Publications by authors named "Christina R Hartigan"

14 Publications

  • Page 1 of 1

Targeting acute myeloid leukemia dependency on VCP-mediated DNA repair through a selective second-generation small-molecule inhibitor.

Sci Transl Med 2021 03;13(587)

INSERM U1186, Gustave-Roussy Cancer Center, Université Paris-Saclay, 94805 Villejuif, France.

The development and survival of cancer cells require adaptive mechanisms to stress. Such adaptations can confer intrinsic vulnerabilities, enabling the selective targeting of cancer cells. Through a pooled in vivo short hairpin RNA (shRNA) screen, we identified the adenosine triphosphatase associated with diverse cellular activities (AAA-ATPase) valosin-containing protein (VCP) as a top stress-related vulnerability in acute myeloid leukemia (AML). We established that AML was the most responsive disease to chemical inhibition of VCP across a panel of 16 cancer types. The sensitivity to VCP inhibition of human AML cell lines, primary patient samples, and syngeneic and xenograft mouse models of AML was validated using -directed shRNAs, overexpression of a dominant-negative VCP mutant, and chemical inhibition. By combining mass spectrometry-based analysis of the VCP interactome and phospho-signaling studies, we determined that VCP is important for ataxia telangiectasia mutated (ATM) kinase activation and subsequent DNA repair through homologous recombination in AML. A second-generation VCP inhibitor, CB-5339, was then developed and characterized. Efficacy and safety of CB-5339 were validated in multiple AML models, including syngeneic and patient-derived xenograft murine models. We further demonstrated that combining DNA-damaging agents, such as anthracyclines, with CB-5339 treatment synergizes to impair leukemic growth in an MLL-AF9-driven AML murine model. These studies support the clinical testing of CB-5339 as a single agent or in combination with standard-of-care DNA-damaging chemotherapy for the treatment of AML.
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http://dx.doi.org/10.1126/scitranslmed.abg1168DOI Listing
March 2021

TIGIT regulates apoptosis of risky memory T cell subsets implicated in belatacept-resistant rejection.

Am J Transplant 2021 Mar 23. Epub 2021 Mar 23.

Emory Transplant Center, Department of Surgery, Emory University School of Medicine, Atlanta, Georgia, USA.

Belatacept confers increased patient and graft survival in renal transplant recipients relative to calcineurin inhibitors, but is associated with an increased rate of acute rejection. Recent immunophenotypic studies comparing pretransplant T cell phenotypes of patients who reject versus those who remain stable on belatacept identified three potential "risky" memory T cell subsets that potentially underlie belatacept-resistant rejection: CD4 CD28 T , CD8 CD28 , and CD4 CD57 PD1 subsets. Here, we compared key phenotypic and functional aspects of these human memory T cell subsets, with the goal of identifying additional potential targets to modulate them. Results demonstrate that TIGIT, an increasingly well-appreciated immune checkpoint receptor, was expressed on all three risky memory T cell subsets in vitro and in vivo in the presence of belatacept. Coculture of human memory CD4 and CD8 T cells with an agonistic anti-TIGIT mAb significantly increased apoptotic cell death of all three risky memory T cell subsets. Mechanistically, TIGIT-mediated apoptosis of risky memory T cells was dependent on FOXP3 Treg, suggesting that agonism of the TIGIT pathway increases FOXP3 Treg suppression of human memory T cell populations. Overall, these data suggest that TIGIT agonism could represent a new therapeutic target to inhibit belatacept-resistant rejection during transplantation.
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http://dx.doi.org/10.1111/ajt.16571DOI Listing
March 2021

The lncRNA lincNMR regulates nucleotide metabolism via a YBX1 - RRM2 axis in cancer.

Nat Commun 2020 06 25;11(1):3214. Epub 2020 Jun 25.

Division of RNA Biology & Cancer, German Cancer Research Center (DKFZ), Heidelberg, Germany.

Long intergenic non-coding RNA-Nucleotide Metabolism Regulator (lincNMR) is a long non-coding RNA (lncRNA) which is induced in hepatocellular carcinoma. Its depletion invokes a proliferation defect, triggers senescence and inhibits colony formation in liver, but also breast and lung cancer cells. Triple-label SILAC proteomics profiles reveal a deregulation of key cell cycle regulators in lincNMR-depleted cells like the key dNTP synthesizing enzymes RRM2, TYMS and TK1, implicating lincNMR in regulating nucleotide metabolism. LincNMR silencing decreases dNTP levels, while exogenous dNTPs rescues the proliferation defect induced by lincNMR depletion. In vivo RNA Antisense Purification (RAP-MS) identifies YBX1 as a direct interaction partner of lincNMR which regulates RRM2, TYMS and TK1 expression and binds to their promoter regions. In a Chick Chorioallantoic Membrane (CAM) in vivo model, lincNMR-depleted tumors are significantly smaller. In summary, we discover a lincRNA, lincNMR, which regulates tumor cell proliferation through a YBX1-RRM2-TYMS-TK1 axis governing nucleotide metabolism.
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http://dx.doi.org/10.1038/s41467-020-17007-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7316977PMC
June 2020

CD28-Dependent CTLA-4 Expression Fine-Tunes the Activation of Human Th17 Cells.

iScience 2020 Apr 14;23(4):100912. Epub 2020 Feb 14.

Emory Transplant Center, Department of Surgery, Emory University School of Medicine, Atlanta, GA, USA. Electronic address:

Previous work has demonstrated that Th17 memory cells but not Th1 cells are resistant to CD28/CTLA-4 blockade with CTLA-4 Ig, leading us to investigate the individual roles of the CD28 and CTLA-4 cosignaling pathways on Th1 versus Th17 cells. We found that selective CD28 blockade with a domain antibody (dAb) inhibited Th1 cells but surprisingly augmented Th17 responses. CD28 agonism resulted in a profound increase in CTLA-4 expression in Th17 cells as compared with Th1 cells. Consistent with these findings, inhibition of the CD28 signaling protein AKT revealed that CTLA-4 expression on Th17 cells was more significantly reduced by AKT inhibition relative to CTLA-4 expression on Th17 cells. Finally, we found that FOXO1 and FOXO3 overexpression restrained high expression of CTLA-4 on Th17 cells but not Th1 cells. This study demonstrates that the heterogeneity of the CD4 T cell compartment has implications for the immunomodulation of pathologic T cell responses.
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http://dx.doi.org/10.1016/j.isci.2020.100912DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7096747PMC
April 2020

Control of human hemoglobin switching by LIN28B-mediated regulation of BCL11A translation.

Nat Genet 2020 02 20;52(2):138-145. Epub 2020 Jan 20.

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

Increased production of fetal hemoglobin (HbF) can ameliorate the severity of sickle cell disease and β-thalassemia. BCL11A represses the genes encoding HbF and regulates human hemoglobin switching through variation in its expression during development. However, the mechanisms underlying the developmental expression of BCL11A remain mysterious. Here we show that BCL11A is regulated at the level of messenger RNA (mRNA) translation during human hematopoietic development. Despite decreased BCL11A protein synthesis earlier in development, BCL11A mRNA continues to be associated with ribosomes. Through unbiased genomic and proteomic analyses, we demonstrate that the RNA-binding protein LIN28B, which is developmentally expressed in a pattern reciprocal to that of BCL11A, directly interacts with ribosomes and BCL11A mRNA. Furthermore, we show that BCL11A mRNA translation is suppressed by LIN28B through direct interactions, independently of its role in regulating let-7 microRNAs, and that BCL11A is the major target of LIN28B-mediated HbF induction. Our results reveal a previously unappreciated mechanism underlying human hemoglobin switching that illuminates new therapeutic opportunities.
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http://dx.doi.org/10.1038/s41588-019-0568-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7031047PMC
February 2020

A large peptidome dataset improves HLA class I epitope prediction across most of the human population.

Nat Biotechnol 2020 02 16;38(2):199-209. Epub 2019 Dec 16.

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

Prediction of HLA epitopes is important for the development of cancer immunotherapies and vaccines. However, current prediction algorithms have limited predictive power, in part because they were not trained on high-quality epitope datasets covering a broad range of HLA alleles. To enable prediction of endogenous HLA class I-associated peptides across a large fraction of the human population, we used mass spectrometry to profile >185,000 peptides eluted from 95 HLA-A, -B, -C and -G mono-allelic cell lines. We identified canonical peptide motifs per HLA allele, unique and shared binding submotifs across alleles and distinct motifs associated with different peptide lengths. By integrating these data with transcript abundance and peptide processing, we developed HLAthena, providing allele-and-length-specific and pan-allele-pan-length prediction models for endogenous peptide presentation. These models predicted endogenous HLA class I-associated ligands with 1.5-fold improvement in positive predictive value compared with existing tools and correctly identified >75% of HLA-bound peptides that were observed experimentally in 11 patient-derived tumor cell lines.
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http://dx.doi.org/10.1038/s41587-019-0322-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7008090PMC
February 2020

Memory T-cell exhaustion and tolerance in transplantation.

Immunol Rev 2019 11 5;292(1):225-242. Epub 2019 Dec 5.

Emory Transplant Center and Department of Surgery, Emory University, Atlanta, GA, USA.

One of the biggest barriers to achieving allograft tolerance is the presence of immunological memory within the recipient, which confers a faster, more robust immune response that is in most cases more resistant to pharmacologic immunosuppression. This review will identify the mechanisms by which alloreactive T cells arise within hosts prior to transplantation, and explore the properties of immunological memory that contribute to allograft rejection. In doing so we will also illuminate how targeting pathways that induce memory T cell exhaustion can promote allograft tolerance. Recent studies demonstrating the impact of the allograft microenvironment on memory cell survival and activation, as well as new therapeutic strategies that are being explored to mitigate memory driven allograft rejection, will also be reviewed.
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http://dx.doi.org/10.1111/imr.12824DOI Listing
November 2019

Domain-specific Quantification of Prion Protein in Cerebrospinal Fluid by Targeted Mass Spectrometry.

Mol Cell Proteomics 2019 12 26;18(12):2388-2400. Epub 2019 Sep 26.

Proteomics Platform, Broad Institute of MIT and Harvard, Cambridge, MA 02142. Electronic address:

Therapies currently in preclinical development for prion disease seek to lower prion protein (PrP) expression in the brain. Trials of such therapies are likely to rely on quantification of PrP in cerebrospinal fluid (CSF) as a pharmacodynamic biomarker and possibly as a trial endpoint. Studies using PrP ELISA kits have shown that CSF PrP is lowered in the symptomatic phase of disease, a potential confounder for reading out the effect of PrP-lowering drugs in symptomatic patients. Because misfolding or proteolytic cleavage could potentially render PrP invisible to ELISA even if its concentration were constant or increasing in disease, we sought to establish an orthogonal method for CSF PrP quantification. We developed a multi-species targeted mass spectrometry method based on multiple reaction monitoring (MRM) of nine PrP tryptic peptides quantified relative to an isotopically labeled recombinant protein standard for human samples, or isotopically labeled synthetic peptides for nonhuman species. Analytical validation experiments showed process replicate coefficients of variation below 15%, good dilution linearity and recovery, and suitable performance for both CSF and brain homogenate and across humans as well as preclinical species of interest. In = 55 CSF samples from individuals referred to prion surveillance centers with rapidly progressive dementia, all six human PrP peptides, spanning the N- and C-terminal domains of PrP, were uniformly reduced in prion disease cases compared with individuals with nonprion diagnoses. Thus, lowered CSF PrP concentration in prion disease is a genuine result of the disease process and not an artifact of ELISA-based measurement. As a result, dose-finding studies for PrP lowering drugs may need to be conducted in presymptomatic at-risk individuals rather than in symptomatic patients. We provide a targeted mass spectrometry-based method suitable for preclinical quantification of CSF PrP as a tool for drug development.
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http://dx.doi.org/10.1074/mcp.RA119.001702DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6885701PMC
December 2019

The NORAD lncRNA assembles a topoisomerase complex critical for genome stability.

Nature 2018 09 27;561(7721):132-136. Epub 2018 Aug 27.

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

The human genome contains thousands of long non-coding RNAs, but specific biological functions and biochemical mechanisms have been discovered for only about a dozen. A specific long non-coding RNA-non-coding RNA activated by DNA damage (NORAD)-has recently been shown to be required for maintaining genomic stability, but its molecular mechanism is unknown. Here we combine RNA antisense purification and quantitative mass spectrometry to identify proteins that directly interact with NORAD in living cells. We show that NORAD interacts with proteins involved in DNA replication and repair in steady-state cells and localizes to the nucleus upon stimulation with replication stress or DNA damage. In particular, NORAD interacts with RBMX, a component of the DNA-damage response, and contains the strongest RBMX-binding site in the transcriptome. We demonstrate that NORAD controls the ability of RBMX to assemble a ribonucleoprotein complex-which we term NORAD-activated ribonucleoprotein complex 1 (NARC1)-that contains the known suppressors of genomic instability topoisomerase I (TOP1), ALYREF and the PRPF19-CDC5L complex. Cells depleted for NORAD or RBMX display an increased frequency of chromosome segregation defects, reduced replication-fork velocity and altered cell-cycle progression-which represent phenotypes that are mechanistically linked to TOP1 and PRPF19-CDC5L function. Expression of NORAD in trans can rescue defects caused by NORAD depletion, but rescue is significantly impaired when the RBMX-binding site in NORAD is deleted. Our results demonstrate that the interaction between NORAD and RBMX is important for NORAD function, and that NORAD is required for the assembly of the previously unknown topoisomerase complex NARC1, which contributes to maintaining genomic stability. In addition, we uncover a previously unknown function for long non-coding RNAs in modulating the ability of an RNA-binding protein to assemble a higher-order ribonucleoprotein complex.
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http://dx.doi.org/10.1038/s41586-018-0453-zDOI Listing
September 2018

Noncanonical translation via deadenylated 3' UTRs maintains primordial germ cells.

Nat Chem Biol 2018 09 9;14(9):844-852. Epub 2018 Jul 9.

Cardiovascular Research Center, Massachusetts General Hospital, Charlestown, MA, USA.

Primordial germ cells (PGCs) form during early embryogenesis with a supply of maternal mRNAs that contain shorter poly(A) tails. How translation of maternal mRNAs is regulated during PGC development remains elusive. Here we describe a small-molecule screen with zebrafish embryos that identified primordazine, a compound that selectively ablates PGCs. Primordazine's effect on PGCs arises from translation repression through primordazine-response elements in the 3' UTRs. Systematic dissection of primordazine's mechanism of action revealed that translation of mRNAs during early embryogenesis occurs by two distinct pathways, depending on the length of their poly(A) tails. In addition to poly(A)-tail-dependent translation (PAT), early embryos perform poly(A)-tail-independent noncanonical translation (PAINT) via deadenylated 3' UTRs. Primordazine inhibits PAINT without inhibiting PAT, an effect that was also observed in quiescent, but not proliferating, mammalian cells. These studies reveal that PAINT is an alternative form of translation in the early embryo and is indispensable for PGC maintenance.
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http://dx.doi.org/10.1038/s41589-018-0098-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6800240PMC
September 2018

Type 2 Diabetes Variants Disrupt Function of SLC16A11 through Two Distinct Mechanisms.

Cell 2017 Jun;170(1):199-212.e20

Broad Institute of Harvard and MIT, Cambridge, MA 02142, USA; Department of Biology, MIT, Cambridge, MA 02139, USA; Department of Systems Biology, Harvard Medical School, Boston, MA 02115, USA. Electronic address:

Type 2 diabetes (T2D) affects Latinos at twice the rate seen in populations of European descent. We recently identified a risk haplotype spanning SLC16A11 that explains ∼20% of the increased T2D prevalence in Mexico. Here, through genetic fine-mapping, we define a set of tightly linked variants likely to contain the causal allele(s). We show that variants on the T2D-associated haplotype have two distinct effects: (1) decreasing SLC16A11 expression in liver and (2) disrupting a key interaction with basigin, thereby reducing cell-surface localization. Both independent mechanisms reduce SLC16A11 function and suggest SLC16A11 is the causal gene at this locus. To gain insight into how SLC16A11 disruption impacts T2D risk, we demonstrate that SLC16A11 is a proton-coupled monocarboxylate transporter and that genetic perturbation of SLC16A11 induces changes in fatty acid and lipid metabolism that are associated with increased T2D risk. Our findings suggest that increasing SLC16A11 function could be therapeutically beneficial for T2D. VIDEO ABSTRACT.
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http://dx.doi.org/10.1016/j.cell.2017.06.011DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5562285PMC
June 2017

Mass Spectrometry Profiling of HLA-Associated Peptidomes in Mono-allelic Cells Enables More Accurate Epitope Prediction.

Immunity 2017 02;46(2):315-326

Broad Institute of MIT and Harvard, Cambridge, MA, USA; Department of Medical Oncology, Dana-Farber Cancer Institute, Boston, MA, 02215, USA; Department of Medicine, Brigham and Women's Hospital, Boston, MA, 02115, USA; Harvard Medical School, Boston, MA, 02115, USA. Electronic address:

Identification of human leukocyte antigen (HLA)-bound peptides by liquid chromatography-tandem mass spectrometry (LC-MS/MS) is poised to provide a deep understanding of rules underlying antigen presentation. However, a key obstacle is the ambiguity that arises from the co-expression of multiple HLA alleles. Here, we have implemented a scalable mono-allelic strategy for profiling the HLA peptidome. By using cell lines expressing a single HLA allele, optimizing immunopurifications, and developing an application-specific spectral search algorithm, we identified thousands of peptides bound to 16 different HLA class I alleles. These data enabled the discovery of subdominant binding motifs and an integrative analysis quantifying the contribution of factors critical to epitope presentation, such as protein cleavage and gene expression. We trained neural-network prediction algorithms with our large dataset (>24,000 peptides) and outperformed algorithms trained on datasets of peptides with measured affinities. We thus demonstrate a strategy for systematically learning the rules of endogenous antigen presentation.
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http://dx.doi.org/10.1016/j.immuni.2017.02.007DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5405381PMC
February 2017

Multicenter evaluation of the thermo scientific prelude for measurement of immunosuppressant drugs using sample preparation liquid chromatography-tandem mass spectrometry.

Ther Drug Monit 2015 Apr;37(2):161-71

*Department of Laboratory Medicine, Boston Children's Hospital, MA; †Thermo Scientific, Franklin, MA; ‡Department of Clinical Pathology, Cleveland Clinic LL Lab, OH; and §Department of Pathology, Johns Hopkins University Hospital, Baltimore, MD.

Immunosuppressant drugs (ISDs) are commonly prescribed to solid organ transplant patients. Their narrow therapeutic index and potential for toxicity necessitates careful monitoring of blood concentrations. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) methods are increasingly used for ISD measurement. However, there remain many challenges with this methodology, particularly regarding interassay variability. The Thermo Scientific Prelude is an online extraction/liquid chromatography platform that uses turbulent flow technology coupled with MS/MS. A multicenter evaluation of the Prelude for the measurement of cyclosporine A, tacrolimus, and sirolimus is described. ISDs were measured at each site using standardized protocols. Sample preparation liquid chromatography-MS/MS was performed using the Prelude coupled to a TSQ Vantage. Chromatography was achieved with a Cyclone-P TurboFlow/Accucore C8 column combination using a multisolvent loading and eluting pump system. Mass spectrometry acquisitions were performed in selective reaction monitoring mode and data processed using TraceFinder (version 3.1). Multisite mean imprecision for cyclosporine A ranged from 8.8% (54 mcg/L) to 9.8% (450 mcg/L); for tacrolimus, 4.7% (15.5 mcg/L) to 12.6% (2.5 mcg/L); for sirolimus, 7.4% (19.9 mcg/L) to 16.5% (2.6 mcg/L). Approximately 110 specimens were used for method comparison. For cyclosporine A, mean bias against the multisite mean ranged from -18% to 1%; for tacrolimus, values ranged from -7% to 4%; for sirolimus, values ranged from -4% to 2%. Comparisons of multisite mean Prelude results with routine ISD method results was also performed for cyclosporine A (slope = 0.7878, intercept = 24.16, r = 0.98), tacrolimus (slope = 0.9391, intercept = 0.1017, r = 98), and sirolimus (slope = 0.9618, intercept = 0.1483, r = 0.97). The Prelude ISD method offers acceptable and comparable multisite performance. This study has also highlighted the importance of adopting standardized protocols and LC-MS/MS methods for better comparability between ISD assays.
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http://dx.doi.org/10.1097/FTD.0000000000000120DOI Listing
April 2015