Efficacy and Safety of Hydroxychloroquine vs Placebo for Pre-exposure SARS-CoV-2 Prophylaxis Among Health Care Workers: A Randomized Clinical Trial.

JAMA Intern Med. 2020 Sep 30;:

Authors: Abella BS, Jolkovsky EL, Biney BT, Uspal JE, Hyman MC, Frank I, Hensley SE, Gill S, Vogl DT, Maillard I, Babushok DV, Huang AC, Nasta SD, Walsh JC, Wiletyo EP, Gimotty PA, Milone MC, Amaravadi RK, and the Prevention and Treatment of COVID-19 With Hydroxychloroquine (PATCH) Investigators

Abstract
Importance: Health care workers (HCWs) caring for patients with coronavirus disease 2019 (COVID-19) are at risk of exposure to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Currently, to our knowledge, there is no effective pharmacologic prophylaxis for individuals at risk.
Objective: To evaluate the efficacy of hydroxychloroquine to prevent transmission of SARS-CoV-2 in hospital-based HCWs with exposure to patients with COVID-19 using a pre-exposure prophylaxis strategy.
Design, Setting, and Participants: This randomized, double-blind, placebo-controlled clinical trial (the Prevention and Treatment of COVID-19 With Hydroxychloroquine Study) was conducted at 2 tertiary urban hospitals, with enrollment from April 9, 2020, to July 14, 2020; follow-up ended August 4, 2020. The trial randomized 132 full-time, hospital-based HCWs (physicians, nurses, certified nursing assistants, emergency technicians, and respiratory therapists), of whom 125 were initially asymptomatic and had negative results for SARS-CoV-2 by nasopharyngeal swab. The trial was terminated early for futility before reaching a planned enrollment of 200 participants.
Interventions: Hydroxychloroquine, 600 mg, daily, or size-matched placebo taken orally for 8 weeks.
Main Outcomes and Measures: The primary outcome was the incidence of SARS-CoV-2 infection as determined by a nasopharyngeal swab during the 8 weeks of treatment. Secondary outcomes included adverse effects, treatment discontinuation, presence of SARS-CoV-2 antibodies, frequency of QTc prolongation, and clinical outcomes for SARS-CoV-2-positive participants.
Results: Of the 132 randomized participants (median age, 33 years [range, 20-66 years]; 91 women [69%]), 125 (94.7%) were evaluable for the primary outcome. There was no significant difference in infection rates in participants randomized to receive hydroxychloroquine compared with placebo (4 of 64 [6.3%] vs 4 of 61 [6.6%]; P > .99). Mild adverse events were more common in participants taking hydroxychloroquine compared with placebo (45% vs 26%; P = .04); rates of treatment discontinuation were similar in both arms (19% vs 16%; P = .81). The median change in QTc (baseline to 4-week evaluation) did not differ between arms (hydroxychloroquine: 4 milliseconds; 95% CI, -9 to 17; vs placebo: 3 milliseconds; 95% CI, -5 to 11; P = .98). Of the 8 participants with positive results for SARS-CoV-2 (6.4%), 6 developed viral symptoms; none required hospitalization, and all clinically recovered.
Conclusions and Relevance: In this randomized clinical trial, although limited by early termination, there was no clinical benefit of hydroxychloroquine administered daily for 8 weeks as pre-exposure prophylaxis in hospital-based HCWs exposed to patients with COVID-19.
Trial Registration: ClinicalTrials.gov Identifier: NCT04329923.

PMID: 33001138 [PubMed - as supplied by publisher]

Repurposing a novel anti-cancer RXR agonist to attenuate acute GVHD and maintain graft-versus-leukemia responses.

Blood. 2020 Sep 24;:

Authors: Thangavelu G, Wang C, Loschi M, Saha A, Osborn M, Furlan SN, Aoyama K, McDonald-Hyman C, Aguilar EG, Janesick AS, Chandraratna RA, Refaeli Y, Panoskaltsis-Mortari A, MacDonald KP, Hill GR, Zeiser R, Maillard I, Serody J, Murphy WJ, Munn DH, Blumberg B, Brown C, Kuchroo VK, Kean LS, Hippen K, Noelle RJ, Blazar BR

Abstract
The nuclear receptors (NR) retinoid X receptors (RXRs) exert immunomodulatory functions to control inflammation and metabolism via homodimers and heterodimers with several other NRs including retinoic acid receptors. IRX4204 is a novel, highly specific RXR agonist in clinical trials that potently and selectively activates RXR homodimers but not heterodimers. Here, we show that in vivo IRX4204 was compared favorably with FK506 in abrogating acute graft-versus-host disease (GVHD), which was associated with inhibiting allogeneic donor T cell proliferation, reducing T helper 1 differentiation and promoting regulatory T cell (Treg) generation. Recipient IRX4204 treatment reduced intestinal injury and decreased IFN-γ and TNF-α serum levels. Transcriptional analysis of donor T cells isolated from intestines of GVHD mice treated with IRX4204 revealed significant decreases in transcripts regulating pro-inflammatory pathways. In vitro, inducible Treg differentiation from naïve CD4+ T cells was enhanced by IRX4204; in vivo, IRX4204 increased the conversion of donor Foxp3- T cells into peripheral Foxp3+ Tregs in GVHD mice. Using Foxp3 lineage tracer mice in which both the origin and current FoxP3 expression of Tregs can be tracked, we demonstrate that IRX4204 supported Treg stability. Despite favoring Tregs and reducing Th1 differentiation, IRX4204-treated recipients maintained graft-versus-leukemia responses against both leukemia and lymphoma cells. Notably, IRX4204 reduced in vitro human T cell proliferation and enhanced Treg generation in mixed lymphocyte reaction cultures. Collectively, these beneficial effects indicate that targeting RXRs with IRX4204 could be used as a novel approach to prevent acute GVHD in the clinic.

PMID: 32976550 [PubMed - as supplied by publisher]

Single-Cell Analyses Identify Brain Mural Cells Expressing CD19 as Potential Off-Tumor Targets for CAR-T Immunotherapies.

Cell. 2020 Oct 01;183(1):126-142.e17

Authors: Parker KR, Migliorini D, Perkey E, Yost KE, Bhaduri A, Bagga P, Haris M, Wilson NE, Liu F, Gabunia K, Scholler J, Montine TJ, Bhoj VG, Reddy R, Mohan S, Maillard I, Kriegstein AR, June CH, Chang HY, Posey AD, Satpathy AT

Abstract
CD19-directed immunotherapies are clinically effective for treating B cell malignancies but also cause a high incidence of neurotoxicity. A subset of patients treated with chimeric antigen receptor (CAR) T cells or bispecific T cell engager (BiTE) antibodies display severe neurotoxicity, including fatal cerebral edema associated with T cell infiltration into the brain. Here, we report that mural cells, which surround the endothelium and are critical for blood-brain-barrier integrity, express CD19. We identify CD19 expression in brain mural cells using single-cell RNA sequencing data and confirm perivascular staining at the protein level. CD19 expression in the brain begins early in development alongside the emergence of mural cell lineages and persists throughout adulthood across brain regions. Mouse mural cells demonstrate lower levels of Cd19 expression, suggesting limitations in preclinical animal models of neurotoxicity. These data suggest an on-target mechanism for neurotoxicity in CD19-directed therapies and highlight the utility of human single-cell atlases for designing immunotherapies.

PMID: 32961131 [PubMed - in process]

Rates of COVID-19-related Outcomes in Cancer compared to non-Cancer Patients.

medRxiv. 2020 Aug 15;:

Authors: Sun L, Sanjna S, Le A, Desai H, Doucette A, Gabriel P, Ritchie M, Rader D, Maillard I, Bange E, Huang A, Vonderheide RH, DeMichele A, Verma A, Mamtani R, Maxwell KN

Abstract
Cancer patients are a vulnerable population postulated to be at higher risk for severe COVID-19 infection. Increased COVID-19 morbidity and mortality in cancer patients may be attributable to age, comorbidities, smoking, healthcare exposure, and cancer treatments, and partially to the cancer itself. Most studies to date have focused on hospitalized patients with severe COVID-19, thereby limiting the generalizability and interpretability of the association between cancer and COVID-19 severity. We compared outcomes of SARS-CoV-2 infection in 323 patients enrolled prior to the pandemic in a large academic biobank (n=67 cancer patients and n=256 non-cancer patients). After adjusting for demographics, smoking status, and comorbidities, a diagnosis of cancer was independently associated with higher odds of hospitalization (OR 2.16, 95% CI 1.12-4.18) and 30-day mortality (OR 5.67, CI 1.49-21.59). These associations were primarily driven by patients with active cancer. These results emphasize the critical importance of preventing SARS-CoV-2 exposure and mitigating infection in cancer patients.

PMID: 32817956 [PubMed]

Notch signaling at the crossroads of innate and adaptive immunity.

J Leukoc Biol. 2020 Jun 18;:

Authors: Vanderbeck A, Maillard I

Abstract
Notch signaling is an evolutionarily conserved cell-to-cell signaling pathway that regulates cellular differentiation and function across multiple tissue types and developmental stages. In this review, we discuss our current understanding of Notch signaling in mammalian innate and adaptive immunity. The importance of Notch signaling is pervasive throughout the immune system, as it elicits lineage and context-dependent effects in a wide repertoire of cells. Although regulation of binary cell fate decisions encompasses many of the functions first ascribed to Notch in the immune system, recent advances in the field have refined and expanded our view of the Notch pathway beyond this initial concept. From establishing T cell identity in the thymus to regulating mature T cell function in the periphery, the Notch pathway is an essential, recurring signal for the T cell lineage. Among B cells, Notch signaling is required for the development and maintenance of marginal zone B cells in the spleen. Emerging roles for Notch signaling in innate and innate-like lineages such as classical dendritic cells and innate lymphoid cells are likewise coming into view. Lastly, we speculate on the molecular underpinnings that shape the activity and versatility of the Notch pathway.

PMID: 32557824 [PubMed - as supplied by publisher]

One-two punch injury to tolerance mechanisms in graft-versus-host disease.

J Clin Invest. 2020 Apr 01;130(4):1625-1628

Authors: Carrington LJ, Maillard I

Abstract
Chronic graft-versus-host disease (GVHD) is a major complication of allogeneic hematopoietic cell transplantation that resembles autoimmunity, with unclear pathogenesis and few effective therapeutic options. In this issue of the JCI, Dertschnig et al. used mouse models to investigate the basis of T cell autoreactivity following GVHD. Notably, GVHD caused irreversible damage to a population of tolerogenic stromal cells that display peripheral tissue-restricted antigens in lymph nodes, which impaired their capacity to purge and suppress autoreactive T cells. Together with damage to central tolerance mechanisms in the thymus, these findings outline a critical one-two punch injury that profoundly disrupts immune tolerance in this devastating disease.

PMID: 32149731 [PubMed - in process]

GCNT1-Mediated O-Glycosylation of the Sialomucin CD43 Is a Sensitive Indicator of Notch Signaling in Activated T Cells.

J Immunol. 2020 03 15;204(6):1674-1688

Authors: Perkey E, Maurice De Sousa D, Carrington L, Chung J, Dils A, Granadier D, Koch U, Radtke F, Ludewig B, Blazar BR, Siebel CW, Brennan TV, Nolz J, Labrecque N, Maillard I

Abstract
Notch signaling is emerging as a critical regulator of T cell activation and function. However, there is no reliable cell surface indicator of Notch signaling across activated T cell subsets. In this study, we show that Notch signals induce upregulated expression of the Gcnt1 glycosyltransferase gene in T cells mediating graft-versus-host disease after allogeneic bone marrow transplantation in mice. To determine if Gcnt1-mediated O-glycosylation could be used as a Notch signaling reporter, we quantified the core-2 O-glycoform of CD43 in multiple T cell subsets during graft-versus-host disease. Pharmacological blockade of Delta-like Notch ligands abrogated core-2 O-glycosylation in a dose-dependent manner after allogeneic bone marrow transplantation, both in donor-derived CD4+ and CD8+ effector T cells and in Foxp3+ regulatory T cells. CD43 core-2 O-glycosylation depended on cell-intrinsic canonical Notch signals and identified CD4+ and CD8+ T cells with high cytokine-producing ability. Gcnt1-deficient T cells still drove lethal alloreactivity, showing that core-2 O-glycosylation predicted, but did not cause, Notch-dependent T cell pathogenicity. Using core-2 O-glycosylation as a marker of Notch signaling, we identified Ccl19-Cre+ fibroblastic stromal cells as critical sources of Delta-like ligands in graft-versus-host responses irrespective of conditioning intensity. Core-2 O-glycosylation also reported Notch signaling in CD8+ T cell responses to dendritic cell immunization, Listeria infection, and viral infection. Thus, we uncovered a role for Notch in controlling core-2 O-glycosylation and identified a cell surface marker to quantify Notch signals in multiple immunological contexts. Our findings will help refine our understanding of the regulation, cellular source, and timing of Notch signals in T cell immunity.

PMID: 32060138 [PubMed - in process]

Menin inhibitor MI-3454 induces remission in MLL1-rearranged and NPM1-mutated models of leukemia.

J Clin Invest. 2020 02 03;130(2):981-997

Authors: Klossowski S, Miao H, Kempinska K, Wu T, Purohit T, Kim E, Linhares BM, Chen D, Jih G, Perkey E, Huang H, He M, Wen B, Wang Y, Yu K, Lee SC, Danet-Desnoyers G, Trotman W, Kandarpa M, Cotton A, Abdel-Wahab O, Lei H, Dou Y, Guzman M, Peterson L, Gruber T, Choi S, Sun D, Ren P, Li LS, Liu Y, Burrows F, Maillard I, Cierpicki T, Grembecka J

Abstract
The protein-protein interaction between menin and mixed lineage leukemia 1 (MLL1) plays a critical role in acute leukemias with translocations of the MLL1 gene or with mutations in the nucleophosmin 1 (NPM1) gene. As a step toward clinical translation of menin-MLL1 inhibitors, we report development of MI-3454, a highly potent and orally bioavailable inhibitor of the menin-MLL1 interaction. MI-3454 profoundly inhibited proliferation and induced differentiation in acute leukemia cells and primary patient samples with MLL1 translocations or NPM1 mutations. When applied as a single agent, MI-3454 induced complete remission or regression of leukemia in mouse models of MLL1-rearranged or NPM1-mutated leukemia, including patient-derived xenograft models, through downregulation of key genes involved in leukemogenesis. We also identified MEIS1 as a potential pharmacodynamic biomarker of treatment response with MI-3454 in leukemia, and demonstrated that this compound is well tolerated and did not impair normal hematopoiesis in mice. Overall, this study demonstrates, for the first time to our knowledge, profound activity of the menin-MLL1 inhibitor as a single agent in clinically relevant PDX models of leukemia. These data provide a strong rationale for clinical translation of MI-3454 or its analogs for leukemia patients with MLL1 rearrangements or NPM1 mutations.

PMID: 31855575 [PubMed - indexed for MEDLINE]

Inhibition of inositol kinase B controls acute and chronic graft-versus-host disease.

Blood. 2020 01 02;135(1):28-40

Authors: Thangavelu G, Du J, Paz KG, Loschi M, Zaiken MC, Flynn R, Taylor PA, Kirchmeier AK, Panoskaltsis-Mortari A, Luznik L, MacDonald KP, Hill GR, Maillard I, Munn DH, Serody JS, Murphy WJ, Miklos D, Cutler CS, Koreth J, Antin JH, Soiffer RJ, Ritz J, Dahlberg C, Miller AT, Blazar BR

Abstract
T-cell activation releases inositol 1,4,5-trisphosphate (IP3), inducing cytoplasmic calcium (Ca2+) influx. In turn, inositol 1,4,5-trisphosphate 3-kinase B (Itpkb) phosphorylates IP3 to negatively regulate and thereby tightly control Ca2+ fluxes that are essential for mature T-cell activation and differentiation and protection from cell death. Itpkb pathway inhibition increases intracellular Ca2+, induces apoptosis of activated T cells, and can control T-cell-mediated autoimmunity. In this study, we employed genetic and pharmacological approaches to inhibit Itpkb signaling as a means of controlling graft-versus-host disease (GVHD). Murine-induced, Itpkb-deleted (Itpkb-/-) T cells attenuated acute GVHD in 2 models without eliminating A20-luciferase B-cell lymphoma graft-versus-leukemia (GVL). A highly potent, selective inhibitor, GNF362, ameliorated acute GVHD without impairing GVL against 2 acute myeloid leukemia lines (MLL-AF9-eGFP and C1498-luciferase). Compared with FK506, GNF362 more selectively deleted donor alloreactive vs nominal antigen-responsive T cells. Consistent with these data and as compared with FK506, GNF362 had favorable acute GVHD and GVL properties against MLL-AF9-eGFP cells. In chronic GVHD preclinical models that have a pathophysiology distinct from acute GVHD, Itpkb-/- donor T cells reduced active chronic GVHD in a multiorgan system model of bronchiolitis obliterans (BO), driven by germinal center reactions and resulting in target organ fibrosis. GNF362 treatment reduced active chronic GVHD in both BO and scleroderma models. Thus, intact Itpkb signaling is essential to drive acute GVHD pathogenesis and sustain active chronic GVHD, pointing toward a novel clinical application to prevent acute or treat chronic GVHD.

PMID: 31697815 [PubMed - indexed for MEDLINE]

Notch signalling in T cell homeostasis and differentiation.

Open Biol. 2019 11 29;9(11):190187

Authors: Brandstadter JD, Maillard I

Abstract
The evolutionarily conserved Notch signalling pathway regulates the differentiation and function of mature T lymphocytes with major context-dependent consequences in host defence, autoimmunity and alloimmunity. The emerging effects of Notch signalling in T cell responses build upon a more established role for Notch in T cell development. Here, we provide a critical review of this burgeoning literature to make sense of what has been learned so far and highlight the experimental strategies that have been most useful in gleaning physiologically relevant information. We outline the functional consequences of Notch signalling in mature T cells in addition to key specific Notch ligand-receptor interactions and downstream molecular signalling pathways. Our goal is to help clarify future directions for this expanding body of work and the best approaches to answer important open questions.

PMID: 31690218 [PubMed - indexed for MEDLINE]

Notch in the niche: new insights into the role of Notch signaling in the bone marrow.

Haematologica. 2019 11;104(11):2117-2119

Authors: Vanderbeck AN, Maillard I

PMID: 31666339 [PubMed - indexed for MEDLINE]

Donor and host B7-H4 expression negatively regulates acute graft-versus-host disease lethality.

JCI Insight. 2019 10 03;4(19):

Authors: Saha A, Taylor PA, Lees CJ, Panoskaltsis-Mortari A, Osborn MJ, Feser CJ, Thangavelu G, Melchinger W, Refaeli Y, Hill GR, Munn DH, Murphy WJ, Serody JS, Maillard I, Kreymborg K, van den Brink M, Dong C, Huang S, Zang X, Allison JP, Zeiser R, Blazar BR

Abstract
B7-H4 is a negative regulatory B7 family member. We investigated the role of host and donor B7-H4 in regulating acute graft-versus-host disease (GVHD). Allogeneic donor T cells infused into B7-H4-/- versus WT recipients markedly accelerated GVHD-induced lethality. Chimera studies pointed toward B7-H4 expression on host hematopoietic cells as more critical than parenchymal cells in controlling GVHD. Rapid mortality in B7-H4-/- recipients was associated with increased donor T cell expansion, gut T cell homing and loss of intestinal epithelial integrity, increased T effector function (proliferation, proinflammatory cytokines, cytolytic molecules), and reduced apoptosis. Higher metabolic demands of rapidly proliferating donor T cells in B7-H4-/- versus WT recipients required multiple metabolic pathways, increased extracellular acidification rates (ECARs) and oxygen consumption rates (OCRs), and increased expression of fuel substrate transporters. During GVHD, B7-H4 expression was upregulated on allogeneic WT donor T cells. B7-H4-/- donor T cells given to WT recipients increased GVHD mortality and had function and biological properties similar to WT T cells from allogeneic B7-H4-/- recipients. Graft-versus-leukemia responses were intact regardless as to whether B7-H4-/- mice were used as hosts or donors. Taken together, these data provide new insights into the negative regulatory processes that control GVHD and provide support for developing therapeutic strategies directed toward the B7-H4 pathway.

PMID: 31578305 [PubMed - indexed for MEDLINE]

Early Notch Signals Induce a Pathogenic Molecular Signature during Priming of Alloantigen-Specific Conventional CD4+ T Cells in Graft-versus-Host Disease.

J Immunol. 2019 07 15;203(2):557-568

Authors: Chung J, Radojcic V, Perkey E, Parnell TJ, Niknafs Y, Jin X, Friedman A, Labrecque N, Blazar BR, Brennan TV, Siebel CW, Maillard I

Abstract
Graft-versus-host disease (GVHD) is the most serious complication of allogeneic hematopoietic cell transplantation. Notch signals delivered during the first 48 h after transplantation drive proinflammatory cytokine production in conventional T cells (Tconv) and inhibit the expansion of regulatory T cells (Tregs). Short-term Notch inhibition induces long-term GVHD protection. However, it remains unknown whether Notch blockade blunts GVHD through its effects on Tconv, Tregs, or both and what early Notch-regulated molecular events occur in alloantigen-specific T cells. To address these questions, we engineered T cell grafts to achieve selective Notch blockade in Tconv versus Tregs and evaluated their capacity to trigger GVHD in mice. Notch blockade in Tconv was essential for GVHD protection as GVHD severity was similar in the recipients of wild-type Tconv combined with Notch-deprived versus wild-type Tregs. To identify the impact of Notch signaling on the earliest steps of T cell activation in vivo, we established a new acute GVHD model mediated by clonal alloantigen-specific 4C CD4+ Tconv. Notch-deprived 4C T cells had preserved early steps of activation, IL-2 production, proliferation, and Th cell polarization. In contrast, Notch inhibition dampened IFN-γ and IL-17 production, diminished mTORC1 and ERK1/2 activation, and impaired transcription of a subset of Myc-regulated genes. The distinct Notch-regulated signature had minimal overlap with known Notch targets in T cell leukemia and developing T cells, highlighting the specific impact of Notch signaling in mature T cells. Our findings uncover a unique molecular program associated with the pathogenic effects of Notch in T cells at the earliest stages of GVHD.

PMID: 31182480 [PubMed - indexed for MEDLINE]

The PAF1c Subunit CDC73 Is Required for Mouse Hematopoietic Stem Cell Maintenance but Displays Leukemia-Specific Gene Regulation.

Stem Cell Reports. 2019 05 14;12(5):1069-1083

Authors: Saha N, Ropa J, Chen L, Hu H, Mysliwski M, Friedman A, Maillard I, Muntean AG

Abstract
The Polymerase Associated Factor 1 complex (PAF1c) functions at the interface of epigenetics and gene transcription. The PAF1c is required for MLL fusion-driven acute myeloid leukemia (AML) through direct regulation of pro-leukemic target genes such as Hoxa9 and Meis1. However, the role of the PAF1c in normal hematopoiesis is unknown. Here, we discovered that the PAF1c subunit, CDC73, is required for both fetal and adult hematopoiesis. Loss of Cdc73 in hematopoietic cells is lethal because of extensive bone marrow failure. Cdc73 has an essential cell-autonomous role for adult hematopoietic stem cell function in vivo, and deletion of Cdc73 results in cell-cycle defects in hematopoietic progenitors. Gene expression profiling indicated a differential regulation of Hoxa9/Meis1 gene programs by CDC73 in progenitors compared with AML cells, suggesting disease-specific functions. Thus, the PAF1c subunit, CDC73 is essential for hematopoietic stem cell function but exhibits leukemia-specific regulation of self-renewal gene programs in AML cells.

PMID: 31031188 [PubMed - indexed for MEDLINE]

Targeting PI3Kδ function for amelioration of murine chronic graft-versus-host disease.

Am J Transplant. 2019 06;19(6):1820-1830

Authors: Paz K, Flynn R, Du J, Tannheimer S, Johnson AJ, Dong S, Stark AK, Okkenhaug K, Panoskaltsis-Mortari A, Sage PT, Sharpe AH, Luznik L, Ritz J, Soiffer RJ, Cutler CS, Koreth J, Antin JH, Miklos DB, MacDonald KP, Hill GR, Maillard I, Serody JS, Murphy WJ, Munn DH, Feser C, Zaiken M, Vanhaesebroeck B, Turka LA, Byrd JC, Blazar BR

Abstract
Chronic graft-versus-host disease (cGVHD) is a leading cause of morbidity and mortality following allotransplant. Activated donor effector T cells can differentiate into pathogenic T helper (Th)-17 cells and germinal center (GC)-promoting T follicular helper (Tfh) cells, resulting in cGVHD. Phosphoinositide-3-kinase-δ (PI3Kδ), a lipid kinase, is critical for activated T cell survival, proliferation, differentiation, and metabolism. We demonstrate PI3Kδ activity in donor T cells that become Tfh cells is required for cGVHD in a nonsclerodermatous multiorgan system disease model that includes bronchiolitis obliterans (BO), dependent upon GC B cells, Tfhs, and counterbalanced by T follicular regulatory cells, each requiring PI3Kδ signaling for function and survival. Although B cells rely on PI3Kδ pathway signaling and GC formation is disrupted resulting in a substantial decrease in Ig production, PI3Kδ kinase-dead mutant donor bone marrow-derived GC B cells still supported BO cGVHD generation. A PI3Kδ-specific inhibitor, compound GS-649443, that has superior potency to idelalisib while maintaining selectivity, reduced cGVHD in mice with active disease. In a Th1-dependent and Th17-associated scleroderma model, GS-649443 effectively treated mice with active cGVHD. These data provide a foundation for clinical trials of US Food and Drug Administration (FDA)-approved PI3Kδ inhibitors for cGVHD therapy in patients.

PMID: 30748099 [PubMed - indexed for MEDLINE]

Small-molecule BCL6 inhibitor effectively treats mice with nonsclerodermatous chronic graft-versus-host disease.

Blood. 2019 01 03;133(1):94-99

Authors: Paz K, Flynn R, Du J, Qi J, Luznik L, Maillard I, MacDonald KP, Hill GR, Serody JS, Murphy WJ, Sage PT, Sharpe AH, Miklos D, Cutler CS, Koreth J, Antin JH, Soiffer RJ, Ritz J, Bradner JE, Melnick AM, Blazar BR

Abstract
Patient outcomes for steroid-dependent or -refractory chronic graft-versus-host diesease (cGVHD) are poor, and only ibrutinib has been US Food and Drug Administration (FDA) approved for this indication. cGVHD is often driven by the germinal center (GC) reaction, in which T follicular helper cells interact with GC B cells to produce antibodies that are associated with disease pathogenesis. The transcriptional corepressor B-cell lymphoma 6 (BCL6) is a member of the Broad-complex, Tramtrack, and Bric-abrac/poxvirus and zinc finger (BTB/POZ) transcription factor family and master regulator of the immune cells in the GC reaction. We demonstrate that BCL6 expression in both donor T cells and B cells is necessary for cGVHD development, pointing to BCL6 as a therapeutic cGVHD target. A small-molecule BCL6 inhibitor reversed active cGVHD in a mouse model of multiorgan system injury with bronchiolitis obliterans associated with a robust GC reaction, but not in cGVHD mice with scleroderma as the prominent manifestation. For cGVHD patients with antibody-driven cGVHD, targeting of BCL6 represents a new approach with specificity for a master GC regulator that would extend the currently available second-line agents.

PMID: 30279226 [PubMed - indexed for MEDLINE]

Notch signaling mediated by Delta-like ligands 1 and 4 controls the pathogenesis of chronic GVHD in mice.

Blood. 2018 11 15;132(20):2188-2200

Authors: Radojcic V, Paz K, Chung J, Du J, Perkey ET, Flynn R, Ivcevic S, Zaiken M, Friedman A, Yan M, Pletneva MA, Sarantopoulos S, Siebel CW, Blazar BR, Maillard I

Abstract
Chronic graft-versus-host disease (cGVHD) is a major complication of allogeneic hematopoietic cell transplantation (allo-HCT) and remains an area of unmet clinical need with few treatment options available. Notch blockade prevents acute GVHD in multiple mouse models, but the impact of Notch signaling on cGVHD remains unknown. Using genetic and antibody-mediated strategies of Notch inhibition, we investigated the role of Notch signaling in complementary mouse cGVHD models that mimic several aspects of human cGVHD in search of candidate therapeutics. In the B10.D2→BALB/c model of sclerodermatous cGVHD, Delta-like ligand 4 (Dll4)-driven Notch signaling was essential for disease development. Antibody-mediated Dll4 inhibition conferred maximum benefits when pursued early in a preventative fashion, with anti-Dll1 enhancing early protection. Notch-deficient alloantigen-specific T cells showed no early defects in proliferation or helper polarization in vivo but subsequently exhibited markedly decreased cytokine secretion and enhanced accumulation of FoxP3+ regulatory T cells. In the B6→B10.BR major histocompatibility complex-mismatched model with multi-organ system cGVHD and prominent bronchiolitis obliterans (BO), but not skin manifestations, absence of Notch signaling in T cells provided long-lasting disease protection that was replicated by systemic targeting of Dll1, Dll4, or both Notch ligands, even during established disease. Notch inhibition decreased target organ damage and germinal center formation. Moreover, decreased BO-cGVHD was observed upon inactivation of Notch1 and/or Notch2 in T cells. Systemic targeting of Notch2 alone was safe and conferred therapeutic benefits. Altogether, Notch ligands and receptors regulate key pathogenic steps in cGVHD and emerge as novel druggable targets to prevent or treat different forms of cGVHD.

PMID: 30181175 [PubMed - indexed for MEDLINE]

Stage-specific roles for Zmiz1 in Notch-dependent steps of early T-cell development.

Blood. 2018 09 20;132(12):1279-1292

Authors: Wang Q, Yan R, Pinnell N, McCarter AC, Oh Y, Liu Y, Sha C, Garber NF, Chen Y, Wu Q, Ku CJ, Tran I, Serna Alarcon A, Kuick R, Engel JD, Maillard I, Cierpicki T, Chiang MY

Abstract
Notch1 signaling must elevate to high levels in order to drive the proliferation of CD4-CD8- double-negative (DN) thymocytes and progression to the CD4+CD8+ double-positive (DP) stage through β-selection. During this critical phase of pre-T-cell development, which is also known as the DN-DP transition, it is unclear whether the Notch1 transcriptional complex strengthens its signal output as a discrete unit or through cofactors. We previously showed that the protein inhibitor of activated STAT-like coactivator Zmiz1 is a context-dependent cofactor of Notch1 in T-cell leukemia. We also showed that withdrawal of Zmiz1 generated an early T-lineage progenitor (ETP) defect. Here, we show that this early defect seems inconsistent with loss-of-Notch1 function. In contrast, at the later pre-T-cell stage, withdrawal of Zmiz1 impaired the DN-DP transition by inhibiting proliferation, like withdrawal of Notch. In pre-T cells, but not ETPs, Zmiz1 cooperatively regulated Notch1 target genes Hes1, Lef1, and Myc. Enforced expression of either activated Notch1 or Myc partially rescued the Zmiz1-deficient DN-DP defect. We identified residues in the tetratricopeptide repeat (TPR) domain of Zmiz1 that bind Notch1. Mutating only a single residue impaired the Zmiz1-Notch1 interaction, Myc induction, the DN-DP transition, and leukemic proliferation. Similar effects were seen using a dominant-negative TPR protein. Our studies identify stage-specific roles of Zmiz1. Zmiz1 is a context-specific cofactor for Notch1 during Notch/Myc-dependent thymocyte proliferation, whether normal or malignant. Finally, we highlight a vulnerability in leukemic cells that originated from a developmentally important Zmiz1-Notch1 interaction that is hijacked during transformation from normal pre-T cells.

PMID: 30076146 [PubMed - indexed for MEDLINE]

The FOSSIL Study: FLAG or standard 7+3 induction therapy in secondary acute myeloid leukemia.

Leuk Res. 2018 07;70:91-96

Authors: Vulaj V, Perissinotti AJ, Uebel JR, Nachar VR, Scappaticci GB, Crouch A, Bixby DL, Burke PW, Maillard I, Talpaz M, Marini BL

Abstract
Patients with secondary acute myeloid leukemia (sAML) have poor outcomes, with CR/CRi rates of 25-35% with standard 7 + 3 induction chemotherapy, while single center non-comparative analyses suggest promising outcomes with FLAG. We conducted a single-center, retrospective cohort study assessing outcomes in treatment-naïve patients with sAML treated with fludarabine, high-dose cytarabine, and granulocyte colony-stimulating factor (FLAG, n = 40) compared with 7 + 3 (n = 66). Median patient age was 63 years (range: 27-82) in the FLAG group and 60 years (range: 21-76) in the 7 + 3 group (P = 0.968). Patients treated with FLAG achieved higher overall response rates (CR + CRi + MLFS) compared to 7 + 3 (70% vs. 48%, P = 0.043). FLAG was well tolerated, with only one induction death (30-day mortality rate, 3% vs. 8%, P = 0.405) and no cases of cerebellar toxicity. Duration of neutropenia was significantly shorter with FLAG (median 16 vs. 23 days, P < 0.001). Half of the FLAG-treated patients proceeded to consolidative therapy compared with only 27% of those who received 7 + 3 (P = 0.022). Overall survival was comparable between groups (8.5 mos, FLAG vs. 9.1 mos, 7 + 3; P = 0.798). Thus, FLAG may represent a low-cost treatment strategy in sAML that produces higher response rates and promising survival outcomes with minimal treatment-related toxicity. Further studies are required to prospectively compare FLAG to the newly FDA-approved CPX-351 in sAML.

PMID: 29908418 [PubMed - indexed for MEDLINE]

An immune-beige adipocyte communication via nicotinic acetylcholine receptor signaling.

Nat Med. 2018 06;24(6):814-822

Authors: Jun H, Yu H, Gong J, Jiang J, Qiao X, Perkey E, Kim DI, Emont MP, Zestos AG, Cho JS, Liu J, Kennedy RT, Maillard I, Xu XZS, Wu J

Abstract
Beige adipocytes have recently been shown to regulate energy dissipation when activated and help organisms defend against hypothermia and obesity. Prior reports indicate that beige-like adipocytes exist in adult humans and that they may present novel opportunities to curb the global epidemic in obesity and metabolic illnesses. In an effort to identify unique features of activated beige adipocytes, we found that expression of the cholinergic receptor nicotinic alpha 2 subunit (Chrna2) was induced in subcutaneous fat during the activation of these cells and that acetylcholine-producing immune cells within this tissue regulated this signaling pathway via paracrine mechanisms. CHRNA2 functioned selectively in uncoupling protein 1 (Ucp1)-positive beige adipocytes, increasing thermogenesis through a cAMP- and protein kinase A-dependent pathway. Furthermore, this signaling via CHRNA2 was conserved and present in human subcutaneous adipocytes. Inactivation of Chrna2 in mice compromised the cold-induced thermogenic response selectively in subcutaneous fat and exacerbated high-fat diet-induced obesity and associated metabolic disorders, indicating that even partial loss of beige fat regulation in vivo had detrimental consequences. Our results reveal a beige-selective immune-adipose interaction mediated through CHRNA2 and identify a novel function of nicotinic acetylcholine receptors in energy metabolism. These findings may lead to identification of therapeutic targets to counteract human obesity.

PMID: 29785025 [PubMed - indexed for MEDLINE]

MIR142 Loss-of-Function Mutations Derepress ASH1L to Increase HOXA Gene Expression and Promote Leukemogenesis.

Cancer Res. 2018 07 01;78(13):3510-3521

Authors: Trissal MC, Wong TN, Yao JC, Ramaswamy R, Kuo I, Baty J, Sun Y, Jih G, Parikh N, Berrien-Elliott MM, Fehniger TA, Ley TJ, Maillard I, Reddy PR, Link DC

Abstract
Point mutations in the seed sequence of miR-142-3p are present in a subset of acute myelogenous leukemia (AML) and in several subtypes of B-cell lymphoma. Here, we show that mutations associated with AML result both in loss of miR-142-3p function and in decreased miR-142-5p expression. Mir142 loss altered the hematopoietic differentiation of multipotent hematopoietic progenitors, enhancing their myeloid potential while suppressing their lymphoid potential. During hematopoietic maturation, loss of Mir142 increased ASH1L protein expression and consequently resulted in the aberrant maintenance of Hoxa gene expression in myeloid-committed hematopoietic progenitors. Mir142 loss also enhanced the disease-initiating activity of IDH2-mutant hematopoietic cells in mice. Together these data suggest a novel model in which miR-142, through repression of ASH1L activity, plays a key role in suppressing HOXA9/A10 expression during normal myeloid differentiation. AML-associated loss-of-function mutations of MIR142 disrupt this negative signaling pathway, resulting in sustained HOXA9/A10 expression in myeloid progenitors/myeloblasts and ultimately contributing to leukemic transformation.Significance: These findings provide mechanistic insights into the role of miRNAs in leukemogenesis and hematopoietic stem cell function. Cancer Res; 78(13); 3510-21. ©2018 AACR.

PMID: 29724719 [PubMed - indexed for MEDLINE]