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Gain-of-function mutations in the histone acetylation "reader" eleven-nineteen-leukemia (ENL), found in acute myeloid leukemia (AML) and Wilms tumor, are known to drive condensate formation and gene activation in cellular systems. However, their role in tumorigenesis remains unclear. Using a conditional knock-in mouse model, we show that mutant ENL perturbs normal hematopoiesis, induces aberrant expansion of myeloid progenitors, and triggers rapid onset of aggressive AML. Mutant ENL alters developmental and inflammatory gene programs in part by remodeling histone modifications. Mutant ENL forms condensates in hematopoietic stem/progenitor cells at key leukemogenic genes, and disrupting condensate formation via mutagenesis impairs its chromatin and oncogenic function. Moreover, treatment with an acetyl-binding inhibitor of the mutant ENL displaces these condensates from target loci, inhibits mutant ENL-induced chromatin changes, and delays AML initiation and progression in vivo. Our study elucidates the function of ENL mutations in chromatin regulation and tumorigenesis and demonstrates the potential of targeting pathogenic condensates in cancer treatment. Significance: A direct link between ENL mutations, condensate formation, and tumorigenesis is lacking. This study elucidates the function and mechanism of ENL mutations in leukemogenesis, establishing these mutations as bona fide oncogenic drivers. Our results also support the role of condensate dysregulation in cancer and reveal strategies to target pathogenic condensates., (©2024 The Authors; Published by the American Association for Cancer Research.)

Non-hematopoietic cells are essential contributors to hematopoiesis. However, heterogeneity and spatial organization of these cells in human bone marrow remain largely uncharacterized. We used single-cell RNA sequencing (scRNA-seq) to profile 29,325 non-hematopoietic cells and discovered nine transcriptionally distinct subtypes. We simultaneously profiled 53,417 hematopoietic cells and predicted their interactions with non-hematopoietic subsets. We employed co-detection by indexing (CODEX) to spatially profile over 1.2 million cells. We integrated scRNA-seq and CODEX data to link predicted cellular signaling with spatial proximity. Our analysis revealed a hyperoxygenated arterio-endosteal neighborhood for early myelopoiesis, and an adipocytic localization for early hematopoietic stem and progenitor cells (HSPCs). We used our CODEX atlas to annotate new images and uncovered mesenchymal stromal cell (MSC) expansion and spatial neighborhoods co-enriched for leukemic blasts and MSCs in acute myeloid leukemia (AML) patient samples. This spatially resolved, multiomic atlas of human bone marrow provides a reference for investigation of cellular interactions that drive hematopoiesis., Competing Interests: Declaration of interests Maillard: Garuda Therapeutics: Membership on an entity's Board of Directors or advisory committees; Regeneron: Research Funding; Genentech: Research Funding. Carroll: Cartography Bioscences: Membership on an entity's Board of Directors or advisory committees; Janssen Pharmaceuticals: Consultancy., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

In multicellular organisms, cell-to-cell communication is critical for the regulation of tissue organization. Notch signaling relies on direct interactions between Notch receptors on signal-receiving cells and Notch ligands on adjacent cells. Notch evolved to mediate local cellular interactions that are responsive to spatial cues via dosage-sensitive short-lived signals. Immune cells utilize these unique properties of Notch signaling to direct their development, differentiation, and function. In this review, we explore how immune cells interact through Notch receptors with stromal cells in specialized niches of lymphohematopoietic organs that express Notch-activating ligands. We emphasize factors that control these interactions and focus on how Notch signals communicate spatial, quantitative, and temporal information to program the function of signal-receiving cells in the immune system., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests:Work on Notch signaling in the Maillard laboratory is supported by National Institutes of Health (R01-AI091627 to I.M.). M.S. is supported by T32-CA009615. I.M. has received research funding from Genentech and Regeneron, and he is a member of Garuda Therapeutics’ scientific advisory board., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

In development, pioneer transcription factors access silent chromatin to reveal lineage-specific gene programs. The structured DNA-binding domains of pioneer factors have been well characterized, but whether and how intrinsically disordered regions affect chromatin and control cell fate is unclear. Here, we report that deletion of an intrinsically disordered region of the pioneer factor TCF-1 (termed L1) leads to an early developmental block in T cells. The few T cells that develop from progenitors expressing TCF-1 lacking L1 exhibit lineage infidelity distinct from the lineage diversion of TCF-1-deficient cells. Mechanistically, L1 is required for activation of T cell genes and repression of GATA2-driven genes, normally reserved to the mast cell and dendritic cell lineages. Underlying this lineage diversion, L1 mediates binding of TCF-1 to its earliest target genes, which are subject to repression as T cells develop. These data suggest that the intrinsically disordered N terminus of TCF-1 maintains T cell lineage fidelity., (© 2023. The Author(s), under exclusive licence to Springer Nature America, Inc.)

Diffuse large B-cell lymphoma (DLBCL) is the most common subtype of non-Hodgkin lymphoma. Up to 40% of patients with DLBCL display refractory disease or relapse after standard chemotherapy treatment (rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisone [R-CHOP]), leading to significant morbidity and mortality. The molecular mechanisms of chemoresistance in DLBCL remain incompletely understood. Using a cullin-really interesting new gene (RING) ligase-based CRISPR-Cas9 library, we identify that inactivation of the E3 ubiquitin ligase KLHL6 promotes DLBCL chemoresistance. Furthermore, proteomic approaches helped identify KLHL6 as a novel master regulator of plasma membrane-associated NOTCH2 via proteasome-dependent degradation. In CHOP-resistant DLBCL tumors, mutations of NOTCH2 result in a protein that escapes the mechanism of ubiquitin-dependent proteolysis, leading to protein stabilization and activation of the oncogenic RAS signaling pathway. Targeting CHOP-resistant DLBCL tumors with the phase 3 clinical trial molecules nirogacestat, a selective γ-secretase inhibitor, and ipatasertib, a pan-AKT inhibitor, synergistically promotes DLBCL destruction. These findings establish the rationale for therapeutic strategies aimed at targeting the oncogenic pathway activated in KLHL6- or NOTCH2-mutated DLBCL., (© 2023 by The American Society of Hematology.)

Nonhematopoietic lymph node stromal cells (LNSCs) regulate lymphocyte trafficking, survival, and function for key roles in host defense, autoimmunity, alloimmunity, and lymphoproliferative disorders. However, the study of LNSCs in human diseases is complicated by a dependence on viable lymphoid tissues, which are most often excised prior to establishment of a specific diagnosis. Here, we demonstrate that cryopreservation can be used to bank lymphoid tissue for the study of LNSCs in human disease. Using human tonsils and lymph nodes (LN), lymphoid tissue fragments were cryopreserved for subsequent enzymatic digestion and recovery of viable nonhematopoietic cells. Flow cytometry and single-cell transcriptomics identified comparable proportions of LN stromal cell types in fresh and cryopreserved tissue. Moreover, cryopreservation had little effect on transcriptional profiles, which showed significant overlap between tonsils and LN. The presence and spatial distribution of transcriptionally defined cell types were confirmed by in situ analyses. Our broadly applicable approach promises to greatly enable research into the roles of LNSCs in human disease., (© 2023 Wiley-VCH GmbH.)

Allogeneic hematopoietic cell transplantation can cure patients with high-risk leukemia through graft-versus-leukemia (GVL) effects, the process by which malignant leukemic cells are cleared by donor-derived immune cells from the graft. The problem of harnessing GVL effects while controlling inflammation and host-organ damage linked with graft-versus-host disease (GVHD) has been the most formidable hurdle facing allogeneic hematopoietic cell transplantation. This powerful, curative-intent therapy remains among the most toxic treatments in the hematologist's armamentarium due to the combined risks of GVHD-related morbidity, infections, and leukemia relapse. In this issue of the JCI, Li, Wang, et al. report that T cell Stat3 deficiency can extricate GVL effects from GVHD through tissue-specific programmed death-ligand 1/programmed cell death protein 1-dependent (PD-L1/PD-1-dependent) bioenergetic alterations that blunt harmful T cell effects in GVHD target organs, while preserving their beneficial antitumor activity in lymphohematopoietic tissues.

Damage to the gastrointestinal tract following allogeneic hematopoietic stem cell transplantation is a significant contributor to the severity and perpetuation of graft-versus-host disease. In preclinical models and clinical trials, we showed that infusing high numbers of regulatory T cells reduces graft-versus-host disease incidence. Despite no change in in vitro suppressive function, transfer of ex vivo expanded regulatory T cells transduced to overexpress G protein-coupled receptor 15 or C-C motif chemokine receptor 9, specific homing receptors for colon or small intestine, respectively, lessened graft-versus-host disease severity in mice. Increased regulatory T cell frequency and retention within the gastrointestinal tissues of mice that received gut homing T cells correlated with lower inflammation and gut damage early post-transplant, decreased graft-versus-host disease severity, and prolonged survival compared with those receiving control transduced regulatory T cells. These data provide evidence that enforced targeting of ex vivo expanded regulatory T cells to the gastrointestinal tract diminishes gut injury and is associated with decreased graft-versus-host disease severity., (Copyright © 2023 American Society of Transplantation & American Society of Transplant Surgeons. Published by Elsevier Inc. All rights reserved.)

Fibroblastic reticular cells (FRCs) direct the interaction and activation of immune cells in discrete microenvironments of lymphoid organs. Despite their important role in steering innate and adaptive immunity, the age- and inflammation-associated changes in the molecular identity and functional properties of human FRCs have remained largely unknown. Here, we show that human tonsillar FRCs undergo dynamic reprogramming during life and respond vigorously to inflammatory perturbation in comparison to other stromal cell types. The peptidase inhibitor 16 (PI16)-expressing reticular cell (PI16 + RC) subset of adult tonsils exhibited the strongest inflammation-associated structural remodeling. Interactome analysis combined with ex vivo and in vitro validation revealed that T cell activity within subepithelial niches is controlled by distinct molecular pathways during PI16 + RC-lymphocyte interaction. In sum, the topological and molecular definition of the human tonsillar stromal cell landscape reveals PI16 + RCs as a specialized FRC niche at the core of mucosal immune responses in the oropharynx., (© 2023. The Author(s).)

Notch signaling promotes T cell pathogenicity and graft-versus-host disease (GVHD) after allogeneic hematopoietic cell transplantation (allo-HCT) in mice, with a dominant role for the Delta-like Notch ligand DLL4. To assess whether Notch's effects are evolutionarily conserved and to identify the mechanisms of Notch signaling inhibition, we studied antibody-mediated DLL4 blockade in a nonhuman primate (NHP) model similar to human allo-HCT. Short-term DLL4 blockade improved posttransplant survival with durable protection from gastrointestinal GVHD in particular. Unlike prior immunosuppressive strategies tested in the NHP GVHD model, anti-DLL4 interfered with a T cell transcriptional program associated with intestinal infiltration. In cross-species investigations, Notch inhibition decreased surface abundance of the gut-homing integrin α4β7 in conventional T cells while preserving α4β7 in regulatory T cells, with findings suggesting increased β1 competition for α4 binding in conventional T cells. Secondary lymphoid organ fibroblastic reticular cells emerged as the critical cellular source of Delta-like Notch ligands for Notch-mediated up-regulation of α4β7 integrin in T cells after allo-HCT. Together, DLL4-Notch blockade decreased effector T cell infiltration into the gut, with increased regulatory to conventional T cell ratios early after allo-HCT. Our results identify a conserved, biologically unique, and targetable role of DLL4-Notch signaling in intestinal GVHD.

The chromatin reader eleven-nineteen leukemia (ENL) has been identified as a critical dependency in acute myeloid leukemia (AML), but its therapeutic potential remains unclear. We describe a potent and orally bioavailable small-molecule inhibitor of ENL, TDI-11055, which displaces ENL from chromatin by blocking its YEATS domain interaction with acylated histones. Cell lines and primary patient samples carrying MLL rearrangements or NPM1 mutations are responsive to TDI-11055. A CRISPR-Cas9-mediated mutagenesis screen uncovers an ENL mutation that confers resistance to TDI-11055, validating the compound's on-target activity. TDI-11055 treatment rapidly decreases chromatin occupancy of ENL-associated complexes and impairs transcription elongation, leading to suppression of key oncogenic gene expression programs and induction of differentiation. In vivo treatment with TDI-11055 blocks disease progression in cell line- and patient-derived xenograft models of MLL-rearranged and NPM1-mutated AML. Our results establish ENL displacement from chromatin as a promising epigenetic therapy for molecularly defined AML subsets and support the clinical translation of this approach., Significance: AML is a poor-prognosis disease for which new therapeutic approaches are desperately needed. We developed an orally bioavailable inhibitor of ENL, demonstrated its potent efficacy in MLL-rearranged and NPM1-mutated AML, and determined its mechanisms of action. These biological and chemical insights will facilitate both basic research and clinical translation. This article is highlighted in the In This Issue feature, p. 2483., (©2022 The Authors; Published by the American Association for Cancer Research.)

Peripheral T-cell lymphomas (PTCL) are agressive lymphomas that develop from mature T cells. The most common PTCLs are genetically, molecularly, and clinically diverse and are generally associated with dismal outcomes. While Notch signaling plays a critically important role in both the development of immature T cells and their malignant transformation, its role in PTCL is poorly understood, despite the increasingly appreciated function of Notch in regulating the proliferation and differentiation of mature T cells. Here, we demonstrate that Notch receptors and their Delta-like family ligands (DLL1/DLL4) play a pathogenic role in PTCL. Notch1 activation was observed in common PTCL subtypes, including PTCL-not otherwise specified (NOS). In a large cohort of PTCL-NOS biopsies, Notch1 activation was significantly associated with surrogate markers of proliferation. Complementary genetically engineered mouse models and spontaneous PTCL models were used to functionally examine the role of Notch signaling, and Notch1/Notch2 blockade and pan-Notch blockade using dominant-negative MAML significantly impaired the proliferation of malignant T cells and PTCL progression in these models. Treatment with DLL1/DLL4 blocking antibodies established that Notch signaling is ligand-dependent. Together, these findings reveal a role for ligand-dependent Notch signaling in driving peripheral T-cell lymphomagenesis., Significance: This work demonstrates that ligand-dependent Notch activation promotes the growth and proliferation of mature T-cell lymphomas, providing new therapeutic strategies for this group of aggressive lymphomas., (©2022 American Association for Cancer Research.)

In lymphopenic environments, secondary lymphoid organs regulate the size of B and T cell compartments by supporting the homeostatic proliferation of mature lymphocytes. The molecular mechanisms underlying these responses and their functional consequences remain incompletely understood. To evaluate homeostasis of the mature B cell pool during lymphopenia, we turned to an adoptive transfer model of purified follicular B cells into Rag2-/- mouse recipients. Highly purified follicular B cells transdifferentiated into marginal zone-like B cells when transferred into Rag2-/- lymphopenic hosts but not into wild-type hosts. In lymphopenic spleens, transferred B cells gradually lost their follicular phenotype and acquired characteristics of marginal zone B cells, as judged by cell surface phenotype, expression of integrins and chemokine receptors, positioning close to the marginal sinus, and an ability to rapidly generate functional plasma cells. Initiation of follicular to marginal zone B cell transdifferentiation preceded proliferation. Furthermore, the transdifferentiation process was dependent on Notch2 receptors in B cells and expression of Delta-like 1 Notch ligands by splenic Ccl19-Cre+ fibroblastic stromal cells. Gene expression analysis showed rapid induction of Notch-regulated transcripts followed by upregulated Myc expression and acquisition of broad transcriptional features of marginal zone B cells. Thus, naive mature B cells are endowed with plastic transdifferentiation potential in response to increased stromal Notch ligand availability during lymphopenia.

Regulatory T cells (Tregs) promote immune homeostasis by maintaining self-tolerance and regulating inflammatory responses. Under certain inflammatory conditions, Tregs can lose their lineage stability and function. Previous studies have reported that ex vivo exposure to retinoic acid (RA) enhances Treg function and stability. However, it is unknown how RA receptor signaling in Tregs influences these processes in vivo. Herein, we employed mouse models in which RA signaling is silenced by the expression of the dominant negative receptor (DN) RARα in all T cells. Despite the fact that DNRARα conventional T cells are hypofunctional, Tregs had increased CD25 expression, STAT5 pathway activation, mTORC1 signaling and supersuppressor function. Furthermore, DNRARα Tregs had increased inhibitory molecule expression, amino acid transporter expression, and metabolic fitness and decreased antiapoptotic proteins. Supersuppressor function was observed when wild-type mice were treated with a pharmacologic pan-RAR antagonist. Unexpectedly, Treg-specific expression of DNRARα resulted in distinct phenotypes, such that a single allele of DNRARα in Tregs heightened their suppressive function, and biallelic expression led to loss of suppression and autoimmunity. The loss of Treg function was not cell intrinsic, as Tregs that developed in a noninflammatory milieu in chimeric mice reconstituted with DNRARα and wild-type bone marrow maintained the enhanced suppressive capacity. Fate mapping suggested that maintaining Treg stability in an inflammatory milieu requires RA signaling. Our findings indicate that RA signaling acts as a rheostat to balance Treg function in inflammatory and noninflammatory conditions in a dose-dependent manner., (© 2022. The Author(s), under exclusive licence to CSI and USTC.)

Despite advances in the field, chronic graft-versus-host-disease (cGVHD) remains a leading cause of morbidity and mortality following allogenic hematopoietic stem cell transplant. Because treatment options remain limited, we tested efficacy of anticancer, chromatin-modifying enzyme inhibitors in a clinically relevant murine model of cGVHD with bronchiolitis obliterans (BO). We observed that the novel enhancer of zeste homolog 2 (EZH2) inhibitor JQ5 and the BET-bromodomain inhibitor JQ1 each improved pulmonary function; impaired the germinal center (GC) reaction, a prerequisite in cGVHD/BO pathogenesis; and JQ5 reduced EZH2-mediated H3K27me3 in donor T cells. Using conditional EZH2 knockout donor cells, we demonstrated that EZH2 is obligatory for the initiation of cGVHD/BO. In a sclerodermatous cGVHD model, JQ5 reduced the severity of cutaneous lesions. To determine how the 2 drugs could lead to the same physiological improvements while targeting unique epigenetic processes, we analyzed the transcriptomes of splenic GCB cells (GCBs) from transplanted mice treated with either drug. Multiple inflammatory and signaling pathways enriched in cGVHD/BO GCBs were reduced by each drug. GCBs from JQ5- but not JQ1-treated mice were enriched for proproliferative pathways also seen in GCBs from bone marrow-only transplanted mice, likely reflecting their underlying biology in the unperturbed state. In conjunction with in vivo data, these insights led us to conclude that epigenetic targeting of the GC is a viable clinical approach for the treatment of cGVHD, and that the EZH2 inhibitor JQ5 and the BET-bromodomain inhibitor JQ1 demonstrated clinical potential for EZH2i and BETi in patients with cGVHD/BO., (© 2022 by The American Society of Hematology.)

X-linked moesin associated immunodeficiency (X-MAID) is a primary immunodeficiency disease in which patients suffer from profound lymphopenia leading to recurrent infections. The disease is caused by a single point mutation leading to a R171W amino acid change in the protein moesin (moesin R171W ). Moesin is a member of the ERM family of proteins, which reversibly link the cortical actin cytoskeleton to the plasma membrane. Here, we describe a novel mouse model with global expression of moesin R171W that recapitulates multiple facets of patient disease, including severe lymphopenia. Further analysis reveals that these mice have diminished numbers of thymocytes and bone marrow precursors. X-MAID mice also exhibit systemic inflammation that is ameliorated by elimination of mature lymphocytes through breeding to a Rag1-deficient background. The few T cells in the periphery of X-MAID mice are highly activated and have mostly lost moesin R171W expression. In contrast, single-positive (SP) thymocytes do not appear activated and retain high expression levels of moesin R171W . Analysis of ex vivo CD4 SP thymocytes reveals defects in chemotactic responses and reduced migration on integrin ligands. While chemokine signaling appears intact, CD4 SP thymocytes from X-MAID mice are unable to polarize and rearrange cytoskeletal elements. This mouse model will be a valuable tool for teasing apart the complexity of the immunodeficiency caused by moesin R171W , and will provide new insights into how the actin cortex regulates lymphocyte function., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Avery, Robertson, Wu, Roy, Chauvin, Perkey, Vanderbeck, Maillard and Burkhardt.)

Non-neuronal cholinergic signaling, mediated by acetylcholine, plays important roles in physiological processes including inflammation and immunity. Our group first discovered evidence of non-neuronal cholinergic circuitry in adipose tissue, whereby immune cells secrete acetylcholine to activate beige adipocytes during adaptive thermogenesis. Here, we reveal that macrophages are the cellular protagonists responsible for secreting acetylcholine to regulate thermogenic activation in subcutaneous fat, and we term these cells cholinergic adipose macrophages (ChAMs). An adaptive increase in ChAM abundance is evident following acute cold exposure, and macrophage-specific deletion of choline acetyltransferase (ChAT), the enzyme for acetylcholine biosynthesis, impairs the cold-induced thermogenic capacity of mice. Further, using pharmacological and genetic approaches, we show that ChAMs are regulated via adrenergic signaling, specifically through the β 2 adrenergic receptor. These findings demonstrate that macrophages are an essential adipose tissue source of acetylcholine for the regulation of adaptive thermogenesis, and may be useful for therapeutic targeting in metabolic diseases., (© 2021 The Authors.)

Purpose: Multiple studies have demonstrated the negative impact of cancer care delays during the COVID-19 pandemic, and transmission mitigation techniques are imperative for continued cancer care delivery. We aimed to gauge the effectiveness of these measures at the University of Pennsylvania., Methods: We conducted a longitudinal study of SARS-CoV-2 antibody seropositivity and seroconversion in patients presenting to infusion centers for cancer-directed therapy between May 21, 2020, and October 8, 2020. Participants completed questionnaires and had up to five serial blood collections., Results: Of 124 enrolled patients, only two (1.6%) had detectable SARS-CoV-2 antibodies on initial blood draw, and no initially seronegative patients developed newly detectable antibodies on subsequent blood draw(s), corresponding to a seroconversion rate of 0% (95% CI, 0.0 TO 4.1%) over 14.8 person-years of follow up, with a median of 13 health care visits per patient., Conclusion: These results suggest that patients with cancer receiving in-person care at a facility with aggressive mitigation efforts have an extremely low likelihood of COVID-19 infection., Competing Interests: Peter GabrielTravel, Accommodations, Expenses: Varian Medical Systems Marylyn RitchieConsulting or Advisory Role: Cipherome, Goldfinch BioUncompensated Relationships: Regeneron Daniel J. RaderStock and Other Ownership Interests: Staten BiotechnologyConsulting or Advisory Role: Alnylam, Novartis, Pfizer Robert H. VonderheidePatents, Royalties, Other Intellectual Property: Receives royalties from Boston Children's Hospital for a licensed research-only monoclonal antibody, inventor on a licensed patent regarding cancer vaccine antigens Lynn M. SchuchterConsulting or Advisory Role: IncyteResearch Funding: GlaxoSmithKline, Merck, Bristol Myers SquibbExpert Testimony: PfizerTravel, Accommodations, Expenses: Stand Up To Cancer (SU2C) Ivan MaillardHonoraria: RegeneronConsulting or Advisory Role: Tobira Therapeutics, Garuda TherapeuticsSpeakers' Bureau: Seattle GeneticsTravel, Accommodations, Expenses: Tobira Therapeutics, Regeneron Ronac MamtaniHonoraria: Flatiron HealthConsulting or Advisory Role: Genentech/Roche, Seattle Genetics/AstellasResearch Funding: Merck Scott E. HensleyConsulting or Advisory Role: Sanofi Pasteur, Lumen, Novavax, Merck Robert GrossConsulting or Advisory Role: PfizerResearch Funding: Merck Alexander C. HuangConsulting or Advisory Role: ImmunaiResearch Funding: Bristol Myers Squibb Angela DeMicheleResearch Funding: Pfizer, Genentech, Calithera Biosciences, NovartisNo other potential conflicts of interest were reported.