[Show abstract][Hide abstract] ABSTRACT: Rapidly cycling fetal and neonatal hematopoietic stem cells (HSCs) generate a pool of quiescent adult HSCs after establishing hematopoiesis in the bone marrow. We report an essential role for the trithorax group gene absent, small, or homeotic 1-like (Ash1l) at this developmental transition. Emergence and expansion of Ash1l-deficient fetal/neonatal HSCs were preserved; however, in young adult animals, HSCs were profoundly depleted. Ash1l-deficient adult HSCs had markedly decreased quiescence and reduced cyclin-dependent kinase inhibitor 1b/c (Cdkn1b/1c) expression and failed to establish long-term trilineage bone marrow hematopoiesis after transplantation to irradiated recipients. Wild-type HSCs could efficiently engraft when transferred to unirradiated, Ash1l-deficient recipients, indicating increased availability of functional HSC niches in these mice. Ash1l deficiency also decreased expression of multiple Hox genes in hematopoietic progenitors. Ash1l cooperated functionally with mixed-lineage leukemia 1 (Mll1), as combined loss of Ash1l and Mll1, but not isolated Ash1l or Mll1 deficiency, induced overt hematopoietic failure. Our results uncover a trithorax group gene network that controls quiescence, niche occupancy, and self-renewal potential in adult HSCs.
The Journal of clinical investigation 04/2015; 125(5). DOI:10.1172/JCI78124 · 13.22 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Chronic Lymphocytic Leukemia (CLL) is defined by a perturbed B-cell receptor-mediated signaling machinery. We aimed to model differential signaling behavior between B cells from CLL and healthy individuals to pinpoint modes of dysregulation.
We developed an experimental methodology combining immunophenotyping, multiplexed phosphospecific flow cytometry, and multifactorial statistical modeling. Utilizing patterns of signaling network covariance, we modeled BCR signaling in 67 CLL patients using Partial Least Squares Regression (PLSR). Results from multidimensional modeling were validated using an independent test cohort of 38 patients.
We identified a dynamic and variable imbalance between proximal (pSYK, pBTK) and distal (pPLCγ2, pBLNK, ppERK) phosphoresponses. PLSR identified the relationship between upstream tyrosine kinase SYK and its target, PLCγ2, as maximally predictive and sufficient to distinguish CLL from healthy samples, pointing to this juncture in the signaling pathway as a hallmark of CLL B cells. Specific BCR pathway signaling signatures that correlate with the disease and its degree of aggressiveness were identified. Heterogeneity in the PLSR response variable within the B cell population is both a characteristic mark of healthy samples and predictive of disease aggressiveness.
Single-cell multidimensional analysis of BCR signaling permitted focused analysis of the variability and heterogeneity of signaling behavior from patient-to-patient, and from cell-to-cell. Disruption of the pSYK/pPLCγ2 relationship is uncovered as a robust hallmark of CLL B cell signaling behavior. Together, these observations implicate novel elements of the BCR signal transduction as potential therapeutic targets.
PLoS ONE 01/2014; 9(1):e79987. DOI:10.1371/journal.pone.0079987 · 3.23 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Here we report a comprehensive characterization of our recently developed inhibitor MM-401 that targets the MLL1 H3K4 methyltransferase activity. MM-401 is able to specifically inhibit MLL1 activity by blocking MLL1-WDR5 interaction and thus the complex assembly. This targeting strategy does not affect other mixed-lineage leukemia (MLL) family histone methyltransferases (HMTs), revealing a unique regulatory feature for the MLL1 complex. Using MM-401 and its enantiomer control MM-NC-401, we show that inhibiting MLL1 methyltransferase activity specifically blocks proliferation of MLL cells by inducing cell-cycle arrest, apoptosis, and myeloid differentiation without general toxicity to normal bone marrow cells or non-MLL cells. More importantly, transcriptome analyses show that MM-401 induces changes in gene expression similar to those of MLL1 deletion, supporting a predominant role of MLL1 activity in regulating MLL1-dependent leukemia transcription program. We envision broad applications for MM-401 in basic and translational research.
[Show abstract][Hide abstract] ABSTRACT: Follicular lymphoma (FL) constitutes the second most common non-Hodgkin lymphoma in the Western world. FL carries characteristic recurrent structural genomic aberrations. However, information regarding the coding genome in FL is still evolving. Here, we describe the results of massively parallel exome sequencing and SNP 6.0 array genomic profiling of 11 highly purified FL cases and one transformed FL case and the validation of selected mutations in 102 FL cases. We report identification of 15 novel recurrently mutated genes in FL. These include frequent mutations in the linker histone genes HIST1H1 B-E (27%) and mutations in OCT2 (also known as POU2F2; 8%), IRF8 (6%), and ARID1A (11%). A subset of the mutations in HIST1H1 B-Eaffected binding to DNMT3B and mutations in HIST1H1 B-E and in EZH2 or ARID1A were largely mutually exclusive, implicating HIST1H1 B-E in epigenetic deregulation in FL. Mutations in OCT2 (POU2F2) affected its transcriptional and functional properties as measured through luciferase assays, the biological analysis of stable transduced cell lines and global expression profiling. Finally, multiple novel mutated genes located within regions of acquired uniparental disomy (aUPD) in FL are identified. In aggregate, these data substantially broaden our understanding of the genomic pathogenesis of FL.
[Show abstract][Hide abstract] ABSTRACT: Follicular lymphoma (FL) is an indolent disease, but 30%-40% of cases undergo histologic transformation to an aggressive malignancy, typically represented by diffuse large B cell lymphoma (DLBCL). The pathogenesis of this process remains largely unknown. Using whole-exome sequencing and copy-number analysis, we show here that the dominant clone of FL and transformed FL (tFL) arise by divergent evolution from a common mutated precursor through the acquisition of distinct genetic events. Mutations in epigenetic modifiers and antiapoptotic genes are introduced early in the common precursor, whereas tFL is specifically associated with alterations deregulating cell-cycle progression and DNA damage responses (CDKN2A/B, MYC, and TP53) as well as aberrant somatic hypermutation. The genomic profile of tFL shares similarities with that of germinal center B cell-type de novo DLBCL but also displays unique combinations of altered genes with diagnostic and therapeutic implications.
[Show abstract][Hide abstract] ABSTRACT: Approximately 80 % of chronic lymphocytic leukemia (CLL) carries somatically acquired genomic copy number aberrations (aCNAs). These include gains of entire chromosomes (trisomy 12) and recurrent genomic losses, including interstitial deletions of various lengths at 13q14, 11q, and of more uniform length at 17p. In addition, approximately 10-15 second-tier aCNAs, with frequencies of 1-5 %, have been identified. In this chapter, we will discuss the biology and clinical significance of these CLL-associated aCNAs in detail and also discuss generic aspects of aCNAs relevant to all cancer cells. The hypothesis is advanced that most if not all aCNAs in CLL deregulate multiple target genes as a consequence of aCNA-associated gene mutations and through stable deregulation of gene expression. The concept of elevated genomic complexity (multiple aCNAs per CLL case) is reinforced as one of the strongest biological traits associated with aggressive CLL with short survival. Further, all inherited polymorphic copy number variations as detected through SNP 6.0 array profiling of T-cell-derived DNA of 255 CLL patients are listed to allow the reader a more critical appraisal of the somatic status of CLL-associated aCNAs as reported in the literature. Finally, given that aCNAs and gene mutations coexist in many CLL cells, we stress the importance of understanding in detail the relative biological and clinical roles each mutation type serves in individual CLL patients; this is a research area in need of more in-depth investigation.
Advances in Experimental Medicine and Biology 09/2013; 792:47-86. DOI:10.1007/978-1-4614-8051-8_3 · 1.96 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Chronic lymphocytic leukemia (CLL) is the most common leukemia in the Western World and remains incurable with conventional chemotherapy treatment approaches. CLL has a highly varied clinical course. The substantial clinical variability in the clinical course of CLL has motivated intense efforts at identifying molecular markers that can be used for CLL prognostication. While many such markers have been proposed, few have stood the test of time; this is due to various reasons outlined in detail in this chapter.Of the reasons that have affected the usefulness and broad applicability of CLL biomarkers a few stand out as recurrent: lack of independent effects of individual markers on prognosis; the use of arbitrary cutoffs when using continuous variables; technical challenges in validity, reproducibility, and reliability (classical test characteristics); and lack of marker validation in prospectively identified CLL patient cohorts.Nonetheless, a few useful prognostic markers (CLL interphase FISH, immunoglobulin heavy chain variable region mutation status) have been identified, and others are still in transition to widespread clinical applications (TP53 mutations, SNP array-based elevated genomic complexity).As CLL therapy transitions from genotoxic combination therapies to targeted therapies, it will be of importance to reestablish the usefulness of our current understanding of individual CLL traits in CLL prognosis. Finally, the identification of predictive markers remains important given the established associations of poor response rates with shortened survival and the ongoing need for more personalized approaches in CLL management.
Advances in Experimental Medicine and Biology 09/2013; 792:193-214. DOI:10.1007/978-1-4614-8051-8_9 · 1.96 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Purpose:
The identification of gene mutations and structural genomic aberrations that are critically involved in chronic lymphocytic leukemia (CLL) pathogenesis is still evolving. One may postulate that genomic driver lesions with effects on CLL cell proliferation, apoptosis thresholds, or chemotherapy resistance should increase in frequency over time when measured sequentially in a large CLL cohort.
We sequentially sampled a large well-characterized CLL cohort at a mean of 4 years between samplings and measured acquired copy number aberrations (aCNA) and LOH using single-nucleotide polymorphism (SNP) 6.0 array profiling and the mutational state of TP53, NOTCH1, and SF3B1 using Sanger sequencing. The paired analysis included 156 patients, of whom 114 remained untreated and 42 received intercurrent therapies, predominantly potent chemoimmunotherapy, during the sampling interval.
We identify a strong effect of intercurrent therapies on the frequency of acquisition of aCNAs in CLL. Importantly, the spectrum of acquired genomic changes was largely similar in patients who did or did not receive intercurrent therapies; therefore, various genomic changes that become part of the dominant clones are often already present in CLL cell populations before therapy. Furthermore, we provide evidence that therapy of CLL with preexisting TP53 mutations results in outgrowth of genomically very complex clones, which dominate at relapse.
Using complementary technologies directed at the detection of genomic events that are present in substantial proportions of the clinically relevant CLL disease bulk, we capture aspects of genomic evolution in CLL over time, including increases in the frequency of genomic complexity, specific recurrent aCNAs, and TP53 mutations.
Clinical Cancer Research 04/2013; 19(11). DOI:10.1158/1078-0432.CCR-13-0138 · 8.72 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The application of SNP array technology to the analysis of cancer genomes has greatly advanced our knowledge of the incidence and functional consequences of acquired genomic copy number aberrations (aCNA) and LOH in various malignancies. The major challenges of using SNP arrays are accurately identifying acquired genomic DNA aberrations in the raw array data with very high sensitivity and specificity and meaningfully assessing the associations between these aberrations and biological characteristics or patient outcomes. Critical to the success and valid interpretation of data derived from SNP array profiling are (1) the purity of cells used as a source of template DNA; (2) the analysis of paired DNA samples (tumor and normal); (3) use of validated software tools for data analysis; (4) access to an acceptable gold standard for aCNA and LOH, including FISH data, cytogenetic results, and Q-PCR data; and (5) statistical support to employ or develop algorithmic approaches to SNP array data analysis. Overcalling of lesions including lack of validation and undercalling of lesions that display low fractional allelic representations are common problems. This guide should help the reader establish this powerful technology in the laboratory and aims to stimulate transition of SNP array profiling into clinical applications.
[Show abstract][Hide abstract] ABSTRACT: Genetic aberrations contribute to acute myeloid leukemia (AML). However, half of AML cases do not contain the well-known aberrations detectable mostly by cytogenetic analysis, and these cases are classified as normal karyotype AML. Different outcomes of normal karyotype AML suggest that this subgroup of AML could be genetically heterogeneous. But lack of genetic markers makes it difficult to further study this subgroup of AML. Using paired-end RNAseq method, we performed a transcriptome analysis in 45 AML cases including 29 normal karyotype AML, 8 abnormal karyotype AML and 8 AML without karyotype informaiton. Our study identified 134 fusion transcripts, all of which were formed between the partner genes adjacent in the same chromosome and distributed at different frequencies in the AML cases. Seven fusions are exclusively present in normal karyotype AML, and the rest fusions are shared between the normal karyotype AML and abnormal karyotype AML. CIITA, a master regulator of MHC class II gene expression and truncated in B-cell lymphoma and Hodgkin disease, is found to fuse with DEXI in 48% of normal karyotype AML cases. The fusion transcripts formed between adjacent genes highlight the possibility that certain such fusions could be involved in oncological process in AML, and provide a new source to identify genetic markers for normal karyotype AML.
PLoS ONE 12/2012; 7(12):e51203. DOI:10.1371/journal.pone.0051203 · 3.23 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The frequent occurrence of persistent or relapsed disease following induction chemotherapy in AML necessitates a better understanding of the clonal relationship of AML in various disease phases. In this study, we employed SNP 6.0 array-based genomic profiling of acquired copy number aberrations (aCNA) and copy neutral LOH (cnLOH) together with sequence analysis of recurrently mutated genes to characterize paired AML genomes. We analyzed 28 AML sample pairs from patients that achieved complete remission with chemotherapy and subsequently relapsed and 11 sample pairs from patients with persistent disease following induction chemotherapy. Through review of aCNA/cnLOH and gene mutation profiles in informative cases we demonstrate that relapsed AML invariably represents reemergence or evolution of a founder clone. Furthermore, all individual aCNA or cnLOH detected at presentation persisted at relapse indicating that this lesion type is proximally involved in AML evolution. Analysis of informative paired persistent AML disease samples uncovered cases with two coexisting dominant clones of which at least one was chemotherapy sensitive and one resistant, respectively. These data support the conclusion that incomplete eradication of AML founder clones rather than stochastic emergence of fully unrelated novel clones underlies AML relapse and persistence with direct implications for clinical AML research.
[Show abstract][Hide abstract] ABSTRACT: Chronic lymphocytic leukemia (CLL) is the most common leukemia in the Western world and remains incurable with conventional chemotherapy treatment approaches. CLL as a disease entity is defined by a relatively parsimonious set of diagnostic criteria and therefore likely constitutes an umbrella term for multiple related illnesses. Of the enduring fundamental biological processes that affect the biology and clinical behavior of CLL, few are as central to the pathogenesis of CLL as recurrent acquired genomic copy number aberrations (aCNA) and recurrent gene mutations. Here, a state-of-the-art overview of the pathological anatomy of the CLL genome is presented, including detailed descriptions of the anatomy of aCNA and gene mutations. Data from SNP array profiling and large-scale sequencing of large CLL cohorts, as well as stimulated karyotyping, are discussed. This review is organized by discussions of the anatomy, underlying pathomechanisms and clinical significance of individual genomic lesions and recurrent gene mutations. Finally, gaps in knowledge regarding the biological and clinical effects of recurrent genomic aberrations or gene mutations on CLL are outlined to provide critical stimuli for future research.
[Show abstract][Hide abstract] ABSTRACT: Tipifarnib (T) exhibits modest activity in elderly adults with newly diagnosed acute myelogenous leukemia (AML). Based on preclinical synergy, a phase 1 trial of T plus etoposide (E) yielded 25% complete remission (CR). We selected 2 comparable dose levels for a randomized phase 2 trial in 84 adults (age range, 70-90 years; median, 76 years) who were not candidates for conventional chemotherapy. Arm A (T 600 mg twice a day × 14 days, E 100 mg days 1-3 and 8-10) and arm B (T 400 mg twice a day × 14 days, E 200 mg days 1-3 and 8-10) yielded similar CR, but arm B had greater toxicity. Total CR was 25%, day 30 death rate 7%. A 2-gene signature of high RASGRP1 and low aprataxin (APTX) expression previously predicted for T response. Assays using blasts from a subset of 40 patients treated with T plus E on this study showed that AMLs with a RASGRP1/APTX ratio of more than 5.2 had a 78% CR rate and negative predictive value 87%. This ratio did not correlate with outcome in 41 patients treated with conventional chemotherapies. The next T-based clinical trials will test the ability of the 2-gene signature to enrich for T responders prospectively. This study is registered at www.clinicaltrials.gov as #NCT00602771.
[Show abstract][Hide abstract] ABSTRACT: Mutations in the chromatin remodeling gene ARID1A have recently been identified in the majority of ovarian clear cell carcinomas (OCCCs). To determine the prevalence of mutations in other tumor types, we evaluated 759 malignant neoplasms including those of the pancreas, breast, colon, stomach, lung, prostate, brain, and blood (leukemias). We identified truncating mutations in 6% of the neoplasms studied; nontruncating somatic mutations were identified in an additional 0.4% of neoplasms. Mutations were most commonly found in gastrointestinal samples with 12 of 119 (10%) colorectal and 10 of 100 (10%) gastric neoplasms, respectively, harboring changes. More than half of the mutated colorectal and gastric cancers displayed microsatellite instability (MSI) and the mutations in these tumors were out-of-frame insertions or deletions at mononucleotide repeats. Mutations were also identified in 2-8% of tumors of the pancreas, breast, brain (medulloblastomas), prostate, and lung, and none of these tumors displayed MSI. These findings suggest that the aberrant chromatin remodeling consequent to ARID1A inactivation contributes to a variety of different types of neoplasms.
Human Mutation 01/2012; 33(1):100-3. DOI:10.1002/humu.21633 · 5.14 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Leukemia is one of the leading journals in hematology and oncology. It is published monthly and covers all aspects of the research and treatment of leukemia and allied diseases. Studies of normal hemopoiesis are covered because of their comparative relevance.
Leukemia: official journal of the Leukemia Society of America, Leukemia Research Fund, U.K 12/2011; 26(5):1108-10. DOI:10.1038/leu.2011.361 · 10.43 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: To further our understanding of the genetic basis of acute myelogenous leukemia (AML), we determined the coding exon sequences of ∼ 18 000 protein-encoding genes in 8 patients with secondary AML. Here we report the discovery of novel somatic mutations in the transcriptional corepressor gene BCORL1 that is located on the X-chromosome. Analysis of BCORL1 in an unselected cohort of 173 AML patients identified a total of 10 mutated cases (6%) with BCORL1 mutations, whereas analysis of 19 AML cell lines uncovered 4 (21%) BCORL1 mutated cell lines. The majority (87%) of the mutations in BCORL1 were predicted to inactivate the gene product as a result of nonsense mutations, splice site mutation, or out-of-frame insertions or deletions. These results indicate that BCORL1 by genetic criteria is a novel candidate tumor suppressor gene, joining the growing list of genes recurrently mutated in AML.