Regulation of RNA polymerase II activity is essential for terminal erythroid maturation.

The terminal maturation of human erythroblasts requires significant changes in gene expression in the context of dramatic nuclear condensation. Defects in this process are associated with inherited anemias and myelodysplastic syndromes. The progressively dense appearance of the condensing nucleus in maturing erythroblasts led to the assumption that heterochromatin accumulation underlies this process, but despite extensive study, the precise mechanisms underlying this essential biologic process remain elusive. To delineate the epigenetic changes associated with the terminal maturation of human erythroblasts, we performed mass spectrometry of histone posttranslational modifications combined with chromatin immunoprecipitation coupled with high-throughput sequencing, Assay for Transposase Accessible Chromatin, and RNA sequencing. Our studies revealed that the terminal maturation of human erythroblasts is associated with a dramatic decline in histone marks associated with active transcription elongation, without accumulation of heterochromatin. Chromatin structure and gene expression were instead correlated with dynamic changes in occupancy of elongation competent RNA polymerase II, suggesting that terminal erythroid maturation is controlled largely at the level of transcription. We further demonstrate that RNA polymerase II "pausing" is highly correlated with transcriptional repression, with elongation competent RNA polymerase II becoming a scare resource in late-stage erythroblasts, allocated to erythroid-specific genes. Functional studies confirmed an essential role for maturation stage-specific regulation of RNA polymerase II activity during erythroid maturation and demonstrate a critical role for HEXIM1 in the regulation of gene expression and RNA polymerase II activity in maturing erythroblasts. Taken together, our findings reveal important insights into the mechanisms that regulate terminal erythroid maturation and provide a novel paradigm for understanding normal and perturbed erythropoiesis.

A Unique Epigenomic Landscape Defines Human Erythropoiesis.

Mammalian erythropoiesis yields a highly specialized cell type, the mature erythrocyte, evolved to meet the organismal needs of increased oxygen-carrying capacity. To better understand the regulation of erythropoiesis, we performed genome-wide studies of chromatin accessibility, DNA methylation, and transcriptomics using a recently developed strategy to obtain highly purified populations of primary human erythroid cells. The integration of gene expression, DNA methylation, and chromatin state dynamics reveals that stage-specific gene regulation during erythropoiesis is a stepwise and hierarchical process involving many cis-regulatory elements. Erythroid-specific, nonpromoter sites of chromatin accessibility are linked to erythroid cell phenotypic variation and inherited disease. Comparative analyses of stage-specific chromatin accessibility indicate that there is limited early chromatin priming of erythroid genes during hematopoiesis. The epigenome of terminally differentiating erythroid cells defines a distinct subset of highly specialized cells that are vastly dissimilar from other hematopoietic and nonhematopoietic cell types. These epigenomic and transcriptome data are powerful tools to study human erythropoiesis.

Hemoglobin C trait accentuates erythrocyte dehydration in hereditary xerocytosis.

A 17-year-old male presented with acute hemolysis with stomatocytosis, elevated mean corpuscular hemoglobin concentration (MCHC), and osmotic gradient ektacytometry consistent with marked erythrocyte dehydration. Erythrocytes from both parents also demonstrated evidence of dehydration with elevated MCHC and abnormal ektacytometry, but neither to the degree of the patient. Genetic studies revealed the patient had hereditary xerocytosis (HX) due to a novel PIEZO1 mutation inherited from his mother and hemoglobin C (HbC) trait inherited from his father. HbC trait accentuated the erythrocyte dehydration of HX. Coinheritance of interrelated disorders and/or modifier alleles should be considered whenever severe erythrocyte dehydration is observed.

CTCF and CohesinSA-1 Mark Active Promoters and Boundaries of Repressive Chromatin Domains in Primary Human Erythroid Cells.

BACKGROUND: CTCF and cohesinSA-1 are regulatory proteins involved in a number of critical cellular processes including transcription, maintenance of chromatin domain architecture, and insulator function. To assess changes in the CTCF and cohesinSA-1 interactomes during erythropoiesis, chromatin immunoprecipitation coupled with high throughput sequencing and mRNA transcriptome analyses via RNA-seq were performed in primary human hematopoietic stem and progenitor cells (HSPC) and primary human erythroid cells from single donors. RESULTS: Sites of CTCF and cohesinSA-1 co-occupancy were enriched in gene promoters in HSPC and erythroid cells compared to single CTCF or cohesin sites. Cell type-specific CTCF sites in erythroid cells were linked to highly expressed genes, with the opposite pattern observed in HSPCs. Chromatin domains were identified by ChIP-seq with antibodies against trimethylated lysine 27 histone H3, a modification associated with repressive chromatin. Repressive chromatin domains increased in both number and size during hematopoiesis, with many more repressive domains in erythroid cells than HSPCs. CTCF and cohesinSA-1 marked the boundaries of these repressive chromatin domains in a cell-type specific manner. CONCLUSION: These genome wide data, changes in sites of protein occupancy, chromatin architecture, and related gene expression, support the hypothesis that CTCF and cohesinSA-1 have multiple roles in the regulation of gene expression during erythropoiesis including transcriptional regulation at gene promoters and maintenance of chromatin architecture. These data from primary human erythroid cells provide a resource for studies of normal and perturbed erythropoiesis.

Brief-exposure to preoperative bevacizumab reveals a TGF-ß signature predictive of response in HER2-negative breast cancers.

To best define biomarkers of response, and to shed insight on mechanism of action of certain clinically important agents for early breast cancer, we used a brief-exposure paradigm in the preoperative setting to study transcriptional changes in patient tumors that occur with one dose of therapy prior to combination chemotherapy. Tumor biopsies from breast cancer patients enrolled in two preoperative clinical trials were obtained at baseline and after one dose of bevacizumab (HER2-negative), trastuzumab (HER2-positive) or nab-paclitaxel, followed by treatment with combination chemo-biologic therapy. RNA-Sequencing based PAM50 subtyping at baseline of 46 HER2-negative patients revealed a strong association between the basal-like subtype and pathologic complete response (pCR) to chemotherapy plus bevacizumab (p ≤ 0.0027), but did not provide sufficient specificity to predict response. However, a single dose of bevacizumab resulted in down-regulation of a well-characterized TGF-ß activity signature in every single breast tumor that achieved pCR (p ≤ 0.004). The TGF-ß signature was confirmed to be a tumor-specific read-out of the canonical TGF-ß pathway using pSMAD2 (p ≤ 0.04), with predictive power unique to brief-exposure to bevacizumab (p ≤ 0.016), but not trastuzumab or nab-paclitaxel. Down-regulation of TGF-ß activity was associated with reduction in tumor hypoxia by transcription and protein levels, suggesting therapy-induced disruption of an autocrine-loop between tumor stroma and malignant cells. Modulation of the TGF-ß pathway upon brief-exposure to bevacizumab may provide an early functional readout of pCR to preoperative anti-angiogenic therapy in HER2-negative breast cancer, thus providing additional avenues for exploration in both preclinical and clinical settings with these agents.

Sirolimus and trastuzumab combination therapy for HER2-positive metastatic breast cancer after progression on prior trastuzumab therapy.

Constitutive activation of the PI3K/Akt/mTOR pathway has been suggested as a mechanism of resistance to trastuzumab therapy. This phase II trial was designed to evaluate the safety and clinical activity of daily oral sirolimus, a mammalian target of rapamycin (mTOR) inhibitor, in combination with trastuzumab in HER2-positive metastatic breast cancer following disease progression on prior trastuzumab therapy. Sirolimus 6 mg oral daily dose was administered with a standard dose and schedule of trastuzumab weekly or every 3 weeks. Pharmacodynamic studies included Western blot analysis of S6K1, phosphoS6K1, and mTOR in peripheral mononuclear cells, circulating tumor cells (CTC), and endothelial cells (CEC). Eleven patients were evaluable for safety; and nine were evaluable for response assessment. Subsequent enrollment was stopped due to slow accrual. Study treatment-related grade 3 toxicity included pneumonitis, myelosuppression (leukopenia/anemia), and dermatologic reactions (mucositis, nail changes and rash), with no grade 4 events. One patient received eight cycles (58 weeks) and achieved a partial response. Five patients treated for a total of 101 weeks (median 12 weeks, range 8-47 weeks) achieved stable disease as best response. Overall response rate was 1/9 (11 %) and clinical benefit rate was 4/9 (44 %). There was no statistically significant correlation between response and post-treatment change in levels of the mTOR pathway biomarkers, CTCs, HER2 CTCs, or CECs. Sirolimus 6 mg administered daily with trastuzumab appears to be well tolerated in patients with metastatic HER2-positive breast cancer following disease progression on prior trastuzumab therapy, with evidence of disease activity. mTOR inhibition may overcome resistance to trastuzumab in some HER2-positive tumors.

Phase 1b study of the mammalian target of rapamycin inhibitor sirolimus in combination with nanoparticle albumin-bound paclitaxel in patients with advanced solid tumors.

BACKGROUND: The optimal weekly oral dose of sirolimus and intravenous nanoparticle albumin-bound paclitaxel (nab-paclitaxel) were evaluated. METHODS: A phase 1b study was performed to evaluate escalating doses of oral sirolimus (5-60 mg) on days 2, 9, and 16 with intravenous nab-paclitaxel (100 mg/m(2) ) on days 1, 8, and 15 in a 28-day cycle. A run-in treatment of nab-paclitaxel (day -14) and sirolimus (day -7) was administered for pharmacokinetic and pharmacodynamic assessments. Clinical trial endpoints included dose-limiting toxicities (DLTs), maximum tolerated doses, and response rates. Pharmacodynamics included immunohistochemistry for phosphatase and tensin homolog, mammalian target of rapamycin (mTOR), AKT, phosphorylated AKT, S6K1, and phosphorylated S6K1; exploratory gene expression analysis; and [(18) F]fludeoxyglucose (FDG) positron emission tomography. RESULTS: Twenty-three patients with advanced solid tumors were treated. Fifteen patients had prior taxane therapy. Twenty-two patients were evaluable for responses. One patient had a complete response, and 5 patients had a partial response (3 confirmed). DLTs were seen in 1 patient each at 10 (grade 3 dyspnea/hypoxia) and 40 mg (grade 4 leukopenia/neutropenia) and in 2 patients at 60 mg (grade 3 fatigue and grade 4 pericardial effusion). Patients with higher expression of posttreatment AKT and a greater decline in FDG activity were more likely to have a treatment response or stable disease. CONCLUSIONS: Sirolimus showed an acceptable safety profile at a weekly dose of 40 mg with weekly intravenous nab-paclitaxel at 100 mg/m(2) on days 1, 8, and 15 every 28 days. The posttreatment AKT score and changes in FDG activity may have roles as early predictors of responses to mTOR inhibitors.

Global transcriptome analysis and enhancer landscape of human primary T follicular helper and T effector lymphocytes.

T follicular helper (Tfh) cells are a subset of CD4(+) T helper cells that migrate into germinal centers and promote B-cell maturation into memory B and plasma cells. Tfh cells are necessary for promotion of protective humoral immunity following pathogen challenge, but when aberrantly regulated, drive pathogenic antibody formation in autoimmunity and undergo neoplastic transformation in angioimmunoblastic T-cell lymphoma and other primary cutaneous T-cell lymphomas. Limited information is available on the expression and regulation of genes in human Tfh cells. Using a fluorescence-activated cell sorting-based strategy, we obtained primary Tfh and non-Tfh T effector cells from tonsils and prepared genome-wide maps of active, intermediate, and poised enhancers determined by chromatin immunoprecipitation-sequencing, with parallel transcriptome analyses determined by RNA sequencing. Tfh cell enhancers were enriched near genes highly expressed in lymphoid cells or involved in lymphoid cell function, with many mapping to sites previously associated with autoimmune disease in genome-wide association studies. A group of active enhancers unique to Tfh cells associated with differentially expressed genes was identified. Fragments from these regions directed expression in reporter gene assays. These data provide a significant resource for studies of T lymphocyte development and differentiation and normal and perturbed Tfh cell function.

Deletion of Mecom in mouse results in early-onset spinal deformity and osteopenia.

Recent studies have indicated a role for a MECOM allele in susceptibility to osteoporotic fractures in humans. We have generated a mutation in Mecom in mouse (termed ME(m1)) via lacZ knock-in into the upstream transcription start site for the gene, resulting in disruption of Mds1 and Mds1-Evi1 transcripts, but not of Evi1 transcripts. We demonstrate that ME(m1/m1) mice have severe kyphoscoliosis that is reminiscent of human congenital or primary kyphoscoliosis. ME(m1/m1) mice appear normal at birth, but by 2weeks, they exhibit a slight lumbar lordosis and narrowed intervertebral space. This progresses to severe lordosis with disc collapse and synostosis, together with kyphoscoliosis. Bone formation and strength testing show that ME(m1/m1) mice have normal bone formation and composition but are osteopenic. While endochondral bone development is normal, it is markedly dysplastic in its organization. Electron micrographs of the 1week postnatal intervertebral discs reveals marked disarray of collagen fibers, consistent with an inherent weakness in the non-osseous connective tissue associated with the spine. These findings indicate that lack of ME leads to a complex defect in both osseous and non-osseous musculoskeletal tissues, including a marked vertebral osteopenia, degeneration of the IVD, and disarray of connective tissues, which is likely due to an inherent inability to establish and/or maintain components of these tissues.

Molecular phenotypes in triple negative breast cancer from African American patients suggest targets for therapy.

Triple negative breast cancer (TNBC) is characterized by high proliferation, poor differentiation and a poor prognosis due to high rates of recurrence. Despite lower overall incidence African American (AA) patients suffer from higher breast cancer mortality in part due to the higher proportion of TNBC cases among AA patients compared to European Americans (EA). It was recently shown that the clinical heterogeneity of TNBC is reflected by distinct transcriptional programs with distinct drug response profiles in preclinical models. In this study, gene expression profiling and immunohistochemistry were used to elucidate potential differences between TNBC tumors of EA and AA patients on a molecular level. In a retrospective cohort of 136 TNBC patients, a major transcriptional signature of proliferation was found to be significantly upregulated in samples of AA ethnicity. Furthermore, transcriptional profiles of AA tumors showed differential activation of insulin-like growth factor 1 (IGF1) and a signature of BRCA1 deficiency in this cohort. Using signatures derived from the meta-analysis of TNBC gene expression carried out by Lehmann et al., tumors from AA patients were more likely of basal-like subtypes whereas transcriptional features of many EA samples corresponded to mesenchymal-like or luminal androgen receptor driven subtypes. These results were validated in The Cancer Genome Atlas mRNA and protein expression data, again showing enrichment of a basal-like phenotype in AA tumors and mesenchymal subtypes in EA tumors. In addition, increased expression of VEGF-activated genes together with elevated microvessel area determined by the AQUA method suggest that AA patients exhibit higher tumor vascularization. This study confirms the existence of distinct transcriptional programs in triple negative breast cancer in two separate cohorts and that these programs differ by racial group. Differences in TNBC subtypes and levels of tumor angiogenesis in AA versus EA patients suggest that targeted therapy choices should be considered in the context of race.

Targeting a DNA binding motif of the EVI1 protein by a pyrrole-imidazole polyamide.

The zinc finger protein EVI1 is causally associated with acute myeloid leukemogenesis, and inhibition of its function with a small molecule therapeutic may provide effective therapy for EVI1-expressing leukemias. In this paper we describe the development of a pyrrole-imidazole polyamide to specifically block EVI1 binding to DNA. We first identify essential domains for leukemogenesis through structure-function studies on both EVI1 and the t(3;21)(q26;q22)-derived RUNX1-MDS1-EVI1 (RME) protein, which revealed that DNA binding to the cognate motif GACAAGATA via the first of two zinc finger domains (ZF1, encompassing fingers 1-7) is essential transforming activity. To inhibit DNA binding via ZF1, we synthesized a pyrrole-imidazole polyamide 1, designed to bind to a subsite within the GACAAGATA motif and thereby block EVI1 binding. DNase I footprinting and electromobility shift assays revealed a specific and high affinity interaction between polyamide 1 and the GACAAGATA motif. In an in vivo CAT reporter assay using NIH-3T3-derived cell line with a chromosome-embedded tet-inducible EVI1-VP16 as well as an EVI1-responsive reporter, polyamide 1 completely blocked EVI1-responsive reporter activity. Growth of a leukemic cell line bearing overexpressed EVI1 was also inhibited by treatment with polyamide 1, while a control cell line lacking EVI1 was not. Finally, colony formation by RME was attenuated by polyamide 1 in a serial replating assay. These studies provide evidence that a cell permeable small molecule may effectively block the activity of a leukemogenic transcription factor and provide a valuable tool to dissect critical functions of EVI1 in leukemogenesis.

PR-domain-containing Mds1-Evi1 is critical for long-term hematopoietic stem cell function.

The Mds1 and Evi1 complex locus (Mecom) gives rise to several alternative transcripts implicated in leukemogenesis. However, the contribution that Mecom-derived gene products make to normal hematopoiesis remains largely unexplored. To investigate the role of the upstream transcription start site of Mecom in adult hematopoiesis, we created a mouse model with a lacZ knock-in at this site, termed ME(m1), which eliminates Mds1-Evi1 (ME), the longer, PR-domain-containing isoform produced by the gene (also known as PRDM3). ß-galactosidase-marking studies revealed that, within hematopoietic cells, ME is exclusively expressed in the stem cell compartment. ME deficiency leads to a reduction in the number of HSCs and a complete loss of long-term repopulation capacity, whereas the stem cell compartment is shifted from quiescence to active cycling. Genetic exploration of the relative roles of endogenous ME and EVI1 isoforms revealed that ME preferentially rescues long-term HSC defects. RNA-seq analysis in Lin(-)Sca-1(+)c-Kit(+) cells (LSKs) of ME(m1) documents near complete silencing of Cdkn1c, encoding negative cell-cycle regulator p57-Kip2. Reintroduction of ME into ME(m1) LSKs leads to normalization of both p57-Kip2 expression and growth control. Our results clearly demonstrate a critical role of PR-domain-containing ME in linking p57-kip2 regulation to long-term HSC function.

GPHMM: an integrated hidden Markov model for identification of copy number alteration and loss of heterozygosity in complex tumor samples using whole genome SNP arrays.

There is an increasing interest in using single nucleotide polymorphism (SNP) genotyping arrays for profiling chromosomal rearrangements in tumors, as they allow simultaneous detection of copy number and loss of heterozygosity with high resolution. Critical issues such as signal baseline shift due to aneuploidy, normal cell contamination, and the presence of GC content bias have been reported to dramatically alter SNP array signals and complicate accurate identification of aberrations in cancer genomes. To address these issues, we propose a novel Global Parameter Hidden Markov Model (GPHMM) to unravel tangled genotyping data generated from tumor samples. In contrast to other HMM methods, a distinct feature of GPHMM is that the issues mentioned above are quantitatively modeled by global parameters and integrated within the statistical framework. We developed an efficient EM algorithm for parameter estimation. We evaluated performance on three data sets and show that GPHMM can correctly identify chromosomal aberrations in tumor samples containing as few as 10% cancer cells. Furthermore, we demonstrated that the estimation of global parameters in GPHMM provides information about the biological characteristics of tumor samples and the quality of genotyping signal from SNP array experiments, which is helpful for data quality control and outlier detection in cohort studies.

Chromosome 7 aneusomy in metaplastic breast carcinomas with chondroid, squamous, and spindle-cell differentiation.

Metaplastic breast carcinomas (MBCs) are basal-like tumors that frequently express epidermal growth factor receptor (EGFR) via an unknown underlying genetic mechanism. In this study, the EGFR/CEP7 gene copy number in 17 MBCs with chondroid, squamous, and spindle-cell differentiation showing EGFR expression by immunohistochemistry was analyzed using fluorescence in situ hybridization. All cases had a balanced EGFR/CEP7 ratio. EGFR gene amplification was not observed in any case. Monosomy was found in 25% and polysomy in 12.5% of carcinomas with chondroid differentiation. All spindle-cell carcinomas and 50% of squamous carcinomas showed trisomy. Comparison with CEP7 copy number revealed aneusomy of chromosome 7, as opposed to increases or decreases specific to the EGFR gene or 7p. Although no direct correlation between EGFR expression by immunohistochemistry and aneusomy was observed, cases with a score of 3+ showed a higher frequency of EGFR gene copy gain. In the absence of EGFR amplification, chromosome 7 aneusomy might be a useful criterion for the determination of potential candidates for EGFR tyrosine kinase inhibitor clinical trials.

Fluorescence in situ hybridization analysis of the PRV-1 gene in polycythemia vera: implications for its role in diagnosis and pathogenesis.

OBJECTIVE: In polycythemia vera (PV) there is no specific clonal marker because the molecular lesion responsible for PV is unknown. The recent demonstration that the PRV-1 gene is overexpressed in granulocytes from patients with PV provided the rationale for the current study to investigate whether PRV-1 is structurally rearranged, thus explaining its aberrant expression. MATERIALS AND METHODS: Fluorescence in situ hybridization was used to determine chromosomal localization of PRV-1 and to study whether the PRV1 gene is rearranged in 26 patients with PV. RESULTS: PRV-1 was localized to chromosome 19, band region q13.12-2. Structural rearrangements of PRV-1 were evaluated in bone marrow cells from 26 patients with PV: 14 with a normal karyotype and 12 with an abnormal karyotype. None of 150 metaphase cells or more than 10,000 interphase cells demonstrated PRV-1 gene deletion, amplification, or separation of the probe signal, which would indicate a PRV-1 rearrangement. CONCLUSION: These findings are consistent with a lack of structural rearrangement of PRV-1 in patients with PV. Thus, overexpression of PRV-1 in granulocytes from patients with PV is related to mechanisms that do not involve structural genetic changes.