Smc3 dosage regulates B cell transit through germinal centers and restricts their malignant transformation.

During the germinal center (GC) reaction, B cells undergo extensive redistribution of cohesin complex and three-dimensional reorganization of their genomes. Yet, the significance of cohesin and architectural programming in the humoral immune response is unknown. Herein we report that homozygous deletion of Smc3, encoding the cohesin ATPase subunit, abrogated GC formation, while, in marked contrast, Smc3 haploinsufficiency resulted in GC hyperplasia, skewing of GC polarity and impaired plasma cell (PC) differentiation. Genome-wide chromosomal conformation and transcriptional profiling revealed defects in GC B cell terminal differentiation programs controlled by the lymphoma epigenetic tumor suppressors Tet2 and Kmt2d and failure of Smc3-haploinsufficient GC B cells to switch from B cell- to PC-defining transcription factors. Smc3 haploinsufficiency preferentially impaired the connectivity of enhancer elements controlling various lymphoma tumor suppressor genes, and, accordingly, Smc3 haploinsufficiency accelerated lymphomagenesis in mice with constitutive Bcl6 expression. Collectively, our data indicate a dose-dependent function for cohesin in humoral immunity to facilitate the B cell to PC phenotypic switch while restricting malignant transformation.

Till SMARCA5 loss do nucleosomes part.

In this issue of Molecular Cell, Bomber et al. demonstrate that acute loss of SMARCA5 in human cells leads to eviction of CTCF and an increase in nucleosome repeat length without direct impact on transcriptional activity.

Subtype-specific 3D genome alteration in acute myeloid leukaemia.

Acute myeloid leukaemia (AML) represents a set of heterogeneous myeloid malignancies, and hallmarks include mutations in epigenetic modifiers, transcription factors and kinases1-5. The extent to which mutations in AML drive alterations in chromatin 3D structure and contribute to myeloid transformation is unclear. Here we use Hi-C and whole-genome sequencing to analyse 25 samples from patients with AML and 7 samples from healthy donors. Recurrent and subtype-specific alterations in A/B compartments, topologically associating domains and chromatin loops were identified. RNA sequencing, ATAC with sequencing and CUT&Tag for CTCF, H3K27ac and H3K27me3 in the same AML samples also revealed extensive and recurrent AML-specific promoter-enhancer and promoter-silencer loops. We validated the role of repressive loops on their target genes by CRISPR deletion and interference. Structural variation-induced enhancer-hijacking and silencer-hijacking events were further identified in AML samples. Hijacked enhancers play a part in AML cell growth, as demonstrated by CRISPR screening, whereas hijacked silencers have a downregulating role, as evidenced by CRISPR-interference-mediated de-repression. Finally, whole-genome bisulfite sequencing of 20 AML and normal samples revealed the delicate relationship between DNA methylation, CTCF binding and 3D genome structure. Treatment of AML cells with a DNA hypomethylating agent and triple knockdown of DNMT1, DNMT3A and DNMT3B enabled the manipulation of DNA methylation to revert 3D genome organization and gene expression. Overall, this study provides a resource for leukaemia studies and highlights the role of repressive loops and hijacked cis elements in human diseases.

Single-cell mutation analysis of clonal evolution in myeloid malignancies.

Myeloid malignancies, including acute myeloid leukaemia (AML), arise from the expansion of haematopoietic stem and progenitor cells that acquire somatic mutations. Bulk molecular profiling has suggested that mutations are acquired in a stepwise fashion: mutant genes with high variant allele frequencies appear early in leukaemogenesis, and mutations with lower variant allele frequencies are thought to be acquired later1-3. Although bulk sequencing can provide information about leukaemia biology and prognosis, it cannot distinguish which mutations occur in the same clone(s), accurately measure clonal complexity, or definitively elucidate the order of mutations. To delineate the clonal framework of myeloid malignancies, we performed single-cell mutational profiling on 146 samples from 123 patients. Here we show that AML is dominated by a small number of clones, which frequently harbour co-occurring mutations in epigenetic regulators. Conversely, mutations in signalling genes often occur more than once in distinct subclones, consistent with increasing clonal diversity. We mapped clonal trajectories for each sample and uncovered combinations of mutations that synergized to promote clonal expansion and dominance. Finally, we combined protein expression with mutational analysis to map somatic genotype and clonal architecture with immunophenotype. Our findings provide insights into the pathogenesis of myeloid transformation and how clonal complexity evolves with disease progression.

A Multiplexed System for Quantitative Comparisons of Chromatin Landscapes.

Genome-wide profiling of histone modifications can provide systematic insight into the regulatory elements and programs engaged in a given cell type. However, conventional chromatin immunoprecipitation and sequencing (ChIP-seq) does not capture quantitative information on histone modification levels, requires large amounts of starting material, and involves tedious processing of each individual sample. Here, we address these limitations with a technology that leverages DNA barcoding to profile chromatin quantitatively and in multiplexed format. We concurrently map relative levels of multiple histone modifications across multiple samples, each comprising as few as a thousand cells. We demonstrate the technology by monitoring dynamic changes following inhibition of p300, EZH2, or KDM5, by linking altered epigenetic landscapes to chromatin regulator mutations, and by mapping active and repressive marks in purified human hematopoietic stem cells. Hence, this technology enables quantitative studies of chromatin state dynamics across rare cell types, genotypes, environmental conditions, and drug treatments.

Aire regulates chromatin looping by evicting CTCF from domain boundaries and favoring accumulation of cohesin on superenhancers.

Aire controls immunological tolerance by driving promiscuous expression of a large swath of the genome in medullary thymic epithelial cells (mTECs). Its molecular mechanism remains enigmatic. High-resolution chromosome-conformation capture (Hi-C) experiments on ex vivo mTECs revealed Aire to have a widespread impact on higher-order chromatin structure, disfavoring architectural loops while favoring transcriptional loops. In the presence of Aire, cohesin complexes concentrated on superenhancers together with mediator complexes, while the CCCTC-binding factor (CTCF) was relatively depleted from structural domain boundaries. In particular, Aire associated with the cohesin loader, NIPBL, strengthening this factor's affiliation with cohesin's enzymatic subunits. mTEC transcripts up-regulated in the presence of Aire corresponded closely to those down-regulated in the absence of one of the cohesin subunits, SA-2. A mechanistic model incorporating these findings explains many of the unusual features of Aire's impact on mTEC transcription, providing molecular insight into tolerance induction.

Molecular predictors of immunophenotypic measurable residual disease clearance in acute myeloid leukemia.

Measurable residual disease (MRD) is a powerful prognostic factor in acute myeloid leukemia (AML). However, pre-treatment molecular predictors of immunophenotypic MRD clearance remain unclear. We analyzed a dataset of 211 patients with pre-treatment next-generation sequencing who received induction chemotherapy and had MRD assessed by serial immunophenotypic monitoring after induction, subsequent therapy, and allogeneic stem cell transplant (allo-SCT). Induction chemotherapy led to MRD- remission, MRD+ remission, and persistent disease in 35%, 27%, and 38% of patients, respectively. With subsequent therapy, 34% of patients with MRD+ and 26% of patients with persistent disease converted to MRD-. Mutations in CEBPA, NRAS, KRAS, and NPM1 predicted high rates of MRD- remission, while mutations in TP53, SF3B1, ASXL1, and RUNX1 and karyotypic abnormalities including inv (3), monosomy 5 or 7 predicted low rates of MRD- remission. Patients with fewer individual clones were more likely to achieve MRD- remission. Among 132 patients who underwent allo-SCT, outcomes were favorable whether patients achieved early MRD- after induction or later MRD- after subsequent therapy prior to allo-SCT. As MRD conversion with chemotherapy prior to allo-SCT is rarely achieved in patients with specific baseline mutational patterns and high clone numbers, upfront inclusion of these patients into clinical trials should be considered.

Driver mutations in acute myeloid leukemia.

PURPOSE OF REVIEW: The mutational landscape of acute myeloid leukemia (AML) has revised diagnostic, prognostic, and therapeutic schemata over the past decade. Recurrently mutated AML genes have functional consequences beyond typical oncogene-driven growth and loss of tumor suppresser function. RECENT FINDINGS: Large-scale genomic sequencing efforts have mapped the complexity of AML and trials of mutation-based targeted therapy has led to several FDA-approved drugs for mutant-specific AML. However, many recurrent mutations have been identified across a spectrum from clonal hematopoiesis to myelodysplasia to overt AML, such as effectors of DNA methylation, chromatin modifiers, and spliceosomal machinery. The functional effects of these mutations are the basis for substantial discovery. SUMMARY: Understanding the molecular and pathophysiologic functions of key genes that exert leukemogenic potential is essential towards translating these findings into better treatment for AML.

Loss of plasmacytoid dendritic cell differentiation is highly predictive for post-induction measurable residual disease and inferior outcomes in acute myeloid leukemia.

Measurable residual disease is associated with inferior outcomes in patients with acute myeloid leukemia (AML). Measurable residual disease monitoring enhances risk stratification and may guide therapeutic intervention. The European LeukemiaNet working party recently came to a consensus recommendation incorporating leukemia associated immunophenotype-based different from normal approach by multi-color flow cytometry for measurable residual disease evaluation. However, the analytical approach is highly expertise-dependent and difficult to standardize. Here we demonstrate that loss of plasmacytoid dendritic cell differentiation after 7+3 induction in AML is highly specific for measurable residual disease positivity (specificity 97.4%) in a uniformly treated patient cohort. Moreover, loss of plasmacytoid dendritic cell differentiation as determined by a blast-to-plasmacytoid dendritic cell ratio >10 was strongly associated with inferior overall and relapse-free survival (RFS) [Hazard ratio 2.79, 95% confidence interval (95%CI): 0.98-7.97; P=0.077) and 3.83 (95%CI: 1.51-9.74; P=0.007), respectively), which is similar in magnitude to measurable residual disease positivity. Importantly, measurable residual disease positive patients who reconstituted plasmacytoid dendritic cell differentiation (blast/ plasmacytoid dendritic cell ratio <10) showed a higher rate of measurable residual disease clearance at later pre-transplant time points compared to patients with loss of plasmacytoid dendritic cell differentiation (blast/ plasmacytoid dendritic cell ratio <10) (6 of 12, 50% vs 2 of 18, 11%; P=0.03). Furthermore pre-transplant plasmacytoid dendritic cell recovery was associated with superior outcome in measurable residual disease positive patients. Our study provides a novel, simple, broadly applicable, and quantitative multi-color flow cytometry approach to risk stratification in AML.

Stability and uniqueness of clonal immunoglobulin CDR3 sequences for MRD tracking in multiple myeloma.

Minimal residual disease (MRD) tracking, by next generation sequencing of immunoglobulin sequences, is moving towards clinical implementation in multiple myeloma. However, there is only sparse information available to address whether clonal sequences remain stable for tracking over time, and to what extent light chain sequences are sufficiently unique for tracking. Here, we analyzed immunoglobulin repertoires from 905 plasma cell myeloma and healthy control samples, focusing on the third complementarity determining region (CDR3). Clonal heavy and/or light chain expression was identified in all patients at baseline, with one or more subclones related to the main clone in 3.2%. In 45 patients with 101 sequential samples, the dominant clonal CDR3 sequences remained identical over time, despite differential clonal evolution by whole exome sequencing in 49% of patients. The low frequency of subclonal CDR3 variants, and absence of evolution over time in active multiple myeloma, indicates that tumor cells at this stage are not under selective pressure to undergo antibody affinity maturation. Next, we establish somatic hypermutation and non-templated insertions as the most important determinants of light chain clonal uniqueness, identifying a potentially trackable sequence in the majority of patients. Taken together, we show that dominant clonal sequences identified at baseline are reliable biomarkers for long-term tracking of the malignant clone, including both IGH and the majority of light chain clones.

Cohesin mutations in myeloid malignancies made simple.

PURPOSE OF REVIEW: Recurrent loss of function mutations within genes of the cohesin complex have been identified in myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML). STAG2 is the most commonly mutated cohesin member in AML as well as solid tumors. STAG2 is recurrently, mutated in Ewing's Sarcoma, bladder cancer, and glioblastoma, and is one of only ten genes known to be recurrently mutated in over four distinct tissue types of human cancer RECENT FINDINGS: The cohesin complex, a multiprotein ring, is canonically known to align and stabilize replicated chromosomes prior to cell division. Although initially thought to lead to unequal chromosomal separation in dividing cells, data in myeloid malignancies show this is not observed in cohesin mutant MDS/AML, either in large patient cohorts or mouse models. Mounting evidence supports a potential alternate mechanism whereby drivers of cell-type specific gene expression and hematopoietic development are impaired through alteration in three-dimensional nuclear organization and gene structure. SUMMARY: Understanding the functional consequences of cohesin mutations in regulating lineage-specific and signal-dependent defects and in myeloid transformation will identify novel pathophysiologic mechanisms of disease and inform the development of novel therapeutic targets.

Racial disparities in the survival of American children, adolescents, and young adults with acute lymphoblastic leukemia, acute myelogenous leukemia, and Hodgkin lymphoma.

BACKGROUND: Race-based survival in children and adolescents with hematologic malignancies has been a national challenge for decades. Large-scale investigations of age- and race-based survival trends over time in these patients have not previously been reported. The objective of this study was to investigate whether race- and age-related differences in pediatric and adolescent and young adult (AYA) leukemia and lymphoma survival persist and to what extent these differences have changed over time. METHODS: Using the Surveillance, Epidemiology, and End Results program, this study investigated the outcomes of black and white (1975-2012; n = 27,369) and white and Hispanic (1992-2012; n = 20,574) children (0-14 years old) and AYAs (15-39 years old) with acute lymphoblastic leukemia (ALL), acute myelogenous leukemia (AML), and Hodgkin lymphoma (HL). Estimates of 5- and 10-year relative survival were compared over time. RESULTS: Trends showed a convergence of survival for white and black children with ALL but a divergence in survival for AYA patients. Hispanic children and AYAs both suffered inferior outcomes. Trends for AML revealed persistent survival differences between black and white children and suggested worsening disparities for AYAs. Survival trends in HL revealed sustained survival differences between black and white AYA patients, whereas no differences were found in Hispanic and white patient outcomes for AML or HL. CONCLUSIONS: Although survival for children and AYAs with ALL, AML, and HL has improved over the past 4 decades, differences persist between black, white, and Hispanic children and AYAs; survival disparities between black and white children with ALL have been nearly eliminated. Strategies aimed at identifying causality and reducing disparities are warranted. Cancer 2016. © 2016 American Cancer Society. Cancer 2016;122:2723-2730. © 2016 American Cancer Society.

Genetic alterations of the cohesin complex genes in myeloid malignancies.

Somatic cohesin mutations have been reported in myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML). To account for the morphologic and cytogenetic diversity of these neoplasms, a well-annotated cohort of 1060 patients with myeloid malignancies including MDS (n = 386), myeloproliferative neoplasms (MPNs) (n = 55), MDS/MPNs (n = 169), and AML (n = 450) were analyzed for cohesin gene mutational status, gene expression, and therapeutic and survival outcomes. Somatic cohesin defects were detected in 12% of patients with myeloid malignancies, whereas low expression of these genes was present in an additional 15% of patients. Mutations of cohesin genes were mutually exclusive and mostly resulted in predicted loss of function. Patients with low cohesin gene expression showed similar expression signatures as those with somatic cohesin mutations. Cross-sectional deep-sequencing analysis for clonal hierarchy demonstrated STAG2, SMC3, and RAD21 mutations to be ancestral in 18%, 18%, and 47% of cases, respectively, and each expanded to clonal dominance concordant with disease transformation. Cohesin mutations were significantly associated with RUNX1, Ras-family oncogenes, and BCOR and ASXL1 mutations and were most prevalent in high-risk MDS and secondary AML. Cohesin defects were associated with poor overall survival (27.2 vs 40 months; P = .023), especially in STAG2 mutant MDS patients surviving >12 months (median survival 35 vs 50 months; P = .017).

Germline predisposition for clonal hematopoiesis.

Clonal hematopoiesis (CH) is an entity hallmarked by skewed hematopoiesis with persistent overrepresentation of cells from a common stem/progenitor lineage harboring single-nucleotide variants and/or insertions/deletions. CH is a common and age-related phenomenon that is associated with an increased risk of hematological malignancies, cardiovascular disease, and all-cause mortality. While CH is a term of the hematological aspect, there exists a complex interaction with other organ systems, especially the cardiovascular system. The strongest factor in the development of CH is aging, however, other multiple factors also affect the development of CH including lifestyle-related factors and co-morbid diseases. In recent years, germline genetic factors have been linked to CH risk. In this review, we synthesize what is currently known about how genetic variation affects the risk of CH, how this genetic architecture intersects with myeloid neoplasms, and future prospects for CH.

Chromatin organization in myelodysplastic syndrome.

Disordered chromatin organization has emerged as a new aspect of the pathogenesis of myelodysplastic syndrome (MDS). Characterized by lineage dysplasia and a high transformation rate to acute myeloid leukemia (AML), the genetic determinant of MDS is thought to be the main driver of the disease's progression. Among the recurrently mutated pathways, alterations in chromatin organization, such as the cohesin complex, have a profound impact on hematopoietic stem cell (HSC) function and lineage commitment. The cohesin complex is a ring-like structure comprised of structural maintenance of chromosomes (SMC), RAD21, and STAG proteins that involve three-dimensional (3D) genome organization via loop extrusion in mammalian cells. The partial loss of the functional cohesin ring leads to altered chromatin accessibility specific to key hematopoietic transcription factors, which is thought to be the molecular mechanism of cohesin dysfunction. Currently, there are no specific targeting agents for cohesin mutant MDS/AML. Potential therapeutic strategies have been proposed based on the current understanding of cohesin mutant leukemogenesis. Here, we will review the recent advances in investigation and targeting approaches against cohesin mutant MDS/AML.

Untangling the loops of STAG2 mutations in myelodysplastic syndrome.

Myelodysplastic syndrome (MDS) is a heterogeneous myeloid neoplasm that is hallmarked by the acquisition of genetic events that disrupt normal trilineage hematopoiesis and results in bone marrow dysfunction. Somatic genes involving transcriptional regulation, signal transduction, DNA methylation, and chromatin modification are often implicated in disease pathogenesis. The cohesin complex, composed of SMC1, SMC3, RAD21, and either STAG1 or STAG2, has been identified as a recurrent mutational target with STAG2 mutations accounting for more than half of all cohesin mutations in myeloid malignancies. In the last decade, STAG2 cohesin biology has been of great interest given its role in transcriptional activation, association with poorer prognosis, and lack of mutation-specific therapies. This review discusses the clinical landscape of cohesin mutant myeloid malignancies, particularly STAG2 mutant MDS, including molecular features of STAG2 mutations, clinical implications of cohesin mutant neoplasms, and the current understanding of the pathophysiological function of STAG2 mutations in MDS.

Noncanonical Activity of Med4 as a Gatekeeper of Metastasis through Epigenetic Control of Integrin Signaling.

Breast cancer metastatic relapse after a latency period, known as metastatic dormancy. Through genetic screening in mice, we identified the mediator complex subunit 4 (Med4) as a novel tumor-cell intrinsic gatekeeper in metastatic reactivation. Med4 downregulation effectively awakened dormant breast cancer cells, prompting macroscopic metastatic outgrowth in the lungs. Med4 depletion results in profound changes in nuclear size and three-dimensional chromatin architecture from compacted to relaxed states in contrast to the canonical function of the Mediator complex. These changes rewire the expression of extracellular matrix proteins, integrins, and signaling components resulting in integrin-mediated mechano-transduction and activation of YAP and MRTF. The assembly of stress fibers pulls on the nuclear membrane and contributes to reinforcing the overall chromatin modifications by Med4 depletion. MED4 gene deletions were observed in patients with metastatic breast cancer, and reduced MED4 expression correlates with worse prognosis, highlighting its significance as a potential biomarker for recurrence.

Yin Yang 1 regulates cohesin complex protein SMC3 in mouse hematopoietic stem cells.

ABSTRACT: Yin Yang 1 (YY1) and structural maintenance of chromosomes 3 (SMC3) are 2 critical chromatin structural factors that mediate long-distance enhancer-promoter interactions and promote developmentally regulated changes in chromatin architecture in hematopoietic stem/progenitor cells (HSPCs). Although YY1 has critical functions in promoting hematopoietic stem cell (HSC) self-renewal and maintaining HSC quiescence, SMC3 is required for proper myeloid lineage differentiation. However, many questions remain unanswered regarding how YY1 and SMC3 interact with each other and affect hematopoiesis. We found that YY1 physically interacts with SMC3 and cooccupies with SMC3 at a large cohort of promoters genome wide, and YY1 deficiency deregulates the genetic network governing cell metabolism. YY1 occupies the Smc3 promoter and represses SMC3 expression in HSPCs. Although deletion of 1 Smc3 allele partially restores HSC numbers and quiescence in YY1 knockout mice, Yy1-/-Smc3+/- HSCs fail to reconstitute blood after bone marrow transplant. YY1 regulates HSC metabolic pathways and maintains proper intracellular reactive oxygen species levels in HSCs, and this regulation is independent of the YY1-SMC3 axis. Our results establish a distinct YY1-SMC3 axis and its impact on HSC quiescence and metabolism.