell-intrinsic in non-hormonal cancers, attributed to such genomic alterations as focal amplification of non-coding regions, aberrant activation of transcription factors, and non-coding mutations creating de novo transcription factor binding sites. Here, using freshly resected primary CRC tumors and patient-matched adjacent normal colon epithelia, we find divergent epigenetic landscapes between primary CRC tumors and CRC cell lines. We identify a unique super-enhancer signature largely absent in cell culture. Intriguingly, this phenomenon extends to highly recurrent aberrant super-enhancers gained in CRC over patient-matched normal epithelium suggesting novel insight into the etiology of enhancer reprogramming in CRC and its downstream relevance to tumor biology. We find one such super-enhancer activated in epithelial cancer cells due to surrounding inflammation in the tumor microenvironment. CRISPR-dcas9-KRAB interference of this super-enhancer identifies PDZK1IP1 as its target gene. PDZK1IP1 is previously observed to be highly up-regulated in CRC. However, the mechanism behind its transcriptional activation is not fully understood. We restore both the super-enhancer and PDZK1IP1 levels following treatment with cytokines or xenotransplantation into nude mice, thus demonstrating its etiology via local tumor microenvironment acquisition. Deletion of inflammatory transcription factors RELA and STAT3 in human CRC cells inhibits PDZK1IP1 induction in xenografts. PDZK1IP1 appears to be critical for CRC growth in the setting of its super-enhancer induction as xenografts, but not in cell culture where the super-enhancer is absent and expression is largely silent. Building on its known role in glucose uptake via SGLT receptors, we demonstrate mechanistically that PDZK1IP1 enhances the reductive capacity CRC cancer cells via the pentose phosphate pathway using polar metabolomic profiling. We show this activation enables efficient growth under oxidative conditions both in vitro and in vivo, challenging the previous notion that PDZK1IP1 acts as a tumor suppressor in CRC. Collectively, these observations highlight the biologic significance of epigenomic profiling on patient-matched primary specimens and identify this microenvironment-acquired super-enhancer as an oncogenic driver in the setting of the inflamed tumor. Citation Format: Royce Zhou, Jia Xu, Tiphaine Martin, Alexis Zachem, John He, Sait Ozturk, Deniz Demircioglu, Ankita Bansal, Andrew Trotta, Bruno Giotti, Berkley Gryder, Yao Shen, Saul Carcamo, Xuewei Wu, Kaitlyn Bosch, Benjamin Hopkins, Alexander Tsankov, Randolph Steinhagen, Drew Jones, John Asara, Jerry Chipuk, Rachel Brody, Steven Itzkowitz, Iok In Christine Chio, Dan Hasson, Emily Bernstein, Ramon Parsons. A local tumor microenvironment acquired super-enhancer induces an oncogenic driver in colorectal carcinoma [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 3481.

The histone variant macroH2A has been implicated as a tumor suppressor in melanoma and other cancers, yet its role in the tumor microenvironment remains unappreciated. Using an autochthonous, immunocompetent mouse model of melanoma, we demonstrate that mice devoid of macroH2A exhibit increased tumor burden compared to wild type counterparts. MacroH2A-deficient tumors display an accumulation of immunosuppressive monocytes and decreased functional cytotoxic T cells, and consistent with this compromised anti-tumor response, exhibit upregulation of pro-inflammatory cytokines including Ccl2, Cxcl1 and Il6. Through single cell transcriptomics of the entire melanoma microenvironment, we identify the source of these pro-tumor myeloid chemoattractants as cancer-associated fibroblasts (CAFs), whose frequency and activation increase in the absence of macroH2A. Furthermore, cytokine genes in CAFs lacking macroH2A are hyper-inducible and their regulatory elements present an altered epigenetic landscape. In sum, we reveal a tumor suppressive role for macroH2A variants through repression of inflammatory gene induction in the tumor stroma.

PRDM (PRDI-BF1 and RIZ homology domain containing) family members are sequence-specific transcriptional regulators involved in cell identity and fate determination, often dysregulated in cancer. The PRDM15 gene is of particular interest, given its low expression in adult tissues and its overexpression in B-cell lymphomas. Despite its well characterized role in stem cell biology and during early development, the role of PRDM15 in cancer remains obscure. Herein, we demonstrate that while PRDM15 is largely dispensable for mouse adult somatic cell homeostasis in vivo, it plays a critical role in B-cell lymphomagenesis. Mechanistically, PRDM15 regulates a transcriptional program that sustains the activity of the PI3K/AKT/mTOR pathway and glycolysis in B-cell lymphomas. Abrogation of PRDM15 induces a metabolic crisis and selective death of lymphoma cells. Collectively, our data demonstrate that PRDM15 fuels the metabolic requirement of B-cell lymphomas and validate it as an attractive and previously unrecognized target in oncology., The transcriptional regulator PRDM15 is expressed at low levels in normal tissues but overexpressed in B-cell lymphomas. Here, the authors show that PRDM15 depletion does not affect adult somatic cell homeostasis but leads to a metabolic crisis which impairs B-cell lymphomagenesis.

Subunits of SWI/SNF chromatin remodeling complexes are frequently mutated in human malignancies. The PBAF complex is composed of multiple subunits, including the tumor-suppressor protein PBRM1 (BAF180), as well as ARID2 (BAF200), that are unique to this SWI/SNF complex. PBRM1 is mutated in various cancers, with a high mutation frequency in clear cell renal cell carcinoma (ccRCC). Here, we integrate RNA-seq, histone modification ChIP-seq, and ATAC-seq data to show that loss of PBRM1 results in de novo gains in H3K4me3 peaks throughout the epigenome, including activation of a retinoic acid biosynthesis and signaling gene signature. We show that one such target gene, ALDH1A1, which regulates a key step in retinoic acid biosynthesis, is consistently upregulated with PBRM1 loss in ccRCC cell lines and primary tumors, as well as non-malignant cells. We further find that ALDH1A1 increases the tumorigenic potential of ccRCC cells. Using biochemical methods, we show that ARID2 remains bound to other PBAF subunits after loss of PBRM1 and is essential for increased ALDH1A1 after loss of PBRM1, whereas other core SWI/SNF components are dispensable, including the ATPase subunit BRG1. In total, this study uses global epigenomic approaches to uncover novel mechanisms of PBRM1 tumor suppression in ccRCC. Implications: This study implicates the SWI/SNF subunit and tumor-suppressor PBRM1 in the regulation of promoter histone modifications and retinoic acid biosynthesis and signaling pathways in ccRCC and functionally validates one such target gene, the aldehyde dehydrogenase ALDH1A1.

MacroH2A variants have been associated with tumor suppression through inhibition of proliferation and metastasis, as well as their role in cellular senescence. However, their role in regulating the dormant state of disseminated cancer cells (DCCs) remains unclear. Here we reveal that solitary dormant DCCs display increased levels of macroH2A variants in head and neck squamous cell carcinoma PDX models and patient samples compared to proliferating primary or metastatic lesions. We further demonstrate that microenvironmental and stress adaptive signals such as TGFβ2 and p38α/β, which induce DCC dormancy, upregulate macroH2A expression. Functionally, we find that overexpression of macroH2A variants is sufficient to induce tumor cells into dormancy and notably, inducible expression of the macroH2A2 variant suppresses the growth of DCCs into overt metastasis. However, this dormant state does not require well-characterized dormancy factors such as DEC2 and NR2F1, suggesting alternate pathways. Our transcriptomic analyses reveal that macroH2A2 overexpression inhibits E2F, RAS and MYC signaling programs, while upregulating inflammatory cytokines commonly secreted by senescent cells. Taken together, our results demonstrate that macroH2A2 enforces a stable dormant phenotype in DCCs by activating a select subset of dormancy and senescence genes that limit metastasis initiation.

Resistance to regeneration of insulin-producing pancreatic β cells is a fundamental challenge for type 1 and type 2 diabetes. Recently, small molecule inhibitors of the kinase DYRK1A have proven effective in inducing adult human β cells to proliferate, but their detailed mechanism of action is incompletely understood. We interrogated our human insulinoma and β cell transcriptomic databases seeking to understand why β cells in insulinomas proliferate, while normal β cells do not. This search reveals the DREAM complex as a central regulator of quiescence in human β cells. The DREAM complex consists of a module of transcriptionally repressive proteins that assemble in response to DYRK1A kinase activity, thereby inducing and maintaining cellular quiescence. In the absence of DYRK1A, DREAM subunits reassemble into the pro-proliferative MMB complex. Here, we demonstrate that small molecule DYRK1A inhibitors induce human β cells to replicate by converting the repressive DREAM complex to its pro-proliferative MMB conformation.

Tumors exhibit widespread enhancer landscape reprogramming compared to normal tissue. The etiology is believed to be largely cell-intrinsic in non-hormonal cancers, attributed to such genomic alterations as focal amplification of non-coding regions, aberrant activation of transcription factors, and non-coding mutations creating de novo transcription factor binding sites. Here, using freshly resected primary CRC tumors and patient-matched adjacent normal colon epithelia, we find divergent epigenetic landscapes between primary CRC tumors and CRC cell lines. We identify a unique super-enhancer signature largely absent in cell culture. Intriguingly, this phenomenon extends to highly recurrent aberrant super-enhancers gained in CRC over patient-matched normal epithelium suggesting novel insight into the etiology of enhancer reprogramming in CRC and its downstream relevance to tumor biology. We find one such super-enhancer activated in epithelial cancer cells due to surrounding inflammation in the tumor microenvironment. We restore this super-enhancer and its expressed gene, PDZK1IP1, following treatment with cytokines or xenotransplantation into nude mice, thus demonstrating its etiology via local tumor microenvironment acquisition. Building on its known role in glucose uptake via SGLT receptors, we demonstrate mechanistically that PDZK1IP1 enhances the reductive capacity CRC cancer cells via the pentose phosphate pathway using polar metabolomic profiling. We show this activation enables efficient growth under oxidative conditions both in vitro and in vivo, challenging the previous notion that PDZK1IP1 acts as a tumor suppressor in CRC. Collectively, these observations highlight the biologic significance of epigenomic profiling on patient-matched primary specimens and identify this microenvironment-acquired super-enhancer as an oncogenic driver in the setting of the inflamed tumor.

Disseminated cancer cells (DCCs) identified in secondary organs, sometimes before the primary tumor becomes detectable and treated, can remain dormant for years to decades before manifesting. Microenvironmental and epigenetic mechanisms may control the onset and escape from dormancy, and here we reveal how a combination of the DNA methylation inhibitor 5-azacytidine (AZA) and retinoic acid receptor ligands all-trans retinoic acid (atRA), orchestrate a novel program of stable dormancy. Treatment of HNSCC tumor cells with AZA+atRA induced a SMAD2/3/4 dependent regulation of downstream transcriptional program that restored the anti-proliferative function of TGFβ signaling. Significantly, AZA+atRA or AZA+AM80, an RARα specific agonist, strongly suppresses lung metastasis formation. The metastatic suppression occurs via the induction and maintenance of phenotypically homogenous dormant SMAD4+/NR2F1+ non-proliferative DCCs. These findings suggest that strategies that maintain or induce dormancy programs may be a viable alternative strategy to improve patient outcomes by preventing or significantly delaying metastasis development.

ARID2 is the most recurrently mutated SWI/SNF complex member in melanoma; however, its tumor-suppressive mechanisms in the context of the chromatin landscape remain to be elucidated. Here, we model ARID2 deficiency in melanoma cells, which results in defective PBAF complex assembly with a concomitant genomic redistribution of the BAF complex. Upon ARID2 depletion, a subset of PBAF and shared BAF-PBAF-occupied regions displays diminished chromatin accessibility and associated gene expression, while BAF-occupied enhancers gain chromatin accessibility and expression of genes linked to the process of invasion. As a function of altered accessibility, the genomic occupancy of melanoma-relevant transcription factors is affected and significantly correlates with the observed transcriptional changes. We further demonstrate that ARID2-deficient cells acquire the ability to colonize distal organs in multiple animal models. Taken together, our results reveal a role for ARID2 in mediating BAF and PBAF subcomplex chromatin dynamics with consequences for melanoma metastasis.

Regulation of RNA polymerase II (RNAPII)-mediated transcription controls cellular phenotypes such as cancer. Phosphatase and tensin homologue deleted on chromosome ten (PTEN), one of the most commonly altered tumor suppressors in cancer, affects transcription via its role in antagonizing the PI3K/AKT signaling pathway. Using co-immunoprecipitations and proximal ligation assays we provide evidence that PTEN interacts with AFF4, RNAPII, CDK9, cyclin T1, XPB and CDK7. Using ChIP-seq, we show that PTEN co-localizes with RNAPII and binds to chromatin in promoter and putative enhancer regions identified by histone modifications. Furthermore, we show that loss of PTEN affects RNAPII occupancy in gene bodies and further correlates with gene expression changes. Interestingly, PTEN binds to promoters and negatively regulates the expression of genes involved in transcription including AFF4 and POL2RA, which encodes a subunit of RNAPII. Loss of PTEN also increased cells’ sensitivity to transcription inhibition via small molecules, which could provide a strategy to target PTEN-deficient cancers. Overall, our work describes a previously unappreciated role of nuclear PTEN, which by interacting with the transcription machinery in the context of chromatin exerts an additional layer of regulatory control on RNAPII-mediated transcription.

Considerable efforts have been made to characterize active enhancer elements, which can be annotated by accessible chromatin and H3 lysine 27 acetylation (H3K27ac). However, apart from poised enhancers that are observed in early stages of development and putative silencers, the functional significance of cis-regulatory elements lacking H3K27ac is poorly understood. Here we show that macroH2A histone variants mark a subset of enhancers in normal and cancer cells, which we coined ‘macroH2A-Bound Enhancers’, that negatively modulate enhancer activity. We find macroH2A variants enriched at enhancer elements that are devoid of H3K27ac in a cell type-specific manner, indicating a role for macroH2A at inactive enhancers to maintain cell identity. In following, reactivation of macro-bound enhancers is associated with oncogenic programs in breast cancer and its repressive role is correlated with the activity of macroH2A2 as a negative regulator of BRD4 chromatin occupancy. Finally, through single cell epigenomic profiling, we show that the loss of macroH2A2 leads to increased cellular heterogeneity that may help to explain the role of macroH2A variants in defining oncogenic transcriptional dependencies.

PurposeThe purpose of this paper is to investigate the possible associations between health-relevant personality traits and adherence; and if these traits predict adherence to a web-based occupational health intervention.Design/methodology/approachIn total, 563 participants were analyzed using the Health-relevant Personality Inventory. Adherence measures were: logins, utilization of self-help exercises and time spent logged in.FindingsHigher levels of antagonism (a facet of agreeableness) and impulsivity (a facet of conscientiousness) correlated to fewer logins, and higher levels of negative affectivity (a facet of neuroticism) and impulsivity correlated to a higher utilization of self-help exercises. Alexithymia (a facet of openness) negatively predicted self-help exercise utilization and antagonism was a positive predictor. Negative affectivity was a positive predictor of time spent logged in to the intervention. There were sex-related differences in outcomes.Originality/valueThis is the first study to investigate health-relevant personality traits in relation to adherence to a web-based occupational health intervention. The practical implications are that intervention developers could benefit from taking personality into consideration to better understand and improve adherence.

Line managers may play a central role in the success of occupational health interventions. However, few studies have focussed on the relationship between line managers' behaviours and the outcomes ...

Hyperacusis, over-sensitivity to sounds, causes distress and disability and the etiology is not fully understood. The study aims to explore possible associations between health-relevant personality traits and hyperacusis. Hyperacusis was assessed using the Hyperacusis Questionnaire (HQ), and clinical uncomfortable loudness levels (ULL). Personality was measured with the Health-relevant Personality (HP5i) Inventory. The study sample was 348 (140 men and 208 women; age 23-71 years). Moderate correlations were found between the personality trait negative affectivity (NA; a facet of neuroticism) and dimensions of the HQ and weak correlations were found with the ULLs. Hedonic capacity (a facet of extraversion) was significantly correlated with the HQ but not with the ULLs. Impulsivity (a facet of conscientiousness) was correlated with the HQ and the ULLs. A significant difference in mean values was found in all hyperacusis measures and different levels of NA - those with higher levels displayed more severe signs of hyperacusis. A multiple logistic regression analysis showed that higher levels of NA increases the odds of having hyperacusis on average 4.6 times for men and 2.4 times for women. These findings imply that health-relevant personality traits should be considered in the diagnosis and treatment of hyperacusis.

Summary Two major functions of the epigenome are to regulate gene expression and to suppress transposons. It is unclear how these functions are balanced during physiological challenges requiring tissue regeneration, where exquisite coordination of gene expression is essential. Transcriptomic analysis of seven time points following partial hepatectomy identified the epigenetic regulator UHRF1, which is essential for DNA methylation, as dynamically expressed during liver regeneration in mice. UHRF1 deletion in hepatocytes (Uhrf1HepKO) caused genome-wide DNA hypomethylation but, surprisingly, had no measurable effect on gene or transposon expression or liver homeostasis. Partial hepatectomy of Uhrf1HepKO livers resulted in early and sustained activation of proregenerative genes and enhanced liver regeneration. This was attributed to redistribution of H3K27me3 from promoters to transposons, effectively silencing them and, consequently, alleviating repression of liver regeneration genes, priming them for expression in Uhrf1HepKO livers. Thus, epigenetic compensation safeguards the genome against transposon activation, indirectly affecting gene regulation.

ATRX alterations occur at high frequency in neuroblastoma of adolescents and young adults. Particularly intriguing are the large N-terminal deletions of ATRX (Alpha Thalassemia/Mental Retardation, X-linked) that generate in-frame fusion (IFF) proteins devoid of key chromatin interaction domains, while retaining the SWI/SNF-like helicase region. We demonstrate that ATRX IFF proteins are redistributed from H3K9me3-enriched chromatin to promoters of active genes and identify REST as an ATRX IFF target whose activation promotes silencing of neuronal differentiation genes. We further show that ATRX IFF cells display sensitivity to EZH2 inhibitors, due to derepression of neurogenesis genes, including a subset of REST targets. Taken together, we demonstrate that ATRX structural alterations are not loss-of-function and put forward EZH2 inhibitors as a potential therapy for ATRX IFF neuroblastoma.

Chromatin immunoprecipitation (ChIP) is becoming the standard method to study genome-wide distribution of histone variants and histone posttranslational modifications (PTMs). In this chapter, we describe a detailed native ChIP protocol and downstream procedures for the preparation of DNA libraries for next-generation sequencing. Compared to cross-linked ChIP, "native" ChIP has been shown to produce occupancy pattern data of histone PTMs and histone variants, with higher resolution and higher signal to noise ratio. We further present an adaptation of this protocol to perform native ChIP from as low as 50,000 cells.

Chromatin immunoprecipitation (ChIP) is becoming the standard method to study genome-wide distribution of histone variants and histone posttranslational modifications (PTMs). In this chapter, we describe a detailed native ChIP protocol and downstream procedures for the preparation of DNA libraries for next-generation sequencing. Compared to cross-linked ChIP, "native" ChIP has been shown to produce occupancy pattern data of histone PTMs and histone variants, with higher resolution and higher signal to noise ratio. We further present an adaptation of this protocol to perform native ChIP from as low as 50,000 cells.

The process of cellular senescence generates a repressive chromatin environment, however, the role of histone tail modifications, histone variants and histone proteolytic cleavage in senescence remains unclear. Using well-characterized senescence models in human primary lung fibroblasts and melanocytes, we are investigating how chromatin alterations contribute to the senescence phenotype. Here we report novel histone H3 tail cleavage events in oncogene-induced and replicative senescence models mediated by the protease Cathepsin L. We find that cleaved forms of H3 are nucleosomal and that H3.3 is the preferred cleaved form of H3 - an H3 histone variant that is deposited independent of DNA replication. Ectopic expression of H3.3 and its cleavage product (H3.3cs1), which lacks the first twenty-one amino acids of the H3 tail, is sufficient to induce senescence. Furthermore, we find that chromatin incorporation of H3.3cs1 is mediated by the HUCA (HIRA/UBN1/CABIN1/ASF1a) histone chaperone complex. Genome-wide transcriptional profiling revealed that H3.3cs1 facilitates transcriptional silencing of cell cycle regulators including RB/E2F target genes, likely via the permanent removal of H3K4me3. Collectively, our studies have identified histone H3.3 and its proteolytically processed forms as key regulators of cellular senescence. Citation Format: Luis F. Duarte, Andrew R. J. Young, Zichen Wang, Hsan-Au Wu, Taniya Panda, Yan Kou, Avnish Kapoor, Dan Hasson, Nicholas R. Mills, Avi Ma'ayan, Masashi Narita, Emily Bernstein. Histone tail alterations in cellular senescence. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr SY10-04. doi:10.1158/1538-7445.AM2015-SY10-04

Bromodomain and extraterminal domain inhibitors (BETi) represent promising therapeutic agents for metastatic melanoma, yet their mechanism of action remains unclear. Here we interrogated the transcriptional effects of BETi and identified AMIGO2, a trans-membrane molecule, as a BET target gene essential for melanoma cell survival. AMIGO2 is upregulated in melanoma cells and tissues compared to human melanocytes and nevi, and AMIGO2 silencing in melanoma cells induces G1/S arrest followed by apoptosis. We identified the pseudokinase PTK7 as an AMIGO2 interactor whose function is regulated by AMIGO2. Epigenomic profiling and genome editing revealed that AMIGO2 is regulated by a melanoma-specific BRD2/4-bound promoter and super-enhancer configuration. Upon BETi treatment, BETs are evicted from these regulatory elements, resulting in AMIGO2 silencing and changes in PTK7 proteolytic processing. Collectively, this study uncovers mechanisms underlying the therapeutic effects of BETi in melanoma and reveals the AMIGO2-PTK7 axis as a targetable pathway for metastatic melanoma.

Little is known about personality in relation to assessments of the psychosocial work environment and leadership. Therefore the objective of this study is to explore possible associations and differences in mean values between employee health-relevant personality traits and assessments of the psychosocial work environment and leadership behaviors.754 survey responses from ten organizations were selected from a large-scale intervention study. The Health-relevant Personality 5 inventory was used to assess personality. Five dimensions of the psychosocial work environment were assessed with 38 items from the QPSPositive correlations were found between Hedonic capacity (facet of Extraversion) and perceptions of the psychosocial work environment and leadership behavior. Negative correlations were found for Negative affectivity (facet of Neuroticism), Antagonism (facet of Agreeableness), Impulsivity (facet of Conscientiousness) and Alexithymia (facet of Openness). There were also significant differences in mean values of all work environment indicators between levels of health-relevant personality traits. Those with higher levels of hedonic capacity had higher (better) perceptions compared to those with lower levels. Those with higher levels of negative affectivity had lower (worse) perceptions compared to those with lower levels.The findings show a clear association between employee health-relevant personality traits and assessments of the psychosocial work environment and leadership behavior. Personality can be important to take into consideration for leaders when interpreting survey results and when designing organizational interventions.

Mutations and structural alterations of the SWI/SNF-like chromatin remodeler ATRX (Alpha Thalassemia/Mental Retardation, X-linked) have been reported at high frequency in a number of adult and pediatric tumors. However, the consequences of ATRX mutations in cancer and their impact on the epigenome remain ill defined. Particularly intriguing are the large N-terminal deletions of ATRX in neuroblastoma that generate in-frame fusion (IFF) proteins devoid of key chromatin interaction domains, while retaining the SWI/SNF-like helicase domain. We hereby demonstrate that ATRX IFF proteins have distinct genomic distribution compared to wild type (WT) ATRX and are absent from H3K9me3-enriched chromatin, yet bound to active promoters. We find REST (RE-1 Silencing Transcription Factor) as one such ATRX IFF target that is activated and promotes silencing of neuronal differentiation genes. Notably, we uncover that ATRX IFF cells display exquisite sensitivity to EZH2 inhibitors (EZH2i) in vitro and in vivo, due in part to derepression of neurogenesis genes, including a subset of REST targets. Examination of the epigenomic landscape of neuroblastoma tumor specimens harboring ATRX IFFs reveals that H3K27me3 occupies a subset of neurogenesis genes that are transcriptionally silenced and sensitive to EZH2i in our cell-based assays. In summary, this study demonstrates that ATRX structural alterations are not loss-of-function as predicted, and supports EZH2i as a key therapeutic strategy for ATRX IFF neuroblastoma.

The centromere is the chromosomal locus that ensures fidelity in genome transmission at cell division. Centromere protein A (CENP-A) is a histone H3 variant that specifies centromere location independently of DNA sequence. Conflicting evidence has emerged regarding the histone composition and stoichiometry of CENP-A nucleosomes. Here we show that the predominant form of the CENP-A particle at human centromeres is an octameric nucleosome. CENP-A nucleosomes are very highly phased on α-satellite 171-base-pair monomers at normal centromeres and also display strong positioning at neocentromeres. At either type of functional centromere, CENP-A nucleosomes exhibit similar DNA-wrapping behavior, as do octameric CENP-A nucleosomes reconstituted with recombinant components, having looser DNA termini than those on conventional nucleosomes containing canonical histone H3. Thus, the fundamental unit of the chromatin that epigenetically specifies centromere location in mammals is an octameric nucleosome with loose termini.