MDM2 inhibitors are promising therapeutics for acute myeloid leukemia (AML) with wild-type TP53. Through an integrated analysis of functional genomic data from primary patient samples, we found that an MDM2 inhibitor, idasanutlin, like venetoclax, is ineffective against monocytic leukemia (French-American-British [FAB] subtype M4/M5). To dissect the underlying resistance mechanisms, we explored both intrinsic and extrinsic factors. We found that monocytic leukemia cells express elevated levels of CEBPB, which promote monocytic differentiation, suppress CASP3 and CASP6, and upregulate MCL1, BCL2A1, and the interleukin (IL-1)/tumor necrosis factor alpha (TNF-α)/NF-κB pathway members, thereby conferring drug resistance to a broad range of MDM2 inhibitors, BH3 mimetics, and venetoclax combinations. In addition, aberrant monocytes in M4/M5 leukemia produce elevated levels of IL-1 and TNF-α, which promote monocytic differentiation and upregulate inflammatory cytokines and receptors, thereby extrinsically protecting leukemia blasts from venetoclax and MDM2 inhibition. Interestingly, IL-1β and TNF-α only increase CEBPB levels and protect M4/M5 cells from these drugs but not M0/M1 leukemia cells. Treatment with venetoclax and idasanutlin induces compensatory upregulation of CEBPB and the IL-1/TNF-α/NF-κB pathway independent of the FAB subtype, indicating drug-induced compensatory protection mechanisms. The combination of venetoclax or idasanutlin with inhibitors that block the IL-1/TNF-α pathway demonstrates synergistic cytotoxicity in M4/M5 AML. As such, we uncovered a targetable positive feedback loop that involves CEBPB, IL-1/TNF-α, and monocyte differentiation in M4/M5 leukemia and promotes both intrinsic and extrinsic drug resistance and drug-induced protection against venetoclax and MDM2 inhibitors.

Despite the approval of several new treatments for patients with B-cell lymphoma, there is still a large unmet need. Diffuse large B-cell lymphoma (DLBCL) and follicular lymphoma (FL) are the 2 most common B-cell lymphoma subtypes, accounting for ∼50% of all cases. EZH2 heterozygous gain-of-function somatic driver mutations are frequently found in germinal center B-cell DLBCLs and FLs. An EZH2 inhibitor has shown durable responses in patients with relapsed or refractory FL, however a considerable fraction of the patients did not show an objective response. To identify alternative therapeutic strategies, we performed CRISPR/Cas9 knockout screens in B-cell lymphoma cells treated with or without an EZH2 inhibitor. This led to the identification of the histone methyltransferase DOT1L as a potential therapeutic target. Specifically, we showed that an EZH2 inhibitor synergizes with a DOT1L inhibitor in a panel of B-cell lymphoma cell lines regardless of the EZH2 mutation status. Mechanistically, we demonstrated that the 2 inhibitors cooperatively suppress DOT1L-regulated cell cycle genes, upregulate genes involved in interferon signaling, including antigen presenting genes, and ultimately drive B-cell differentiation by derepressing EZH2-regulated plasma cell signature genes. Furthermore, we demonstrated the effectiveness of this epigenetic combination strategy in a xenograft model, which led to significant abrogation of tumor growth. Together, our studies provide preclinical proof-of-concept for an epigenetic combination therapy to overcome resistance and improve durability of response for the treatment of B-cell lymphoma, warranting clinical investigation and illustrating an important convergent role of EZH2 and DOT1L in B-cell lymphomagenesis.

T-cell-recruiting bispecific antibodies (BsAbs) are in clinical development for relapsed/refractory acute myeloid leukemia (AML). Despite promising results, early clinical trials have failed to demonstrate durable responses. We investigated whether activation of the innate immune system through stimulator of interferon (IFN) genes (STING) can enhance target cell killing by a BsAb targeting CD33 (CD33 bispecific T-cell engager molecule; AMG 330). Indeed, we show that cytotoxicity against AML mediated by AMG 330 can be greatly enhanced when combined with the STING agonist 2',3'-cyclic guanosine monophosphate-adenosine monophosphate (cGAMP) or diamidobenzimidazole (diABZI). We used in vitro cytotoxicity assays, immunoblotting, transcriptomic analyses, and extensive CRISPR-Cas9 knockout experiments to investigate the enhancing effect of a STING agonist on the cytotoxicity of AMG 330 against AML. Importantly, we validated our findings with primary AML cells and in a xenograft AML model. Mechanistically, in addition to direct cytotoxic effects of STING activation on AML cells, activated T cells render AML cells more susceptible to STING activation through their effector cytokines, IFN-γ and tumor necrosis factor, resulting in enhanced type I IFN production and induction of IFN-stimulated genes. This feeds back to the T cells, leading to a further increase in effector cytokines and an overall cytotoxic T-cell phenotype, contributing to the beneficial effect of cGAMP/diABZI in enhancing AMG 330-mediated lysis. We established a key role for IFN-γ in AMG 330-mediated cytotoxicity against AML cells and in rendering AML cells responsive to STING agonism. Here, we propose to improve the efficacy of CD33-targeting BsAbs by combining them with a STING agonist.

The transglutaminase coagulation factor XIII (FXIII) is critical for the stability and function of intravascular fibrin clots. Prorepair extravascular fibrin(ogen) deposits are potentially subject to cross-linking by FXIII and other transglutaminases not typically resident in plasma. However, the impact of these alternative modifiers on fibrin(ogen) structure and function is not known. We tested the hypothesis that tissue transglutaminase (TG2) modifies FXIII-directed fibrin(ogen) cross-linking in vitro and within injured tissue. Global proteomic analysis after experimental acetaminophen (APAP)-induced acute liver injury revealed that intrahepatic fibrin(ogen) deposition was associated with hepatic TG2 levels that exceeded that of FXIII. Mass spectrometry-based cross-link mapping of in vitro fibrin matrices uncovered, to our knowledge, the first evidence of synergistic fibrin(ogen) α-α cross-linking catalyzed by both transglutaminases. Fibrin(ogen) cross-linking was increased in livers from patients with APAP-induced acute liver failure. APAP-challenged TG2-/- mice displayed an altered pattern of FXIII-dependent fibrin(ogen)-γ and fibrin(ogen)-α chain cross-linking aligned with the impact of TG2 on fibrin cross-linking in vitro. This shift in fibrin(ogen) cross-linking exacerbated pathologies including hepatic necrosis and sinusoidal congestion. The results, to our knowledge, are the first to indicate that TG2 impacts FXIII-directed fibrin(ogen) cross-linking, both in vitro and in vivo. The results suggest that TG2 functions to dynamically alter the structure of extravascular fibrin(ogen) to mitigate liver damage, a novel mechanism likely applicable across types of tissue injury.

Microbial dysbiosis and metabolite changes in the gastrointestinal (GI) tract have been linked to pathogenesis and severity of many diseases, including graft-versus-host disease (GVHD), the major complication of allogeneic hematopoietic stem cell transplantation. However, published studies have only considered the microbiome and metabolome of excreted stool and do not provide insight into the variability of the microbial community and metabolite composition throughout the GI tract or the unique temporal dynamics associated with different gut locations. Because such geographical variations are known to influence disease processes, we used a multi-omics approach to characterize the microbiome and metabolite profiles of gut contents from different intestinal regions in well-characterized mouse models of GVHD. Our analysis validated analyses from excreted stool, but importantly, uncovered new biological insights from the microbial and metabolite changes between syngeneic and allogeneic hosts that varied by GI location and time after transplantation. Our integrated analysis confirmed the involvement of known metabolic pathways, including short-chain fatty acid synthesis and bile acid metabolism, and identified additional functional genes, pathways, and metabolites, such as amino acids, fatty acids, and sphingolipids, linked to GI GVHD. Finally, we validated a biological relevance for one such newly identified microbial metabolite, phenyl lactate, that heretofore had not been linked to GI GVHD. Thus, our analysis of the geographic variability in the intestinal microbiome and metabolome offers new insights into GI GVHD pathogenesis and potential for novel therapeutics.

Refractory and/or relapsing T-cell acute lymphoblastic leukemia (T-ALL) remains a major therapeutic challenge. The pre-T-cell receptor (TCR) pathway has recently emerged as a therapeutic target via LCK inhibition in this context. However, there is a need for simple and quickly assessable biomarkers to predict sensitivity to LCK inhibitors. Moreover, targeting LCK alone by tyrosine kinase inhibitors, such as dasatinib, often results in transient clinical responses, emphasizing the need for efficient combination strategies. Here, we assessed pre-TCR α chain (pTα) surface expression by flow cytometry in a unique series of 50 adult T-ALL patient-derived xenografts (PDXs). We show that cases displaying a cortical phenotype often express high levels of surface pTα (pre-TCR+ T-ALL) and that the latter associates with LCK activation. Furthermore, we show that ectopic interleukin-7 receptor (IL-7R) expression can rescue pre-TCR+ T-ALL from dasatinib cytotoxicity (5 PDXs). We tested whether coinhibition of pre-TCR and IL-7R signaling pathways could be synergetic in pre-TCR+ IL-7R+ T-ALL (11 PDXs). Combination of JAK inhibitors, ruxolitinib or tofacitinib, with dasatinib elicited strong and specific synergy in IL-7R+ pre-TCR+ T-ALL in vitro, including in the relapse setting (4 of 28 patient-derived primary samples). Using 3 adult-PDX models, we show that in vivo treatment with this combination significantly delayed leukemic progression and prolonged survival compared with either monotherapy. This preclinical study thus proposes the use of pTα as a biomarker of LCK-inhibitor sensitivity in T-ALL, and suggests that dual targeting of IL-7R and pre-TCR signaling pathways may be a relevant therapeutic strategy in a substantial proportion of adult T-ALL.

Chronic graft-versus-host disease (cGVHD) remains the leading cause of nonrelapse morbidity and mortality after allogeneic hematopoietic cell transplantation (HCT). Effective therapeutic agents targeting dysregulated cytokines including interleukin-17 (IL-17) and colony-stimulating factor 1 (CSF-1) have been defined in preclinical models of cGVHD, and efficacy in subsequent clinical trials has led to their recent US Food and Drug Administration approval. Despite this, these agents are effective in only a subset of patients, expensive, difficult to access outside the United States, and used in a trial-and-error fashion. The ability to readily discern druggable, dysregulated immunity in these patients is desperately needed to facilitate the selection of appropriate treatment and to potentially identify high-risk individuals for preemptive therapy. We used single-cell sequencing-based approaches in our informative preclinical cGVHD models to "reverse engineer" temporal IL-17 and CSF-1 signatures in mouse blood that could be used to interrogate patients. We defined distinct, nonintuitive IL-17 and CSF-1 signatures in mouse blood monocytes that could be identified in relevant monocyte populations within 70% of patients at diagnosis of cGVHD and in half of patients at day +100 after HCT who subsequently developed cGVHD. These signatures can now be evaluated prospectively in clinical studies to help delineate potential responder and nonresponders to relevant therapeutics targeting these pathways.

Patients with thrombocytopenia requiring ongoing platelet transfusion support may develop inadequate platelet count increments, referred to as platelet refractoriness (PR), which further complicates their care. The underlying etiologies of PR can be broadly divided into immune and nonimmune causes. A high index of suspicion is required to initiate testing for alloimmunization, and the leading culprit in immune PR is the development of class I HLA antibodies. The approach to diagnosis of immune PR has changed over recent years with new technologies, but questions regarding the clinical significance and interpretation of these methods have not been conclusively answered. The provision of HLA-matched platelets requires close and timely coordination between transfusion services and clinical teams; however, the true impact of their provision on clinical outcomes is not clear. This paper reviews diagnostic and management challenges, appraises the existing data available to support treatment options, and identifies research gaps.

Immune cell functionality is highly dependent on the actin cytoskeleton. The actin cytoskeleton is regulated by a complex molecular machinery that involves multiple genes. Mutations in these genes can cause inborn errors of immunity, also termed immunoactinopathies, of which Wiskott-Aldrich syndrome is the best-characterized entity. Currently, mutations in 23 genes can be considered causative of immunoactinopathies. Immunoactinopathies are rare disease entities with complex combinations of clinical manifestations, including immunodeficiency, immune dysregulation, malignancies, atopy, thrombocytopenia and bleeding, skin involvement, or congenital defects. Prompt diagnosis is crucial, because hematopoietic stem cell transplantation in an early phase can offer cure and prevent further complications. This review provides a detailed summary of the clinical experience with immunoactinopathies so far, elaborates on the most distinguishing features of immunoactinopathies by providing a clinical categorization, and links this information to the underlying biological pathways. This information may be of help to clinicians in the diagnosis of patients and to eventually improve patient care.

For patients with myelodysplastic neoplasm/syndrome (MDS), allogeneic hematopoietic cell transplantation (allo-HCT) represents the only potentially curative treatment, capable of eradicating disease-related mutant hematopoietic cells and establishing normal donor hematopoiesis. Biologic-assignment clinical trials have indicated that in eligible patients, allo-HCT is associated with superior clinical outcomes compared with nontransplant therapy. However, this therapeutic option is only available to a subset of patients, and the outcome is influenced by multiple factors inherent to the patient, the MDS subtype, and the allo-HCT procedure itself. In 2017, the European Society for Blood and Marrow Transplantation (EBMT) published recommendations for allo-HCT in MDS to guide practical decision making. In the contemporary era, genomic profiling has become routine clinical practice in many centers, and the most recent classification systems include MDS entities that are defined by genetic abnormalities. In particular, the molecular International Prognostic Scoring System offers more precise prognostication across all clinical end points and currently represents the standard tool for estimating patient survival in the absence of disease-modifying treatment. Evidence from multiple sources increasingly indicates that allo-HCT should be considered at the time of diagnosis in all eligible patients with MDS. Therefore, genomic profiling for somatic mutations and testing for germ line predisposition variants are integral to determining a patient's eligibility for transplantation. Although all patients with higher-risk MDS are potential candidates for immediate transplantation, a subset of those with lower-risk MDS may also derive benefit from this procedure at an earlier disease stage. Comprehensive recommendations on behalf of an expert international panel for clinical practice and future clinical studies of relevance are presented.