Autologous CD19-targeting CAR-T has transformed management of relapsed/refractory adult B-cell acute lymphoblastic leukaemia(B-ALL) but relapse post-allogeneic stem cell transplant(allo-SCT) is frequently accompanied by profound lymphopenia, impaired T-cell fitness and aggressive disease requiring urgent treatment, making autologous CAR-T challenging to deliver. We developed an allogeneic matched-donor CD19CAR product (CAR-DLI) for adult B-ALL following allo-SCT failure. Here we evaluate the risks/benefits of pre-CAR-DLI lymphodepleting chemotherapy (LD), and the efficacy of repeat CAR-DLI dosing as per conventional DLI scheduling/protocols. Patients aged 16-70y with r/r B-ALL post-allo-SCT were eligible. Primary outcomes were toxicity and feasibility of CAR-DLI manufacture; secondary outcomes included CAR-DLI engraftment/ expansion/ persistence. 17 allo-SCT donors were leukapheresed and 14 patients (median age,43y) were infused. Median disease burden at registration was 63.5% bone marrow blasts (range, MRD-100%). Patients 1-7 received CAR-DLI-alone; patients 8-14 received CAR-DLI+LD with fludarabine/cyclophosphamide. CAR-DLI+LD vs CAR-DLI-alone was associated with superior peak CAR-DLI engraftment (93,134 vs. 8010 copies/ug gDNA), expansion (858,101 vs. 39,038 copies/ug gDNA/28d) and persistence (median 197 days vs. 32 days). CAR-DLI+LD was not associated with more immunotoxicity than CAR-DLI-alone, and GvHD (grade-1/skin) affected only 2/14(14%) patients. CAR-DLI+LD vs CAR-DLI-alone conferred superior event-free-survival (EFS) and overall-survival (OS) at 12m (57% vs 29%; 83% vs 29%). Repeat CAR-DLI dosing was administered to 8/14(57%) patients with morphological/MRD+ relapse, but with minimal engraftment/expansion or toxicity/efficacy. CAR-DLI+LD has a tolerable safety profile without significant GvHD and is associated with significantly better outcomes than CAR-DLI-alone. Repeat CAR-DLI dosing beyond dose 1 was not found to be effective in this analysis. NCT02893189.

The bone marrow microenvironment is intimately linked to the biology that underpins the development and progression of multiple myeloma. However, the complex cellular and molecular features that form bone marrow niches are poorly defined. Here, we used subcellular spatial transcriptomics to profile the expression of 5,001 genes in human bone marrow in the context of multiple myeloma. Using this approach, we explored the plasma cell and stroma ecosystem in bone marrow trephines from 21 individuals, including 7 with pre-malignant disease and 10 with newly diagnosed multiple myeloma. Using spatial transcriptomics in conjunction with an optimised trephine biobanking methodology, we could resolve major components of the human bone marrow microenvironment and reliably characterise distinct plasma cell populations in samples from healthy, pre-malignant disease and active myeloma. When plasma cells were visualised in the context of location, we detected spatially restricted subpopulations of plasma cells in five of ten newly diagnosed myeloma trephines. Surprisingly, the composition of haematopoietic and stromal microenvironments varied significantly between newly diagnosed myeloma trephines. Furthermore, these differences in microenvironments were also observed within trephines that had spatially restricted plasma cell subpopulations. Thus, these data are not consistent with the hypothesis that a universal bone marrow microenvironment supports the expansion of malignant plasma cells in myeloma. Instead, we propose that myeloma subpopulations form distinct microenvironments and can vary between both patients and spatial location.

Venous thromboembolism (VTE) affects approximately 1 in 200 hospitalized children. The diagnosis of pulmonary embolism (PE), the most severe clinical presentation of VTE, has increased dramatically by ~200% over the past two decades, disproportionately affecting adolescents, and is associated with adverse long-term post-PE sequelae. Nevertheless, the management of pediatric PE remains highly variable. This review focuses on significant advances in pediatric PE with a focus on published studies within the past decade. Using a representative case, we: (1) summarize existing risk prediction tools for acute pediatric PE and a shift in clinical practice in the management of acute PE with the implementation of pediatric PE Response Teams and multidisciplinary decision-making for severe pediatric PE; (2) describe recently completed clinical trials of anticoagulation in children and adolescents and ongoing work to elucidate the appropriate duration of therapy for children and adolescents with PE; (3) review advances in understanding post-PE syndrome and the need for continued refinement of evaluation tools and management approaches. Many unanswered questions remain despite the significant advances in pediatric thrombosis over the past decade.

Chronic Lymphocytic Leukemia (CLL) is the most common chronic blood cancer in adults. Active NOTCH signaling in CLL is associated with poorer prognosis. Importantly, CLL patients with NOTCH1 non-coding mutations in the 3-prime untranslated region (3'UTR) manifested with a more aggressive disease course even compared to those with mutations in the NOTCH1 coding region. Here, we comprehensively characterize a cryptic splice acceptor site in 3'UTR of the NOTCH1 gene being converted into a stronger site. The functional consequences of the resulting NOTCH1 protein variants depend on the exact localization of the splice site, the used open reading frame and the appearance of the next STOP codon. The most frequent 3'UTR mutation (g.139390152, A>G) generates a novel NOTCH1 protein, lacking the PEST domain but expressing an altered C-terminus consisting of 68 amino acids. Mechanistically, we show that this splice variant (NOTCH1 152) is transcriptionally less active and dysregulates the regular ubiquitination dependent degradation of the wild type NOTCH1 intracellular domain (NICD) in trans. Thus, the NOTCH1 152 variant acts as a "sponge" protein in a novel mechanism of oncogenic NOTCH signaling activation, explaining the detrimental disease outcome of CLL patients with non-coding NOTCH1 mutations. We propose that the detection of NOTCH1 152 protein by specific antibodies is a useful prognostic marker for CLL patients.

Chimeric antigen receptor (CAR) T cells exhibit high response rates in B cell malignancies, but most patients eventually relapse. A key mechanism of treatment failure is the loss or downregulation of tumor antigen expression, yet strategies to modulate cell surface levels of CAR T cell targets remain largely unexplored. Here we identify B cell maturation antigen (BCMA), a central CAR T cell target in multiple myeloma (MM), as a highly short-lived protein that undergoes K48-linked polyubiquitylation at the plasma membrane, leading to its p97-dependent degradation via the ubiquitin-proteasome system (UPS). This previously unprecedented mechanism of plasma membrane protein regulation enables significant enhancement of BCMA expression via proteasome inhibitors (PI). The clinically approved PI carfilzomib (CFZ) significantly enhances the efficacy of BCMA-directed CAR T cells against both PI-sensitive and refractory MM cells in vitro and in vivo. Notably, treatment of ten patients with CFZ under the compassionate use CarCAR protocol - after relapse following BCMA CAR T cell therapy - resulted in increased BCMA expression in all patients. However, clinical responses were observed only in those with residual and/or expanding CAR T cells, suggesting restored CAR T cell function. These findings provide a rationale for the use of CFZ treatment in relapsed or refractory MM following BCMA CAR T therapy, advocate for future trials combining CFZ with BCMA CAR T cells and provide a framework for exploring UPS-dependent degradation of other immunotherapy antigens.

The contact system includes factor XII (FXII), factor XI (FXI), prekallikrein (PK) and high-molecular weight kininogen (HK) and has received increased interest as a potential target in immunothrombotic and inflammatory diseases. This system activates two distinct pathways, the intrinsic pathway of coagulation via cleavage of factor IX (FIX) and inflammation via HK cleavage resulting in bradykinin (BK) generation. HK is central to the function of both arms of the system as a substrate for plasma kallikrein (PKa) and critical cofactor which forms interactions with cell receptors and activators. Both FXI and PK circulate in complex with HK and both can be activated by factor XIIa (FXIIa). Reciprocal activation and continuous consumption of PK and FXII is a feature of the contact system. On endothelial cells, PK and FXII become activated but only in the presence of secreted receptor for the globular domain of C1q (gC1qR) and Zn2+ ions. A second mechanism exists on endothelial cells whereby prolylcarboxypeptidase activates the PK-HK complex to generate BK in a FXII independent manner. On platelets, FXI can be cleaved by thrombin, but only in the presence of secreted polyphosphate. This review explores the 3D structure of the contact factors and examines the molecular mechanisms underlying contact activation. We focus on conformational changes that expose cleavage sites and exosites in FXII, PK, and FXI. We also discuss contact factor protein-protein interactions, recognition of polyanions and the role of HK and Zn²⁺ in contact system assembly.

Checkpoint inhibitors (CPIs) have shown remarkable efficacy in Hodgkin lymphoma (HL), and are now used routinely. While allogeneic hematopoietic cell transplantation (allo-HCT) remains a curative option for HL, there are concerns prior CPIs may exacerbate post-allo-HCT complications, particularly graft-versus-host disease (GVHD), and lead to worse outcomes. Given the relative paucity of data, we performed a Center for International Blood and Marrow Transplant Research/European Society for Blood and Marrow Transplantation study to examine the impact of prior CPIs in allo-HCT. We included 2186 adult patients aged >18 years who received a first allo-HCT using a matched related, unrelated, or haploidentical donor from 2008 to 2023. Twenty-seven percent of patients received prior CPIs. GVHD prophylaxis was posttransplant cyclophosphamide (PTCy) in 55.8% of patients in the CPI cohort, and 35% in the non-CPI cohort. Median follow-up among survivors was longer for the non-CPI (39 months) than CPI cohort (16.5 months). In multivariate analysis, prior CPI exposure did not affect overall survival (OS) or nonrelapse mortality, but resulted in improved progression-free survival (non-CPI vs CPI hazard ratio [HR], 0.81; 0.67-0.98; P = .03) and lower relapse incidence (HR, 0.58; 0.45-0.76; P < 001). While grade 2 to 4 (HR, 1.26; 1.04-1.53; P = .02) and 3 to 4 (HR, 1.41; 1.04-1.92; P = .03) acute GVHD (aGVHD) were increased, differences in chronic GVHD (cGVHD) were not significant. PTCy-based GVHD prophylaxis resulted in improved OS, lower grade 2 to 4 aGVHD, and cGVHD in patients with prior CPI exposure. In summary, allo-HCT should still be considered a curative option for patients with HL in the era of CPIs.

The pathogenesis of multiple myeloma (MM) and its precursor monoclonal gammopathy of undetermined significance (MGUS) is linked to an aging immune system. Chronic activation of B/plasma cells may contribute to the origin of MGUS, which is frequent in the older individuals. However, only 1% of individuals with MGUS annually experience progression to MM. The immune system can specifically recognize MGUS lesions, and preclinical MM models provide evidence for both innate and adaptive immune surveillance. Multiomic studies have identified several systemic alterations at the MGUS stage, suggesting accelerated immune aging prior to evolution into clinical malignancy. MM is further associated with spatial alterations in patterns of tumor growth and in situ regulation of regional immunity. Both tumor and microenvironment-related factors contribute to immune paresis, which facilitates the dissemination of clonal plasma cells, and increases the risk of infections in patients with MM. Immune profiles in blood or marrow exhibit considerable heterogeneity, and have been linked to outcomes following immune therapies, including T-cell redirection. Understanding how underlying systemic immune changes impact in vivo function and durability of natural or synthetic tumor/antigen-specific immunity needs further study. Preserving or restoring immune function may be critical for long-term outcomes both in the context of prevention of clinical MM and of treating active disease. Benchmarking of immune biomarkers followed by its prospective integration into current risk models, together with improved understanding of mechanisms underlying tumor immunity in vivo, are needed to optimize immune approaches and improve outcomes in MM.

TP53-Y220C is a recurrent hotspot mutation in cancers and leukemias. It is observed predominantly in acute myeloid leukemia (AML)/myelodysplastic syndromes among hematological malignancies and is associated with poor outcome. The mutation creates a structural pocket in the p53 protein. PC14586 (rezatapopt) is a small molecule designed to bind to this pocket and thus restore a p53-wild type (p53-WT) conformation. We demonstrate that PC14586 converts p53-Y220C into a p53-WT conformation and activates p53 transcriptional targets, but surprisingly induces limited/no apoptosis in TP53-Y220C AML. Mechanistically, MDM2 induced by PC14586-activated conformational p53-WT and the nuclear exporter XPO1 reduce the transcriptional activities of p53, which are fully restored by inhibition of MDM2 and/or XPO1. Importantly, p53-WT protein can bind to BCL-2, competing with BAX in the BH3 binding pocket of BCL-2 and also binds to BCL-xL and MCL-1. However, such binding by PC14586-activated conformational p53-WT is not detected. Pharmacological inhibition of the BCL-2/BAX interaction with venetoclax fully compensates for this deficiency, induces massive cell death in AML cells and stem/progenitor cells in vitro and prolongs survival of TP53-Y220C AML xenografts in vivo. Collectively, we identified transcription-dependent and -independent mechanisms that limit the apoptogenic activities of reactivated conformational p53-WT and suggest approaches to optimize apoptosis induction in TP53-mutant leukemia. A clinical trial of PC14586 in TP53-Y220C AML/myelodysplastic syndromes has recently been initiated (NCT06616636).

The development of multiple myeloma is typically associated with various cytogenetic abnormalities; however, these genetic changes alone do not fully account for the observed heterogeneity in patient prognosis and treatment response. Recent studies leveraging next-generation sequencing and genomic approaches have shown that epigenetic alterations are crucial in myeloma development and therapeutic resistance. These changes contribute to high levels of transcriptomic instability and enables cellular adaptation to targeted therapies and immunotherapies through diverse evolutionary trajectories. In this regard, aberrations of histone modifications and chromatin remodeling affect various cellular processes such as DNA repair, DNA damage response, cellular survival, and apoptosis signaling, which provides a strong rationale for developing epigenetic-targeted therapies for myeloma treatment. In this review, we focus on recent advances and research gaps in understanding the deregulation of histone acetylation, a widespread and versatile process of histone modification occurring at lysine residues at the N-terminus of histone tails, and its intimate interplay with chromatin remodeling complexes in orchestrating dynamic chromatin functional states and transcriptional outputs. We also provide an updated review of epigenetic modulatory drugs targeting HDAC, CBP/p300, and bromodomain and extraterminal proteins, along with a discussion of their limitations and future perspectives in myeloma treatment.