Publications by authors named "Peter D Aplan"

87 Publications

CENP-A overexpression promotes aneuploidy with karyotypic heterogeneity.

J Cell Biol 2021 Apr;220(4)

Genetics Branch, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD.

Chromosomal instability (CIN) is a hallmark of many cancers. Restricting the localization of centromeric histone H3 variant CENP-A to centromeres prevents CIN. CENP-A overexpression (OE) and mislocalization have been observed in cancers and correlate with poor prognosis; however, the molecular consequences of CENP-A OE on CIN and aneuploidy have not been defined. Here, we show that CENP-A OE leads to its mislocalization and CIN with lagging chromosomes and micronuclei in pseudodiploid DLD1 cells and xenograft mouse model. CIN is due to reduced localization of proteins to the kinetochore, resulting in defects in kinetochore integrity and unstable kinetochore-microtubule attachments. CENP-A OE contributes to reduced expression of cell adhesion genes and higher invasion of DLD1 cells. We show that CENP-A OE contributes to aneuploidy with karyotypic heterogeneity in human cells and xenograft mouse model. In summary, our results provide a molecular link between CENP-A OE and aneuploidy, and suggest that karyotypic heterogeneity may contribute to the aggressive phenotype of CENP-A-overexpressing cancers.
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http://dx.doi.org/10.1083/jcb.202007195DOI Listing
April 2021

Clinical and molecular consequences of fusion genes in myeloid malignancies.

Stem Cells 2020 11 26;38(11):1366-1374. Epub 2020 Aug 26.

Genetics Branch, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, Maryland, USA.

Leukemias are heterogeneous diseases characterized by aberrant hematopoietic stem and progenitor cells (HSPCs). Oncogenic fusion genes and proteins, produced via gross chromosomal rearrangements, such as chromosomal translocation, insertion, and inversion, play important roles in hematologic malignancies. These oncoproteins alter fundamental cellular properties, such as self-renewal, differentiation, and proliferation, and confer leukemogenic potential to HSPCs. In addition to providing fundamental insights into the process of leukemic transformation, these fusion genes provide targets for treatment and monitoring of myeloid leukemias. Furthermore, new technologies such as next-generation sequencing have allowed additional insights into the nature of leukemic fusion genes. In this review, we discuss the history, biologic effect, and clinical impact of fusion genes in the field of myeloid leukemias.
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http://dx.doi.org/10.1002/stem.3263DOI Listing
November 2020

Mechanistic insights into chromatin targeting by leukemic NUP98-PHF23 fusion.

Nat Commun 2020 07 3;11(1):3339. Epub 2020 Jul 3.

Department of Pharmacology, University of Colorado School of Medicine, Aurora, CO, 80045, USA.

Chromosomal NUP98-PHF23 translocation is associated with an aggressive form of acute myeloid leukemia (AML) and poor survival rate. Here, we report the molecular mechanisms by which NUP98-PHF23 recognizes the histone mark H3K4me3 and is inhibited by small molecule compounds, including disulfiram that directly targets the PHD finger of PHF23 (PHF23PHD). Our data support a critical role for the PHD fingers of NUP98-PHF23, and related NUP98-KDM5A and NUP98-BPTF fusions in driving leukemogenesis, and demonstrate that blocking this interaction in NUP98-PHF23 expressing AML cells leads to cell death through necrotic and late apoptosis pathways. An overlap of NUP98-KDM5A oncoprotein binding sites and H3K4me3-positive loci at the Hoxa/b gene clusters and Meis1 in ChIP-seq, together with NMR analysis of the H3K4me3-binding sites of the PHD fingers from PHF23, KDM5A and BPTF, suggests a common PHD finger-dependent mechanism that promotes leukemogenesis by this type of NUP98 fusions. Our findings highlight the direct correlation between the abilities of NUP98-PHD finger fusion chimeras to associate with H3K4me3-enriched chromatin and leukemic transformation.
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http://dx.doi.org/10.1038/s41467-020-17098-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7335091PMC
July 2020

Thymic precursor cells generate acute myeloid leukemia in NUP98-PHF23/NUP98-HOXD13 double transgenic mice.

Sci Rep 2019 11 20;9(1):17213. Epub 2019 Nov 20.

Genetics Branch, Center for cancer Research, NCI/NIH, Bethesda, MD, USA.

Transgenic mice that express either a NUP98-PHF23 (NP23) or NUP98-HOXD13 (NHD13) fusion in the hematopoietic compartment develop a wide spectrum of leukemias, including myeloid, erythroid, megakaryocytic and lymphoid, at age 9-14 months. NP23-NHD13 double transgenic mice were generated by interbreeding NP23 and NHD13 mice. Remarkably, 100% of the NP23-NHD13 double transgenic mice developed acute myeloid leukemia (AML) within three months, characterized by replacement of the thymus with leukemic myeloblasts. The marked infiltration of thymus led to the intriguing hypothesis that AML generated in NP23-NHD13 mice arose in the thymus, as opposed to the bone marrow (BM). Transplantation of CD4-CD8- double negative (DN) thymocytes (which were also negative for Mac1 and Gr1) from leukemic NHD13/NP23 mice demonstrated that DN thymocytes could transmit AML, and limiting dilution studies showed that leukemia initiating cells were increased 14-fold in the thymus compared to BM. Further thymocyte fractionation demonstrated that DN1 and DN2, but not DN3 or DN4 fractions transmitted AML, and a marked expansion (100-fold) of Lineage-Sca1 + Kit + (LSK) cells in the thymus of the NP23-NHD13 mice. Taken together, these results show that the thymus of NP23-NHD13 mice acts as a reservoir for AML initiating cells and that thymic progenitors can transmit AML.
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http://dx.doi.org/10.1038/s41598-019-53610-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6868234PMC
November 2019

A unique mutator phenotype reveals complementary oncogenic lesions leading to acute leukemia.

JCI Insight 2019 12 5;4(23). Epub 2019 Dec 5.

Genetics Branch, Center for Cancer Research, National Cancer Institute, NIH, Bethesda, Maryland, USA.

Mice homozygous for a hypomorphic allele of DNA replication factor minichromosome maintenance protein 2 (designated Mcm2cre/cre) develop precursor T cell lymphoblastic leukemia/lymphoma (pre-T LBL) with 4-32 small interstitial deletions per tumor. Mice that express a NUP98-HOXD13 (NHD13) transgene develop multiple types of leukemia, including myeloid and T and B lymphocyte. All Mcm2cre/cre NHD13+ mice develop pre-T LBL, and 26% develop an unrelated, concurrent B cell precursor acute lymphoblastic leukemia (BCP-ALL). Copy number alteration (CNA) analysis demonstrated that pre-T LBLs were characterized by homozygous deletions of Pten and Tcf3 and partial deletions of Notch1 leading to Notch1 activation. In contrast, BCP-ALLs were characterized by recurrent deletions involving Pax5 and Ptpn1 and copy number gain of Abl1 and Nup214 resulting in a Nup214-Abl1 fusion. We present a model in which Mcm2 deficiency leads to replicative stress, DNA double strand breaks (DSBs), and resultant CNAs due to errors in DNA DSB repair. CNAs that involve critical oncogenic pathways are then selected in vivo as malignant lymphoblasts because of a fitness advantage. Some CNAs, such as those involving Abl1 and Notch1, represent attractive targets for therapy.
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http://dx.doi.org/10.1172/jci.insight.131434DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6962024PMC
December 2019

HoxA9 binds and represses the Cebpa +8 kb enhancer.

PLoS One 2019 23;14(5):e0217604. Epub 2019 May 23.

Division of Pediatric Oncology, Johns Hopkins University, Baltimore, Maryland, United States of America.

C/EBPα plays a key role in specifying myeloid lineage development. HoxA9 is expressed in myeloid progenitors, with its level diminishing during myeloid maturation, and HOXA9 is over-expressed in a majority of acute myeloid leukemia cases, including those expressing NUP98-HOXD13. The objective of this study was to determine whether HoxA9 directly represses Cebpa gene expression. We find 4-fold increased HoxA9 and 5-fold reduced Cebpa in marrow common myeloid and LSK progenitors from Vav-NUP98-HOXD13 transgenic mice. Conversely, HoxA9 decreases 5-fold while Cebpa increases during granulocytic differentiation of 32Dcl3 myeloid cells. Activation of exogenous HoxA9-ER in 32Dcl3 cells reduces Cebpa mRNA even in the presence of cycloheximide, suggesting direct repression. Cebpa transcription in murine myeloid cells is regulated by a hematopoietic-specific +37 kb enhancer and by a more widely active +8 kb enhancer. ChIP-Seq analysis of primary myeloid progenitor cells expressing exogenous HoxA9 or HoxA9-ER demonstrates that HoxA9 localizes to both the +8 kb and +37 kb Cebpa enhancers. Gel shift analysis demonstrates HoxA9 binding to three consensus sites in the +8 kb enhancer, but no affinity for the single near-consensus site present in the +37 kb enhancer. Activity of a Cebpa +8 kb enhancer/promoter-luciferase reporter in 32Dcl3 or MOLM14 myeloid cells is increased ~2-fold by mutation of its three HOXA9-binding sites, suggesting that endogenous HoxA9 represses +8 kb Cebpa enhancer activity. In contrast, mutation of five C/EBPα-binding sites in the +8 kb enhancer reduces activity 3-fold. Finally, expression of a +37 kb enhancer/promoter-hCD4 transgene reporter is reduced ~2-fold in marrow common myeloid progenitors when the Vav-NUP98-HOXD13 transgene is introduced. Overall, these data support the conclusion that HoxA9 represses Cebpa expression, at least in part via inhibition of its +8 kb enhancer, potentially allowing normal myeloid progenitors to maintain immaturity and contributing to the pathogenesis of acute myeloid leukemia associated with increased HOXA9.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0217604PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6532930PMC
February 2020

Engineered Bcor mutations lead to acute leukemia of progenitor B-1 lymphocyte origin in a sensitized background.

Blood 2019 06 16;133(24):2610-2614. Epub 2019 Apr 16.

Genetics Branch, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD; and.

Approximately 10% of mice develop an aggressive acute lymphoblastic leukemia of B-1 lymphocyte progenitor origin (pro-B1 ALL), accompanied by somatic frameshift mutations of the BCL6 interacting corepressor () gene, most commonly within a 9-bp "hotspot" in exon 8. To determine whether experimentally engineered mutations would lead to pro-B1 ALL, we used clustered, regularly interspaced, short palindromic repeats-associated protein 9 to introduce a frameshift mutation into hematopoietic stem and progenitor cells through the use of small guide RNAs ( sgRNAs). Recipient mice transplanted with bone marrow or fetal liver cells that had been transduced with a sgRNA developed pro-B1 ALL, characterized by a B-1 progenitor immunophenotype, clonal Igh gene rearrangement, and indel mutation, whereas control recipients did not. Similar to a subset of human B-cell precursor ALL, the murine pro-B1 ALL had acquired somatic mutations in Jak kinase genes. JAK inhibitors (ruxolitinib and tofacitinib) inhibited the growth of pro-B1 ALL cell lines established from Bcor sgRNA/NP23 recipients at clinically achievable concentrations (100 nM). Our results demonstrate that mutations collaborate with to induce pro-B1 ALL, and that JAK inhibitors are potential therapies for pro-B1 ALL.
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http://dx.doi.org/10.1182/blood.2018864173DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6566592PMC
June 2019

Use of Hematopoietic Stem Cell Transplantation to Assess the Origin of Myelodysplastic Syndrome.

J Vis Exp 2018 10 3(140). Epub 2018 Oct 3.

Genetics Branch, Center for Cancer Research, National Cancer Institute, National Institutes of Health.

Myelodysplastic syndromes (MDS) are a diverse group of hematopoietic stem cell disorders that are defined by ineffective hematopoiesis, peripheral blood cytopenias, dysplasia, and a propensity for transformation to acute leukemia. NUP98-HOXD13 (NHD13) transgenic mice recapitulate human MDS in terms of peripheral blood cytopenias, dysplasia, and transformation to acute leukemia. We previously demonstrated that MDS could be transferred from a genetically engineered mouse with MDS to wild-type recipients by transplanting MDS bone marrow nucleated cells (BMNC). To more clearly understand the MDS cell of origin, we have developed approaches to transplant specific, immunophenotypically defined hematopoietic subsets. In this article, we describe the process of isolating and transplanting specific populations of hematopoietic stem and progenitor cells. Following transplantation, we describe approaches to assess the efficiency of transplantation and persistence of the donor MDS cells.
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http://dx.doi.org/10.3791/58140DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6235411PMC
October 2018

Hedgehog/GLI1 activation leads to leukemic transformation of myelodysplastic syndrome in vivo and GLI1 inhibition results in antitumor activity.

Oncogene 2019 01 31;38(5):687-698. Epub 2018 Aug 31.

The Sidney Kimmel Comprehensive Cancer Center, Johns Hopkins University School of Medicine, Baltimore, MD, 21287, USA.

Myelodysplastic syndromes (MDSs) are stem cell disorders with risk of transformation to acute myeloid leukemia (AML). Gene expression profiling reveals transcriptional expression of GLI1, of Hedgehog (Hh) signaling, in poor-risk MDS/AML. Using a murine model of MDS we demonstrated that constitutive Hh/Gli1 activation accelerated leukemic transformation and decreased overall survival. Hh/Gli1 activation resulted in clonal expansion of phenotypically defined granulocyte macrophage progenitors (GMPs) and acquisition of self-renewal potential in a non-self-renewing progenitor compartment. Transcriptome analysis of GMPs revealed enrichment in gene signatures of self-renewal pathways, operating via direct Gli1 activation. Using human cell lines we demonstrated that in addition to canonical Hh signaling, GLI1 is activated in a Smoothened-independent manner. GLI1 knockdown or inhibition with GANT61 resulted in decreased proliferation and clonogenic potential. Our data suggest that GLI1 activation is frequent in MDS during disease progression and inhibition of GLI1 is an attractive therapeutic target for a subset of patients.
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http://dx.doi.org/10.1038/s41388-018-0431-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6358463PMC
January 2019

SIRT1 Activation Disrupts Maintenance of Myelodysplastic Syndrome Stem and Progenitor Cells by Restoring TET2 Function.

Cell Stem Cell 2018 09 23;23(3):355-369.e9. Epub 2018 Aug 23.

Department of Hematological Malignancies Translational Science, Gehr Family Center for Leukemia Research, Hematologic Malignancies and Stem Cell Transplantation Institute, Beckman Research Institute, City of Hope Medical Center, Duarte, CA 91010, USA. Electronic address:

Myelodysplastic syndrome (MDS), a largely incurable hematological malignancy, is derived from aberrant clonal hematopoietic stem/progenitor cells (HSPCs) that persist after conventional therapies. Defining the mechanisms underlying MDS HSPC maintenance is critical for developing MDS therapy. The deacetylase SIRT1 regulates stem cell proliferation, survival, and self-renewal by deacetylating downstream proteins. Here we show that SIRT1 protein levels were downregulated in MDS HSPCs. Genetic or pharmacological activation of SIRT1 inhibited MDS HSPC functions, whereas SIRT1 deficiency enhanced MDS HSPC self-renewal. Mechanistically, the inhibitory effects of SIRT1 were dependent on TET2, a safeguard against HSPC transformation. SIRT1 deacetylated TET2 at conserved lysine residues in its catalytic domain, enhancing TET2 activity. Our genome-wide analysis identified cancer-related genes regulated by the SIRT1/TET2 axis. SIRT1 activation also inhibited functions of MDS HSPCs from patients with TET2 heterozygous mutations. Altogether, our results indicate that restoring TET2 function through SIRT1 activation represents a promising means to target MDS HSPCs.
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http://dx.doi.org/10.1016/j.stem.2018.07.018DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6143172PMC
September 2018

Dual Roles of Poly(dA:dT) Tracts in Replication Initiation and Fork Collapse.

Cell 2018 08 2;174(5):1127-1142.e19. Epub 2018 Aug 2.

Laboratory of Genome Integrity, National Cancer Institute, NIH, Bethesda, MD, USA. Electronic address:

Replication origins, fragile sites, and rDNA have been implicated as sources of chromosomal instability. However, the defining genomic features of replication origins and fragile sites are among the least understood elements of eukaryote genomes. Here, we map sites of replication initiation and breakage in primary cells at high resolution. We find that replication initiates between transcribed genes within nucleosome-depleted structures established by long asymmetrical poly(dA:dT) tracts flanking the initiation site. Paradoxically, long (>20 bp) (dA:dT) tracts are also preferential sites of polar replication fork stalling and collapse within early-replicating fragile sites (ERFSs) and late-replicating common fragile sites (CFSs) and at the rDNA replication fork barrier. Poly(dA:dT) sequences are fragile because long single-strand poly(dA) stretches at the replication fork are unprotected by the replication protein A (RPA). We propose that the evolutionary expansion of poly(dA:dT) tracts in eukaryotic genomes promotes replication initiation, but at the cost of chromosome fragility.
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http://dx.doi.org/10.1016/j.cell.2018.07.011DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6591735PMC
August 2018

Mutant IL-7Rα and mutant NRas are sufficient to induce murine T cell acute lymphoblastic leukemia.

Leukemia 2018 08 15;32(8):1795-1882. Epub 2018 Feb 15.

Cytokines and Immunity Section, Cancer and Inflammation Program, National Cancer Institute (NCI), National Institutes of Health (NIH), Frederick, MD, USA.

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http://dx.doi.org/10.1038/s41375-017-0001-0DOI Listing
August 2018

Progenitor B-1 B-cell acute lymphoblastic leukemia is associated with collaborative mutations in 3 critical pathways.

Blood Adv 2017 Sep 8;1(20):1749-1759. Epub 2017 Sep 8.

Genetics Branch and.

B-1 and B-2 lymphocytes are derived from distinct developmental pathways and represent layered arms of the innate and adaptive immune systems, respectively. In contrast to a majority of murine B-cell malignancies, which stain positive with the B220 antibody, we discovered a novel form of B-cell leukemia in - () transgenic mice. The immunophenotype (Lin B220 CD19 AA4.1) was identical to that of progenitor (pro) B-1 cells, and V gene usage was skewed toward 3' V regions, similar to murine fetal liver B cells. Moreover, the gene expression profile of these leukemias was most similar to that of fetal liver pro-B fraction BC, a known source of B-1 B cells, further supporting a pro-B-1 origin of these leukemias. The pro-B-1 acute lymphoblastic leukemias (ALLs) acquired spontaneous mutations in both and Janus kinase () pathway (// and ) genes, supporting a hypothesis that mutations in 3 critical pathways (stem-cell self-renewal, B-cell differentiation, and cytokine signaling) collaborate to induce B-cell precursor (BCP) ALL. Finally, the thymic stromal lymphopoietin (Tslp) cytokine is required for murine B-1 development, and chromosomal rearrangements resulting in overexpression of the TSLP receptor (CRLF2) are present in some patients with high-risk BCP-ALL (referred to as r ALL). Gene expression profiles of pro-B-1 ALL were more similar to that of r ALL than non-r ALL, and analysis of V gene usage from patients with ALL demonstrated preferential usage of V regions used by human B-1 B cells, leading to the suggestion that this subset of patients with BCP-ALL has a malignancy of B-1, rather than B-2, B-cell origin.
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http://dx.doi.org/10.1182/bloodadvances.2017009837DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5728343PMC
September 2017

Myeloablative hematopoietic stem cell transplantation improves survival but is not curative in a pre-clinical model of myelodysplastic syndrome.

PLoS One 2017 27;12(9):e0185219. Epub 2017 Sep 27.

Genetics Branch, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, Maryland, United States of America.

Allogeneic hematopoietic stem cell transplantation (A-HSCT) remains the only curative option for patients with myelodysplastic syndrome (MDS). We used the NUP98-HOXD13 (NHD13) murine model for MDS to study HSCT in a pre-clinical setting. NHD13 recipients transplanted with syngeneic bone marrow (S-HSCT) following myeloablative irradiation showed disease remission, with normalization of peripheral blood parameters and marked decrease in circulating leukocytes derived from the MDS clone. Despite the disease remission and improved survival compared to non-transplanted NHD13 controls, all mice eventually relapsed, indicating persistence of a long-lived radio-resistant MDS clone. In an effort to induce a graft versus leukemia (GVL) effect, A-HSCT with donor bone marrow that was mismatched at minor histocompatibility loci was compared to S-HSCT. Although recipients in the A-HSCT showed a lower early relapse rate than in S-HSCT, all mice in both groups eventually relapsed and died by 54 weeks post-transplant. To obtain a more significant GVL effect, donor splenocytes containing reactive T-cells were transplanted with allogeneic bone marrow. Although the relapse rate was only 20% at post-transplantation week 38, suggesting a GVL effect, this was accompanied by a severe graft versus host disease (GVHD) Taken together, these findings indicate that a myeloablative dose of ionizing radiation is insufficient to eradicate the MDS initiating cell, and that transplantation of donor splenocytes leads to decreased relapse rates, at the cost of severe GVHD. We suggest that NHD13 mice represent a feasible pre-clinical model for the study of HSCT for MDS.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0185219PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5617185PMC
October 2017

Genome Organization Drives Chromosome Fragility.

Cell 2017 Jul 20;170(3):507-521.e18. Epub 2017 Jul 20.

Laboratory of Genome Integrity, National Cancer Institute, NIH, Bethesda, MD, USA. Electronic address:

In this study, we show that evolutionarily conserved chromosome loop anchors bound by CCCTC-binding factor (CTCF) and cohesin are vulnerable to DNA double strand breaks (DSBs) mediated by topoisomerase 2B (TOP2B). Polymorphisms in the genome that redistribute CTCF/cohesin occupancy rewire DNA cleavage sites to novel loop anchors. While transcription- and replication-coupled genomic rearrangements have been well documented, we demonstrate that DSBs formed at loop anchors are largely transcription-, replication-, and cell-type-independent. DSBs are continuously formed throughout interphase, are enriched on both sides of strong topological domain borders, and frequently occur at breakpoint clusters commonly translocated in cancer. Thus, loop anchors serve as fragile sites that generate DSBs and chromosomal rearrangements. VIDEO ABSTRACT.
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http://dx.doi.org/10.1016/j.cell.2017.06.034DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6133249PMC
July 2017

Generation of Gross Chromosomal Rearrangements by a Single Engineered DNA Double Strand Break.

Sci Rep 2017 02 22;7:43156. Epub 2017 Feb 22.

Genetics Branch National Cancer Institute, National Institutes of Health, Bethesda, MD 20892, USA.

Gross chromosomal rearrangements (GCRs), including translocations, inversions amplifications, and deletions, can be causal events leading to malignant transformation. GCRs are thought to be triggered by DNA double strand breaks (DSBs), which in turn can be spontaneous or induced by external agents (eg. cytotoxic chemotherapy, ionizing radiation). It has been shown that induction of DNA DSBs at two defined loci can produce stable balanced chromosomal translocations, however, a single engineered DNA DSB could not. Herein, we report that although a single engineered DNA DSB in H2AX "knockdown" cells did not generate GCRs, repair of a single engineered DNA DSB in fibroblasts that had ablated H2ax did produce clonal, stable GCRs, including balanced translocations and megabase-pair inversions. Upon correction of the H2ax deficiency, cells no longer generated GCRs following a single engineered DNA DSB. These findings demonstrate that clonal, stable GCRs can be produced by a single engineered DNA DSB in H2ax knockout cells, and that the production of these GCRs is ameliorated by H2ax expression.
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http://dx.doi.org/10.1038/srep43156DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5320478PMC
February 2017

Somatic mutations in murine models of leukemia and lymphoma: Disease specificity and clinical relevance.

Genes Chromosomes Cancer 2017 Jun 31;56(6):472-483. Epub 2017 Mar 31.

Genetics Branch, Center for Cancer Research, National Cancer Institute, NIH, Bethesda, Maryland.

Malignant transformation is a multistep process that is dictated by the acquisition of multiple genomic aberrations that provide growth and survival advantage. During the post genomic era, high throughput genomic sequencing has advanced exponentially, leading to identification of countless cancer associated mutations with potential for targeted therapy. Mouse models of cancer serve as excellent tools to examine the functionality of gene mutations and their contribution to the malignant process. However, it remains unclear whether the genetic events that occur during transformation are similar in mice and humans. To address that, we chose several transgenic mouse models of hematopoietic malignancies and identified acquired mutations in these mice by means of targeted re-sequencing of known cancer-associated genes as well as whole exome sequencing. We found that mutations that are typically found in acute myeloid leukemia or T cell acute lymphoblastic leukemia patients are also common in mouse models of the respective disease. Moreover, we found that the most frequent mutations found in a mouse model of lymphoma occur in a set of epigenetic modifier genes, implicating this pathway in the generation of lymphoma. These results demonstrate that genetically engineered mouse models (GEMM) mimic the genetic evolution of human cancer and serve as excellent platforms for target discovery and validation.
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http://dx.doi.org/10.1002/gcc.22451DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5399546PMC
June 2017

Templated Sequence Insertion Polymorphisms in the Human Genome.

Front Chem 2016 16;4:43. Epub 2016 Nov 16.

Genetics Branch, National Cancer Institute, National Institutes of Health Bethesda, MD, USA.

Templated Sequence Insertion Polymorphism (TSIP) is a recently described form of polymorphism recognized in the human genome, in which a sequence that is templated from a distant genomic region is inserted into the genome, seemingly at random. TSIPs can be grouped into two classes based on nucleotide sequence features at the insertion junctions; Class 1 TSIPs show features of insertions that are mediated via the LINE-1 ORF2 protein, including (1) target-site duplication (TSD), (2) polyadenylation 10-30 nucleotides downstream of a "cryptic" polyadenylation signal, and (3) preference for insertion at a 5'-TTTT/A-3' sequence. In contrast, class 2 TSIPs show features consistent with repair of a DNA double-strand break (DSB) via insertion of a DNA "patch" that is derived from a distant genomic region. Survey of a large number of normal human volunteers demonstrates that most individuals have 25-30 TSIPs, and that these TSIPs track with specific geographic regions. Similar to other forms of human polymorphism, we suspect that these TSIPs may be important for the generation of human diversity and genetic diseases.
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http://dx.doi.org/10.3389/fchem.2016.00043DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5110952PMC
November 2016

Accurate Medicine: Indirect Targeting of NPM1-Mutated AML.

Cancer Discov 2016 10;6(10):1087-1089

Leukemia Biology Section, Genetics Branch, National Cancer Institute, National Institutes of Health, Bethesda, Maryland.

Acute myeloid leukemia (AML) is now recognized to be an imprecise term that refers to a range of myeloid malignancies that have different genetical etiologies, clinical characteristics, and therapeutic sensitivities. Targeting the MLL1 and DOT1L histone modification complexes, both alone and in combination, showed activity against AML driven by a mutant NPM1 protein in several preclinical models and may represent a new treatment direction for this devastating disease. Cancer Discov; 6(10); 1087-9 ©2016 AACR.See related article by Kühn and colleagues p. 1166.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5074693PMC
http://dx.doi.org/10.1158/2159-8290.CD-16-0917DOI Listing
October 2016

Therapeutic targeting of IL-7Rα signaling pathways in ALL treatment.

Blood 2016 07 6;128(4):473-8. Epub 2016 Jun 6.

Cytokines and Immunity Section, Cancer and Inflammation Program, National Cancer Institute, National Institutes of Health, Frederick, MD;

Increased understanding of pediatric acute lymphoblastic leukemia (ALL) pathobiology has led to dramatic improvements in patient survival. However, there is still a need to develop targeted therapies to enable reduced chemotherapy intensity and to treat relapsed patients. The interleukin-7 receptor α (IL-7Rα) signaling pathways are prime therapeutic targets because these pathways harbor genetic aberrations in both T-cell ALL and B-cell precursor ALL. Therapeutic targeting of the IL-7Rα signaling pathways may lead to improved outcomes in a subset of patients.
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http://dx.doi.org/10.1182/blood-2016-03-679209DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4965903PMC
July 2016

All-trans retinoic acid synergizes with FLT3 inhibition to eliminate FLT3/ITD+ leukemia stem cells in vitro and in vivo.

Blood 2016 06 21;127(23):2867-78. Epub 2016 Apr 21.

Department of Oncology and Department of Pediatrics, Johns Hopkins University School of Medicine, Baltimore, MD.

FMS-like tyrosine kinase 3 (FLT3)-mutant acute myeloid leukemia (AML) portends a poor prognosis, and ineffective targeting of the leukemic stem cell (LSC) population remains one of several obstacles in treating this disease. All-trans retinoic acid (ATRA) has been used in several clinical trials for the treatment of nonpromyelocytic AML with limited clinical activity observed. FLT3 tyrosine kinase inhibitors (TKIs) used as monotherapy also achieve limited clinical responses and are thus far unable to affect cure rates in AML patients. We explored the efficacy of combining ATRA and FLT3 TKIs to eliminate FLT3/internal tandem duplication (ITD)(+) LSCs. Our studies reveal highly synergistic drug activity, preferentially inducing apoptosis in FLT3/ITD(+) cell lines and patient samples. Colony-forming unit assays further demonstrate decreased clonogenicity of FLT3/ITD(+) cells upon treatment with ATRA and TKI. Most importantly, the drug combination depletes FLT3/ITD(+) LSCs in a genetic mouse model of AML, and prolongs survival of leukemic mice. Furthermore, engraftment of primary FLT3/ITD(+) patient samples is reduced in mice following treatment with FLT3 TKI and ATRA in combination, with evidence of cellular differentiation occurring in vivo. Mechanistically, we provide evidence that the synergism of ATRA and FLT3 TKIs is at least in part due to the observation that FLT3 TKI treatment upregulates the antiapoptotic protein Bcl6, limiting the drug's apoptotic effect. However, cotreatment with ATRA reduces Bcl6 expression to baseline levels through suppression of interleukin-6 receptor signaling. These studies provide evidence of the potential of this drug combination to eliminate FLT3/ITD(+) LSCs and reduce the rate of relapse in AML patients with FLT3 mutations.
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http://dx.doi.org/10.1182/blood-2015-05-646786DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4900954PMC
June 2016

Far Upstream Element Binding Protein Plays a Crucial Role in Embryonic Development, Hematopoiesis, and Stabilizing Myc Expression Levels.

Am J Pathol 2016 Mar 14;186(3):701-15. Epub 2016 Jan 14.

Laboratory of Pathology, Center for Cancer Research, National Cancer Institute, Bethesda, Maryland. Electronic address:

The transcription factor far upstream element binding protein (FBP) binds and activates the MYC promoter when far upstream element is via TFIIH helicase activity early in the transcription cycle. The fundamental biology and pathology of FBP are complex. In some tumors FBP seems pro-oncogenic, whereas in others it is a tumor suppressor. We generated an FBP knockout (Fubp1(-/-)) mouse to study FBP deficiency. FBP is embryo lethal from embryonic day 10.5 to birth. A spectrum of pathology is associated with FBP loss; besides cerebral hyperplasia and pulmonary hypoplasia, pale livers, hypoplastic spleen, thymus, and bone marrow, cardiac hypertrophy, placental distress, and small size were all indicative of anemia. Immunophenotyping of hematopoietic cells in wild-type versus knockout livers revealed irregular trilineage anemia, with deficits in colony formation. Despite normal numbers of hematopoietic stem cells, transplantation of Fubp1(-/-) hematopoietic stem cells into irradiated mice entirely failed to reconstitute hematopoiesis. In competitive transplantation assays against wild-type donor bone marrow, Fubp1(-/-) hematopoietic stem cells functioned only sporadically at a low level. Although cultures of wild-type mouse embryo fibroblasts set Myc levels precisely, Myc levels of mouse varied wildly between fibroblasts harvested from different Fubp1(-/-) embryos, suggesting that FBP contributes to Myc set point fixation. FBP helps to hold multiple physiologic processes to close tolerances, at least in part by constraining Myc expression.
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http://dx.doi.org/10.1016/j.ajpath.2015.10.028DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4816710PMC
March 2016

A robust in vivo model for B cell precursor acute lymphoblastic leukemia.

J Clin Invest 2015 Sep 24;125(9):3427-9. Epub 2015 Aug 24.

B cell precursor acute lymphoblastic leukemia (BCP ALL) is the most common malignancy in children. While treatments have improved remarkably over the past four decades, resistant disease and late effects that result from cytotoxic chemotherapy remain serious problems for individuals with BCP ALL. Improved genetic tools have led to the discovery of numerous somatic mutations associated with BCP ALL, leading to a framework for the genetic classification of BCP ALL. In this issue of the JCI, Duque-Afonso et al. develop an accurate in vivo model for BCP ALL that recapitulates the key features of human disease, including acquired mutations in genes encoding PAX5 and components of the JAK/STAT pathway. The authors further show, as proof of principle, that this model can be used to evaluate the efficacy of drugs designed to target specific acquired mutations in patients with BCP ALL.
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http://dx.doi.org/10.1172/JCI83799DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4588242PMC
September 2015

Thymic expression of a T-cell receptor targeting a tumor-associated antigen coexpressed in the thymus induces T-ALL.

Blood 2015 May 26;125(19):2958-67. Epub 2015 Mar 26.

Pediatric Oncology Branch and.

T-cell receptors (TCRs) and chimeric antigen receptors recognizing tumor-associated antigens (TAAs) can now be engineered to be expressed on a wide array of immune effectors. Engineered receptors targeting TAAs have most commonly been expressed on mature T cells, however, some have postulated that receptor expression on immune progenitors could yield T cells with enhanced potency. We generated mice (survivin-TCR-transgenic [Sur-TCR-Tg]) expressing a TCR recognizing the immunodominant epitope (Sur20-28) of murine survivin during early stages of thymopoiesis. Spontaneous T-cell acute lymphoblastic leukemia (T-ALL) occurred in 100% of Sur-TCR-Tg mice derived from 3 separate founders. The leukemias expressed the Sur-TCR and signaled in response to the Sur20-28 peptide. In preleukemic mice, we observed increased cycling of double-negative thymocytes expressing the Sur-TCR and increased nuclear translocation of nuclear factor of activated T cells, consistent with TCR signaling induced by survivin expression in the murine thymus. β2M(-/-) Sur-TCR-Tg mice, which cannot effectively present survivin peptides on class I major histocompatibility complex, had significantly diminished rates of leukemia. We conclude that TCR signaling during the early stages of thymopoiesis mediates an oncogenic signal, and therefore expression of signaling receptors on developing thymocytes with specificity for TAAs expressed in the thymus could pose a risk for neoplasia, independent of insertional mutagenesis.
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http://dx.doi.org/10.1182/blood-2014-10-609271DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4424417PMC
May 2015

Landscape of insertion polymorphisms in the human genome.

Genome Biol Evol 2015 Mar 4;7(4):960-8. Epub 2015 Mar 4.

Genetics Branch, National Cancer Institute, National Institutes of Health, Bethesda, Maryland

Nucleotide substitutions, small (<50 bp) insertions or deletions (indels), and large (>50 bp) deletions are well-known causes of genetic variation within the human genome. We recently reported a previously unrecognized form of polymorphic insertions, termed templated sequence insertion polymorphism (TSIP), in which the inserted sequence was templated from a distant genomic region, and was inserted in the genome through reverse transcription of an RNA intermediate. TSIPs can be grouped into two classes based on nucleotide sequence features at the insertion junctions; class 1 TSIPs show target site duplication, polyadenylation, and preference for insertion at a 5'-TTTT/A-3' sequence, suggesting a LINE-1 based insertion mechanism, whereas class 2 TSIPs show features consistent with repair of a DNA double strand break by nonhomologous end joining. To gain a more complete picture of TSIPs throughout the human population, we evaluated whole-genome sequence from 52 individuals, and identified 171 TSIPs. Most individuals had 25-30 TSIPs, and common (present in >20% of individuals) TSIPs were found in individuals throughout the world, whereas rare TSIPs tended to cluster in specific geographic regions. The number of rare TSIPs was greater than the number of common TSIPs, suggesting that TSIP generation is an ongoing process. Intriguingly, mitochondrial sequences were a frequent template for class 2 insertions, used more commonly than any nuclear chromosome. Similar to single nucleotide polymorphisms and indels, we suspect that these TSIPs may be important for the generation of human diversity and genetic diseases, and can be useful in tracking historical migration of populations.
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http://dx.doi.org/10.1093/gbe/evv043DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4419790PMC
March 2015

DNA-damage-induced differentiation of leukaemic cells as an anti-cancer barrier.

Nature 2014 Oct 27;514(7520):107-11. Epub 2014 Jul 27.

Laboratory of Genome Integrity, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20892, USA.

Self-renewal is the hallmark feature both of normal stem cells and cancer stem cells. Since the regenerative capacity of normal haematopoietic stem cells is limited by the accumulation of reactive oxygen species and DNA double-strand breaks, we speculated that DNA damage might also constrain leukaemic self-renewal and malignant haematopoiesis. Here we show that the histone methyl-transferase MLL4, a suppressor of B-cell lymphoma, is required for stem-cell activity and an aggressive form of acute myeloid leukaemia harbouring the MLL-AF9 oncogene. Deletion of MLL4 enhances myelopoiesis and myeloid differentiation of leukaemic blasts, which protects mice from death related to acute myeloid leukaemia. MLL4 exerts its function by regulating transcriptional programs associated with the antioxidant response. Addition of reactive oxygen species scavengers or ectopic expression of FOXO3 protects MLL4(-/-) MLL-AF9 cells from DNA damage and inhibits myeloid maturation. Similar to MLL4 deficiency, loss of ATM or BRCA1 sensitizes transformed cells to differentiation, suggesting that myeloid differentiation is promoted by loss of genome integrity. Indeed, we show that restriction-enzyme-induced double-strand breaks are sufficient to induce differentiation of MLL-AF9 blasts, which requires cyclin-dependent kinase inhibitor p21(Cip1) (Cdkn1a) activity. In summary, we have uncovered an unexpected tumour-promoting role of genome guardians in enforcing the oncogene-induced differentiation blockade in acute myeloid leukaemia.
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http://dx.doi.org/10.1038/nature13483DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4410707PMC
October 2014

Repair of DNA double-strand breaks by templated nucleotide sequence insertions derived from distant regions of the genome.

Proc Natl Acad Sci U S A 2014 May 12;111(21):7729-34. Epub 2014 May 12.

Genetics Branch, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892; and

We used the I-SceI endonuclease to produce DNA double-strand breaks (DSBs) and observed that a fraction of these DSBs were repaired by insertion of sequences, which we termed "templated sequence insertions" (TSIs), derived from distant regions of the genome. These TSIs were derived from genic, retrotransposon, or telomere sequences and were not deleted from the donor site in the genome, leading to the hypothesis that they were derived from reverse-transcribed RNA. Cotransfection of RNA and an I-SceI expression vector demonstrated insertion of RNA-derived sequences at the DNA-DSB site, and TSIs were suppressed by reverse-transcriptase inhibitors. Both observations support the hypothesis that TSIs were derived from RNA templates. In addition, similar insertions were detected at sites of DNA DSBs induced by transcription activator-like effector nuclease proteins. Whole-genome sequencing of myeloma cell lines revealed additional TSIs, demonstrating that repair of DNA DSBs via insertion was not restricted to experimentally produced DNA DSBs. Analysis of publicly available databases revealed that many of these TSIs are polymorphic in the human genome. Taken together, these results indicate that insertional events should be considered as alternatives to gross chromosomal rearrangements in the interpretation of whole-genome sequence data and that this mutagenic form of DNA repair may play a role in genetic disease, exon shuffling, and mammalian evolution.
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http://dx.doi.org/10.1073/pnas.1321889111DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4040595PMC
May 2014

NUP98-PHF23 is a chromatin-modifying oncoprotein that causes a wide array of leukemias sensitive to inhibition of PHD histone reader function.

Cancer Discov 2014 May 17;4(5):564-77. Epub 2014 Feb 17.

1Genetics Branch, Center for Cancer Research, National Cancer Institute, NIH, Bethesda; 2Department of Pathology, Johns Hopkins University, Baltimore, Maryland; 3Department of Biomolecular Chemistry, University of Wisconsin, Madison, Wisconsin; and 4Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill School of Medicine, Chapel Hill, North Carolina.

In this report, we show that expression of a NUP98-PHF23 (NP23) fusion, associated with acute myeloid leukemia (AML) in humans, leads to myeloid, erythroid, T-cell, and B-cell leukemia in mice. The leukemic and preleukemic tissues display a stem cell-like expression signature, including Hoxa, Hoxb, and Meis1 genes. The PHF23 plant homeodomain (PHD) motif is known to bind to H3K4me3 residues, and chromatin immunoprecipitation experiments demonstrated that the NP23 protein binds to chromatin at a specific subset of H3K4me3 sites, including at Hoxa, Hoxb, and Meis1. Treatment of NP23 cells with disulfiram, which inhibits the binding of PHD motifs to H3K4me3, rapidly and selectively killed NP23-expressing myeloblasts; cell death was preceded by decreased expression of Hoxa, Hoxb, and Meis1. Furthermore, AML driven by a related fusion gene, NUP98-JARID1A (NJL), was also sensitive to disulfiram. Thus, the NP23 mouse provides a platform to evaluate compounds that disrupt binding of oncogenic PHD proteins to H3K4me3.
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http://dx.doi.org/10.1158/2159-8290.CD-13-0419DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4018760PMC
May 2014

Age-related epigenetic drift in the pathogenesis of MDS and AML.

Genome Res 2014 Apr 10;24(4):580-91. Epub 2014 Jan 10.

Fels Institute for Cancer Research and Molecular Biology, Temple University, Philadelphia, Pennsylvania 19140, USA;

The myelodysplastic syndrome (MDS) is a clonal hematologic disorder that frequently evolves to acute myeloid leukemia (AML). Its pathogenesis remains unclear, but mutations in epigenetic modifiers are common and the disease often responds to DNA methylation inhibitors. We analyzed DNA methylation in the bone marrow and spleen in two mouse models of MDS/AML, the NUP98-HOXD13 (NHD13) mouse and the RUNX1 mutant mouse model. Methylation array analysis showed an average of 512/3445 (14.9%) genes hypermethylated in NHD13 MDS, and 331 (9.6%) genes hypermethylated in RUNX1 MDS. Thirty-two percent of genes in common between the two models (2/3 NHD13 mice and 2/3 RUNX1 mice) were also hypermethylated in at least two of 19 human MDS samples. Detailed analysis of 41 genes in mice showed progressive drift in DNA methylation from young to old normal bone marrow and spleen; to MDS, where we detected accelerated age-related methylation; and finally to AML, which markedly extends DNA methylation abnormalities. Most of these genes showed similar patterns in human MDS and AML. Repeat element hypomethylation was rare in MDS but marked the transition to AML in some cases. Our data show consistency in patterns of aberrant DNA methylation in human and mouse MDS and suggest that epigenetically, MDS displays an accelerated aging phenotype.
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http://dx.doi.org/10.1101/gr.157529.113DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3975058PMC
April 2014