Publications by authors named "Richard Koche"

66 Publications

Therapeutic Efficacy of Combined JAK1/2, Pan-PIM, and CDK4/6 Inhibition in Myeloproliferative Neoplasms.

Clin Cancer Res 2021 Mar 29. Epub 2021 Mar 29.

Human Oncology and Pathogenesis Program, Memorial Sloan Kettering Cancer Center

Purpose: The JAK1/2 inhibitor ruxolitinib has demonstrated significant benefits for patients with Myeloproliferative Neoplasms (MPNs). However, patients often lose response to ruxolitinib or suffer disease progression despite therapy with ruxolitinib. These observations have prompted efforts to devise treatment strategies to improve therapeutic efficacy in combination with ruxolitinib therapy. Activation of JAK-STAT signaling results in dysregulation of key downstream pathways, notably increased expression of cell cycle mediators including CDC25A and the PIM kinases.

Experimental Design: Given the involvement of cell cycle mediators in MPNs, we sought to examine the efficacy of therapy combining ruxolitinib with a CDK4/6 inhibitor (LEE011) and a PIM kinase inhibitor (PIM447). We utilized JAK2 mutant cell lines, murine models, and primary MPN patient samples for these studies.

Results: Exposure of JAK2 mutant cell lines to the triple combination of ruxolitinib, LEE011 and PIM447 resulted in expected on-target pharmacodynamic effects, as well as increased apoptosis and a decrease in the proportion of cells in S-phase, compared with ruxolitinib. As compared to ruxolitinib monotherapy, combination therapy led to reductions in spleen and liver size, reduction of bone marrow reticulin fibrosis, improved overall survival, and elimination of disease-initiating capacity of treated bone marrow, in murine models of MPN. Finally, the triple combination reduced colony formation capacity of primary MPN patient samples to a greater extent than ruxolitinib.

Conclusion: The triple combination of ruxolitinib, LEE011 and PIM447 represents a promising therapeutic strategy with the potential to increase therapeutic responses in MPN patients.
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http://dx.doi.org/10.1158/1078-0432.CCR-20-4898DOI Listing
March 2021

Complex-dependent histone acetyltransferase activity of KAT8 determines its role in transcription and cellular homeostasis.

Mol Cell 2021 Apr 2;81(8):1749-1765.e8. Epub 2021 Mar 2.

Cell Biology Program and Center for Epigenetics, Memorial Sloan Kettering Cancer Center, New York, NY 10065, USA; Biotech Research and Innovation Centre (BRIC), University of Copenhagen, Copenhagen N 2200, Denmark; The Novo Nordisk Foundation Center for Stem Cell Biology (Danstem), University of Copenhagen, Copenhagen N 2200, Denmark. Electronic address:

Acetylation of lysine 16 on histone H4 (H4K16ac) is catalyzed by histone acetyltransferase KAT8 and can prevent chromatin compaction in vitro. Although extensively studied in Drosophila, the functions of H4K16ac and two KAT8-containing protein complexes (NSL and MSL) are not well understood in mammals. Here, we demonstrate a surprising complex-dependent activity of KAT8: it catalyzes H4K5ac and H4K8ac as part of the NSL complex, whereas it catalyzes the bulk of H4K16ac as part of the MSL complex. Furthermore, we show that MSL complex proteins and H4K16ac are not required for cell proliferation and chromatin accessibility, whereas the NSL complex is essential for cell survival, as it stimulates transcription initiation at the promoters of housekeeping genes. In summary, we show that KAT8 switches catalytic activity and function depending on its associated proteins and that, when in the NSL complex, it catalyzes H4K5ac and H4K8ac required for the expression of essential genes.
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http://dx.doi.org/10.1016/j.molcel.2021.02.012DOI Listing
April 2021

LKB1/STK11 is a tumor suppressor in the progression of myeloproliferative neoplasms.

Cancer Discov 2021 Feb 12. Epub 2021 Feb 12.

Division of Experimental Hematology, St. Jude Children's Research Hospital

The myeloproliferative neoplasms frequently progress to blast phase disease, an aggressive form of acute myeloid leukemia. To identify genes that suppress disease progression, we performed a focused CRISPR/Cas9 screen and discovered that depletion of LKB1/Stk11 led to enhanced in vitro self-renewal of murine MPN cells. Deletion of Stk11 in a mouse MPN model caused rapid lethality with enhanced fibrosis, osteosclerosis and an accumulation immature cells in the bone marrow, as well as enhanced engraftment of primary human MPN cells in vivo. LKB1 loss was associated with increased mitochondrial ROS and stabilization of HIF1a, and downregulation of LKB1 and increased levels of HIF1a were observed in human blast phase MPN specimens. Of note, we observed strong concordance of pathways that were enriched in murine MPN cells with LKB1 loss with those enriched in blast phase MPN patient specimens, supporting the conclusion that STK11 is a tumor suppressor in the MPNs.
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http://dx.doi.org/10.1158/2159-8290.CD-20-1353DOI Listing
February 2021

A gene-environment-induced epigenetic program initiates tumorigenesis.

Nature 2021 02 3;590(7847):642-648. Epub 2021 Feb 3.

Cancer Biology and Genetics Program, Memorial Sloan Kettering Cancer Center, New York, NY, USA.

Tissue damage increases the risk of cancer through poorly understood mechanisms. In mouse models of pancreatic cancer, pancreatitis associated with tissue injury collaborates with activating mutations in the Kras oncogene to markedly accelerate the formation of early neoplastic lesions and, ultimately, adenocarcinoma. Here, by integrating genomics, single-cell chromatin assays and spatiotemporally controlled functional perturbations in autochthonous mouse models, we show that the combination of Kras mutation and tissue damage promotes a unique chromatin state in the pancreatic epithelium that distinguishes neoplastic transformation from normal regeneration and is selected for throughout malignant evolution. This cancer-associated epigenetic state emerges within 48 hours of pancreatic injury, and involves an 'acinar-to-neoplasia' chromatin switch that contributes to the early dysregulation of genes that define human pancreatic cancer. Among the factors that are most rapidly activated after tissue damage in the pre-malignant pancreatic epithelium is the alarmin cytokine interleukin 33, which recapitulates the effects of injury in cooperating with mutant Kras to unleash the epigenetic remodelling program of early neoplasia and neoplastic transformation. Collectively, our study demonstrates how gene-environment interactions can rapidly produce gene-regulatory programs that dictate early neoplastic commitment, and provides a molecular framework for understanding the interplay between genetic and environmental cues in the initiation of cancer.
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http://dx.doi.org/10.1038/s41586-020-03147-xDOI Listing
February 2021

Convergent organization of aberrant MYB complex controls oncogenic gene expression in acute myeloid leukemia.

Elife 2021 Feb 2;10. Epub 2021 Feb 2.

Molecular Pharmacology Program, Sloan Kettering Institute, New York, United States.

Dysregulated gene expression contributes to most prevalent features in human cancers. Here, we show that most subtypes of acute myeloid leukemia (AML) depend on the aberrant assembly of MYB transcriptional co-activator complex. By rapid and selective peptidomimetic interference with the binding of CBP/P300 to MYB, but not CREB or MLL1, we find that the leukemic functions of MYB are mediated by CBP/P300 co-activation of a distinct set of transcription factor complexes. These MYB complexes assemble aberrantly with LYL1, E2A, C/EBP family members, LMO2, and SATB1. They are organized convergently in genetically diverse subtypes of AML and are at least in part associated with inappropriate transcription factor co-expression. Peptidomimetic remodeling of oncogenic MYB complexes is accompanied by specific proteolysis and dynamic redistribution of CBP/P300 with alternative transcription factors such as RUNX1 to induce myeloid differentiation and apoptosis. Thus, aberrant assembly and sequestration of MYB:CBP/P300 complexes provide a unifying mechanism of oncogenic gene expression in AML. This work establishes a compelling strategy for their pharmacologic reprogramming and therapeutic targeting for diverse leukemias and possibly other human cancers caused by dysregulated gene control.
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http://dx.doi.org/10.7554/eLife.65905DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7886351PMC
February 2021

SWI/SNF Complex Mutations Promote Thyroid Tumor Progression and Insensitivity to Redifferentiation Therapies.

Cancer Discov 2020 Dec 14. Epub 2020 Dec 14.

Human Oncology and Pathogenesis Program, Memorial Sloan Kettering Cancer Center, New York, New York.

Mutations of subunits of the SWI/SNF chromatin remodeling complexes occur commonly in cancers of different lineages, including advanced thyroid cancers. Here we show that thyroid-specific loss of , or in mouse BRAF-mutant tumors promotes disease progression and decreased survival, associated with lesion-specific effects on chromatin accessibility and differentiation. As compared with normal thyrocytes, BRAF-mutant mouse papillary thyroid cancers have decreased lineage transcription factor expression and accessibility to their target DNA binding sites, leading to impairment of thyroid-differentiated gene expression and radioiodine incorporation, which is rescued by MAPK inhibition. Loss of individual SWI/SNF subunits in BRAF tumors leads to a repressive chromatin state that cannot be reversed by MAPK pathway blockade, rendering them insensitive to its redifferentiation effects. Our results show that SWI/SNF complexes are central to the maintenance of differentiated function in thyroid cancers, and their loss confers radioiodine refractoriness and resistance to MAPK inhibitor-based redifferentiation therapies. SIGNIFICANCE: Reprogramming cancer differentiation confers therapeutic benefit in various disease contexts. Oncogenic BRAF silences genes required for radioiodine responsiveness in thyroid cancer. Mutations in SWI/SNF genes result in loss of chromatin accessibility at thyroid lineage specification genes in -mutant thyroid tumors, rendering them insensitive to the redifferentiation effects of MAPK blockade.
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http://dx.doi.org/10.1158/2159-8290.CD-20-0735DOI Listing
December 2020

Enhancer hijacking determines extrachromosomal circular MYCN amplicon architecture in neuroblastoma.

Nat Commun 2020 11 16;11(1):5823. Epub 2020 Nov 16.

Berlin Institute of Health, Anna-Louisa-Karsch-Str. 2, 10178, Berlin, Germany.

MYCN amplification drives one in six cases of neuroblastoma. The supernumerary gene copies are commonly found on highly rearranged, extrachromosomal circular DNA (ecDNA). The exact amplicon structure has not been described thus far and the functional relevance of its rearrangements is unknown. Here, we analyze the MYCN amplicon structure using short-read and Nanopore sequencing and its chromatin landscape using ChIP-seq, ATAC-seq and Hi-C. This reveals two distinct classes of amplicons which explain the regulatory requirements for MYCN overexpression. The first class always co-amplifies a proximal enhancer driven by the noradrenergic core regulatory circuit (CRC). The second class of MYCN amplicons is characterized by high structural complexity, lacks key local enhancers, and instead contains distal chromosomal fragments harboring CRC-driven enhancers. Thus, ectopic enhancer hijacking can compensate for the loss of local gene regulatory elements and explains a large component of the structural diversity observed in MYCN amplification.
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http://dx.doi.org/10.1038/s41467-020-19452-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7669906PMC
November 2020

FOXA1 Mutations Reveal Distinct Chromatin Profiles and Influence Therapeutic Response in Breast Cancer.

Cancer Cell 2020 10 3;38(4):534-550.e9. Epub 2020 Sep 3.

Human Oncology & Pathogenesis Program (HOPP), Memorial Sloan Kettering Cancer Center, New York, NY 10065, USA; Department of Pathology, Memorial Sloan Kettering Cancer Center, New York, NY 10065, USA. Electronic address:

Mutations in the pioneer transcription factor FOXA1 are a hallmark of estrogen receptor-positive (ER) breast cancers. Examining FOXA1 in ∼5,000 breast cancer patients identifies several hotspot mutations in the Wing2 region and a breast cancer-specific mutation SY242CS, located in the third β strand. Using a clinico-genomically curated cohort, together with breast cancer models, we find that FOXA1 mutations associate with a lower response to aromatase inhibitors. Mechanistically, Wing2 mutations display increased chromatin binding at ER loci upon estrogen stimulation, and an enhanced ER-mediated transcription without changes in chromatin accessibility. In contrast, SY242CS shows neomorphic properties that include the ability to open distinct chromatin regions and activate an alternative cistrome and transcriptome. Structural modeling predicts that SY242CS confers a conformational change that mediates stable binding to a non-canonical DNA motif. Taken together, our results provide insights into how FOXA1 mutations perturb its function to dictate cancer progression and therapeutic response.
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http://dx.doi.org/10.1016/j.ccell.2020.08.003DOI Listing
October 2020

Plasmacytoid dendritic cell expansion defines a distinct subset of RUNX1-mutated acute myeloid leukemia.

Blood 2021 Mar;137(10):1377-1391

Human Oncology and Pathogenesis Program, Molecular Cancer Medicine Service.

Plasmacytoid dendritic cells (pDCs) are the principal natural type I interferon-producing dendritic cells. Neoplastic expansion of pDCs and pDC precursors leads to blastic plasmacytoid dendritic cell neoplasm (BPDCN), and clonal expansion of mature pDCs has been described in chronic myelomonocytic leukemia. The role of pDC expansion in acute myeloid leukemia (AML) is poorly studied. Here, we characterize patients with AML with pDC expansion (pDC-AML), which we observe in ∼5% of AML cases. pDC-AMLs often possess cross-lineage antigen expression and have adverse risk stratification with poor outcome. RUNX1 mutations are the most common somatic alterations in pDC-AML (>70%) and are much more common than in AML without pDC expansion and BPDCN. We demonstrate that pDCs are clonally related to, as well as originate from, leukemic blasts in pDC-AML. We further demonstrate that leukemic blasts from RUNX1-mutated AML upregulate a pDC transcriptional program, poising the cells toward pDC differentiation and expansion. Finally, tagraxofusp, a targeted therapy directed to CD123, reduces leukemic burden and eliminates pDCs in a patient-derived xenograft model. In conclusion, pDC-AML is characterized by a high frequency of RUNX1 mutations and increased expression of a pDC transcriptional program. CD123 targeting represents a potential treatment approach for pDC-AML.
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http://dx.doi.org/10.1182/blood.2020007897DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7955409PMC
March 2021

PRMT5 Inhibition Modulates E2F1 Methylation and Gene-Regulatory Networks Leading to Therapeutic Efficacy in JAK2-Mutant MPN.

Cancer Discov 2020 Nov 15;10(11):1742-1757. Epub 2020 Jul 15.

Molecular Cancer Medicine Service, Human Oncology and Pathogenesis Program, Memorial Sloan Kettering Cancer Center, New York, New York.

We investigated the role of PRMT5 in myeloproliferative neoplasm (MPN) pathogenesis and aimed to elucidate key PRMT5 targets contributing to MPN maintenance. PRMT5 is overexpressed in primary MPN cells, and PRMT5 inhibition potently reduced MPN cell proliferation . PRMT5 inhibition was efficacious at reversing elevated hematocrit, leukocytosis, and splenomegaly in a model of JAK2 polycythemia vera and leukocyte and platelet counts, hepatosplenomegaly, and fibrosis in the MPL model of myelofibrosis. Dual targeting of JAK and PRMT5 was superior to JAK or PRMT5 inhibitor monotherapy, further decreasing elevated counts and extramedullary hematopoiesis PRMT5 inhibition reduced expression of E2F targets and altered the methylation status of E2F1 leading to attenuated DNA damage repair, cell-cycle arrest, and increased apoptosis. Our data link PRMT5 to E2F1 regulatory function and MPN cell survival and provide a strong mechanistic rationale for clinical trials of PRMT5 inhibitors in MPN. SIGNIFICANCE: Expression of PRMT5 and E2F targets is increased in JAK2 MPN. Pharmacologic inhibition of PRMT5 alters the methylation status of E2F1 and shows efficacy in JAK2/MPL MPN models and primary samples. PRMT5 represents a potential novel therapeutic target for MPN, which is now being clinically evaluated..
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http://dx.doi.org/10.1158/2159-8290.CD-20-0026DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7642059PMC
November 2020

L1CAM defines the regenerative origin of metastasis-initiating cells in colorectal cancer.

Nat Cancer 2020 Jan 13;1(1):28-45. Epub 2020 Jan 13.

Cancer Biology and Genetics Program, Sloan Kettering Institute, Memorial Sloan Kettering Cancer Center, New York, NY, USA.

Metastasis-initiating cells with stem-like properties drive cancer lethality, yet their origins and relationship to primary-tumor-initiating stem cells are not known. We show that L1CAM cells in human colorectal cancer (CRC) have metastasis-initiating capacity, and we define their relationship to tissue regeneration. L1CAM is not expressed in the homeostatic intestinal epithelium, but is induced and required for epithelial regeneration following colitis and in CRC organoid growth. By using human tissues and mouse models, we show that L1CAM is dispensable for adenoma initiation but required for orthotopic carcinoma propagation, liver metastatic colonization and chemoresistance. L1CAM cells partially overlap with LGR5 stem-like cells in human CRC organoids. Disruption of intercellular epithelial contacts causes E-cadherin-REST transcriptional derepression of L1CAM, switching chemoresistant CRC progenitors from an L1CAM to an L1CAM state. Thus, L1CAM dependency emerges in regenerative intestinal cells when epithelial integrity is lost, a phenotype of wound healing deployed in metastasis-initiating cells.
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http://dx.doi.org/10.1038/s43018-019-0006-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7351134PMC
January 2020

Mutant FOXL2 Hijacks SMAD4 and SMAD2/3 to Drive Adult Granulosa Cell Tumors.

Cancer Res 2020 09 8;80(17):3466-3479. Epub 2020 Jul 8.

Biotech Research and Innovation Centre (BRIC), University of Copenhagen, Copenhagen N, Denmark.

The mutant protein FOXL2 is expressed in at least 95% of adult-type ovarian granulosa cell tumors (AGCT) and is considered to be a driver of oncogenesis in this disease. However, the molecular mechanism by which FOXL2 contributes to tumorigenesis is not known. Here, we show that mutant FOXL2 acquires the ability to bind SMAD4, forming a FOXL2/SMAD4/SMAD2/3 complex that binds a novel hybrid DNA motif AGHCAHAA, unique to the FOXL2 mutant. This binding induced an enhancer-like chromatin state, leading to transcription of nearby genes, many of which are characteristic of epithelial-to-mesenchymal transition. FOXL2 also bound hybrid loci in primary AGCT. Ablation of SMAD4 or SMAD2/3 resulted in strong reduction of FOXL2 binding at hybrid sites and decreased expression of associated genes. Accordingly, inhibition of TGFβ mitigated the transcriptional effect of FOXL2. Our results provide mechanistic insight into AGCT pathogenesis, identifying FOXL2 and its interaction with SMAD4 as potential therapeutic targets to this condition. SIGNIFICANCE: FOXL2 hijacks SMAD4 and leads to the expression of genes involved in EMT, stemness, and oncogenesis in AGCT, making FOXL2 and the TGFβ pathway therapeutic targets in this condition. GRAPHICAL ABSTRACT: http://cancerres.aacrjournals.org/content/canres/80/17/3466/F1.large.jpg.
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http://dx.doi.org/10.1158/0008-5472.CAN-20-0259DOI Listing
September 2020

Leukemia Cell of Origin Influences Apoptotic Priming and Sensitivity to LSD1 Inhibition.

Cancer Discov 2020 Oct 30;10(10):1500-1513. Epub 2020 Jun 30.

Department of Pediatric Oncology, Dana-Farber Cancer Institute and Division of Hematology/Oncology, Boston Children's Hospital, Harvard Medical School, Boston, Massachusetts.

The cell of origin of oncogenic transformation is a determinant of therapeutic sensitivity, but the mechanisms governing cell-of-origin-driven differences in therapeutic response have not been delineated. Leukemias initiating in hematopoietic stem cells (HSC) are less sensitive to chemotherapy and highly express the transcription factor (EVI1) compared with leukemias derived from myeloid progenitors. Here, we compared leukemias initiated in either HSCs or myeloid progenitors to reveal a novel function for EVI1 in modulating p53 protein abundance and activity. HSC-derived leukemias exhibit decreased apoptotic priming, attenuated p53 transcriptional output, and resistance to lysine-specific demethylase 1 (LSD1) inhibitors in addition to classical genotoxic stresses. p53 loss of function in progenitor-derived leukemias induces resistance to LSD1 inhibition, and EVI1 leukemias are sensitized to LSD1 inhibition by venetoclax. Our findings demonstrate a role for in p53 wild-type cancers in reducing p53 function and provide a strategy to circumvent drug resistance in chemoresistant acute myeloid leukemia. SIGNIFICANCE: We demonstrate that the cell of origin of leukemia initiation influences p53 activity and dictates therapeutic sensitivity to pharmacologic LSD1 inhibitors via the transcription factor EVI1. We show that drug resistance could be overcome in HSC-derived leukemias by combining LSD1 inhibition with venetoclax...
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http://dx.doi.org/10.1158/2159-8290.CD-19-1469DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7584353PMC
October 2020

Synergistic targeting of FLT3 mutations in AML via combined menin-MLL and FLT3 inhibition.

Blood 2020 11;136(21):2442-2456

Department of Hematology, Medical Oncology, and Pulmonary Medicine, University Medical Center, Johannes Gutenberg-University, Mainz, Germany.

The interaction of menin (MEN1) and MLL (MLL1, KMT2A) is a dependency and provides a potential opportunity for treatment of NPM1-mutant (NPM1mut) and MLL-rearranged (MLL-r) leukemias. Concomitant activating driver mutations in the gene encoding the tyrosine kinase FLT3 occur in both leukemias and are particularly common in the NPM1mut subtype. In this study, transcriptional profiling after pharmacological inhibition of the menin-MLL complex revealed specific changes in gene expression, with downregulation of the MEIS1 transcription factor and its transcriptional target gene FLT3 being the most pronounced. Combining menin-MLL inhibition with specific small-molecule kinase inhibitors of FLT3 phosphorylation resulted in a significantly superior reduction of phosphorylated FLT3 and transcriptional suppression of genes downstream of FLT3 signaling. The drug combination induced synergistic inhibition of proliferation, as well as enhanced apoptosis, compared with single-drug treatment in models of human and murine NPM1mut and MLL-r leukemias harboring an FLT3 mutation. Primary acute myeloid leukemia (AML) cells harvested from patients with NPM1mutFLT3mut AML showed significantly better responses to combined menin and FLT3 inhibition than to single-drug or vehicle control treatment, whereas AML cells with wild-type NPM1, MLL, and FLT3 were not affected by either of the 2 drugs. In vivo treatment of leukemic animals with MLL-r FLT3mut leukemia reduced leukemia burden significantly and prolonged survival compared with results in the single-drug and vehicle control groups. Our data suggest that combined menin-MLL and FLT3 inhibition represents a novel and promising therapeutic strategy for patients with NPM1mut or MLL-r leukemia and concurrent FLT3 mutation.
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http://dx.doi.org/10.1182/blood.2020005037DOI Listing
November 2020

Loss of H3K36 Methyltransferase SETD2 Impairs V(D)J Recombination during Lymphoid Development.

iScience 2020 Mar 27;23(3):100941. Epub 2020 Feb 27.

Department of Pediatric Oncology, Dana Farber Cancer Institute, and Division of Hematology/Oncology, Boston Children's Hospital, 450 Brookline Avenue, Boston, MA 02215-5450, USA. Electronic address:

Repair of DNA double-stranded breaks (DSBs) during lymphocyte development is essential for V(D)J recombination and forms the basis of immunoglobulin variable region diversity. Understanding of this process in lymphogenesis has historically been centered on the study of RAG1/2 recombinases and a set of classical non-homologous end-joining factors. Much less has been reported regarding the role of chromatin modifications on this process. Here, we show a role for the non-redundant histone H3 lysine methyltransferase, Setd2, and its modification of lysine-36 trimethylation (H3K36me3), in the processing and joining of DNA ends during V(D)J recombination. Loss leads to mis-repair of Rag-induced DNA DSBs, especially when combined with loss of Atm kinase activity. Furthermore, loss reduces immune repertoire and a severe block in lymphogenesis as well as causes post-mitotic neuronal apoptosis. Together, these studies are suggestive of an important role of Setd2/H3K36me3 in these two mammalian developmental processes that are influenced by double-stranded break repair.
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http://dx.doi.org/10.1016/j.isci.2020.100941DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7066224PMC
March 2020

ARID1A determines luminal identity and therapeutic response in estrogen-receptor-positive breast cancer.

Nat Genet 2020 02 13;52(2):198-207. Epub 2020 Jan 13.

Human Oncology and Pathogenesis Program, Memorial Sloan Kettering Cancer Center, New York, NY, USA.

Mutations in ARID1A, a subunit of the SWI/SNF chromatin remodeling complex, are the most common alterations of the SWI/SNF complex in estrogen-receptor-positive (ER) breast cancer. We identify that ARID1A inactivating mutations are present at a high frequency in advanced endocrine-resistant ER breast cancer. An epigenome CRISPR-CAS9 knockout (KO) screen identifies ARID1A as the top candidate whose loss determines resistance to the ER degrader fulvestrant. ARID1A inactivation in cells and in patients leads to resistance to ER degraders by facilitating a switch from ER-dependent luminal cells to ER-independent basal-like cells. Cellular plasticity is mediated by loss of ARID1A-dependent SWI/SNF complex targeting to genomic sites of the luminal lineage-determining transcription factors including ER, forkhead box protein A1 (FOXA1) and GATA-binding factor 3 (GATA3). ARID1A also regulates genome-wide ER-FOXA1 chromatin interactions and ER-dependent transcription. Altogether, we uncover a critical role for ARID1A in maintaining luminal cell identity and endocrine therapeutic response in ER breast cancer.
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http://dx.doi.org/10.1038/s41588-019-0554-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7341683PMC
February 2020

Extrachromosomal circular DNA drives oncogenic genome remodeling in neuroblastoma.

Nat Genet 2020 01 16;52(1):29-34. Epub 2019 Dec 16.

Department of Pediatric Oncology/Hematology, Charité-Universitätsmedizin Berlin, Berlin, Germany.

Extrachromosomal circularization of DNA is an important genomic feature in cancer. However, the structure, composition and genome-wide frequency of extrachromosomal circular DNA have not yet been profiled extensively. Here, we combine genomic and transcriptomic approaches to describe the landscape of extrachromosomal circular DNA in neuroblastoma, a tumor arising in childhood from primitive cells of the sympathetic nervous system. Our analysis identifies and characterizes a wide catalog of somatically acquired and undescribed extrachromosomal circular DNAs. Moreover, we find that extrachromosomal circular DNAs are an unanticipated major source of somatic rearrangements, contributing to oncogenic remodeling through chimeric circularization and reintegration of circular DNA into the linear genome. Cancer-causing lesions can emerge out of circle-derived rearrangements and are associated with adverse clinical outcome. It is highly probable that circle-derived rearrangements represent an ongoing mutagenic process. Thus, extrachromosomal circular DNAs represent a multihit mutagenic process, with important functional and clinical implications for the origins of genomic remodeling in cancer.
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http://dx.doi.org/10.1038/s41588-019-0547-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7008131PMC
January 2020

α-Ketoglutarate links p53 to cell fate during tumour suppression.

Nature 2019 09 18;573(7775):595-599. Epub 2019 Sep 18.

Cancer Biology and Genetics Program, Memorial Sloan Kettering Cancer Center, New York, NY, USA.

The tumour suppressor TP53 is mutated in the majority of human cancers, and in over 70% of pancreatic ductal adenocarcinoma (PDAC). Wild-type p53 accumulates in response to cellular stress, and regulates gene expression to alter cell fate and prevent tumour development. Wild-type p53 is also known to modulate cellular metabolic pathways, although p53-dependent metabolic alterations that constrain cancer progression remain poorly understood. Here we find that p53 remodels cancer-cell metabolism to enforce changes in chromatin and gene expression that favour a premalignant cell fate. Restoring p53 function in cancer cells derived from KRAS-mutant mouse models of PDAC leads to the accumulation of α-ketoglutarate (αKG, also known as 2-oxoglutarate), a metabolite that also serves as an obligate substrate for a subset of chromatin-modifying enzymes. p53 induces transcriptional programs that are characteristic of premalignant differentiation, and this effect can be partially recapitulated by the addition of cell-permeable αKG. Increased levels of the αKG-dependent chromatin modification 5-hydroxymethylcytosine (5hmC) accompany the tumour-cell differentiation that is triggered by p53, whereas decreased 5hmC characterizes the transition from premalignant to de-differentiated malignant lesions that is associated with mutations in Trp53. Enforcing the accumulation of αKG in p53-deficient PDAC cells through the inhibition of oxoglutarate dehydrogenase-an enzyme of the tricarboxylic acid cycle-specifically results in increased 5hmC, tumour-cell differentiation and decreased tumour-cell fitness. Conversely, increasing the intracellular levels of succinate (a competitive inhibitor of αKG-dependent dioxygenases) blunts p53-driven tumour suppression. These data suggest that αKG is an effector of p53-mediated tumour suppression, and that the accumulation of αKG in p53-deficient tumours can drive tumour-cell differentiation and antagonize malignant progression.
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http://dx.doi.org/10.1038/s41586-019-1577-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6830448PMC
September 2019

Cohesin Members Stag1 and Stag2 Display Distinct Roles in Chromatin Accessibility and Topological Control of HSC Self-Renewal and Differentiation.

Cell Stem Cell 2019 11 5;25(5):682-696.e8. Epub 2019 Sep 5.

Human Oncology and Pathogenesis Program and Center for Hematologic Malignancies, Memorial Sloan Kettering Cancer Center, New York, NY 10065, USA; Department of Medicine, Leukemia Service, Memorial Sloan Kettering Cancer Center, New York, NY 10065, USA; Center for Epigenetics Research, Memorial Sloan Kettering Cancer Center, New York, NY 10065, USA. Electronic address:

Transcriptional regulators, including the cohesin complex member STAG2, are recurrently mutated in cancer. The role of STAG2 in gene regulation, hematopoiesis, and tumor suppression remains unresolved. We show that Stag2 deletion in hematopoietic stem and progenitor cells (HSPCs) results in altered hematopoietic function, increased self-renewal, and impaired differentiation. Chromatin immunoprecipitation (ChIP) sequencing revealed that, although Stag2 and Stag1 bind a shared set of genomic loci, a component of Stag2 binding sites is unoccupied by Stag1, even in Stag2-deficient HSPCs. Although concurrent loss of Stag2 and Stag1 abrogated hematopoiesis, Stag2 loss alone decreased chromatin accessibility and transcription of lineage-specification genes, including Ebf1 and Pax5, leading to increased self-renewal and reduced HSPC commitment to the B cell lineage. Our data illustrate a role for Stag2 in transformation and transcriptional dysregulation distinct from its shared role with Stag1 in chromosomal segregation.
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http://dx.doi.org/10.1016/j.stem.2019.08.003DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6842438PMC
November 2019

2-hydroxyglutarate inhibits MyoD-mediated differentiation by preventing H3K9 demethylation.

Proc Natl Acad Sci U S A 2019 06 10;116(26):12851-12856. Epub 2019 Jun 10.

Cancer Biology & Genetics Program, Memorial Sloan Kettering Cancer Center, New York, NY 10065;

Oncogenic IDH1/2 mutations produce 2-hydroxyglutarate (2HG), resulting in competitive inhibition of DNA and protein demethylation. IDH-mutant cancer cells show an inability to differentiate but whether 2HG accumulation is sufficient to perturb differentiation directed by lineage-specifying transcription factors is unknown. A MyoD-driven model was used to study the role of IDH mutations in the differentiation of mesenchymal cells. The presence of 2HG produced by oncogenic IDH2 blocks the ability of MyoD to drive differentiation into myotubes. DNA 5mC hypermethylation is dispensable while H3K9 hypermethylation is required for this differentiation block. IDH2-R172K mutation results in H3K9 hypermethylation and impaired accessibility at myogenic chromatin regions but does not result in genome-wide decrease in accessibility. The results demonstrate the ability of the oncometabolite 2HG to block transcription factor-mediated differentiation in a molecularly defined system.
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http://dx.doi.org/10.1073/pnas.1817662116DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6600935PMC
June 2019

Genome-scale screens identify JNK-JUN signaling as a barrier for pluripotency exit and endoderm differentiation.

Nat Genet 2019 06 20;51(6):999-1010. Epub 2019 May 20.

Sloan Kettering Institute, New York, NY, USA.

Human embryonic stem cells (ESCs) and human induced pluripotent stem cells hold great promise for cell-based therapies and drug discovery. However, homogeneous differentiation remains a major challenge, highlighting the need for understanding developmental mechanisms. We performed genome-scale CRISPR screens to uncover regulators of definitive endoderm (DE) differentiation, which unexpectedly uncovered five Jun N-terminal kinase (JNK)-JUN family genes as key barriers of DE differentiation. The JNK-JUN pathway does not act through directly inhibiting the DE enhancers. Instead, JUN co-occupies ESC enhancers with OCT4, NANOG, SMAD2 and SMAD3, and specifically inhibits the exit from the pluripotent state by impeding the decommissioning of ESC enhancers and inhibiting the reconfiguration of SMAD2 and SMAD3 chromatin binding from ESC to DE enhancers. Therefore, the JNK-JUN pathway safeguards pluripotency from precocious DE differentiation. Direct pharmacological inhibition of JNK significantly improves the efficiencies of generating DE and DE-derived pancreatic and lung progenitor cells, highlighting the potential of harnessing the knowledge from developmental studies for regenerative medicine.
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http://dx.doi.org/10.1038/s41588-019-0408-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6545159PMC
June 2019

A Gain-of-Function p53-Mutant Oncogene Promotes Cell Fate Plasticity and Myeloid Leukemia through the Pluripotency Factor FOXH1.

Cancer Discov 2019 07 8;9(7):962-979. Epub 2019 May 8.

Cancer Biology and Genetics Program, Sloan Kettering Institute, Memorial Sloan Kettering Cancer Center, New York, New York.

Mutations in the tumor suppressor gene are common in many cancer types, including the acute myeloid leukemia (AML) subtype known as complex karyotype AML (CK-AML). Here, we identify a gain-of-function (GOF) mutation that accelerates CK-AML initiation beyond p53 loss and, surprisingly, is required for disease maintenance. The mutation ( in humans) exhibits a neomorphic function by promoting aberrant self-renewal in leukemic cells, a phenotype that is present in hematopoietic stem and progenitor cells (HSPC) even prior to their transformation. We identify FOXH1 as a critical mediator of mutant p53 function that binds to and regulates stem cell-associated genes and transcriptional programs. Our results identify a context where mutant p53 acts as a bona fide oncogene that contributes to the pathogenesis of CK-AML and suggests a common biological theme for GOF in cancer. SIGNIFICANCE: Our study demonstrates how a GOF p53 mutant can hijack an embryonic transcription factor to promote aberrant self-renewal. In this context, mutant functions as an oncogene to both initiate and sustain myeloid leukemia and suggests a potential convergent activity of mutant across cancer types..
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http://dx.doi.org/10.1158/2159-8290.CD-18-1391DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6606372PMC
July 2019

PI3K Inhibition Activates SGK1 via a Feedback Loop to Promote Chromatin-Based Regulation of ER-Dependent Gene Expression.

Cell Rep 2019 04;27(1):294-306.e5

Human Oncology and Pathogenesis Program (HOPP), Memorial Sloan Kettering Cancer Center, 1275 York Avenue, Box 20, New York, NY 10065, USA; Department of Medicine, Memorial Sloan Kettering Cancer Center, New York, NY 10065, USA; Research & Development Oncology, AstraZeneca Pharmaceuticals, Gaithersburg, MD 20878, USA. Electronic address:

The PI3K pathway integrates extracellular stimuli to phosphorylate effectors such as AKT and serum-and-glucocorticoid-regulated kinase (SGK1). We have previously reported that the PI3K pathway regulates estrogen receptor (ER)-dependent transcription in breast cancer through the phosphorylation of the lysine methyltransferase KMT2D by AKT. Here, we show that PI3Kα inhibition, via a negative-feedback loop, activates SGK1 to promote chromatin-based regulation of ER-dependent transcription. PI3K/AKT inhibitors activate ER, which promotes SGK1 transcription through direct binding to its promoter. Elevated SGK1, in turn, phosphorylates KMT2D, suppressing its function, leading to a loss of methylation of lysine 4 on histone H3 (H3K4) and a repressive chromatin state at ER loci to attenuate ER activity. Thus, SGK1 regulates the chromatin landscape and ER-dependent transcription via the direct phosphorylation of KMT2D. These findings reveal an ER-SGK1-KMT2D signaling circuit aimed to attenuate ER response through a role for SGK1 to program chromatin and ER transcriptional output.
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http://dx.doi.org/10.1016/j.celrep.2019.02.111DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6503687PMC
April 2019

Targeting the CALR interactome in myeloproliferative neoplasms.

JCI Insight 2018 11 15;3(22). Epub 2018 Nov 15.

Human Oncology and Pathogenesis Program.

Mutations in the ER chaperone calreticulin (CALR) are common in myeloproliferative neoplasm (MPN) patients, activate the thrombopoietin receptor (MPL), and mediate constitutive JAK/STAT signaling. The mechanisms by which CALR mutations cause myeloid transformation are incompletely defined. We used mass spectrometry proteomics to identify CALR-mutant interacting proteins. Mutant CALR caused mislocalization of binding partners and increased recruitment of FLI1, ERP57, and CALR to the MPL promoter to enhance transcription. Consistent with a critical role for CALR-mediated JAK/STAT activation, we confirmed the efficacy of JAK2 inhibition on CALR-mutant cells in vitro and in vivo. Due to the altered interactome induced by CALR mutations, we hypothesized that CALR-mutant MPNs may be vulnerable to disruption of aberrant CALR protein complexes. A synthetic peptide designed to competitively inhibit the carboxy terminal of CALR specifically abrogated MPL/JAK/STAT signaling in cell lines and primary samples and improved the efficacy of JAK kinase inhibitors. These findings reveal what to our knowledge is a novel potential therapeutic approach for patients with CALR-mutant MPN.
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http://dx.doi.org/10.1172/jci.insight.122703DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6302938PMC
November 2018

HOXA9 Reprograms the Enhancer Landscape to Promote Leukemogenesis.

Cancer Cell 2018 10 27;34(4):643-658.e5. Epub 2018 Sep 27.

Department of Pathology and Laboratory Medicine, Indiana University School of Medicine, Indianapolis, IN 46202, USA. Electronic address:

Aberrant expression of HOXA9 is a prominent feature of acute leukemia driven by diverse oncogenes. Here we show that HOXA9 overexpression in myeloid and B progenitor cells leads to significant enhancer reorganizations with prominent emergence of leukemia-specific de novo enhancers. Alterations in the enhancer landscape lead to activation of an ectopic embryonic gene program. We show that HOXA9 functions as a pioneer factor at de novo enhancers and recruits CEBPα and the MLL3/MLL4 complex. Genetic deletion of MLL3/MLL4 blocks histone H3K4 methylation at de novo enhancers and inhibits HOXA9/MEIS1-mediated leukemogenesis in vivo. These results suggest that therapeutic targeting of HOXA9-dependent enhancer reorganization can be an effective therapeutic strategy in acute leukemia with HOXA9 overexpression.
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http://dx.doi.org/10.1016/j.ccell.2018.08.018DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6179449PMC
October 2018

Inhibition of MEK and ATR is effective in a B-cell acute lymphoblastic leukemia model driven by and activated .

Blood Adv 2018 10;2(19):2478-2490

Department of Pediatric Oncology, Dana-Farber Cancer Institute, Boston, MA.

Infant B-cell acute lymphoblastic leukemias (B-ALLs) that harbor rearrangements are associated with a poor prognosis. One important obstacle to progress for this patient population is the lack of immunocompetent models that faithfully recapitulate the short latency and aggressiveness of this disease. Recent whole-genome sequencing of B-ALL samples revealed a high frequency of activating mutations; however, single-agent targeting of downstream effectors of the pathway in these mutated r B-ALLs has demonstrated limited and nondurable antileukemic effects. Here, we demonstrate that the expression of activating mutant cooperates with to generate a highly aggressive serially transplantable B-ALL in mice. We used our novel mouse model to test the sensitivity of leukemia to small molecule inhibitors and found potent and synergistic preclinical efficacy of dual targeting of the Mek and Atr pathways in mouse- and patient-derived xenografts with both mutations in vivo, suggesting this combination as an attractive therapeutic opportunity that might be used to treat patients with these mutations. Our studies indicate that this mouse model of B-ALL is a powerful tool to explore the molecular and genetic pathogenesis of this disease subtype, as well as a preclinical discovery platform for novel therapeutic strategies.
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http://dx.doi.org/10.1182/bloodadvances.2018021592DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6177652PMC
October 2018

DICER1 Is Essential for Self-Renewal of Human Embryonic Stem Cells.

Stem Cell Reports 2018 09 23;11(3):616-625. Epub 2018 Aug 23.

Developmental Biology Program, Memorial Sloan Kettering Cancer Center, 1275 York Avenue, New York, NY 10065, USA. Electronic address:

MicroRNAs (miRNAs) are the effectors of a conserved gene-silencing system with broad roles in post-transcriptional regulation. Due to functional overlaps, assigning specific functions to individual miRNAs has been challenging. DICER1 cleaves pre-miRNA hairpins into mature miRNAs, and previously Dicer1 knockout mouse embryonic stem cells have been generated to study miRNA function in early mouse development. Here we report an essential requirement of DICER1 for the self-renewal of human embryonic stem cells (hESCs). Utilizing a conditional knockout approach, we found that DICER1 deletion led to increased death receptor-mediated apoptosis and failure of hESC self-renewal. We further devised a targeted miRNA screening strategy and uncovered essential pro-survival roles of members of the mir-302-367 and mir-371-373 clusters that bear the seed sequence AAGUGC. This platform is uniquely suitable for dissecting the roles of individual miRNAs in hESC self-renewal and differentiation, which may help us better understand the early development of human embryos.
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http://dx.doi.org/10.1016/j.stemcr.2018.07.013DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6135725PMC
September 2018

Genetic and epigenetic evolution as a contributor to WT1-mutant leukemogenesis.

Blood 2018 09 31;132(12):1265-1278. Epub 2018 Jul 31.

Human Oncology and Pathogenesis Program.

Genetic studies have identified recurrent somatic mutations in acute myeloid leukemia (AML) patients, including in the Wilms' tumor 1 () gene. The molecular mechanisms by which mutations contribute to leukemogenesis have not yet been fully elucidated. We investigated the role of gene dosage in steady-state and pathologic hematopoiesis. heterozygous loss enhanced stem cell self-renewal in an age-dependent manner, which increased stem cell function over time and resulted in age-dependent leukemic transformation. -haploinsufficient leukemias were characterized by progressive genetic and epigenetic alterations, including those in known leukemia-associated alleles, demonstrating a requirement for additional events to promote hematopoietic transformation. Consistent with this observation, we found that depletion cooperates with mutation to induce fully penetrant AML. Our studies provide insight into mechanisms of -loss leukemogenesis and into the evolutionary events required to induce transformation of -haploinsufficient stem/progenitor cells.
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http://dx.doi.org/10.1182/blood-2018-03-837468DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6148447PMC
September 2018