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 (CPI) have shown remarkable efficacy in patients with Hodgkin lymphoma (HL) and are now used routinely for this disease. While allogeneic hematopoietic cell transplantation (alloHCT) remains a curative option for HL, there are concerns that prior CPI may exacerbate post alloHCT complications, particularly graft-versus-host disease (GVHD), and lead to worse outcomes. Given the relative paucity of data, we performed a CIBMTR/EBMT study to examine the impact of prior CPI in alloHCT outcomes. We included 2186 adult patients > 18 years who received a first alloHCT using a matched related, unrelated or haploidentical donor from 2008-2023. Twenty-seven percent of patients received prior CPI. GVHD prophylaxis was post-transplant cyclophosphamide in 55.8% 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 the multivariate analysis, prior CPI exposure did not affect overall survival or non-relapse mortality but resulted in improved progression-free survival (non-CPI vs. CPI HR 0.81, 0.67-0.98, p=0.03) and lower incidence of relapse (HR 0.58, 0.45-0.76, p<0001). While grade II-IV (HR1.26, 1.04-1.53; p=0.02) and III-IV (HR1.41, 1.04-1.92; p=0.03) acute GVHD were increased differences in chronic GVHD were not significant. Use of post-transplant cyclophosphamide based GVHD prophylaxis resulted in improved OS, lower grade II-IV acute GVHD and chronic GVHD 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 checkpoint inhibitors.

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 elderly. 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 MM patients. 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.

Marstacimab is a monoclonal antibody that targets the tissue factor pathway inhibitor to rebalance hemostasis. Previous phase 1 and 2 trials established marstacimab safety and efficacy in adults with severe hemophilia A or B. BASIS is an open-label, phase 3 trial of marstacimab in males aged 12-74 years with severe hemophilia A (factor VIII <1%) or moderately severe to severe hemophilia B (factor IX ≤2%). Participants without inhibitors received on-demand (OD) or routine prophylaxis (RP) factor replacement during a 6-month observational phase (OP) before receiving once-weekly subcutaneous 150 mg marstacimab prophylaxis during a 12-month active treatment phase (ATP). Primary endpoints were annualized bleeding rate (ABR) for treated bleeds vs prior OD or RP during the OP and safety. Of 128 participants enrolled in the OP, 116 received marstacimab in the ATP. In the OD group (n=33), mean ABR (95% CI) decreased from 39.86 (33.05-48.07) in the OP to 3.20 (2.10-4.88) in the ATP, demonstrating superiority of marstacimab (estimated ABR ratio, 0.080 [0.057-0.113]; P < .0001). In the RP group (n=83), mean ABR decreased from 7.90 (5.14-10.66) in the OP to 5.09 (3.40-6.78) in the ATP, demonstrating noninferiority and superiority of marstacimab (estimated ABR difference, -2.81 [-5.42 to -0.20]; P = .0349). There were no deaths or thromboembolic events. Weekly subcutaneous marstacimab reduced ABR compared with OD or RP therapy in the OP in individuals with severe hemophilia A or moderately severe to severe hemophilia B without inhibitors. Marstacimab was safe and well tolerated with no unanticipated side effects. This trial was registered at www.clinicaltrials.gov as # NCT03938792.