Publications by authors named "Benjamin L Ebert"

227 Publications

Dnmt3a-mutated clonal hematopoiesis promotes osteoporosis.

J Exp Med 2021 Dec 26;218(12). Epub 2021 Oct 26.

Department of Medical Oncology, Dana Farber Cancer Institute, Boston, MA.

Osteoporosis is caused by an imbalance of osteoclasts and osteoblasts, occurring in close proximity to hematopoietic cells in the bone marrow. Recurrent somatic mutations that lead to an expanded population of mutant blood cells is termed clonal hematopoiesis of indeterminate potential (CHIP). Analyzing exome sequencing data from the UK Biobank, we found CHIP to be associated with increased incident osteoporosis diagnoses and decreased bone mineral density. In murine models, hematopoietic-specific mutations in Dnmt3a, the most commonly mutated gene in CHIP, decreased bone mass via increased osteoclastogenesis. Dnmt3a-/- demethylation opened chromatin and altered activity of inflammatory transcription factors. Bone loss was driven by proinflammatory cytokines, including Irf3-NF-κB-mediated IL-20 expression from Dnmt3a mutant macrophages. Increased osteoclastogenesis due to the Dnmt3a mutations was ameliorated by alendronate or IL-20 neutralization. These results demonstrate a novel source of osteoporosis-inducing inflammation.
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http://dx.doi.org/10.1084/jem.20211872DOI Listing
December 2021

Congenital X-linked Neutropenia with Myelodysplasia and Somatic Tetraploidy due to a Germline Mutation in SEPT6.

Am J Hematol 2021 Oct 22. Epub 2021 Oct 22.

Division of Hematology-Oncology, Boston Children's Hospital, Harvard Medical School, Boston, USA.

Septins play key roles in mammalian cell division and cytokinesis but have not previously been implicated in a germline human disorder. A male infant with severe neutropenia and progressive dysmyelopoiesis with tetraploid myeloid precursors was identified. No known genetic etiologies for neutropenia or bone marrow failure were found. However, next-generation sequencing of germline samples from the patient revealed a novel, de novo germline stop-loss mutation in the X-linked gene SEPT6 that resulted in reduced SEPT6 staining in BM granulocyte precursors and megakaryocytes. Patient skin fibroblast-derived induced pluripotent stem cells (iPSCs) produced reduced myeloid colonies, particularly of the granulocyte lineage. CRISPR/Cas9 knock-in of the patient's mutation or complete knock-out of SEPT6 was not tolerated in non-patient derived iPSCs or human myeloid cell lines, but SEPT6 knock-out was successful in an erythroid cell line and resulting clones revealed a propensity to multinucleation. In silico analysis predicts the mutated protein hinders the dimerization of SEPT6 coiled coils in both parallel and antiparallel arrangements, which could in turn impair filament formation. These data demonstrate a critical role for SEPT6 in chromosomal segregation in myeloid progenitors that can account for the unusual predisposition to aneuploidy and dysmyelopoiesis. This article is protected by copyright. All rights reserved.
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http://dx.doi.org/10.1002/ajh.26382DOI Listing
October 2021

Distinction of lymphoid and myeloid clonal hematopoiesis.

Nat Med 2021 Oct 18. Epub 2021 Oct 18.

Broad Institute of MIT and Harvard, Cambridge, MA, USA.

Clonal hematopoiesis (CH) results from somatic genomic alterations that drive clonal expansion of blood cells. Somatic gene mutations associated with hematologic malignancies detected in hematopoietic cells of healthy individuals, referred to as CH of indeterminate potential (CHIP), have been associated with myeloid malignancies, while mosaic chromosomal alterations (mCAs) have been associated with lymphoid malignancies. Here, we analyzed CHIP in 55,383 individuals and autosomal mCAs in 420,969 individuals with no history of hematologic malignancies in the UK Biobank and Mass General Brigham Biobank. We distinguished myeloid and lymphoid somatic gene mutations, as well as myeloid and lymphoid mCAs, and found both to be associated with risk of lineage-specific hematologic malignancies. Further, we performed an integrated analysis of somatic alterations with peripheral blood count parameters to stratify the risk of incident myeloid and lymphoid malignancies. These genetic alterations can be readily detected in clinical sequencing panels and used with blood count parameters to identify individuals at high risk of developing hematologic malignancies.
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http://dx.doi.org/10.1038/s41591-021-01521-4DOI Listing
October 2021

is mutated in clonal hematopoiesis and myelodysplastic syndromes and impacts RNA splicing.

Blood Cancer Discov 2021 Sep 14;2(5):500-517. Epub 2021 Jul 14.

The Charles Bronfman Institute for Personalized Medicine, Icahn School of Medicine at Mount Sinai, New York, NY, USA.

Clonal hematopoiesis results from somatic mutations in cancer driver genes in hematopoietic stem cells. We sought to identify novel drivers of clonal expansion using an unbiased analysis of sequencing data from 84,683 persons and identified common mutations in the 5-methylcytosine reader, , as well as in , , and . We also identified these mutations at low frequency in myelodysplastic syndrome patients. edited mouse hematopoietic stem and progenitor cells exhibited a competitive advantage and increased genome-wide intron retention. mutations potentially link DNA methylation and RNA splicing, the two most commonly mutated pathways in clonal hematopoiesis and MDS.
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http://dx.doi.org/10.1158/2643-3230.BCD-20-0224DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8462124PMC
September 2021

Systematic profiling of DNMT3A variants reveals protein instability mediated by the DCAF8 E3 ubiquitin ligase adaptor.

Cancer Discov 2021 Aug 24. Epub 2021 Aug 24.

Molecular and Cellular Biology, Baylor College of Medicine, Houston

Clonal hematopoiesis is a prevalent age-related condition associated with greatly increased risk of hematologic disease; mutations in DNA methyltransferase 3A (DNMT3A) are the most common driver of this state. DNMT3A variants occur across the gene with some particularly associated with malignancy, but the functional relevance and mechanisms of pathogenesis of the majority of mutations is unknown. Here, we systematically investigated the methyltransferase activity and protein stability of 253 disease-associated DNMT3A mutations, finding that 74% were loss-of-function mutations. Half of these variants exhibited reduced protein stability and, as a class, correlated with greater clonal expansion and AML development. We investigated the mechanisms underlying the instability using a CRISPR screen and uncovered regulated destruction of DNMT3A mediated by the DCAF8 E3 ubiquitin ligase adaptor. We establish a new paradigm to classify novel variants that has prognostic and potential therapeutic significance for patients with hematologic disease.
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http://dx.doi.org/10.1158/2159-8290.CD-21-0560DOI Listing
August 2021

Clonal hematopoiesis in patients receiving chimeric antigen receptor T-cell therapy.

Blood Adv 2021 08;5(15):2982-2986

Department of Medical Oncology, Dana-Farber Cancer Institute, Harvard Medical School, Boston, MA.

Chimeric antigen receptor (CAR) T-cells have emerged as an efficacious modality in patients with non-Hodgkin lymphoma (NHL) and multiple myeloma (MM). Clonal hematopoiesis of indeterminate potential (CHIP), a state in which mutations in hematopoietic cells give rise to a clonal population of cells, is more common in patients exposed to cytotoxic therapies, has been shown to influence inflammatory immune programs, and is associated with an adverse prognosis in patients with NHL and MM receiving autologous transplantation. We therefore hypothesized that CHIP could influence clinical outcomes in patients receiving CAR T-cell therapy. In a cohort of 154 patients with NHL or MM receiving CAR T-cells, we found that CHIP was present in 48% of patients and associated with increased rates of complete response and cytokine release syndrome severity, but only in patients younger than age 60 years. Despite these differences, CHIP was not associated with a difference in progression-free or overall survival, regardless of age. Our data suggest that CHIP can influence CAR T-cell biology and clinical outcomes, but, in contrast to autologous transplantation, CHIP was not associated with worse survival and should not be a reason to exclude individuals from receiving this potentially life-prolonging treatment.
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http://dx.doi.org/10.1182/bloodadvances.2021004554DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8361461PMC
August 2021

Supplemental Association of Clonal Hematopoiesis With Incident Heart Failure.

J Am Coll Cardiol 2021 07;78(1):42-52

Department of Epidemiology, Brown University, Providence, Rhode Island, USA; Care New England, Center for Primary Care and Prevention, Pawtucket, Rhode Island, USA; Department of Family Medicine, Warren Alpert Medical School of Brown University, Providence, Rhode Island, USA. Electronic address:

Background: Age-related clonal hematopoiesis of indeterminate potential (CHIP), defined as clonally expanded leukemogenic sequence variations (particularly in DNMT3A, TET2, ASXL1, and JAK2) in asymptomatic individuals, is associated with cardiovascular events, including recurrent heart failure (HF).

Objectives: This study sought to evaluate whether CHIP is associated with incident HF.

Methods: CHIP status was obtained from whole exome or genome sequencing of blood DNA in participants without prevalent HF or hematological malignancy from 5 cohorts. Cox proportional hazards models were performed within each cohort, adjusting for demographic and clinical risk factors, followed by fixed-effect meta-analyses. Large CHIP clones (defined as variant allele frequency >10%), HF with or without baseline coronary heart disease, and left ventricular ejection fraction were evaluated in secondary analyses.

Results: Of 56,597 individuals (59% women, mean age 58 years at baseline), 3,406 (6%) had CHIP, and 4,694 developed HF (8.3%) over up to 20 years of follow-up. CHIP was prospectively associated with a 25% increased risk of HF in meta-analysis (hazard ratio: 1.25; 95% confidence interval: 1.13-1.38) with consistent associations across cohorts. ASXL1, TET2, and JAK2 sequence variations were each associated with an increased risk of HF, whereas DNMT3A sequence variations were not associated with HF. Secondary analyses suggested large CHIP was associated with a greater risk of HF (hazard ratio: 1.29; 95% confidence interval: 1.15-1.44), and the associations for CHIP on HF with and without prior coronary heart disease were homogenous. ASXL1 sequence variations were associated with reduced left ventricular ejection fraction.

Conclusions: CHIP, particularly sequence variations in ASXL1, TET2, and JAK2, represents a new risk factor for HF.
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http://dx.doi.org/10.1016/j.jacc.2021.04.085DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8313294PMC
July 2021

Association of Diet Quality With Prevalence of Clonal Hematopoiesis and Adverse Cardiovascular Events.

JAMA Cardiol 2021 Sep;6(9):1069-1077

Cardiovascular Research Center, Massachusetts General Hospital, Boston.

Importance: Clonal hematopoiesis of indeterminate potential (CHIP), the expansion of somatic leukemogenic variations in hematopoietic stem cells, has been associated with atherosclerotic cardiovascular disease. Because the inherited risk of developing CHIP is low, lifestyle elements such as dietary factors may be associated with the development and outcomes of CHIP.

Objective: To examine whether there is an association between diet quality and the prevalence of CHIP.

Design, Setting, And Participants: This retrospective cohort study used data from participants in the UK Biobank, an ongoing population-based study in the United Kingdom that examines whole-exome sequencing data and survey-based information on health-associated behaviors. Individuals from the UK Biobank were recruited between 2006 and 2010 and followed up prospectively with linkage to health data records through May 2020. The present study included 44 111 participants in the UK Biobank who were age 40 to 70 years, had data available from whole-exome sequencing of blood DNA, and were free of coronary artery disease (CAD) or hematologic cancer at baseline.

Exposures: Diet quality was categorized as unhealthy if the intake of healthy elements (fruits and vegetables) was lower than the median of all survey responses, and the intake of unhealthy elements (red meat, processed food, and added salt) was higher than the median. Diets were classified as healthy if the intake of healthy elements was higher than the median, and the intake of unhealthy elements was lower than the median. The presence of CHIP was detected by data from whole-exome sequencing of blood DNA.

Main Outcomes And Measures: The primary outcome was CHIP prevalence. Multivariable logistic regression analysis was used to examine the association between diet quality and the presence of CHIP. Multivariable Cox proportional hazards models were used to assess the association of incident events (acute coronary syndromes, coronary revascularization, or death) in each diet quality category stratified by the presence of CHIP.

Results: Among 44 111 participants (mean [SD] age at time of blood sample collection, 56.3 [8.0] years; 24 507 women [55.6%]), 2271 individuals (5.1%) had an unhealthy diet, 38 552 individuals (87.4%) had an intermediate diet, and 3288 individuals (7.5%) had a healthy diet. A total of 2507 individuals (5.7%) had CHIP, and the prevalence of CHIP decreased as diet quality improved from unhealthy (162 of 2271 participants [7.1%]) to intermediate (2177 of 38 552 participants [5.7%]) to healthy (168 of 3288 participants [5.1%]; P = .003 for trend). Compared with individuals without CHIP who had an intermediate diet, the rates of incident cardiovascular events progressively decreased among those with CHIP who had an unhealthy diet (hazard ratio [HR], 1.52; 95% CI, 1.04-2.22) and those with CHIP who had a healthy diet (HR, 0.99; 95% CI, 0.62-1.58) over a median of 10.0 years (interquartile range, 9.6-10.4 years) of follow-up.

Conclusions And Relevance: This cohort study suggests that an unhealthy diet quality may be associated with a higher prevalence of CHIP and higher rates of adverse cardiovascular events and death independent of CHIP status.
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http://dx.doi.org/10.1001/jamacardio.2021.1678DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8190703PMC
September 2021

Hematopoietic mosaic chromosomal alterations increase the risk for diverse types of infection.

Nat Med 2021 06 7;27(6):1012-1024. Epub 2021 Jun 7.

Institute for Molecular Medicine Finland, Helsinki, Finland.

Age is the dominant risk factor for infectious diseases, but the mechanisms linking age to infectious disease risk are incompletely understood. Age-related mosaic chromosomal alterations (mCAs) detected from genotyping of blood-derived DNA, are structural somatic variants indicative of clonal hematopoiesis, and are associated with aberrant leukocyte cell counts, hematological malignancy, and mortality. Here, we show that mCAs predispose to diverse types of infections. We analyzed mCAs from 768,762 individuals without hematological cancer at the time of DNA acquisition across five biobanks. Expanded autosomal mCAs were associated with diverse incident infections (hazard ratio (HR) 1.25; 95% confidence interval (CI) = 1.15-1.36; P = 1.8 × 10), including sepsis (HR 2.68; 95% CI = 2.25-3.19; P = 3.1 × 10), pneumonia (HR 1.76; 95% CI = 1.53-2.03; P = 2.3 × 10), digestive system infections (HR 1.51; 95% CI = 1.32-1.73; P = 2.2 × 10) and genitourinary infections (HR 1.25; 95% CI = 1.11-1.41; P = 3.7 × 10). A genome-wide association study of expanded mCAs identified 63 loci, which were enriched at transcriptional regulatory sites for immune cells. These results suggest that mCAs are a marker of impaired immunity and confer increased predisposition to infections.
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http://dx.doi.org/10.1038/s41591-021-01371-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8245201PMC
June 2021

Avadomide induces degradation of ZMYM2 fusion oncoproteins in hematologic malignancies.

Blood Cancer Discov 2021 May 10;2(3):250-265. Epub 2021 Mar 10.

Broad Institute of MIT and Harvard, Cambridge, Massachusetts, USA.

Thalidomide analogs exert their therapeutic effects by binding to the CRL4 E3 ubiquitin ligase, promoting ubiquitination and subsequent proteasomal degradation of specific protein substrates. Drug-induced degradation of IKZF1 and IKZF3 in B-cell malignancies demonstrates the clinical utility of targeting disease-relevant transcription factors for degradation. Here, we found that avadomide (CC-122) induces CRBN-dependent ubiquitination and proteasomal degradation of ZMYM2 (ZNF198), a transcription factor involved in balanced chromosomal rearrangements with and in aggressive forms of hematologic malignancies. The minimal drug-responsive element of ZMYM2 is a zinc-chelating MYM domain and is contained in the N-terminal portion of ZMYM2 that is universally included in the derived fusion proteins. We demonstrate that avadomide has the ability to induce proteasomal degradation of ZMYM2-FGFR1 and ZMYM2-FLT3 chimeric oncoproteins, both and . Our findings suggest that patients with hematologic malignancies harboring these fusion proteins may benefit from avadomide treatment.
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http://dx.doi.org/10.1158/2643-3230.bcd-20-0105DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8133393PMC
May 2021

Whole exome sequencing of a breast tumor in a patient with Diamond Blackfan anemia.

Blood Cells Mol Dis 2021 07 1;89:102566. Epub 2021 Apr 1.

Blueprint Medicines, United States of America.

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http://dx.doi.org/10.1016/j.bcmd.2021.102566DOI Listing
July 2021

Cancer therapies based on targeted protein degradation - lessons learned with lenalidomide.

Nat Rev Clin Oncol 2021 07 2;18(7):401-417. Epub 2021 Mar 2.

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

For decades, anticancer targeted therapies have been designed to inhibit kinases or other enzyme classes and have profoundly benefited many patients. However, novel approaches are required to target transcription factors, scaffolding proteins and other proteins central to cancer biology that typically lack catalytic activity and have remained mostly recalcitrant to drug development. The selective degradation of target proteins is an attractive approach to expand the druggable proteome, and the selective oestrogen receptor degrader fulvestrant served as an early example of this concept. Following a long and tragic history in the clinic, the immunomodulatory imide drug (IMiD) thalidomide was discovered to exert its therapeutic activity via a novel and unexpected mechanism of action: targeting proteins to an E3 ubiquitin ligase for subsequent proteasomal degradation. This discovery has paralleled and directly catalysed myriad breakthroughs in drug development, leading to the rapid maturation of generalizable chemical platforms for the targeted degradation of previously undruggable proteins. Decades of clinical experience have established front-line roles for thalidomide analogues, including lenalidomide and pomalidomide, in the treatment of haematological malignancies. With a new generation of 'degrader' drugs currently in development, this experience provides crucial insights into class-wide features of degraders, including a unique pharmacology, mechanisms of resistance and emerging therapeutic opportunities. Herein, we review these past experiences and discuss their application in the clinical development of novel degrader therapies.
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http://dx.doi.org/10.1038/s41571-021-00479-zDOI Listing
July 2021

CBL mutations drive PI3K/AKT signaling via increased interaction with LYN and PIK3R1.

Blood 2021 04;137(16):2209-2220

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

Casitas B-lineage lymphoma (CBL) encodes an E3 ubiquitin ligase and signaling adaptor that regulates receptor and nonreceptor tyrosine kinases. Recurrent CBL mutations occur in myeloid neoplasms, including 10% to 20% of chronic myelomonocytic leukemia (CMML) cases, and selectively disrupt the protein's E3 ubiquitin ligase activity. CBL mutations have been associated with poor prognosis, but the oncogenic mechanisms and therapeutic implications of CBL mutations remain incompletely understood. We combined functional assays and global mass spectrometry to define the phosphoproteome, CBL interactome, and mechanism of signaling activation in a panel of cell lines expressing an allelic series of CBL mutations. Our analyses revealed that increased LYN activation and interaction with mutant CBL are key drivers of enhanced CBL phosphorylation, phosphoinositide-3-kinase regulatory subunit 1 (PIK3R1) recruitment, and downstream phosphatidylinositol 3-kinase (PI3K)/AKT signaling in CBL-mutant cells. Signaling adaptor domains of CBL, including the tyrosine kinase-binding domain, proline-rich region, and C-terminal phosphotyrosine sites, were all required for the oncogenic function of CBL mutants. Genetic ablation or dasatinib-mediated inhibition of LYN reduced CBL phosphorylation, CBL-PIK3R1 interaction, and PI3K/AKT signaling. Furthermore, we demonstrated in vitro and in vivo antiproliferative efficacy of dasatinib in CBL-mutant cell lines and primary CMML. Overall, these mechanistic insights into the molecular function of CBL mutations provide rationale to explore the therapeutic potential of LYN inhibition in CBL-mutant myeloid malignancies.
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http://dx.doi.org/10.1182/blood.2020006528DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8063090PMC
April 2021

Modeling and targeting of erythroleukemia by hematopoietic genome editing.

Blood 2021 03;137(12):1628-1640

Department of Pathology, St Jude Children's Research Hospital, Memphis, TN.

Acute erythroid leukemia (AEL) is characterized by a distinct morphology, mutational spectrum, lack of preclinical models, and poor prognosis. Here, using multiplexed genome editing of mouse hematopoietic stem and progenitor cells and transplant assays, we developed preclinical models of AEL and non-erythroid acute leukemia and describe the central role of mutational cooperativity in determining leukemia lineage. Different combination of mutations in Trp53, Bcor, Dnmt3a, Rb1, and Nfix resulted in the development of leukemia with an erythroid phenotype, accompanied by the acquisition of alterations in signaling and transcription factor genes that recapitulate human AEL by cross-species genomic analysis. Clonal expansion during tumor evolution was driven by mutational cooccurrence, with clones harboring a higher number of founder and secondary lesions (eg, mutations in signaling genes) showing greater evolutionary fitness. Mouse and human AEL exhibited deregulation of genes regulating erythroid development, notably Gata1, Klf1, and Nfe2, driven by the interaction of mutations of the epigenetic modifiers Dnmt3a and Tet2 that perturbed methylation and thus expression of lineage-specific transcription factors. The established mouse leukemias were used as a platform for drug screening. Drug sensitivity was associated with the leukemia genotype, with the poly (ADP-ribose) polymerase inhibitor talazoparib and the demethylating agent decitabine efficacious in Trp53/Bcor-mutant AEL, CDK7/9 inhibitors in Trp53/Bcor/Dnmt3a-mutant AEL, and gemcitabine and bromodomain inhibitors in NUP98-KDM5A leukemia. In conclusion, combinatorial genome editing has shown the interplay of founding and secondary genetic alterations in phenotype and clonal evolution, epigenetic regulation of lineage-specific transcription factors, and therapeutic tractability in erythroid leukemogenesis.
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http://dx.doi.org/10.1182/blood.2020009103DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7995291PMC
March 2021

Reversible ON- and OFF-switch chimeric antigen receptors controlled by lenalidomide.

Sci Transl Med 2021 01;13(575)

Broad Institute of Harvard and MIT, Cambridge, MA 02142, USA.

Cell-based therapies are emerging as effective agents against cancer and other diseases. As autonomous "living drugs," these therapies lack precise control. Chimeric antigen receptor (CAR) T cells effectively target hematologic malignancies but can proliferate rapidly and cause toxicity. We developed ON and OFF switches for CAR T cells using the clinically approved drug lenalidomide, which mediates the proteasomal degradation of several target proteins by inducing interactions between the CRL4 E3 ubiquitin ligase and a C2H2 zinc finger degron motif. We performed a systematic screen to identify "super-degron" tags with enhanced sensitivity to lenalidomide-induced degradation and used these degradable tags to generate OFF-switch degradable CARs. To create an ON switch, we engineered a lenalidomide-inducible dimerization system and developed split CARs that required both lenalidomide and target antigen for activation. Subtherapeutic lenalidomide concentrations controlled the effector functions of ON- and OFF-switch CAR T cells. In vivo, ON-switch split CARs demonstrated lenalidomide-dependent antitumor activity, and OFF-switch degradable CARs were depleted by drug treatment to limit inflammatory cytokine production while retaining antitumor efficacy. Together, the data showed that these lenalidomide-gated switches are rapid, reversible, and clinically suitable systems to control transgene function in diverse gene- and cell-based therapies.
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http://dx.doi.org/10.1126/scitranslmed.abb6295DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8045771PMC
January 2021

Functional Genomics Identify Distinct and Overlapping Genes Mediating Resistance to Different Classes of Heterobifunctional Degraders of Oncoproteins.

Cell Rep 2021 01;34(1):108532

Department of Medical Oncology, Dana-Farber Cancer Institute, Boston, MA, USA; Harvard Medical School, Boston, MA, USA; Broad Institute of MIT and Harvard, Cambridge, MA, USA.

Heterobifunctional proteolysis-targeting chimeric compounds leverage the activity of E3 ligases to induce degradation of target oncoproteins and exhibit potent preclinical antitumor activity. To dissect the mechanisms regulating tumor cell sensitivity to different classes of pharmacological "degraders" of oncoproteins, we performed genome-scale CRISPR-Cas9-based gene editing studies. We observed that myeloma cell resistance to degraders of different targets (BET bromodomain proteins, CDK9) and operating through CRBN (degronimids) or VHL is primarily mediated by prevention of, rather than adaptation to, breakdown of the target oncoprotein; and this involves loss of function of the cognate E3 ligase or interactors/regulators of the respective cullin-RING ligase (CRL) complex. The substantial gene-level differences for resistance mechanisms to CRBN- versus VHL-based degraders explains mechanistically the lack of cross-resistance with sequential administration of these two degrader classes. Development of degraders leveraging more diverse E3 ligases/CRLs may facilitate sequential/alternating versus combined uses of these agents toward potentially delaying or preventing resistance.
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http://dx.doi.org/10.1016/j.celrep.2020.108532DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8485877PMC
January 2021

Cohesin mutations alter DNA damage repair and chromatin structure and create therapeutic vulnerabilities in MDS/AML.

JCI Insight 2021 02 8;6(3). Epub 2021 Feb 8.

Department of Medical Oncology, Dana-Farber Cancer Institute, Boston, Massachusetts, USA.

The cohesin complex plays an essential role in chromosome maintenance and transcriptional regulation. Recurrent somatic mutations in the cohesin complex are frequent genetic drivers in cancer, including myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML). Here, using genetic dependency screens of stromal antigen 2-mutant (STAG2-mutant) AML, we identified DNA damage repair and replication as genetic dependencies in cohesin-mutant cells. We demonstrated increased levels of DNA damage and sensitivity of cohesin-mutant cells to poly(ADP-ribose) polymerase (PARP) inhibition. We developed a mouse model of MDS in which Stag2 mutations arose as clonal secondary lesions in the background of clonal hematopoiesis driven by tet methylcytosine dioxygenase 2 (Tet2) mutations and demonstrated selective depletion of cohesin-mutant cells with PARP inhibition in vivo. Finally, we demonstrated a shift from STAG2- to STAG1-containing cohesin complexes in cohesin-mutant cells, which was associated with longer DNA loop extrusion, more intermixing of chromatin compartments, and increased interaction with PARP and replication protein A complex. Our findings inform the biology and therapeutic opportunities for cohesin-mutant malignancies.
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http://dx.doi.org/10.1172/jci.insight.142149DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7934867PMC
February 2021

Hematopoietic mosaic chromosomal alterations and risk for infection among 767,891 individuals without blood cancer.

medRxiv 2020 Nov 16. Epub 2020 Nov 16.

Age is the dominant risk factor for infectious diseases, but the mechanisms linking the two are incompletely understood . Age-related mosaic chromosomal alterations (mCAs) detected from blood-derived DNA genotyping, are structural somatic variants associated with aberrant leukocyte cell counts, hematological malignancy, and mortality . Whether mCAs represent independent risk factors for infection is unknown. Here we use genome-wide genotyping of blood DNA to show that mCAs predispose to diverse infectious diseases. We analyzed mCAs from 767,891 individuals without hematological cancer at DNA acquisition across four countries. Expanded mCA (cell fraction >10%) prevalence approached 4% by 60 years of age and was associated with diverse incident infections, including sepsis, pneumonia, and coronavirus disease 2019 (COVID-19) hospitalization. A genome-wide association study of expanded mCAs identified 63 significant loci. Germline genetic alleles associated with expanded mCAs were enriched at transcriptional regulatory sites for immune cells. Our results link mCAs with impaired immunity and predisposition to infections. Furthermore, these findings may also have important implications for the ongoing COVID-19 pandemic, particularly in prioritizing individual preventive strategies and evaluating immunization responses.
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http://dx.doi.org/10.1101/2020.11.12.20230821DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7685330PMC
November 2020

Small-molecule-induced polymerization triggers degradation of BCL6.

Nature 2020 12 18;588(7836):164-168. Epub 2020 Nov 18.

Broad Institute of MIT and Harvard, Cambridge, MA, USA.

Effective and sustained inhibition of non-enzymatic oncogenic driver proteins is a major pharmacological challenge. The clinical success of thalidomide analogues demonstrates the therapeutic efficacy of drug-induced degradation of transcription factors and other cancer targets, but a substantial subset of proteins are resistant to targeted degradation using existing approaches. Here we report an alternative mechanism of targeted protein degradation, in which a small molecule induces the highly specific, reversible polymerization of a target protein, followed by its sequestration into cellular foci and subsequent degradation. BI-3802 is a small molecule that binds to the Broad-complex, Tramtrack and Bric-à-brac (BTB) domain of the oncogenic transcription factor B cell lymphoma 6 (BCL6) and leads to the proteasomal degradation of BCL6. We use cryo-electron microscopy to reveal how the solvent-exposed moiety of a BCL6-binding molecule contributes to a composite ligand-protein surface that engages BCL6 homodimers to form a supramolecular structure. Drug-induced formation of BCL6 filaments facilitates ubiquitination by the SIAH1 E3 ubiquitin ligase. Our findings demonstrate that a small molecule such as BI-3802 can induce polymerization coupled to highly specific protein degradation, which in the case of BCL6 leads to increased pharmacological activity compared to the effects induced by other BCL6 inhibitors. These findings open new avenues for the development of therapeutic agents and synthetic biology.
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http://dx.doi.org/10.1038/s41586-020-2925-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7816212PMC
December 2020

Premature Menopause, Clonal Hematopoiesis, and Coronary Artery Disease in Postmenopausal Women.

Circulation 2021 Feb 9;143(5):410-423. Epub 2020 Nov 9.

Cardiology Division (M.C.H., J.P.P., P.N.), Massachusetts General Hospital, Harvard Medical School, Boston.

Background: Premature menopause is an independent risk factor for cardiovascular disease in women, but mechanisms underlying this association remain unclear. Clonal hematopoiesis of indeterminate potential (CHIP), the age-related expansion of hematopoietic cells with leukemogenic mutations without detectable malignancy, is associated with accelerated atherosclerosis. Whether premature menopause is associated with CHIP is unknown.

Methods: We included postmenopausal women from the UK Biobank (n=11 495) aged 40 to 70 years with whole exome sequences and from the Women's Health Initiative (n=8111) aged 50 to 79 years with whole genome sequences. Premature menopause was defined as natural or surgical menopause occurring before age 40 years. Co-primary outcomes were the presence of any CHIP and CHIP with variant allele frequency >0.1. Logistic regression tested the association of premature menopause with CHIP, adjusted for age, race, the first 10 principal components of ancestry, smoking, diabetes, and hormone therapy use. Secondary analyses considered natural versus surgical premature menopause and gene-specific CHIP subtypes. Multivariable-adjusted Cox models tested the association between CHIP and incident coronary artery disease.

Results: The sample included 19 606 women, including 418 (2.1%) with natural premature menopause and 887 (4.5%) with surgical premature menopause. Across cohorts, CHIP prevalence in postmenopausal women with versus without a history of premature menopause was 8.8% versus 5.5% (<0.001), respectively. After multivariable adjustment, premature menopause was independently associated with CHIP (all CHIP: odds ratio, 1.36 [95% 1.10-1.68]; =0.004; CHIP with variant allele frequency >0.1: odds ratio, 1.40 [95% CI, 1.10-1.79]; =0.007). Associations were larger for natural premature menopause (all CHIP: odds ratio, 1.73 [95% CI, 1.23-2.44]; =0.001; CHIP with variant allele frequency >0.1: odds ratio, 1.91 [95% CI, 1.30-2.80]; <0.001) but smaller and nonsignificant for surgical premature menopause. In gene-specific analyses, only CHIP was significantly associated with premature menopause. Among postmenopausal middle-aged women, CHIP was independently associated with incident coronary artery disease (hazard ratio associated with all CHIP: 1.36 [95% CI, 1.07-1.73]; =0.012; hazard ratio associated with CHIP with variant allele frequency >0.1: 1.48 [95% CI, 1.13-1.94]; =0.005).

Conclusions: Premature menopause, especially natural premature menopause, is independently associated with CHIP among postmenopausal women. Natural premature menopause may serve as a risk signal for predilection to develop CHIP and CHIP-associated cardiovascular disease.
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http://dx.doi.org/10.1161/CIRCULATIONAHA.120.051775DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7911856PMC
February 2021

Cancer therapy shapes the fitness landscape of clonal hematopoiesis.

Nat Genet 2020 11 26;52(11):1219-1226. Epub 2020 Oct 26.

Department of Oncology, University of Cambridge, Addenbrooke's Hospital, Cambridge, UK.

Acquired mutations are pervasive across normal tissues. However, understanding of the processes that drive transformation of certain clones to cancer is limited. Here we study this phenomenon in the context of clonal hematopoiesis (CH) and the development of therapy-related myeloid neoplasms (tMNs). We find that mutations are selected differentially based on exposures. Mutations in ASXL1 are enriched in current or former smokers, whereas cancer therapy with radiation, platinum and topoisomerase II inhibitors preferentially selects for mutations in DNA damage response genes (TP53, PPM1D, CHEK2). Sequential sampling provides definitive evidence that DNA damage response clones outcompete other clones when exposed to certain therapies. Among cases in which CH was previously detected, the CH mutation was present at tMN diagnosis. We identify the molecular characteristics of CH that increase risk of tMN. The increasing implementation of clinical sequencing at diagnosis provides an opportunity to identify patients at risk of tMN for prevention strategies.
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http://dx.doi.org/10.1038/s41588-020-00710-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7891089PMC
November 2020

Calmodulin inhibitors improve erythropoiesis in Diamond-Blackfan anemia.

Sci Transl Med 2020 10;12(566)

Stem Cell Program, Boston Children's Hospital and Harvard Stem Cell Institute, Boston, MA 02115, USA.

Diamond-Blackfan anemia (DBA) is a rare hematopoietic disease characterized by a block in red cell differentiation. Most DBA cases are caused by mutations in ribosomal proteins and characterized by higher than normal activity of the tumor suppressor p53. Higher p53 activity is thought to contribute to DBA phenotypes by inducing apoptosis during red blood cell differentiation. Currently, there are few therapies available for patients with DBA. We performed a chemical screen using zebrafish ribosomal small subunit protein 29 () mutant embryos that have a p53-dependent anemia and identified calmodulin inhibitors that rescued the phenotype. Our studies demonstrated that calmodulin inhibitors attenuated p53 protein amount and activity. Treatment with calmodulin inhibitors led to decreased p53 translation and accumulation but does not affect p53 stability. A U.S. Food and Drug Administration-approved calmodulin inhibitor, trifluoperazine, rescued hematopoietic phenotypes of DBA models in vivo in zebrafish and mouse models. In addition, trifluoperazine rescued these phenotypes in human CD34 hematopoietic stem and progenitor cells. Erythroid differentiation was also improved in CD34 cells isolated from a patient with DBA. This work uncovers a potential avenue of therapeutic development for patients with DBA.
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http://dx.doi.org/10.1126/scitranslmed.abb5831DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7709865PMC
October 2020

Inherited causes of clonal haematopoiesis in 97,691 whole genomes.

Nature 2020 10 14;586(7831):763-768. Epub 2020 Oct 14.

Department of Epidemiology, School of Public Health, University of Michigan, Ann Arbor, MI, USA.

Age is the dominant risk factor for most chronic human diseases, but the mechanisms through which ageing confers this risk are largely unknown. The age-related acquisition of somatic mutations that lead to clonal expansion in regenerating haematopoietic stem cell populations has recently been associated with both haematological cancer and coronary heart disease-this phenomenon is termed clonal haematopoiesis of indeterminate potential (CHIP). Simultaneous analyses of germline and somatic whole-genome sequences provide the opportunity to identify root causes of CHIP. Here we analyse high-coverage whole-genome sequences from 97,691 participants of diverse ancestries in the National Heart, Lung, and Blood Institute Trans-omics for Precision Medicine (TOPMed) programme, and identify 4,229 individuals with CHIP. We identify associations with blood cell, lipid and inflammatory traits that are specific to different CHIP driver genes. Association of a genome-wide set of germline genetic variants enabled the identification of three genetic loci associated with CHIP status, including one locus at TET2 that was specific to individuals of African ancestry. In silico-informed in vitro evaluation of the TET2 germline locus enabled the identification of a causal variant that disrupts a TET2 distal enhancer, resulting in increased self-renewal of haematopoietic stem cells. Overall, we observe that germline genetic variation shapes haematopoietic stem cell function, leading to CHIP through mechanisms that are specific to clonal haematopoiesis as well as shared mechanisms that lead to somatic mutations across tissues.
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http://dx.doi.org/10.1038/s41586-020-2819-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7944936PMC
October 2020

Fitness Landscape of Clonal Hematopoiesis Under Selective Pressure of Immune Checkpoint Blockade.

JCO Precis Oncol 2020 9;4. Epub 2020 Sep 9.

Department of Medical Oncology, Dana-Farber Cancer Institute, Harvard Medical School, Boston, MA.

Purpose: Conventional cytotoxic therapies increase the risk of clonal hematopoiesis and select for -mutant clones, which carry a high risk for transformation to therapy-related myelodysplastic neoplasms. In contrast, the effect of immune checkpoint blockade (ICB) on clonal hematopoiesis is unknown.

Methods: Paired peripheral-blood samples taken before and after treatment with ICB were obtained for 91 patients with either cutaneous melanoma or basal cell carcinoma. Error-corrected sequencing of a targeted panel of genes recurrently mutated in clonal hematopoiesis was performed on peripheral-blood genomic DNA.

Results: The average interval between acquisition of the paired samples was 180 days. Forty-one percent of the patients had clonal hematopoiesis at a variant allele frequency (VAF) > 0.01 in the pretreatment sample. There was near-complete agreement in the distribution and burden of clonal hematopoiesis mutations in the paired blood samples, with 87 of 88 mutations identified across the cohort present in paired samples, regardless of the duration between sample collection. The VAF in the paired samples also showed a high correlation, with an = 0.95 ( < .0001). In contrast to cytotoxic therapy, exposure to ICB did not lead to selection of - or -mutant clones. However, consistent with the known effects of DNA-damaging therapy, we identified one patient who had eight unique mutations in the posttreatment blood sample after receiving two courses of radiation therapy.

Conclusion: There was no expansion of hematopoietic clones or selection for clones at high risk for malignant transformation in patients who received ICB, observations that warrant further validation in larger cohorts. These findings highlight an important difference between ICB and conventional cytotoxic therapies and their respective impacts on premalignant genetic lesions.
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http://dx.doi.org/10.1200/PO.20.00186DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7529531PMC
September 2020

An induced pluripotent stem cell model of Fanconi anemia reveals mechanisms of p53-driven progenitor cell differentiation.

Blood Adv 2020 10;4(19):4679-4692

Stem Cell Program, Boston Children's Hospital, Boston, MA.

Fanconi anemia (FA) is a disorder of DNA repair that manifests as bone marrow (BM) failure. The lack of accurate murine models of FA has refocused efforts toward differentiation of patient-derived induced pluripotent stem cells (IPSCs) to hematopoietic progenitor cells (HPCs). However, an intact FA DNA repair pathway is required for efficient IPSC derivation, hindering these efforts. To overcome this barrier, we used inducible complementation of FANCA-deficient IPSCs, which permitted robust maintenance of IPSCs. Modulation of FANCA during directed differentiation to HPCs enabled the production of FANCA-deficient human HPCs that recapitulated FA genotoxicity and hematopoietic phenotypes relative to isogenic FANCA-expressing HPCs. FANCA-deficient human HPCs underwent accelerated terminal differentiation driven by activation of p53/p21. We identified growth arrest specific 6 (GAS6) as a novel target of activated p53 in FANCA-deficient HPCs and modulate GAS6 signaling to rescue hematopoiesis in FANCA-deficient cells. This study validates our strategy to derive a sustainable, highly faithful human model of FA, uncovers a mechanism of HPC exhaustion in FA, and advances toward future cell therapy in FA.
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http://dx.doi.org/10.1182/bloodadvances.2020001593DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7556119PMC
October 2020

Contribution of clonal hematopoiesis to adult-onset hemophagocytic lymphohistiocytosis.

Blood 2020 12;136(26):3051-3055

Department of Medical Oncology, Dana-Farber Cancer Institute.

Adult-onset hemophagocytic lymphohistiocytosis (HLH) is a rare, life-threatening disease of immune hyperactivation. Unlike pediatric HLH, adult HLH is rarely driven by germline genetic variants. Although numerous precipitating etiologies have been identified, the reason that HLH occurs in only a subset of individuals and how other factors contribute to the disease remains unknown. We hypothesized that clonal hematopoiesis (CH), a state in which somatic mutations in blood cells cause an expanded population of mutant hematopoietic cells and drive an aberrant inflammatory state, could contribute to adult-onset HLH. In a highly annotated cohort of older adults with HLH we found that CH was more prevalent than in control cohorts. Using the adult-onset HLH mouse model in which repeated treatments of the TLR9 agonist, ODN1826, was delivered to the mouse, we observed that macrophages carrying mutations in Tet2, one of the most commonly mutated genes in CH, have an enhanced inflammatory response to TLR9 agonism. Finally, mice carrying Tet2 mutations in the hematopoietic compartment (a common model for CH) displayed an exaggerated response to TLR9 agonism, including worse splenomegaly and anemia. Our data suggest that CH is more common in individuals with adult-onset HLH and can contribute to the pathophysiology of this disease.
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http://dx.doi.org/10.1182/blood.2020008206DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7770567PMC
December 2020
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