Publications by authors named "Aharon G Freud"

59 Publications

Unraveling the Role of Innate Lymphoid Cells in AcuteMyeloid Leukemia.

Cancers (Basel) 2021 Jan 17;13(2). Epub 2021 Jan 17.

Comprehensive Cancer Center, The Ohio State University, Columbus, OH 43210, USA.

Over the past 50 years, few therapeutic advances have been made in treating acute myeloid leukemia (AML), an aggressive form of blood cancer, despite vast improvements in our ability to classify the disease. Emerging evidence suggests the immune system is important in controlling AML progression and in determining prognosis. Natural killer (NK) cells are important cytotoxic effector cells of the innate lymphoid cell (ILC) family that have been shown to have potent anti-leukemic functions. Recent studies are now revealing impairment or dysregulation of other ILCs in various types of cancers, including AML, which limits the effectiveness of NK cells in controlling cancer progression. NK cell development and function are inhibited in AML patients, which results in worse clinical outcomes; however, the specific roles of other ILC populations in AML are just now beginning to be unraveled. In this review, we summarize what is known about the role of ILC populations in AML.
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http://dx.doi.org/10.3390/cancers13020320DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7830843PMC
January 2021

Established and emergent roles for Ikaros transcription factors in lymphoid cell development and function.

Immunol Rev 2021 Mar 17;300(1):82-99. Epub 2020 Dec 17.

Department of Microbial Infection and Immunity, The Ohio State University College of Medicine and Wexner Medical Center, Columbus, OH, USA.

Ikaros zinc finger transcription factors are important regulators of the gene programs underlying the development of hematopoietic cell lineages. The family consists of five members: Ikaros, Helios, Aiolos, Eos, and Pegasus, which engage in both homo- and heterotypic intrafamilial interactions to exert diverse functional effects. Pioneering studies focused on the role of these factors in early lymphoid development, as their absence resulted in severe defects in lymphocyte populations. More recent work has now begun to define nuanced, stage-specific roles for Ikaros family members in the differentiation and function of mature T, B, and innate lymphoid cell populations including natural killer (NK) cells. The precise transcriptional mechanisms by which these factors function, both independently and collaboratively, is an area of active investigation. However, several key themes appear to be emerging regarding the pathways influenced by Ikaros family members, including the end-to-end regulation of cytokine signaling. Here, we review roles for Ikaros factors in lymphoid cell development, differentiation, and function, including a discussion of the current understanding of the transcriptional mechanisms they employ and considerations for the future study of this important transcription factor family.
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http://dx.doi.org/10.1111/imr.12936DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8015388PMC
March 2021

Notch Regulates Innate Lymphoid Cell Plasticity during Human NK Cell Development.

J Immunol 2020 11 5;205(10):2679-2693. Epub 2020 Oct 5.

Comprehensive Cancer Center, The Ohio State University, Columbus, OH 43210;

Human NK cells develop in tonsils through discrete NK cell developmental intermediates (NKDIs), yet the mechanistic regulation of this process is unclear. We demonstrate that Notch activation in human tonsil-derived stage 3 (CD34CD117CD94NKp80) and 4A (CD34CD117CD94NKp80) NKDIs promoted non-NK innate lymphoid cell differentiation at the expense of NK cell differentiation. In contrast, stage 4B (CD34CD117CD94NKp80) NKDIs were NK cell lineage committed despite Notch activation. Interestingly, whereas NK cell functional maturation from stage 3 and 4A NKDIs was independent of Notch activation, the latter was required for high NKp80 expression and a stage 4B-like phenotype by the NKDI-derived NK cells. The Notch-dependent effects required simultaneous engagement with OP9 stromal cells and were also stage-specific, with NOTCH1 and NOTCH2 receptors regulating stage 3 NKDIs and NOTCH1 primarily regulating stage 4A NKDIs. These data establish stage-specific and stromal-dependent roles for Notch in regulating human NK cell developmental plasticity and maturation.
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http://dx.doi.org/10.4049/jimmunol.2000434DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7658047PMC
November 2020

Editorial: Molecular and Cellular Pathways in NK Cell Development.

Front Immunol 2020 14;11:1448. Epub 2020 Jul 14.

Department of Pediatrics, Columbia University Irving Medical Center, New York, NY, United States.

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http://dx.doi.org/10.3389/fimmu.2020.01448DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7371939PMC
April 2021

Clinical outcomes in T-cell large granular lymphocytic leukaemia: prognostic factors and treatment response.

Br J Haematol 2021 Feb 9;192(3):484-493. Epub 2020 Jun 9.

Division of Hematology, Department of Internal Medicine, James Comprehensive Cancer Center, The Ohio State University, Columbus, OH, USA.

T-cell large granular lymphocytic leukaemia (T-LGLL) is an incurable leukaemia characterised by clonal proliferation of abnormal cytotoxic T cells that can result in severe neutropenia, transfusion-dependent anaemia and pancytopenia requiring treatment. The most commonly used agents, methotrexate (MTX), cyclophosphamide (Cy) and cyclosporine primarily produce partial remissions (PRs), with few complete responses (CRs). We evaluated the clinical course and treatment response of 60 consecutive patients with T-LGLL to evaluate clinical outcomes and future potential treatment directions. Impaired overall survival was noted among male patients, patients with elevated lactate dehydrogenase, and those without rheumatoid arthritis. Cy was the most efficacious second-line agent, with a 70% overall response rate (ORR; three CR, four PR). All patients who failed frontline MTX responded to second-line Cy. In the relapsed or Cy-refractory setting, alemtuzumab (n = 4) and pentostatin (n = 3) had an ORR of 50% and 66%, respectively, while duvelisib induced a long-term response in one patient. In this large, retrospective analysis, our results suggest Cy is a highly effective therapy for second-line treatment in T-LGLL and should be considered a strong candidate for up-front therapy in select high-risk patients. Prospective studies evaluating pentostatin, alemtuzumab and novel agents, such as duvelisib, are needed for patients with relapsed/refractory T-LGLL.
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http://dx.doi.org/10.1111/bjh.16808DOI Listing
February 2021

Efficacy of FGFR Inhibitors and Combination Therapies for Acquired Resistance in FGFR2-Fusion Cholangiocarcinoma.

Mol Cancer Ther 2020 03 7;19(3):847-857. Epub 2020 Jan 7.

Comprehensive Cancer Center, The Ohio State University, Columbus, Ohio.

The fibroblast growth factor receptor (FGFR) signaling pathway is aberrantly activated in approximately 15% to 20% of patients with intrahepatic cholangiocarcinoma. Currently, several FGFR kinase inhibitors are being assessed in clinical trials for patients with FGFR-altered cholangiocarcinoma. Despite evidence of initial responses and disease control, virtually all patients eventually develop acquired resistance. Thus, there is a critical need for the development of innovative therapeutic strategies to overcome acquired drug resistance. Here, we present findings from a patient with FGFR2-altered metastatic cholangiocarcinoma who enrolled in a phase II clinical trial of the FGFR inhibitor, infigratinib (BGJ398). Treatment was initially effective as demonstrated by imaging and tumor marker response; however, after 8 months on trial, the patient exhibited tumor regrowth and disease progression. Targeted sequencing of tumor DNA after disease progression revealed the kinase domain p.E565A and p.L617M single-nucleotide variants (SNV) hypothesized to drive acquired resistance to infigratinib. The sensitivities of these SNVs, which were detected post-infigratinib therapy, were extended to include clinically relevant FGFR inhibitors, including AZD4547, erdafitinib (JNJ-42756493), dovitinib, ponatinib, and TAS120, and were evaluated Through a proteomics approach, we identified upregulation of the PI3K/AKT/mTOR signaling pathway in cells harboring the p.E565A mutation and demonstrated that combination therapy strategies with FGFR and mTOR inhibitors may be used to overcome resistance to FGFR inhibition, specific to infigratinib. Collectively, these studies support the development of novel combination therapeutic strategies in addition to the next generation of FGFR inhibitors to overcome acquired resistance in patients.
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http://dx.doi.org/10.1158/1535-7163.MCT-19-0631DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7359896PMC
March 2020

EGFL7 Antagonizes NOTCH Signaling and Represents a Novel Therapeutic Target in Acute Myeloid Leukemia.

Clin Cancer Res 2020 02 31;26(3):669-678. Epub 2019 Oct 31.

The Ohio State University Comprehensive Cancer Center, Columbus, Ohio.

Purpose: EGF-like domain 7 (EGFL7) is a secreted protein and recently has been shown to play an important role in acute myeloid leukemia (AML); however, the underlying mechanism by which EGFL7 promotes leukemogenesis is largely unknown.

Experimental Design: Using an antibody interaction array, we measured the ability of EGFL7 to bind directly approximately 400 proteins expressed by primary AML blasts. Primary patient samples were stimulated with recombinant EGFL7 (rEGFL7) or anti-EGFL7 blocking antibody to assess alterations in downstream signaling and the ability to effect blast differentiation and survival. We treated three independent AML models with anti-EGFL7 or IgG1 control to determine whether anti-EGFL7 could prolong survival .

Results: We found EGFL7 significantly binds several signaling proteins important for normal and malignant hematopoiesis including NOTCH. Stimulation of AML blasts with rEGFL7 reduced NOTCH intracellular domain and NOTCH target gene expression while treatment with an anti-EGFL7 blocking antibody resulted in reactivation of NOTCH signaling, increased differentiation, and apoptosis. Competitive ligand-binding assays showed rEGFL7 inhibits DELTA-like (DLL) 4-mediated NOTCH activation while anti-EGFL7 combined with DLL4 significantly increased NOTCH activation and induced apoptosis. Using three different AML mouse models, we demonstrated that treatment with anti-EGFL7 alone results in increased survival.

Conclusions: Our data demonstrate that EGFL7 contributes to NOTCH silencing in AML by antagonizing canonical NOTCH ligand binding. Reactivation of NOTCH signaling using anti-EGFL7 results in prolonged survival of leukemic mice, supporting the use of EGFL7 as a novel therapeutic target in AML.
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http://dx.doi.org/10.1158/1078-0432.CCR-19-2479DOI Listing
February 2020

True Detective: Unraveling Group 1 Innate Lymphocyte Heterogeneity.

Trends Immunol 2019 10 6;40(10):909-921. Epub 2019 Sep 6.

Department of Microbiology, Immunology, and Molecular Genetics, David Geffen School of Medicine at UCLA, Los Angeles, CA 900953, USA; Molecular Biology Institute, University of California, Los Angeles, Los Angeles, CA 90095, USA. Electronic address:

Innate lymphoid cells (ILCs) consist of a heterogeneous family of lymphocytes that regulate tissue homeostasis and can contribute to pathology in mice and humans. Mammalian group 1 ILCs are defined by the production of interferon (IFN)-γ and the functional dependence on the transcription factor T-bet. While recent studies demonstrate that group 1 ILCs consist of circulating mature natural killer (NK) cells and tissue-resident ILC1s, the functional, phenotypic, and developmental properties that distinguish these two cell lineages are often confusing and difficult to dissect. In this review, we critically evaluate the current knowledge of mammalian group 1 ILC heterogeneity and propose new inclusive nomenclature to clarify the roles of ILC1s and NK cells during homeostasis and disease.
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http://dx.doi.org/10.1016/j.it.2019.08.005DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6823149PMC
October 2019

Tumor heterogeneity and acquired drug resistance in FGFR2-fusion-positive cholangiocarcinoma through rapid research autopsy.

Cold Spring Harb Mol Case Stud 2019 08 1;5(4). Epub 2019 Aug 1.

Comprehensive Cancer Center, The Ohio State University, Columbus, Ohio 43210, USA.

Cholangiocarcinoma is a highly aggressive and lethal malignancy, with limited treatment options available. Recently, FGFR inhibitors have been developed and utilized in FGFR-mutant cholangiocarcinoma; however, resistance often develops and the genomic determinants of resistance are not fully characterized. We completed whole-exome sequencing (WES) of 11 unique tumor samples obtained from a rapid research autopsy on a patient with FGFR-fusion-positive cholangiocarcinoma who initially responded to the pan-FGFR inhibitor, INCB054828. In vitro studies were carried out to characterize the novel FGFR alteration and secondary mutation identified. Multisite WES and analysis of tumor heterogeneity through subclonal inference identified four genetically distinct cancer cell populations, two of which were only observed after treatment. Additionally, WES revealed an N549H mutation hypothesized to confer resistance to the FGFR inhibitor INCB054828 in a single tumor sample. This hypothesis was corroborated with in vitro cell-based studies in which cells expressing fusion were sensitive to INCB054828 (IC value of 10.16 nM), whereas cells with the addition of the N549H mutation were resistant to INCB054828 (IC value of 1527.57 nM). Furthermore, the N549H secondary mutation displayed cross-resistance to other selective FGFR inhibitors, but remained sensitive to the nonselective inhibitor, ponatinib. Rapid research autopsy has the potential to provide unprecedented insights into the clonal evolution of cancer throughout the course of the disease. In this study, we demonstrate the emergence of a drug resistance mutation and characterize the evolution of tumor subclones within a cholangiocarcinoma disease course.
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http://dx.doi.org/10.1101/mcs.a004002DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6672025PMC
August 2019

Characterization of a KLK2-FGFR2 fusion gene in two cases of metastatic prostate cancer.

Prostate Cancer Prostatic Dis 2019 12 1;22(4):624-632. Epub 2019 May 1.

Comprehensive Cancer Center, The Ohio State University, Columbus, OH, 43210, USA.

Background: The fibroblast growth factor receptor (FGFR) signaling pathway is activated in multiple tumor types through gene amplifications, single base substitutions, or gene fusions. Multiple small molecule kinase inhibitors targeting FGFR are currently being evaluated in clinical trials for patients with FGFR chromosomal translocations. Patients with novel gene fusions involving FGFR may represent candidates for kinase inhibitors.

Methods: A targeted RNA-sequencing assay identified a KLK2-FGFR2 fusion gene in two patients with metastatic prostate cancer. NIH3T3 cells were transduced to express the KLK2-FGFR2 fusion. Migration assays, Western blots, and drug sensitivity assays were performed to functionally characterize the fusion.

Results: Expression of the KLK2-FGFR2 fusion protein in NIH3T3 cells induced a profound morphological change promoting enhanced migration and activation of downstream proteins in FGFR signaling pathways. The KLK2-FGFR2 fusion protein was determined to be highly sensitive to the selective FGFR inhibitors AZD-4547, BGJ398, JNJ-42756943, the irreversible inhibitor TAS-120, and the non-selective inhibitor Ponatinib. The KLK2-FGFR2 fusion did not exhibit sensitivity to the non-selective inhibitor Dovitinib.

Conclusions: Importantly, the KLK2-FGFR2 fusion represents a novel target for precision therapies and should be screened for in men with prostate cancer.
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http://dx.doi.org/10.1038/s41391-019-0145-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6824932PMC
December 2019

Genomic characterization of metastatic ultra-hypermutated interdigitating dendritic cell sarcoma through rapid research autopsy.

Oncotarget 2019 Jan 8;10(3):277-288. Epub 2019 Jan 8.

Department of Internal Medicine, Division of Medical Oncology, Comprehensive Cancer Center, The Ohio State University, Columbus, Ohio, USA.

Interdigitating dendritic cell sarcoma (IDCS) is an extremely rare cancer of dendritic cell origin that lacks a standardized treatment approach. Here, we performed genomic characterization of metastatic IDCS through whole exome sequencing (WES) of tumor tissues procured from a patient who underwent research autopsy. WES was also performed on a treatment-naïve tumor biopsy sample obtained from prior surgical resection. Our analyses revealed ultra-hypermutation, defined as >100 mutations per megabase, in this patient's cancer, which was further characterized by the presence of three distinct mutational signatures including UV radiation and APOBEC signatures. To characterize clonal heterogeneity, we used the bioinformatics tool Canopy to leverage single nucleotide and copy number variants to catalog six subclones across various metastatic tumors. Truncal alterations, defined as being present in all clonal tumor cell populations, in this patient's cancer include point mutations in and and amplifications of and , which are likely driver mutations. In summary, we have performed genomic characterization evaluating tumor mutational burden (TMB) and heterogeneity in a patient with metastatic IDCS. Despite ultra-hypermutation, this patient's cancer was not responsive to treatment with PD-1 inhibition. Our results underscore the importance of characterizing clonal heterogeneity in TMB-high cancers.
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http://dx.doi.org/10.18632/oncotarget.26352DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6349455PMC
January 2019

Cellular pathways in the development of human and murine innate lymphoid cells.

Curr Opin Immunol 2019 02 19;56:100-106. Epub 2018 Dec 19.

Department of Hematology, City of Hope National Medical Center, Los Angeles, CA, United States. Electronic address:

Innate lymphoid cells (ILCs) are critical to effective immune surveillance against pathogens, have malignant counterparts, and contribute to disease. Thus, it is important to understand ILC development. All ILCs are derived from the common lymphoid progenitor cell; however, the exact mechanisms and signals that initiate their divergence from T cells, B cells and one and other are incompletely understood. Evidence now supports a stepwise developmental process that includes distinct cellular intermediates, progressively narrowed differentiation, and some plasticity. While the current models of human and murine ILC development share many similarities, they also include some distinct differences. Together these findings have established a working dynamic model of ILC development.
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http://dx.doi.org/10.1016/j.coi.2018.11.003DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7285385PMC
February 2019

The IL-15-AKT-XBP1s signaling pathway contributes to effector functions and survival in human NK cells.

Nat Immunol 2019 01 10;20(1):10-17. Epub 2018 Dec 10.

The Ohio State University Comprehensive Cancer Center, Columbus, OH, USA.

Interleukin 15 (IL-15) is one of the most important cytokines that regulate the biology of natural killer (NK) cells. Here we identified a signaling pathway-involving the serine-threonine kinase AKT and the transcription factor XBP1s, which regulates unfolded protein response genes-that was activated in response to IL-15 in human NK cells. IL-15 induced the phosphorylation of AKT, which led to the deubiquitination, increased stability and nuclear accumulation of XBP1s protein. XBP1s bound to and recruited the transcription factor T-BET to the gene encoding granzyme B, leading to increased transcription. XBP1s positively regulated the cytolytic activity of NK cells against leukemia cells and was also required for IL-15-mediated NK cell survival through an anti-apoptotic mechanism. Thus, the newly identified IL-15-AKT-XBP1s signaling pathway contributes to enhanced effector functions and survival of human NK cells.
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http://dx.doi.org/10.1038/s41590-018-0265-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6293989PMC
January 2019

CD56 Expression Marks Human Group 2 Innate Lymphoid Cell Divergence from a Shared NK Cell and Group 3 Innate Lymphoid Cell Developmental Pathway.

Immunity 2018 09 4;49(3):464-476.e4. Epub 2018 Sep 4.

Department of Pathology, The James Cancer Hospital and Solove Research Institute, The Ohio State University, Columbus, OH 43210 USA; Comprehensive Cancer Center, The James Cancer Hospital and Solove Research Institute, The Ohio State University, Columbus, OH 43210 USA. Electronic address:

According to the established model of murine innate lymphoid cell (ILC) development, helper ILCs develop separately from natural killer (NK) cells. However, it is unclear how helper ILCs and NK cells develop in humans. Here we elucidated key steps of NK cell, ILC2, and ILC3 development within human tonsils using ex vivo molecular and functional profiling and lineage differentiation assays. We demonstrated that while tonsillar NK cells, ILC2s, and ILC3s originated from a common CD34CD117 ILC precursor pool, final steps of ILC2 development deviated independently and became mutually exclusive from those of NK cells and ILC3s, whose developmental pathways overlapped. Moreover, we identified a CD34CD117 ILC precursor population that expressed CD56 and gave rise to NK cells and ILC3s but not to ILC2s. These data support a model of human ILC development distinct from the mouse, whereby human NK cells and ILC3s share a common developmental pathway separate from ILC2s.
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http://dx.doi.org/10.1016/j.immuni.2018.08.010DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6148384PMC
September 2018

SMAD4 promotes TGF-β-independent NK cell homeostasis and maturation and antitumor immunity.

J Clin Invest 2018 11 15;128(11):5123-5136. Epub 2018 Oct 15.

Division of Hematology, Department of Internal Medicine, College of Medicine, The Ohio State University, Columbus, Ohio, USA.

SMAD4 is the only common SMAD in TGF-β signaling that usually impedes immune cell activation in the tumor microenvironment. However, we demonstrated here that selective deletion of Smad4 in NK cells actually led to dramatically reduced tumor cell rejection and augmented tumor cell metastases, reduced murine CMV clearance, as well as impeded NK cell homeostasis and maturation. This was associated with a downregulation of granzyme B (Gzmb), Kit, and Prdm1 in Smad4-deficient NK cells. We further unveiled the mechanism by which SMAD4 promotes Gzmb expression. Gzmb was identified as a direct target of a transcriptional complex formed by SMAD4 and JUNB. A JUNB binding site distinct from that for SMAD4 in the proximal Gzmb promoter was required for transcriptional activation by the SMAD4-JUNB complex. In a Tgfbr2 and Smad4 NK cell-specific double-conditional KO model, SMAD4-mediated events were found to be independent of canonical TGF-β signaling. Our study identifies and mechanistically characterizes unusual functions and pathways for SMAD4 in governing innate immune responses to cancer and viral infection, as well as NK cell development.
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http://dx.doi.org/10.1172/JCI121227DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6205382PMC
November 2018

Human AML activates the aryl hydrocarbon receptor pathway to impair NK cell development and function.

Blood 2018 10 29;132(17):1792-1804. Epub 2018 Aug 29.

Comprehensive Cancer Center.

Acute myeloid leukemia (AML) can evade the mouse and human innate immune system by suppressing natural killer (NK) cell development and NK cell function. This is driven in part by the overexpression of microRNA (miR)-29b in the NK cells of AML patients, but how this occurs is unknown. In the current study, we demonstrate that the transcription factor aryl hydrocarbon receptor (AHR) directly regulates miR-29b expression. We show that human AML blasts activate the AHR pathway and induce miR-29b expression in NK cells, thereby impairing NK cell maturation and NK cell function, which can be reversed by treating NK cells with an AHR antagonist. Finally, we show that inhibition of constitutive AHR activation in AML blasts lowers their threshold for apoptosis and decreases their resistance to NK cell cytotoxicity. Together, these results identify the AHR pathway as a molecular mechanism by which AML impairs NK cell development and function. The results lay the groundwork in establishing AHR antagonists as potential therapeutic agents for clinical development in the treatment of AML.
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http://dx.doi.org/10.1182/blood-2018-03-838474DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6202909PMC
October 2018

MicroRNA regulation of natural killer cell development and function in leukemia.

Mol Immunol 2019 11 9;115:12-20. Epub 2018 Aug 9.

Division of Hematology, Department of Internal Medicine, The Ohio State University, Columbus, Ohio, United States; Comprehensive Cancer Center and The James Cancer Hospital and Solove Research Institute, The Ohio State University, Columbus, Ohio, United States. Electronic address:

MicroRNAs (miRNAs) are now recognized as important regulators of all cellular processes, including immune function and cancer survival. These evolutionary preserved, single-stranded, non-coding RNA molecules mediate important functional effects primarily through post-transcriptional regulation of protein expression. MiRNAs are known to mediate multiple oncogenic pathways in tumor cells, both tumor promoting and tumor suppressing. In addition to a direct tumor cell effect, miRNAs have also been shown to play a critical role in immune cell development, function and survival. Here we expand on previous reports to evaluate miRNA regulation in natural killer (NK) cells primarily in humans and focus on their influence on NK cell development and function in the setting of hematologic malignancies. In addition, we highlight the most recent miRNA discoveries in hematologic malignancies and discuss areas of future exploration relevant to the translational field of innate immunology and miRNA-based therapeutic intervention.
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http://dx.doi.org/10.1016/j.molimm.2018.07.022DOI Listing
November 2019

Epigenetic and Posttranscriptional Regulation of CD16 Expression during Human NK Cell Development.

J Immunol 2018 01 11;200(2):565-572. Epub 2017 Dec 11.

The Ohio State University Comprehensive Cancer Center, Arthur G. James Cancer Hospital and Richard J. Solove Research Institute, Columbus, OH 43210;

The surface receptor FcγRIIIA (CD16a) is encoded by the gene and is acquired by human NK cells during maturation. NK cells bind the Fc portion of IgG via CD16a and execute Ab-dependent cell-mediated cytotoxicity, which is critical for the effectiveness of several antitumor mAb therapies. The role of epigenetic regulatory mechanisms controlling transcriptional and posttranscriptional CD16 expression in NK cells is unknown. In this study, we compared specific patterns of DNA methylation and expression of with , which differ in cell type-specific expression despite displaying nearly identical genomic sequences. We identified a sequence within the promoter that selectively exhibits reduced methylation in CD16a NK cells versus CD16a NK cells and neutrophils. This region contained the transcriptional start site of the most highly expressed CD16a isoform in NK cells. Luciferase assays revealed remarkable cell-type specificity and methylation-dependent activity of versus -derived sequences. Genomic differences between and are enriched at CpG dinucleotides, and mutation of variant CpGs reversed cell-type specificity. We further identified miR-218 as a posttranscriptional negative regulator of CD16a in NK cells. Forced overexpression of miR-218 in NK cells knocked down CD16a mRNA and protein expression. Moreover, miR-218 was highly expressed in CD16a NK cells compared with CD16a NK cells. Taken together, we propose a system of regulation in human NK cells in which CpG dinucleotide sequences and concurrent DNA methylation confer developmental and cell type-specific transcriptional regulation, whereas miR-218 provides an additional layer of posttranscriptional regulation during the maturation process.
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http://dx.doi.org/10.4049/jimmunol.1701128DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5881939PMC
January 2018

The Broad Spectrum of Human Natural Killer Cell Diversity.

Immunity 2017 11;47(5):820-833

Division of Hematology, Department of Internal Medicine, The Ohio State University, Columbus, OH 43210, USA; Comprehensive Cancer Center and The James Cancer Hospital and Solove Research Institute, The Ohio State University, Columbus, OH 43210, USA. Electronic address:

Natural killer (NK) cells provide protection against infectious pathogens and cancer. For decades it has been appreciated that two major NK cell subsets (CD56 and CD56) exist in humans and have distinct anatomical localization patterns, phenotypes, and functions in immunity. In light of this traditional NK cell dichotomy, it is now clear that the spectrum of human NK cell diversity is much broader than originally appreciated as a result of variegated surface receptor, intracellular signaling molecule, and transcription factor expression; tissue-specific imprinting; and foreign antigen exposure. The recent discoveries of tissue-resident NK cell developmental intermediates, non-NK innate lymphoid cells, and the capacity for NK cells to adapt and differentiate into long-lived memory cells has added further complexity to this field. Here we review our current understanding of the breadth and generation of human NK cell diversity.
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http://dx.doi.org/10.1016/j.immuni.2017.10.008DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5728700PMC
November 2017

Analytic validation and real-time clinical application of an amplicon-based targeted gene panel for advanced cancer.

Oncotarget 2017 Sep 1;8(44):75822-75833. Epub 2017 Sep 1.

Comprehensive Cancer Center, The Ohio State University, Columbus, OH, USA.

Multiplex somatic testing has emerged as a strategy to test patients with advanced cancer. We demonstrate our analytic validation approach for a gene hotspot panel and real-time prospective clinical application for any cancer type. The TruSight Tumor 26 assay amplifies 85 somatic hotspot regions across 26 genes. Using cell line and tumor mixes, we observed that 100% of the 14,715 targeted bases had at least 1000x raw coverage. We determined the sensitivity (100%, 95% CI: 96-100%), positive predictive value (100%, 95% CI: 96-100%), reproducibility (100% concordance), and limit of detection (3% variant allele frequency at 1000x read depth) of this assay to detect single nucleotide variants and small insertions and deletions. Next, we applied the assay prospectively in a clinical tumor sequencing study to evaluate 174 patients with metastatic or advanced cancer, including frozen tumors, formalin-fixed tumors, and enriched peripheral blood mononuclear cells in hematologic cancers. We reported one or more somatic mutations in 89 (53%) of the sequenced tumors (167 passing quality filters). Forty-three of these patients (26%) had mutations that would enable eligibility for targeted therapies. This study demonstrates the validity and feasibility of applying TruSight Tumor 26 for pan-cancer testing using multiple specimen types.
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http://dx.doi.org/10.18632/oncotarget.20616DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5652665PMC
September 2017

IL-18 Drives ILC3 Proliferation and Promotes IL-22 Production via NF-κB.

J Immunol 2017 10 25;199(7):2333-2342. Epub 2017 Aug 25.

The James Cancer Hospital and Solove Research Institute, The Ohio State University Comprehensive Cancer Center, Columbus, OH 43210;

Group 3 innate lymphoid cells (ILC3s) are important regulators of the immune system, maintaining homeostasis in the presence of commensal bacteria, but activating immune defenses in response to microbial pathogens. ILC3s are a robust source of IL-22, a cytokine critical for stimulating the antimicrobial response. We sought to identify cytokines that can promote proliferation and induce or maintain IL-22 production by ILC3s and determine a molecular mechanism for this process. We identified IL-18 as a cytokine that cooperates with an ILC3 survival factor, IL-15, to induce proliferation of human ILC3s, as well as induce and maintain IL-22 production. To determine a mechanism of action, we examined the NF-κB pathway, which is activated by IL-18 signaling. We found that the NF-κB complex signaling component, p65, binds to the proximal region of the promoter and promotes transcriptional activity. Finally, we observed that CD11c dendritic cells expressing IL-18 are found in close proximity to ILC3s in human tonsils in situ. Therefore, we identify a new mechanism by which human ILC3s proliferate and produce IL-22, and identify NF-κB as a potential therapeutic target to be considered in pathologic states characterized by overproduction of IL-18 and/or IL-22.
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http://dx.doi.org/10.4049/jimmunol.1601554DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5624342PMC
October 2017

Validation of a Targeted RNA Sequencing Assay for Kinase Fusion Detection in Solid Tumors.

J Mol Diagn 2017 09 9;19(5):682-696. Epub 2017 Aug 9.

Comprehensive Cancer Center, The Ohio State University, Columbus, Ohio; Department of Internal Medicine, Division of Medical Oncology, The Ohio State University, Columbus, Ohio. Electronic address:

Kinase gene fusions are important drivers of oncogenic transformation and can be inhibited with targeted therapies. Clinical grade diagnostics using RNA sequencing to detect gene rearrangements in solid tumors are limited, and the few that are available require prior knowledge of fusion break points. To address this, we have analytically validated a targeted RNA sequencing assay (OSU-SpARKFuse) for fusion detection that interrogates complete transcripts from 93 kinase and transcription factor genes. From a total of 74 positive and 36 negative control samples, OSU-SpARKFuse had 93.3% sensitivity and 100% specificity for fusion detection. Assessment of repeatability and reproducibility revealed 96.3% and 94.4% concordance between intrarun and interrun technical replicates, respectively. Application of this assay on prospective patient samples uncovered OLFM4 as a novel RET fusion partner in a small-bowel cancer and led to the discovery of a KLK2-FGFR2 fusion in a patient with prostate cancer who subsequently underwent treatment with a pan-fibroblast growth factor receptor inhibitor. Beyond fusion detection, OSU-SpARKFuse has built-in capabilities for discovery research, including gene expression analysis, detection of single-nucleotide variants, and identification of alternative splicing events.
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http://dx.doi.org/10.1016/j.jmoldx.2017.05.006DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5975628PMC
September 2017

Emerging insights on the pathogenesis and treatment of extranodal NK/T cell lymphomas (ENKTL).

Discov Med 2017 03;23(126):189-199

Division of Hematologic Malignancies and Hematopoietic Stem Cell Transplantation, Department of Medical Oncology, Sidney Kimmel Cancer Center, Thomas Jefferson University, Philadelphia, PA 19107, USA.

Extranodal NK/T-cell lymphoma (ENKTL) is a rare aggressive extranodal non-Hodgkin lymphoma (NHL) universally associated with Epstein-Barr virus (EBV). ENKTL most commonly occurs in non-elderly immune competent males in Asia and South America. A number of antecedent lymphoproliferative disorders (LPDs) have been described in Asian and South American patients, but the majority of Caucasian ENKTL patients have no known preceding LPD or underlying immunodeficiency. Other than EBV, no environmental or extrinsic factor has been implicated in oncogenesis. The precise mechanisms by which EBV infects NK or T cells and the virus' role in the pathogenesis of ENKTL have not been fully deciphered. However, a number of recent discoveries including disturbances in cell signaling and mutations in tumor suppressor genes have been identified, which are providing insights into the pathogenesis of ENKTL. In this review, we highlight the molecular, viral, and genetic underpinnings of ENKTL and discuss potential therapeutic implications.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5585079PMC
March 2017

Modeling Human Natural Killer Cell Development in the Era of Innate Lymphoid Cells.

Front Immunol 2017 27;8:360. Epub 2017 Mar 27.

Comprehensive Cancer Center, The James Cancer Hospital and Solove Research Institute, The Ohio State University, Columbus, OH, USA; Division of Hematology and Oncology, Department of Internal Medicine, The James Cancer Hospital and Solove Research Institute, The Ohio State University, Columbus, OH, USA.

Decades after the discovery of natural killer (NK) cells, their developmental pathways in mice and humans have not yet been completely deciphered. Accumulating evidence indicates that NK cells can develop in multiple tissues throughout the body. Moreover, detailed and comprehensive models of NK cell development were proposed soon after the turn of the century. However, with the recent identification and characterization of other subtypes of innate lymphoid cells (ILCs), which show some overlapping functional and phenotypic features with NK cell developmental intermediates, the distinct stages through which human NK cells develop from early hematopoietic progenitor cells remain unclear. Thus, there is a need to reassess and refine older models of NK cell development in the context of new data and in the era of ILCs. Our group has focused on elucidating the developmental pathway of human NK cells in secondary lymphoid tissues (SLTs), including tonsils and lymph nodes. Here, we provide an update of recent progress that has been made with regard to human NK cell development in SLTs, and we discuss these new findings in the context of contemporary models of ILC development.
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http://dx.doi.org/10.3389/fimmu.2017.00360DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5366880PMC
March 2017

Frequency and clinical correlates of elevated plasma Epstein-Barr virus DNA at diagnosis in peripheral T-cell lymphomas.

Int J Cancer 2017 04;140(8):1899-1906

Division of Hematology, The Ohio State University, Columbus, OH.

Epstein-Barr virus (EBV)-encoded RNAs (EBER) in tumor tissue and cell-free plasma EBV-DNA (pEBVd) are detected in EBV-associated lymphomas. Studies have suggested that EBER+ peripheral T-cell lymphomas (PTCL) have worse prognosis but the role of EBV in these neoplasms remains unclear. pEBVd is quantitative and more easily amenable to standardization than EBER, but frequency of pEBVd detection, clinical impact and agreement with EBER status in PTCL are unknown. We retrospectively assessed frequency of detectable pre-treatment pEBVd, presence of EBER in tumor tissue, and outcomes in 61 of 135 EBV-assessable PTCL patients. Fifteen of 61 patients (24.5%, 95% CI: 14-37%) were pre-treatment pEBVd+, with no significant differences in baseline characteristics or treatment between pEBVd+ and pEBVd- patients. EBER-ISH was performed on 10 pEBVd+ and 35 pEBVd- tumors. All 10 pEBVd+ patients were EBER+, but 9 pEBVd- patients were also EBER+. With median follow up of 24 months (range 1-96), overall survival (OS) was shorter in pEBVd+ compared to pEBVd- patients (13 vs. 72 months; p = 0.04). In our retrospective study, pre-treatment pEBVd was elevated in 25% of PTCL patients, was highly specific for EBER+ tumors, and was associated with shorter survival. pEBVd should be further explored as a prognostic variable and tumor biomarker in PTCL.
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http://dx.doi.org/10.1002/ijc.30566DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5323329PMC
April 2017

Biallelic mutations in IRF8 impair human NK cell maturation and function.

J Clin Invest 2017 01 28;127(1):306-320. Epub 2016 Nov 28.

Human NK cell deficiencies are rare yet result in severe and often fatal disease, particularly as a result of viral susceptibility. NK cells develop from hematopoietic stem cells, and few monogenic errors that specifically interrupt NK cell development have been reported. Here we have described biallelic mutations in IRF8, which encodes an interferon regulatory factor, as a cause of familial NK cell deficiency that results in fatal and severe viral disease. Compound heterozygous or homozygous mutations in IRF8 in 3 unrelated families resulted in a paucity of mature CD56dim NK cells and an increase in the frequency of the immature CD56bright NK cells, and this impairment in terminal maturation was also observed in Irf8-/-, but not Irf8+/-, mice. We then determined that impaired maturation was NK cell intrinsic, and gene expression analysis of human NK cell developmental subsets showed that multiple genes were dysregulated by IRF8 mutation. The phenotype was accompanied by deficient NK cell function and was stable over time. Together, these data indicate that human NK cells require IRF8 for development and functional maturation and that dysregulation of this function results in severe human disease, thereby emphasizing a critical role for NK cells in human antiviral defense.
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http://dx.doi.org/10.1172/JCI86276DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5199714PMC
January 2017

Extranodal NK/T Cell Lymphoma, Nasal Type (ENKTL-NT): An Update on Epidemiology, Clinical Presentation, and Natural History in North American and European Cases.

Curr Hematol Malig Rep 2016 12;11(6):514-527

Division of Hematology and Comprehensive Cancer Center, Ohio State University, 320 West 10th Avenue, Columbus, OH, 43210, USA.

Purpose Of Review: Extranodal NK/T cell lymphoma, nasal type (ENKTL-NT) is an aggressive extranodal non-Hodgkin lymphoma most commonly occurring in East Asia and Latin America but with increasing incidence in the United States. Data on epidemiology, disease presentation, and outcome for European and North American ("Western") cases are very limited. We review published landmark clinical studies on ENKTL-NT in the West and report in detail recent data, including our institutional experience.

Recent Findings: We highlight key observations in its epidemiology, natural history, and trends in clinical management. In the USA, ENKTL-NT is more common among Asian Pacific Islanders (API) and Hispanics compared to non-Hispanic whites. Published studies indicate less heterogeneity in clinical presentation in Western ENKTL-NT compared to Asian patients. While there is variation in age at diagnosis, presence of antecedent lymphoproliferative disorders, and outcomes among racial/ethnic groups, the universal association of ENKTL-NT with EBV and the poor response of this neoplasm to anthracycline-based therapy is consistent across all geographic areas. Data on epidemiology, disease presentation, and clinical outcomes in mature T cell and NK cell (T/NK cell) neoplasms, including ENKTL-NT, in Europe and North America are very limited. As the classification and diagnostic characterization of the currently recognized T/NK cell lymphoma disease entities continue to evolve, gaps and inconsistencies in data reporting across different studies are being recognized. Despite these limitations, several studies from the USA suggest that the incidence of ENKTL-NT is higher in Asian Pacific Islanders (API) and non-white Hispanics and that outcomes may be worse in non-whites. However, the universal association of ENKTL-NT with Epstein-Barr virus (EBV) across all ethnic groups suggests a common pathogenesis. Given the overlap between the entities included in the category of T/NK cell neoplasms, there is a need to further define biological and clinical differences that may affect diagnosis, treatment, and outcome.
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http://dx.doi.org/10.1007/s11899-016-0355-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5199232PMC
December 2016