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Background Acute graft-versus-host disease (GVHD) is a common and life-threatening complication of allogeneic haematopoietic stem cell transplantation (HSCT); there is an urgent unmet need for effective therapies. We aimed to evaluate the Janus kinase 1 inhibitor itacitinib versus placebo, both in combination with corticosteroids, for initial treatment of acute GVHD. Methods GRAVITAS-301 was an international, double-blind, adaptive (group sequential design) phase 3 study conducted at 129 hospitals and community practices in 19 countries. Eligible patients were aged 18 years or older, had previously received allogeneic HSCT for a haematological malignancy, developed grades II-IV acute GVHD, and received up to 2 days of systemic corticosteroids. Patients were stratified by clinical standard-risk or high-risk acute GVHD and randomly assigned (1:1), via a centralised interactive voice response system, to receive either oral itacitinib (200 mg) or placebo once daily, both in addition to corticosteroids. The primary endpoint was overall response rate (ORR) at day 28 (defined as the proportion of patients with complete response, very good partial response, or partial response 28 days after the start of treatment). For sample size determination, an absolute improvement in ORR at day 28 over standard therapy of 16% was considered clinically meaningful. Efficacy analyses were performed in the intention-to-treat population; safety analyses included patients who received at least one dose of study drug. GRAVITAS-301 is registered with ClinicalTrials.gov (NCT03139604) and is complete. Findings Between July 19, 2017, and Oct 3, 2019, 439 patients were randomly assigned to receive either itacitinib plus corticosteroids (n=219; itacitinib group) or placebo plus corticosteroids (n=220; placebo group). 173 (39%) patients were female and 390 (89%) were White. At baseline, 107 (24%) of 439 patients (itacitinib 51 [23%] of 219; placebo 56 [25%] of 220) had clinical high-risk acute GVHD. The ORR at day 28 was 74% (95% CI 67middot6-79middot7; 162 of 219; complete response 53% [116 of 219]) for itacitinib and 66% (59middot7-72middot6; 146 of 220; complete response, 40% [89 of 220]) for placebo (odds ratio for ORR 1middot45, 95% CI 0middot96-2middot20; two-sided p=0middot078). Grade 3 or worse adverse events occurred in 185 (86%) of 215 itacitinib recipients and 178 (82%) of 216 placebo recipients, and most commonly included thrombocytopenia or platelet count decreased (78 [36%] vs 68 [31%]), neutropenia or neutrophil count decreased (49 [23%] vs 45 [21%]), anaemia (42 [20%] vs 26 [12%]), and hyperglycaemia (26 [12%] vs 28 [13%]). Treatment related deaths occurred in three of 215 patients (1%) in the itacitinib group and four of 216 (2%) in the placebo group. Interpretation The observed improvement in ORR at day 28 with the addition of itacitinib versus placebo to corticosteroids did not reach the prespecified significance level. Further studies might provide additional insight into the utility of selective JAK1 inhibition for the treatment of acute GVHD. Funding Incyte. Copyright (c) 2021 Elsevier Ltd. All rights reserved.

Edited by Associate Editor Robert Zeiser, this How I Treat series focuses on post–allogeneic transplant management of high-risk patients. These patients are at risk for relapse of malignancy and severe graft-versus-host disease (GVHD). Two articles in the series discuss adoptive cellular immunotherapy and maintenance therapy to preempt relapse of acute myeloid leukemia. The third article addresses the state of the art for preventing GVHD in this subset of stem cell transplant recipients.

Chronic graft-versus-host disease (cGVHD) is a major immunologic complication of allogeneic hematopoietic cell transplantation. cGVHD involves multiple organs, reduces quality of life, and often requires prolonged therapy with glucocorticoids, causing severe side effects. After 4 decades of testing multiple therapeutic approaches, ibrutinib, belumosudil, and ruxolitinib were US Food and Drug Administration approved for cGVHD in the last 4 years. Here we put a spotlight on their mechanisms of action, studies that led to approval, and their future role in cGVHD.

Successful treatment of acute myeloid leukemia (AML) with chimeric antigen receptor (CAR) T cells is hampered by toxicity on normal hematopoietic progenitor cells and low CAR T cell persistence. Here, we develop third-generation anti-CD123 CAR T cells with a humanized CSL362-based ScFv and a CD28-OX40-CD3ζ intracellular signaling domain. This CAR demonstrates anti-AML activity without affecting the healthy hematopoietic system, or causing epithelial tissue damage in a xenograft model. CD123 expression on leukemia cells increases upon 5′-Azacitidine (AZA) treatment. AZA treatment of leukemia-bearing mice causes an increase in CTLA-4negative anti-CD123 CAR T cell numbers following infusion. Functionally, the CTLA-4negative anti-CD123 CAR T cells exhibit superior cytotoxicity against AML cells, accompanied by higher TNFα production and enhanced downstream phosphorylation of key T cell activation molecules. Our findings indicate that AZA increases the immunogenicity of AML cells, enhancing recognition and elimination of malignant cells by highly efficient CTLA-4negative anti-CD123 CAR T cells., The success of CAR-T cells for treating acute myeloid leukaemia (AML) is hampered by toxicity to normal cells and low CAR-T cell persistence. Here, the authors show that the demethylating compound 5′-Azacitdine increases anti-CD123 CAR-T cell cytotoxicity against AML.

Purpose Allogeneic stem cell transplantation would benefit from re-engineering care towards an integrated eHealth-facilitated care model. With this paper we aim to: (1) describe the development of an integrated care model (ICM) in allogeneic SteM-cell-transplantatIon faciLitated by eHealth (SMILe) by combining implementation, behavioral, and computer science methods (e.g., contextual analysis, Behavior Change Wheel, and user-centered design combined with agile software development); and (2) describe that model’s characteristics and its application in clinical practice. Methods The SMILe intervention’s development consisted of four steps, with implementation science methods informing each: (1) planning its set-up within a theoretical foundation; (2) using behavioral science methods to develop the content; (3) choosing and developing its delivery method (human/technology) using behavioral and computer science methods; and (4) describing its characteristics and application in clinical practice. Results The SMILe intervention is embedded within the eHealth enhanced Chronic Care Model, entailing four self-management intervention modules, targeting monitoring and follow-up of important medical and symptom-related parameters, infection prevention, medication adherence, and physical activity. Interventions are delivered partly face-to-face by a care coordinator embedded within the transplant team, and partly via the SMILeApp that connects patients to the transplant team, who can monitor and rapidly respond to any relevant changes within 1 year post-transplant. Conclusion This paper provides stepwise guidance on how implementation, behavioral, and computer science methods can be used to develop interventions aiming to improve care for stem cell transplant patients in real-world clinical settings. This new care model is currently being tested in a hybrid I effectiveness-implementation trial.

BACKGROUND Standard first-line treatment of cGVHD includes systemic corticosteroids; however, about 50% of patients (pts) are steroid refractory or dependent (SR/D) and require additional treatment. The best second-line therapy option has not yet been defined. RUX, an oral JAK1/2 inhibitor, was superior to BAT in SR acute GVHD (aGVHD) in a phase 3 study (REACH2). Here we present the primary analysis of the REACH3 study (NCT03112603), a phase 3, open-label, randomized study evaluating RUX vs BAT in pts with SR/D cGVHD. METHODS Eligible pts were ≥ 12 years old, had received allogeneic hematopoietic cell transplant, and had developed moderate or severe SR/D cGVHD. Pts transitioning from aGVHD to cGVHD without tapering steroids were excluded. Pts treated with JAK inhibitors for aGVHD were allowed if they achieved complete response (CR) or partial response (PR) and had been off JAK inhibitor treatment for ≥ 8 weeks prior to cycle 1 day 1. Those treated with ≥ 2 prior lines of systemic therapy for cGVHD in addition to corticosteroids ± calcineurin inhibitors (CNI) were ineligible. Pts were randomized (1:1) to RUX 10 mg bid or investigator-selected BAT (10 options) and were treated for 6 cycles (cycle = 28 days). Pts continued receiving their regimen of corticosteroids ± CNI. Viral prophylaxis and antibiotics were allowed as needed for infection prevention and treatment. Addition or initiation of a new BAT was allowed only after lack of response, intolerable toxicity, or cGVHD flare and was considered treatment failure. Crossover from BAT to RUX was allowed on or after cycle 7 day 1 (C7D1) in pts who did not achieve or maintain CR/PR, developed toxicity to BAT, or had a cGVHD flare. The primary endpoint was overall response rate (ORR) at C7D1. ORR was defined as the proportion of pts achieving CR or PR, per NIH consensus criteria. Key secondary endpoints were failure-free survival (FFS; defined as time to the earliest of recurrence of underlying disease, start of new systemic treatment for cGVHD, or death) and improvement in symptoms based on change in the modified Lee symptom score (mLSS; 0 [no symptoms] to 100 [worst symptoms]) at C7D1. An mLSS responder was defined as having achieved a ≥ 7-point reduction from baseline in the total symptom score. The primary and key secondary endpoints were α-controlled via an overall hierarchical testing procedure. RESULTS A total of 329 pts were randomized to RUX (n = 165) or BAT (n = 164). Baseline characteristics were balanced in the 2 arms; 61% were male, and median age was 49 years (range, 12-76 years). Twelve pts were < 18 years old. Overall, 48% and 52% of pts had moderate and severe cGVHD, respectively. At data cutoff (May 8, 2020), 125 pts (38%) were on randomized treatment (RUX, 50%; BAT, 26%). Eighty-two (50%) and 122 (74%) pts discontinued RUX and BAT, respectively; 61 (37%) had crossed over to RUX. Reasons for discontinuation included lack of efficacy (15% RUX vs 43% BAT), adverse events (AEs; 17% vs 5%), and relapse (5% vs 4%). The study met its primary endpoint with the efficacy boundary crossed at interim analysis (ORR, P = 0.0003). At C7D1, ORR was significantly higher in the RUX arm vs BAT (50% vs 26%; odds ratio, 2.99; P < 0.0001a); the CR rate was higher with RUX (7% vs 3%; Table). Both key secondary endpoints showed superiority of RUX vs BAT. FFS was significantly longer for RUX-treated pts (median FFS, not reached vs 5.7 months; HR, 0.370 [95% CI, 0.268-0.510]; P < 0.0001; Figure), and the mLSS responder rate was higher (24% vs 10%; odds ratio, 3.00; P = 0.0003). Overall, 31 RUX (19%) and 27 BAT (16%) pts died; the main cause of death was cGVHD (RUX, n = 22 [13%]; BAT, n = 13 [8%], including 2 deaths after crossover to RUX). Rates of AEs up to C7D1 were comparable in the 2 arms (RUX, 98% [grade ≥ 3, 57%]; BAT, 92% [grade ≥ 3, 58%]. The most common AEs (≥ 15%) in the RUX vs BAT arms were anemia (29% vs 13%), hypertension (16% vs 13%), pyrexia (16% vs 9%), and ALT increase (15% vs 4%). Infections of any type occurred in 64% of RUX and 56% of BAT pts (19% vs 18% grade 3, grading based on Cordonnier et al 2006), and included fungal (12% vs 6%), viral (34% vs 29%), and bacterial (28% vs 26%) infections. CONCLUSIONS This is the first successful randomized phase 3 trial of RUX in pts with SR/D cGVHD. RUX demonstrated superior efficacy vs BAT, measured by a higher ORR, longer FFS, and greater symptom improvement. RUX was effective for moderate or severe SR/D cGVHD and its safety profile is consistent with that expected for this drug and this population. Disclosures Zeiser: Malinckrodt: Honoraria; Novartis: Honoraria; Incyte: Honoraria. Polverelli:Novartis: Consultancy. Chakraverty:Mallinckrodt Pharmaceuticals (Therakos (UK) Ltd): Honoraria; Neovii: Honoraria. Flowers:Novartis: Current Employment. Dawkins:Incyte: Current Employment. Hollaender:Novartis Pharma AG: Current Employment. Gowda:Novartis Pharmaceuticals Corporation: Current Employment. Stefanelli:Novartis AG: Current Employment, Current equity holder in publicly-traded company. Lee:Takeda: Research Funding; Pfizer: Consultancy, Research Funding; Novartis: Research Funding; Amgen: Research Funding; Kadmon: Research Funding; Incyte: Consultancy, Research Funding; Syndax: Research Funding; AstraZeneca: Research Funding. Teshima:Sharp & Dohme Corp: Consultancy, Honoraria; Astellas Pharma Inc.: Research Funding; Bristol-Myers Squibb: Honoraria; Japan Society for the Promotion of Science KAKENHI (17H04206): Other; The Center of Innovation Program from Japan Science and Technology Agency: Other; Kyowa Kirin Co., Ltd.: Honoraria, Research Funding; Merck: Consultancy, Honoraria; Janssen Pharmaceutical K.K.: Other; NIPPON SHINYAKU CO., LTD.: Honoraria; Fuji Pharma Co., Ltd.: Honoraria; TEIJIN PHARMA LIMITED: Honoraria; Sanofi K.K.: Research Funding; Chugai Pharmaceutical Co., Ltd.: Research Funding; Novartis Pharma K.K.: Consultancy, Other: Manuscript preparation, Research Funding; Pfizer Japan Inc.: Honoraria; Takeda Pharmaceutical Company Limited: Consultancy, Honoraria. Locatelli:Jazz Pharmaceeutical: Speakers Bureau; Medac: Speakers Bureau; Miltenyi: Speakers Bureau; Bellicum Pharmaceutical: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Amgen: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. OffLabel Disclosure: Ruxolitinib for the treatment of chronic GVHD.

Extracorporeal Photopheresis for Autoimmune Colitis Extracorporeal photopheresis has been an effective treatment for graft-versus-host disease. In this case, it was used to treat a patient with sev...

Microbial invasion into the intestinal mucosa after allogeneic hematopoietic cell transplantation (allo-HCT) triggers neutrophil activation and requires antibiotic interventions to prevent sepsis. However, antibiotics lead to a loss of microbiota diversity, which is connected to a higher incidence of acute graft-versus-host disease (aGVHD). Antimicrobial therapies that eliminate invading bacteria and reduce neutrophil-mediated damage without reducing the diversity of the microbiota are therefore highly desirable. A potential solution would be the use of antimicrobial antibodies that target invading pathogens, ultimately leading to their elimination by innate immune cells. In a mouse model of aGVHD, we investigated the potency of active and passive immunization against the conserved microbial surface polysaccharide poly-N-acetylglucosamine (PNAG) that is expressed on numerous pathogens. Treatment with monoclonal or polyclonal antibodies to PNAG (anti-PNAG) or vaccination against PNAG reduced aGVHD-related mortality. Anti-PNAG treatment did not change the intestinal microbial diversity as determined by 16S ribosomal DNA sequencing. Anti-PNAG treatment reduced myeloperoxidase activation and proliferation of neutrophil granulocytes (neutrophils) in the ileum of mice developing GVHD. In vitro, anti-PNAG treatment showed high antimicrobial activity. The functional role of neutrophils was confirmed by using neutrophil-deficient LysM(cre) Mcl1(fl/fl) mice that had no survival advantage under anti-PNAG treatment. In summary, the control of invading bacteria by anti-PNAG treatment could be a novel approach to reduce the uncontrolled neutrophil activation that promotes early GVHD and opens a new avenue to interfere with aGVHD without affecting commensal intestinal microbial diversity.

Autophagy is a genetically regulated process of adaptation to metabolic stress and was recently shown to be involved in the treatment response of chronic myeloid leukemia (CML). However, in vivo data are limited and the molecular mechanism of autophagy regulators in the process of leukemogenesis is not completely understood. Here we show that Beclin-1 knockdown, but not Atg5 deletion in a murine CML model leads to a reduced leukemic burden and results in a significantly prolonged median survival of targeted mice. Further analyses of murine cell lines and primary patient material indicate that active BCR-ABL directly interacts with BECLIN-1 and phosphorylates its tyrosine residues 233 and 352, resulting in autophagy suppression. By using phosphorylation-deficient and phosphorylation-mimic mutants, we identify BCR-ABL induced BECLIN-1 phosphorylation as a crucial mechanism for BECLIN-1 complex formation: interaction analyses exhibit diminished binding of the positive autophagy regulators UVRAG, VPS15, ATG14 and VPS34 and enhanced binding of the negative regulator Rubicon to BCR-ABL-phosphorylated BECLIN-1. Taken together, our findings show interaction of BCR-ABL and BECLIN-1 thereby highlighting the importance of BECLIN-1-mediated autophagy in BCR-ABL+ cells.

Patients with acute myeloid leukaemia (AML) often achieve remission after therapy, but subsequently die of relapse1 that is driven by chemotherapy-resistant leukaemic stem cells (LSCs)2,3. LSCs are defined by their capacity to initiate leukaemia in immunocompromised mice4. However, this precludes analyses of their interaction with lymphocytes as components of anti-tumour immunity5, which LSCs must escape to induce cancer. Here we demonstrate that stemness and immune evasion are closely intertwined in AML. Using xenografts of human AML as well as syngeneic mouse models of leukaemia, we show that ligands of the danger detector NKG2D—a critical mediator of anti-tumour immunity by cytotoxic lymphocytes, such as NK cells6–9—are generally expressed on bulk AML cells but not on LSCs. AML cells with LSC properties can be isolated by their lack of expression of NKG2D ligands (NKG2DLs) in both CD34-expressing and non-CD34-expressing cases of AML. AML cells that express NKG2DLs are cleared by NK cells, whereas NKG2DL-negative leukaemic cells isolated from the same individual escape cell killing by NK cells. These NKG2DL-negative AML cells show an immature morphology, display molecular and functional stemness characteristics, and can initiate serially re-transplantable leukaemia and survive chemotherapy in patient-derived xenotransplant models. Mechanistically, poly-ADP-ribose polymerase 1 (PARP1) represses expression of NKG2DLs. Genetic or pharmacologic inhibition of PARP1 induces NKG2DLs on the LSC surface but not on healthy or pre-leukaemic cells. Treatment with PARP1 inhibitors, followed by transfer of polyclonal NK cells, suppresses leukaemogenesis in patient-derived xenotransplant models. In summary, our data link the LSC concept to immune escape and provide a strong rationale for targeting therapy-resistant LSCs by PARP1 inhibition, which renders them amenable to control by NK cells in vivo. Leukaemic stem cells in acute myeloid leukaemia are defined by a lack of expression of NKG2D ligands, which mediates their ability to evade surveillance by NK cells.

The curative potential of allogeneic hematopoietic cell transplantation (allo-HCT) in the treatment of acute myelogenous leukemia (AML) and myelodysplastic syndrome (MDS) relies mainly on the graft-versus-leukemia effect. Relapse after allo-HCT occurs in a considerable proportion of patients and has a dismal prognosis, with still very limited curative potential. This review provides an overview of the established and evolving approaches to preventing or treating relapse of AML and MDS after allo-HCT, in the context of novel insight into the biology of relapse. Established prophylactic measures to prevent relapse include optimized conditioning and graft-versus-host disease (GVHD) prophylaxis, as well as donor lymphocyte infusion (DLI) for high-risk patients; novel immunomodulatory interventions and maintenance approaches are still experimental. Improved diagnostics can detect persistent or recurring disease at a molecular level, enabling early preemptive interventions. Established options include hypomethylating agents and DLI. Standard treatments for hematologic relapse include chemotherapy, cessation of immunosuppressive treatment, and DLI. Experimental approaches include molecular targeted therapies, novel immunomodulatory treatments, and second allo-HCT. For all interventions, the potential risks, including occurrence of GVHD, must be weighed against the benefits individually in each patient. Concurrently, prevention and treatment of relapse after allo-HCT remain challenging and unmet medical needs.

Copy number gains, point mutations and epigenetic silencing events are increasingly observed in genes encoding elements of the Ras/Raf/MEK/ERK signaling axis in human breast cancer. The three Raf kinases A-Raf, B-Raf, and Raf-1 have an important role as gatekeepers in ERK pathway activation and are often dysregulated by somatic alterations of their genes or by the aberrant activity of receptor tyrosine kinases (RTKs) and Ras-GTPases. B-Raf represents the most potent Raf isoform and a critical effector downstream of RTKs and RAS proteins. Aberrant RTK signaling is mimicked by the polyoma middle T antigen (PyMT), which activates various oncogenic signaling pathways, incl. the RAS/ERK axis, in a similar manner as RTKs in human breast cancer. Mammary epithelial cell directed expression of PyMT in mice by the MMTV-PyMT transgene induces mammary hyperplasia progressing over adenoma to metastatic breast cancer with an almost complete penetrance. To understand the functional role of B-Raf in this model for luminal type B breast cancer, we crossed MMTV-PyMT mice with animals that either lack B-Raf expression in the mammary gland or express the signaling impaired B-RafAVKA mutant. The AVKA mutation prevents phosphorylation of T599 and S602 in the B-Raf activation loop and thereby activation of the kinase by upstream signals. We demonstrate for the first time that B-Raf expression and activation is important for tumor initiation in vivo as well as for lung metastasis. Isogenic tumor cell lines generated from conditional Braf knock-out or knock-in mice displayed a reduction in EGF-induced ERK pathway activity as well as in proliferation and invasive growth in three-dimensional matrigel cultures. Our results suggest that B-Raf, which has been hardly studied in the context of breast cancer, represents a critical effector of the PyMT oncoprotein and invite for an assessment of its functional role in human breast cancer.

MicroRNAs (miR) are small noncoding RNAs that regulate gene expression, posttranscription, and manipulate immune responses in different types of cancers. In this study, we identify miR-146a as a negative regulator of immune activation, comparable to immune-checkpoint molecules. miR-146a levels were increased in melanoma microenvironmental tissue, and miR-146a−/− mice survived longer and developed less metastases in comparison with wild-type melanoma-bearing mice. T cells isolated from miR-146a−/− mice revealed higher expression levels of the miR-146a target gene Stat1 and the Stat1-regulated cytokine IFNγ. Neutralization of IFNγ in miR-146a−/− mice decreased survival and increased melanoma metastasis patterns to those of wild-type mice. In vitro, IFNγ reduced melanoma cell migration, cell-cycle activity, and basal metabolic rate. Conversely, IFNγ also increased PD-L1 levels on the melanoma cells, which may counterbalance some of the beneficial effects increasing immune escape in vivo. Combined treatment with a miR-146a antagomiR and anti–PD-1 resulted in improved survival over isotype control or anti–PD-1 treatment alone. In summary, these data show that miR-146a plays a central role within the STAT1/IFNγ axis in the melanoma microenvironment, affecting melanoma migration, proliferation, and mitochondrial fitness as well as PD-L1 levels. Additionally, combined inhibition of PD-1 and miR-146a could be a novel strategy to enhance antitumor immune response elicited by checkpoint therapy. Significance: These findings identify a microRNA–based mechanism by which melanoma cells escape the immune system, providing a new therapeutic strategy to improve the current management of patients with melanoma.

This article has been retracted: please see Elsevier Policy on Article Withdrawal (https://www.elsevier.com/about/our-business/policies/article-withdrawal). This article has been retracted at the request of the Editor-in-Chief. Following the publication of the above article, the Editor was notified that an error occurred in which all images were published with incorrect versions. The Editor has taken the decision that the manuscript is no longer acceptable in its current form, nor with a corrigendum, as the extensive changes to the figures and publication would lead to ambiguity for our readers. We have therefore made the decision to retract this manuscript from Cancer Letters with the possibility of resubmission and republication of the manuscript in its corrected form after peer review.

Metastatic small cell lung cancer (SCLC) is not curable. While SCLC is initially sensitive to chemotherapy, remissions are short-lived. The relapse is induced by chemotherapy-selected tumor stem cells, which express the AC133 epitope of the CD133 stem cell marker. We studied the effectiveness of AC133-specific CAR T cells post-chemotherapy using human primary SCLC and an orthotopic xenograft mouse model. AC133-specific CAR T cells migrated to SCLC tumor lesions, reduced the tumor burden, and prolonged survival in a humanized orthotopic SCLC model, but were not able to entirely eliminate tumors. We identified CD73 and PD-L1 as immune-escape mechanisms and combined PD-1-inhibition and CD73-inhibition with CAR T cell treatment. This triple-immunotherapy induced cures in 25% of the mice, without signs of graft-versus-host disease or bone marrow failure. AC133

Significance Fever is a common symptom of infections and inflammation. We observed CD8+ T cells exposed to febrile-range temperature during activation, augmented mitochondrial mass, and metabolic activity. An adoptive transfer of these cells into tumor-bearing mice led to enhanced antitumor responses, suggesting that exposure to febrile temperature during activation contributes to the development of robust T cell responses. We identified that mitochondrial translation is integral to the enhanced mitochondrial mass and function of CD8+ T cells exposed to febrile temperature. Collectively, this research raises the possibility that fever may be beneficial for optimizing metabolic and functional responses in CD8+ T cells., Fever can provide a survival advantage during infection. Metabolic processes are sensitive to environmental conditions, but the effect of fever on T cell metabolism is not well characterized. We show that in activated CD8+ T cells, exposure to febrile temperature (39 °C) augmented metabolic activity and T cell effector functions, despite having a limited effect on proliferation or activation marker expression. Transcriptional profiling revealed an up-regulation of mitochondrial pathways, which was consistent with increased mass and metabolism observed in T cells exposed to 39 °C. Through in vitro and in vivo models, we determined that mitochondrial translation is integral to the enhanced metabolic activity and function of CD8+ T cells exposed to febrile temperature. Transiently exposing donor lymphocytes to 39 °C prior to infusion in a myeloid leukemia mouse model conferred enhanced therapeutic efficacy, raising the possibility that exposure of T cells to febrile temperatures could have clinical potential.

Fever can provide a survival advantage during infection. Metabolic processes are sensitive to environmental conditions, but the effect of fever on T cell metabolism is not well characterized. We show that in activated CD8+ T cells, exposure to febrile temperature (39°C) augmented metabolic activity and T cell effector functions, despite having a limited effect on proliferation or activation marker expression. Transcriptional profiling revealed an up-regulation of mitochondrial pathways, which was consistent with increased mass and metabolism observed in T cells exposed to 39°C. Through in vitro and in vivo models, we determined that mitochondrial translation is integral to the enhanced metabolic activity and function of CD8+ T cells exposed to febrile temperature. Transiently exposing donor lymphocytes to 39°C prior to infusion in a myeloid leukemia mouse model conferred enhanced therapeutic efficacy, raising the possibility that exposure of T cells to febrile temperatures could have clinical potential.

As a result of impressive increases in our knowledge of rodent and human immunology, the understanding of the pathophysiologic mechanisms underlying graft-versus-host disease (GVHD) has dramatically improved in the past 15 years. Despite improved knowledge, translation to clinical care has not proceeded rapidly, and results from experimental models have been inconsistent in their ability to predict the clinical utility of new therapeutic agents. In parallel, new tools in immunology have allowed in-depth analyses of the human system and have recently been applied in the field of clinical GVHD. Notwithstanding these advances, there is a relative paucity of mechanistic insights into human translational research, and this remains an area of high unmet need. Here we review selected recent advances in both preclinical experimental transplantation and translational human studies, including new insights into human immunology, the microbiome, and regenerative medicine. We focus on the fact that both approaches can interactively improve our understanding of both acute and chronic GVHD biology and open the door to improved therapeutics and successes.

Allogeneic hematopoietic stem cell transplantation (allo-HSCT) is potentially curative for acute myeloid leukemia (AML). The inherent graft-versus-leukemia activity (GvL) may be optimized by donor lymphocyte infusions (DLI). Here we present our single-center experience of DLI use patterns and effectiveness, based on 342 consecutive adult patients receiving a first allo-HSCT for AML between 2009 and 2017. The median age at transplantation was 57 years (range 19–79), and the pre-transplant status was active disease in 58% and complete remission (CR) in 42% of cases. In a combined landmark analysis, patients in CR on day +30 and alive on day +100 were included. In this cohort (n=292), 93 patients received cryopreserved aliquots of peripheral blood-derived grafts for DLI (32%) and median survival was 55.7 months (2-year/5-year probability: 62%/49%). Median survival for patients receiving a first dose of DLI “preemptively,” in the absence of relapse and guided by risk marker monitoring (preDLI; n=42), or only after hematological relapse (relDLI; n=51) was 40.9 months (2-year/5-year: 64%/43%) vs 10.4 months (2-year/5-year: 26%/10%), respectively. Survival was inferior when preDLI was initiated at a time of genetic risk marker detection vs mixed chimerism or clinical risk only. Time to first-dose preDLI vs time to first-dose relDLI was similar, suggesting that early warning and intrinsically lower dynamics of AML recurrence may contribute to effectiveness of preDLI-modified GvL activity. Future refinements of the preemptive DLI concept will benefit from collaborative efforts to diagnose measurable residual disease more reliably across the heterogeneous genomic spectrum of AML.

Key Points Index case of nivolumab response associated with altered circulating T-cell composition and heterogeneous PD-L1 expression on AML blasts.Single-cell approaches provide complementary insight into cellular mechanisms of response and resistance to transplant/checkpoint blockade., Visual Abstract, Relapse of myeloproliferative neoplasms (MPNs) after allogeneic hematopoietic stem cell transplantation (HSCT) is associated with poor outcomes, as therapeutic approaches to reinstate effective graft-versus-leukemia (GVL) responses remain suboptimal. Immune escape through overexpression of PD-L1 in JAK2V617F-mutated MPN provides a rationale for therapeutic PD-1 blockade, and indeed, clinical activity of nivolumab in relapsed MPN post-HSCT has been observed. Elucidation of the features of response following PD-1 blockade in such patients could inform novel therapeutic concepts that enhance GVL. Here, we report an integrated high-dimensional analysis using single-cell RNA sequencing, T-cell receptor sequencing, cellular indexing of transcriptomes and epitopes by sequencing (CITE-seq), and assay for transposase-accessible chromatin using sequencing (scATAC-seq), together with mass cytometry, in peripheral blood mononuclear cells collected at 6 timepoints before, during, and after transient response to PD-1 blockade from an index case of relapsed MPN following HSCT. Before nivolumab infusion, acute myeloid leukemia (AML) blasts demonstrated high expression of chemokines, and T cells were characterized by expression of interferon-response genes. This baseline inflammatory signature disappeared after nivolumab infusion. Clinical response was characterized by transient expansion of a polyclonal CD4+ T-cell population and contraction of an AML subpopulation that exhibited megakaryocytic features and elevated PD-L1 expression. At relapse, the proportion of the AML subpopulation with progenitor-like features progressively increased, suggesting coevolution of AML blasts and donor-derived T cells. We thus demonstrate how single-cell technologies can provide complementary insight into cellular mechanisms underlying response to PD-1 blockade, motivating future longitudinal high-dimensional single-cell studies of GVL responses in relapsed myeloid disease.

As substantial constituents of the multiple myeloma (MM) microenvironment, pro-inflammatory macrophages have emerged as key promoters of disease progression, bone destruction, and immune impairment. We identify beta-2-microglobulin (β2m) as a driver in initiating inflammation in myeloma-associated macrophages (MAMs). Lysosomal accumulation of phagocytosed β2m promotes β2m amyloid aggregation in MAMs, resulting in lysosomal rupture and ultimately production of active interleukin-1β (IL-1β) and IL-18. This process depends on activation of the NLRP3 inflammasome after β2m accumulation, as macrophages from NLRP3-deficient mice lack efficient β2m-induced IL-1β production. Moreover, depletion or silencing of β2m in MM cells abrogates inflammasome activation in a murine MM model. Finally, we demonstrate that disruption of NLRP3 or IL-18 diminishes tumor growth and osteolytic bone destruction normally promoted by β2m-induced inflammasome signaling. Our results provide mechanistic evidence for β2m's role as an NLRP3 inflammasome activator during MM pathogenesis. Moreover, inhibition of NLRP3 represents a potential therapeutic approach in MM.

Introduction: Antitumor activity of the neddylation inhibitor pevonedistat has been documented in several hematologic and non-hematologic malignancies. Unexpectedly, Zhou et al (PNAS, 2016) discovered a dose-dependent biphasic effect of pevonedistat in solid tumor cell lines. While micromolar concentrations inhibited tumor cell growth, low nanomolar concentrations significantly increased cell proliferation and tumor stem cell self-renewal both in vitro and in vivo. The effect of low-dose pevonedistat has not yet been explored in the field of hematopoietic stem cell transplantation. Therefore, we evaluated how pevonedistat affects the viabiilty, growth and proportions of CD34 + cell subpopulations. In view of the emerging role of neddylation in the regulation of both innate and adaptive immunity, we also investigated the influence of pevonedistat on T-cell activation to explore a potentially beneficial effect on posttransplant immune complications. Methods and Results: Using the WST-1 assay we confirmed the biphasic effect of pevonedistat on normal mobilized CD34 + cells. Incubation for 72 h with 0.1 µM pevonedistat significantly increased metabolic activity as a surrogate parameter for proliferation, while 1.0 µM pevonedistat showed a cytotoxic effect. We explored the underlying mechanism for the low-dose effect. Since Zhou et al. previously showed that pevonedistat can promote tumor stem cell proliferation by inducing EGFR homodimerization, we used a proximity ligation assay and found that 0.1 µM pevonedistat induced EGFR homodimerization in normal mobilized CD34 + cells, too. In addition to homodimerization, we also looked at phosphorylation at Tyr1068, a marker of EGFR activation. By flow cytometry, we showed that phosphorylation was increased by 0.01 µM and 0.1 µM pevonedistat. Using an ELISA-based transcription assay, we also observed a biphasic effect of pevonedistat on c-Myc expression, which is regarded as a marker of 'stemness'. Incubation with pevonedistat for 72 hrs at 0.01 and 0.1 µM stimulated expression of c-Myc, whereas incubation at 1.0 µM downregulated c-Myc. Fractions of hematopoietic stem and progenitor cell (HSPC) subpopulations were measured in CD34 + cells from cord blood after incubation with 0.01, 0.1 and 1.0 µM pevonedistat. Flow cytometry was performed using antibodies against CD34, CD45RA and CD133, as well as 7-AAD for testing cell viability. Exposure to pevonedistat for 72 hrs at 0.1 µM caused an increase in the number of CD34 + cells compared to vehicle-treated CD34+ cells at 72 h as well as compared to initial number of CD34+ cells, whereas 1.0 µM caused a significant decrease. The absolute number of multipotent progenitors (MPP) (CD34 +CD133 +CD45RA -) remained relatively stable at all concentrations, while lympho-myeloid progenitors (LMPP) (CD34+CD133+CD45RA+) and late progenitors (LP) (CD34+CD133-CD45RA+) increased slightly with 0.1 µM pevonedistat compared with controls. However, a significant decrease in LMPP and LP cell numbers was observed at 1.0 µM. Different concentrations of pevonedistat were tested for their capability to modulate allogeneically stimulated T cell activation in a multi-donor mixed lymphocyte reaction (mdMLR) assay in vitro. Mesenchymal stromal cells (MSCs)-derived extracellular vesicles (MSC-EV) were used as internal immuno-modulatory and non-immuno-modulatory controls in the assay. After 5 days, alterations in the immune cell composition were analyzed by flow cytometry. Pevonedistat was not toxic for MNCs in the mdMLR. However, it decreased the number of activated (CD25high CD54+) CD4+ cells and CD8+ cells. Conclusions: One of the problems in the post-transplant period is a rapid decline in MPP numbers, associated with increased risk of engraftment failure. We showed that low-dose pevonedistat (0.1 µM) is capable of increasing the number of CD34 + cells in vitro while keeping the absolute number of MPPs stable. This finding, together with the observed increase in c-Myc expression, suggests that pevonedistat may help to preserve 'stemness' of CD34+ donor cells, thus supporting engraftment of hematopoietic stem and progenitor cells. Furthermore, the immunosuppressive effects revealed by mdMLR suggest that low-dose pevonedistat may also play a useful immunomodulatory role in the post-transplant setting to potentially reduce the risk of graft-versus-host disease. Figure 1 Figure 1. Disclosures Majidi: Takeda: Research Funding. Germing: Jazz Pharmaceuticals: Honoraria; Bristol-Myers Squibb: Honoraria, Other: advisory activity, Research Funding; Celgene: Honoraria; Novartis: Honoraria, Research Funding; Janssen: Honoraria. Zeiser: Incyte, Mallinckrodt, Novartis: Honoraria, Speakers Bureau. Gattermann: Celgene: Honoraria; Takeda: Research Funding; Novartis: Honoraria.

As significant constituents of the tumor microenvironment in multiple Myeloma (MM), pro-inflammatory macrophages are key promoters of disease progression, bone destruction, and immune-impairment. Consequently, the identification of endogenous mediators of these inflammatory processes can open novel therapeutic avenues against major pathological features of MM. Here, we identify beta-2-microglobulin (β2m) as an important driver in the initiation of inflammation in myeloma-associated macrophages (MAMs). Lysosomal accumulation of phagocytosed β2m in patient-derived MAMs promoted β2m amyloid aggregation, resulting in lysosomal rupture and ultimately in the production of active interleukin (IL)-1β and IL-18. Interestingly, this process strictly depended on the activation of the NLRP3 inflammasome after β2m accumulation, as macrophages from NLRP3-deficient mice lacked efficient β2m-induced IL-1β production. Moreover, depletion or silencing of β2m in MM cells abrogated inflammasome activation in a murine MM model. Finally, we demonstrate that specific disruption of NLRP3 or IL-18 diminished tumor growth and osteolytic bone destruction normally promoted by β2m-induced inflammasome signaling. Taken together our results provide novel mechanistic evidence for β2m’s role as an NLRP3 inflammasome activator during MM pathogenesis. Moreover, inhibition of NLRP3 is a potential novel therapeutic approach to combat this severe malignancy.

Introduction: Siremadlin is an oral (PO), potent and selective 2 nd generation inhibitor of Murine Double Minute-2 (MDM2i), resulting in a p53-dependent anti-proliferative effect. Siremadlin has a well-characterized safety profile and encouraging single-agent anti-leukemic activity in relapsed refractory (R/R) acute myeloid leukemia (AML) (Stein 2021, provisionally accepted, NCT02143635). More recently, MDM2i including siremadlin, have been shown to possess potent immunomodulatory effects (IME) in murine AML and solid tumor models (Wang 2021, Fang 2019). These data suggest that IME of siremadlin could be exploited to prevent relapse in AML patients (pts) in remission after allogeneic stem cell transplantation (SCT). Here we report the results of an exploratory post-hoc analysis of safety and preliminary efficacy in a subset of pts with relapsed AML post SCT and subsequently treated with siremadlin monotherapy. Methods: From the first-in-human study, CHDM201X2101 (NCT02143635), among 91 R/R AML pts treated with siremadlin monotherapy, a subset of pts (n= 19) who had undergone SCT before study entry were identified for inclusion in this analysis. Eligible pts were ≥ 18 years with wild-type TP53 AML have failed prior therapies. Siremadlin was administered PO according to 4 treatment regimens: 1A, 250 mg or 350 mg or 400 mg on day 1 of a 3-week (-W) cycle (n= 6); 1B, 120 mg or 150 mg on days 1 and 8 of a 4-W cycle (n= 4); 2A, 20 mg or 30 mg daily for the first 2 weeks of a 4-W cycle (n= 2); and 2C, 45 mg daily for the first week of a 4-W cycle (n= 7). Grading of adverse events (AEs) was per CTCAE version 4.03. Response evaluation was per investigator's assessment based on the International Working Group for AML (Cheson 2003). Results: Nineteen pts with R/R AML after ≥1 SCT had received siremadlin as salvage therapy for active disease. The median age was 64 years (range: 30-72), and median interval from most recent SCT to start of siremadlin was 308 days (range, 119-1105). Median number of prior lines of antineoplastic therapy between most recent SCT and study entry was 1 (range, 0-3), 3 patients had received prior donor lymphocyte infusion [Table 1]. The median duration of exposure to siremadlin was 60 days (range, 21-203), median dose intensity was 52.5 mg/day (range, 13.4-200), and the median cumulative dose was 630 mg (range, 250-1710). Sixteen (84.2%) and 14 (73.7%) pts experienced grade (G) ≥3 AEs and treatment-related AEs, respectively; 14 (73.7%) and 12 (63.2%) pts experienced serious AEs (SAEs) and treatment-related SAEs, respectively. Nine pts had AEs leading to siremadlin dose adjustment or interruption, and 1 pt had an AE leading to treatment discontinuation (fungal sinusitis, G3, not suspected to be related to siremadlin). The most common G 3/4 non-hematological AEs were febrile neutropenia (n=11, 57.9%), hypokalemia (n=5, 26.3%), and tumor lysis syndrome (n=3, 15.8%). Graft vs host disease (GvHD) was observed in 4 pts (21.1%): G3, n=2 (10.5%); G2 and G1, each n=1 (5.3%) [Table 2]. Three of these 4 pts had a history of GvHD prior to commencement of siremadlin. Siremadlin was continued safely at full dose (n=3) or dose-reduced (n=1) in all four pts. For 1 pt treated with 400 mg in Reg 1A, chronic GvHD was declared a dose-limiting toxicity. Overall, the safety profile was consistent with what was observed in the overall X2101 study population of hematologic malignancies (n=93) including n=91 R/R AML pts [Table 3]. Of note, 3 out of 19 patients achieved a major clinical response: 2 achieved complete remission (CR) and 1 CR with incomplete hematologic recovery (CRi). Conclusions: The safety of siremadlin monotherapy in pts with relapsed AML following prior SCT(s) appears to be consistent with that previously defined in R/R AML. No excessive GvHD was reported and siremadlin could be continued safely at full or reduced dose. Preliminary efficacy data indicate anti-leukemic activity. Despite the limitation of this post-hoc analysis in a small cohort of patients, these results and recently identified IME provide evidence to support further exploration of siremadlin post SCT as a potential maintenance or preemptive treatment in AML pts with high risk of relapse to enhance the allogeneic graft vs leukemia, an important and potent therapy against AML relapse. Figure 1 Figure 1. Disclosures Stein: Blueprint Medicines: Consultancy; Daiichi Sankyo: Consultancy; PinotBio: Consultancy; Syros Pharmaceuticals, Inc.: Consultancy; Novartis: Consultancy; Celgene: Consultancy; Bristol Myers Squibb: Consultancy; Agios Pharmaceuticals, Inc: Consultancy; Jazz Pharmaceuticals: Consultancy; Foghorn Therapeutics: Consultancy; Syndax Pharmaceuticals: Consultancy; Genentech: Consultancy; Gilead Sciences, Inc.: Consultancy; Astellas: Consultancy; Abbvie: Consultancy; Janssen Pharmaceuticals: Consultancy. Carpio: Regeneron, TAKEDA, Celgene, Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees, Other. Kiladjian: Novartis: Membership on an entity's Board of Directors or advisory committees; Incyte Corporation: Membership on an entity's Board of Directors or advisory committees; Taiho Oncology, Inc.: Research Funding; PharmaEssentia: Other: Personal fees; Bristol Myers Squibb: Membership on an entity's Board of Directors or advisory committees; AOP Orphan: Membership on an entity's Board of Directors or advisory committees; AbbVie: Membership on an entity's Board of Directors or advisory committees. Curti: Abbvie: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees; Pfizer: Membership on an entity's Board of Directors or advisory committees; Jazz Pharma: Membership on an entity's Board of Directors or advisory committees. Craddock: Novartis Pharmaceuticals: Other: Advisory Board ; Celgene/BMS: Membership on an entity's Board of Directors or advisory committees, Research Funding. Zeiser: Incyte, Mallinckrodt, Novartis: Honoraria, Speakers Bureau. Eldjerou: Novartis Pharmaceuticals: Current Employment. Weber: Novartis Pharma AG: Current Employment, Current equity holder in publicly-traded company. Fabre: Novartis: Current Employment, Current holder of individual stocks in a privately-held company, Current holder of stock options in a privately-held company. Scott: Novartis Pharmaceuticals: Current Employment. DeAngelo: Abbvie: Research Funding; Takeda: Consultancy; Servier: Consultancy; Glycomimetrics: Research Funding; Blueprint: Research Funding; Jazz: Consultancy; Pfizer: Consultancy; Novartis: Consultancy, Research Funding; Incyte: Consultancy; Amgen: Consultancy; Agios: Consultancy; Forty-Seven: Consultancy; Autolus: Consultancy.

Recipients of allogeneic stem cell transplantation (alloSCT) are at high risk for contracting infectious diseases with high morbidity and mortality. Coronavirus disease 2019 (COVID-19) is a viral respiratory disease that can lead to severe pneumonia and acute respiratory distress syndrome, with a potentially fatal outcome. In this retrospective study conducted on behalf of the German Cooperative Transplant Study Group, we aimed to analyze risk factors, disease course, and outcomes of COVID-19 in patients who underwent alloSCT. AlloSCT recipients who became infected with SARS-CoV-2 at German and Austrian transplant centers between February 2020 and July 2021 were included. Classification of COVID-19 severity into mild, moderate-severe, or critical disease and division of the course of the pandemic into 4 phases were done according to the German Robert Koch Institute. The main endpoint was overall mortality at the end of follow-up. We further analyzed the need for treatment in an intensive care unit (ICU) and the severity of disease. Risk factors were evaluated using univariate and multivariate analyses, and survival analysis was performed using Kaplan-Meier method. The study cohort comprised 130 patients from 14 transplant centers, with a median age at diagnosis of COVID-19 of 59 years (range, 20 to 81 years) and a median interval between alloSCT and COVID-19 of 787 days (range, 19 to 8138 days). The most common underlying diseases were acute myeloid leukemia (45.4%) and lymphoma (10.8%). The majority of patients (84.9%) were infected in the later phases of the pandemic; 20.8% had moderate-severe disease, 12.3% had critical disease, and 19.2% were treated in an ICU. After a median follow-up of 127 days, overall mortality was 16.2%, 52.0% among patients treated in an ICU. Risk factors for mortality in multivariate analysis were active disease (odds ratio [OR], 4.46), infection with SARS-CoV-2 ≤365 days after alloSCT (OR, 5.60), age60 years (OR, 5.39), and ongoing immunosuppression with cyclosporine (OR, 8.55). Risk factors for developing moderate-severe or critical disease were concurrent immunosuppression (OR, 4.06) and age40 years (OR, 4.08). Patients after alloSCT exhibit a substantially increased mortality risk after COVID-19 infection compared with the normal population, without considerable improvement over the course of the pandemic. Risk factors include age, early infection post-alloSCT, and active immunosuppression. Further studies are needed to improve prevention and treatment in this high-risk patient group.

Oncogenic KRAS mutations are the most frequent mutations in human cancer, but most difficult to target. While sustained proliferation caused by oncogenic KRAS-downstream signalling is a main driver of carcinogenesis, there is increasing evidence that it also mediates autocrine effects and crosstalk with the tumour microenvironment (TME). Here, we discuss recent reports connecting KRAS mutations with tumour-promoting inflammation and immune modulation caused by KRAS that leads to immune escape in the TME. We discuss the preclinical work on KRAS-induced inflammation and immune modulation in the context of currently ongoing clinical trials targeting cancer entities that carry KRAS mutations and strategies to overcome the oncogene-induced effects on the immune system., Oncogenic signalling has been historically associated with sustained cancer cell-intrinsic proliferation, however its role in promoting tumour immunoresistance has also become evident. Here, Hamarsheh and colleagues review and discuss the preclinical work on the immune modulatory effects of oncogenic KRAS and the potential clinical application.

Graft-versus-host disease (GvHD) was first described in 1959, since then major efforts have been made in order to understand its physiopathology and animal models have played a key role. Three steps, involving different pathways, have been recognised in either acute and chronic GvHD, identifying them as two distinct entities. In order to reduce GvHD incidence and severity, prophylactic measures were added to transplant protocols. The combination of a calcineurin inhibitor (CNI) plus an antimetabolite remains the standard of care. Better knowledge of GvHD pathophysiology has moved this field forward and nowadays different drugs are being used on a daily basis. Improving GvHD prophylaxis is a major goal as it would translate into less non-relapse mortality and better overall survival. As compared to CNI plus methotrexate the combination of CNI plus mycophenolate mophetil (MMF) allows us to obtain similar results in terms of GvHD incidence but a lower toxicity rate in terms of neutropenia or mucositis. The use of ATG has been related to a lower risk of acute and chronic GvHD in prospective randomized trials as well as the use of posttransplant Cyclophosphamide, with no or marginal impact on overall survival but with an improvement in GvHD-relapse free survival (GRFS). The use of sirolimus has been related to a lower risk of acute GvHD and significantly influenced overall survival in one prospective randomized trial. Other prospective trials have evaluated the use of receptors such as CCR5 or α4β7 to avoid T-cells trafficking into GvHD target organs, cytokine blockers or immune check point agonists. Also, epigenetic modifiers have shown promising results in phase II trials. Attention should be paid to graft-versus-leukemia, infections and immune recovery before bringing new prophylactic strategies to clinical practice. Although the list of novel agents for GvHD prophylaxis is growing, randomized trials are still lacking for many of them.

CD8+ effector T (TE) cell proliferation and cytokine production depends on enhanced glucose metabolism. However, circulating T cells continuously adapt to glucose fluctuations caused by diet and inter-organ metabolite exchange. Here we show that transient glucose restriction (TGR) in activated CD8+ TE cells metabolically primes effector functions and enhances tumour clearance in mice. Tumour-specific TGR CD8+ TE cells co-cultured with tumour spheroids in replete conditions display enhanced effector molecule expression, and adoptive transfer of these cells in a murine lymphoma model leads to greater numbers of immunologically functional circulating donor cells and complete tumour clearance. Mechanistically, TE cells treated with TGR undergo metabolic remodelling that, after glucose re-exposure, supports enhanced glucose uptake, increased carbon allocation to the pentose phosphate pathway (PPP) and a cellular redox shift towards a more reduced state—all indicators of a more anabolic programme to support their enhanced functionality. Thus, metabolic conditioning could be used to promote efficiency of T-cell products for adoptive cellular therapy.

Despite undergoing allogeneic hematopoietic stem cell transplantation (HCT), patients with acute myeloid leukemia (AML) with internal tandem duplication mutation in theIn a randomized, placebo-controlled, double-blind phase II trial (SORMAIN; German Clinical Trials Register: DRKS00000591), 83 adult patients withWith a median follow-up of 41.8 months, the hazard ratio (HR) for relapse or death in the sorafenib group versus placebo group was 0.39 (95% CI, 0.18 to 0.85; log-rankSorafenib maintenance therapy reduces the risk of relapse and death after HCT for

Glucocorticoid-refractory (SR) chronic (c) graft-versus-host disease (GVHD) is a multisystem immunological disease and the leading cause of non-relapse mortality (NRM) in patients surviving longer than 2 years after allogeneic hematopoietic cell transplantation. Both ruxolitinib (RUX) and extracorporeal photopheresis (ECP) have shown activity for SR-cGVHD which motivated us to treat refractory cGHVD patients with the RUX-ECP combination. In this retrospective survey, 23 patients received RUX-ECP as salvage therapy for SR-cGVHD. The best response (CR or PR) at any time point during treatment was 74% (17/23) including 9% (2/23) CR and 65% (15/23) PR. The 24-months-survival was 75% (CI 56.0-94.1). Newly diagnosed cytopenia occurred in 22% (5/23) and CMV reactivation was observed in 26% (6/23) of the patients. Serum levels of soluble interleukin-2 receptor (sIL-2R) correlated with response. Our retrospective analysis shows that the RUX-ECP combination is safe and has activity in a fraction of patients with SR-cGVHD, which needs validation in a prospective trial.

Graft-versus-host disease (GVHD) remains a major limitation of allogeneic hematopoietic stem cell transplantation. Only half of patients with severe acute GVHD respond to first-line treatment with corticosteroids and, for several decades, there was no optimal second-line treatment of patients with corticosteroid-refractory acute GVHD. Ruxolitinib was recently approved for the treatment of corticosteroid-refractory acute GVHD in adult and pediatric patients 12 years and older. Thus, it is important to define the patient population that would now be considered as refractory to ruxolitinib vs ruxolitinib dependent. Here, we propose to define ruxolitinib-refractory acute GVHD as disease that shows: (1) progression of GVHD compared with baseline after at least 5 to 10 days of treatment with ruxolitinib, based either on objective increase in stage/grade, or new organ involvement; (2) lack of improvement in GVHD (partial response or better) compared with baseline after ≥14 days of treatment with ruxolitinib; or (3) loss of response, defined as objective worsening of GVHD determined by increase in stage, grade, or new organ involvement at any time after initial improvement. GVHD manifestations that persist without improvement in patients who had a grade ≥3 treatment-emergent and ruxolitinib-attributed adverse event that did not resolve within 7 days of discontinuing ruxolitinib would serve as a clinical indication for additional treatment. In addition, absence of complete response or very good partial response at day 28 after ruxolitinib could be considered as an eligibility criterion.

Acute graft-versus-host disease (aGVHD) is a life-threatening complication after allogeneic hematopoietic cell transplantation (allo-HCT). Despite the use of prophylactic immunosuppression including calcineurin inhibitors, antimetabolites, antithymocyte globulin, or posttransplant cyclophosphamide, patients still develop severe aGVHD. In particular, patients with glucocorticoid-refractory GVHD (SR-GVHD) have a dismal prognosis with a low 1-year post–allo-HCT survival rate. Most classical drugs used to prevent or treat aGVHD target 1 specific pathway such as calcineurin inhibitors or mammalian target of rapamycin inhibitors, or they interfere with fast-dividing activated cells (eg, methotrexate, mycophenolate, and cyclophosphamide). In contrast to these drugs, inhibition-of-signaling molecules, used by multiple immune cells and critical for signal transduction of multiple proinflammatory cytokines, could be more efficacious at blocking GVHD. Ruxolitinib blocks Janus kinases 1 and 2, which are required to mediate the downstream signaling of multiple cytokine receptors. Recently, a multicenter phase 3 clinical trial showed that ruxolitinib led to significant improvements in efficacy outcomes compared to best available therapy, which will lead to a paradigm shift in the treatment of SR-GVHD.

Oncogenic Ras mutations occur in various leukemias. It was unclear if, besides the direct transforming effect via constant RAS/MEK/ERK signaling, an inflammation-related effect of KRAS contributes to the disease. Here, we identify a functional link between oncogenic KrasG12D and NLRP3 inflammasome activation in murine and human cells. Mice expressing active KrasG12D in the hematopoietic system developed myeloproliferation and cytopenia, which is reversed in KrasG12D mice lacking NLRP3 in the hematopoietic system. Therapeutic IL-1-receptor blockade or NLRP3-inhibition reduces myeloproliferation and improves hematopoiesis. Mechanistically, KrasG12D-RAC1 activation induces reactive oxygen species (ROS) production causing NLRP3 inflammasome-activation. In agreement with our observations in mice, patient-derived myeloid leukemia cells exhibit KRAS/RAC1/ROS/NLRP3/IL-1β axis activity. Our findings indicate that oncogenic KRAS not only act via its canonical oncogenic driver function, but also enhances the activation of the pro-inflammatory RAC1/ROS/NLRP3/IL-1β axis. This paves the way for a therapeutic approach based on immune modulation via NLRP3 blockade in KRAS-mutant myeloid malignancies.

Acute myeloid leukemia (AML) relapse after allogeneic hematopoietic cell transplantation (allo-HCT) has a dismal prognosis. We found that T cells of patients relapsing with AML after allo-HCT exhibited reduced glycolysis and interferon-γ production. Functional studies in multiple mouse models of leukemia showed that leukemia-derived lactic acid (LA) interfered with T cell glycolysis and proliferation. Mechanistically, LA reduced intracellular pH in T cells, led to lower transcription of glycolysis-related enzymes, and decreased activity of essential metabolic pathways. Metabolic reprogramming by sodium bicarbonate (NaBi) reversed the LA-induced low intracellular pH, restored metabolite concentrations, led to incorporation of LA into the tricarboxylic acid cycle as an additional energy source, and enhanced graft-versus-leukemia activity of murine and human T cells. NaBi treatment of post–allo-HCT patients with relapsed AML improved metabolic fitness and interferon-γ production in T cells. Overall, we show that metabolic reprogramming of donor T cells is a pharmacological strategy for patients with relapsed AML after allo-HCT.

The bone marrow microenvironment (BMME) is key player in regulation and maintenance of hematopoiesis. Oncogenic

Immune checkpoint inhibitor (ICI) therapy has shown a significant benefit in the treatment of a variety of cancer entities. However, immune-related adverse events (irAEs) occur frequently and can lead to ICI treatment termination. MicroRNA-146a (miR-146a) has regulatory functions in immune cells. We observed that mice lacking miR-146a developed markedly more severe irAEs compared with WT mice in several irAE target organs in 2 different murine models. miR-146a(–/–) mice exhibited increased T cell activation and effector function upon ICI treatment. Moreover, neutrophil numbers in the spleen and the inflamed intestine were highly increased in ICI-treated miR-146a(–/–) mice. Therapeutic administration of a miR-146a mimic reduced irAE severity. To validate our preclinical findings in patients, we analyzed the effect of a SNP in the MIR146A gene on irAE severity in 167 patients treated with ICIs. We found that the SNP rs2910164 leading to reduced miR-146a expression was associated with an increased risk of developing severe irAEs, reduced progression-free survival, and increased neutrophil counts both at baseline and during ICI therapy. In conclusion, we characterized miR-146a as a molecular target for preventing ICI-mediated autoimmune dysregulation. Furthermore, we identified the MIR146A SNP rs2910164 as a biomarker to predict severe irAE development in ICI-treated patients.

Acute graft-versus-host disease (GVHD) is a life-threatening complication after allogeneic hematopoietic cell transplantation (allo-HCT). Although currently used GVHD treatment regimens target the donor immune system, we explored here an approach that aims at protecting and regenerating Paneth cells (PCs) and intestinal stem cells (ISCs). Glucagon-like-peptide-2 (GLP-2) is an enteroendocrine tissue hormone produced by intestinal L cells. We observed that acute GVHD reduced intestinal GLP-2 levels in mice and patients developing GVHD. Treatment with the GLP-2 agonist, teduglutide, reduced de novo acute GVHD and steroid-refractory GVHD, without compromising graft-versus-leukemia (GVL) effects in multiple mouse models. Mechanistically GLP-2 substitution promoted regeneration of PCs and ISCs, which enhanced production of antimicrobial peptides and caused microbiome changes. GLP-2 expanded intestinal organoids and reduced expression of apoptosis-related genes. Low numbers of L cells in intestinal biopsies and high serum levels of GLP-2 were associated with a higher incidence of nonrelapse mortality in patients undergoing allo-HCT. Our findings indicate that L cells are a target of GVHD and that GLP-2–based treatment of acute GVHD restores intestinal homeostasis via an increase of ISCs and PCs without impairing GVL effects. Teduglutide could become a novel combination partner for immunosuppressive GVHD therapy to be tested in clinical trials.

PURPOSE Despite undergoing allogeneic hematopoietic stem cell transplantation (HCT), patients with acute myeloid leukemia (AML) with internal tandem duplication mutation in the FMS-like tyrosine kinase 3 gene ( FLT3-ITD) have a poor prognosis, frequently relapse, and die as a result of AML. It is currently unknown whether a maintenance therapy using FLT3 inhibitors, such as the multitargeted tyrosine kinase inhibitor sorafenib, improves outcome after HCT. PATIENTS AND METHODS In a randomized, placebo-controlled, double-blind phase II trial (SORMAIN; German Clinical Trials Register: DRKS00000591), 83 adult patients with FLT3-ITD–positive AML in complete hematologic remission after HCT were randomly assigned to receive for 24 months either the multitargeted and FLT3-kinase inhibitor sorafenib (n = 43) or placebo (n = 40 placebo). Relapse-free survival (RFS) was the primary endpoint of this trial. Relapse was defined as relapse or death, whatever occurred first. RESULTS With a median follow-up of 41.8 months, the hazard ratio (HR) for relapse or death in the sorafenib group versus placebo group was 0.39 (95% CI, 0.18 to 0.85; log-rank P = .013). The 24-month RFS probability was 53.3% (95% CI, 0.36 to 0.68) with placebo versus 85.0% (95% CI, 0.70 to 0.93) with sorafenib (HR, 0.256; 95% CI, 0.10 to 0.65; log-rank P = .002). Exploratory data show that patients with undetectable minimal residual disease (MRD) before HCT and those with detectable MRD after HCT derive the strongest benefit from sorafenib. CONCLUSION Sorafenib maintenance therapy reduces the risk of relapse and death after HCT for FLT3-ITD–positive AML.