The regulated secretion of von Willebrand factor (VWF) from Weibel-Palade bodies (WPB) in endothelial cells is fundamental to hemostasis. This process relies on recruiting Rab GTPases and their effectors to the WPB membrane, with the guanine nucleotide exchange factor (GEF) MAP-kinase activating death domain (MADD) playing a central role. Biallelic variants in MADD lead to a pleiotropic neurological and developmental disorder that can include bleeding abnormalities. This study investigates the impact of pathogenic MADD variants on VWF secretion using patient-derived endothelial cells. We isolated endothelial colony forming cells (ECFCs) from three pediatric patients with biallelic MADD variants and unaffected heterozygous family members. All patients exhibited low VWF plasma levels (22-30 IU/dL). Proteomic analysis of patient-derived ECFCs revealed an absence of MADD peptides, reduced VWF, and downregulation of proteins involved in the exocytotic machinery, including Rab3D and the Rab3/27 effector Slp4-a. Functional assays demonstrated diminished Rab27A and Rab3D activity and their failure to localize to WPBs in patient cells. Biochemical and live-imaging studies showed that histamine-induced VWF and VWFpp secretion were significantly reduced in patient cells due to delayed and reduced degranulation of WPBs. Our findings demonstrate the critical role of MADD in maintaining the secretion competence of WPBs and the magnitude of VWF secretion by regulating the recruitment of the endothelial exocytotic machinery. This study highlights the in vivo significance of WPB exocytosis in maintaining plasma VWF levels and establishes MADD as the first causal gene for quantitative von Willebrand Disease (VWD) in patients without pathogenic VWF variants.

The treatment landscape of B-cell non-Hodgkin lymphomas is rapidly evolving. However, few advances have occurred in marginal zone lymphoma (MZL) with a single FDA-approved agent impacting the treatment landscape. Multiple factors are associated with this slower pace of progress, with a lower MZL incidence representing a significant factor. Pivotal randomized indolent lymphoma clinical trials analyzed MZL subsets without the appropriate power to capture differences between treatment arms. Furthermore, the current Lugano Classification may not fully capture the presentation or treatment responses of some subtypes, preventing access to clinical trials and limiting an efficacy assessment across the disease spectrum. Thus, current MZL treatment is largely informed by single-arm studies with relatively empiric treatment sequencing among available agents. While frontline strategies in early- and advanced-stage MZL can achieve prolonged disease control, few options exist in the relapsed/refractory setting capable of achieving similar results. Emerging data demonstrate the encouraging efficacy of CD3xCD20 bispecific antibodies and antibody-drug conjugates in achieving deep responses, as well as the potential of circulating tumor DNA in risk stratification and molecular response monitoring. Compounding all these considerations, it is essential to recognize MZL as a heterogeneous group of diseases characterized by unique biology, clinical presentation, treatment response, toxicity, and survival. Nonetheless, a common characteristic across MZL subtypes is their general indolent disease course, emphasizing the need to incorporate patient-centered assessment in clinical trials to better inform the decision-making process.

Epcoritamab and glofitamab are CD20-directed bispecific antibodies approved in the US for relapsed or refractory (R/R) diffuse large B-cell lymphoma (DLBCL). Limited data exist for patients treated outside of trials. Patients with R/R DLBCL receiving commercial epcoritamab or glofitamab between January 1, 2023 and October 15, 2024 were collected from 21 US institutions. Among 245 patients, 156 received epcoritamab and 89 received glofitamab, 113 were refractory to front-line therapy, 40 had MYC and BCL2 and/or BCL6 rearrangements, 147 received prior chimeric antigen receptor T-cell therapy, and 174 patients would have been ineligible for registrational trials. The overall response rate (ORR) for epcoritamab and glofitamab was 51% (23% complete response, [CR]) and 53% (30% CR), respectively. Median progression-free survival (mPFS) was 2.6 months (95% confidence interval [CI] 2.0 to 3.8 months), and median overall survival (mOS) was 7.8 months (95% CI 6.2 to 11.0 months). The 6-month PFS was 36% (95% CI 30% to 44%) and the 6-month OS was 60% (95% CI 54% to 67%). Both trial ineligibility and undetectable CD20 pre-bispecific portended shorter PFS and OS. Of 17 individuals with paired biopsies, 15 (88.2%) lost CD20 expression after bispecifics with a median time to progression of 3.7 months. This analysis including R/R DLBCL patients shows the ORR to CD3/CD20 BsAbs was comparable to pivotal trials, although PFS and OS were lower. Baseline undetectable levels of CD20 was associated with poor outcomes. These results demonstrate the activity of BsAbs in R/R DLBCL and underscore the importance of target antigen expression.

Acute myeloid leukemia (AML), an aggressive hematological malignancy, is driven by oncogenic mutations in stem and progenitor cells that give rise to AML blasts. While these mutations are well-characterized, their impact on healthy hematopoiesis-those blood cells exposed to AML but not mutated-has not been well-characterized. As the marrow is the major site for granulopoiesis, neutrophils are heavily influenced by AML pathobiology. Indeed, most AML patients report neutropenia, rendering them susceptible to infections. However, since AML studies use peripheral blood mononuclear cells devoid of neutrophils, the characterization of neutrophil dysfunction remains poorly understood. To investigate AML-exposed neutrophils, a pre-clinical AML mouse model was used where primary leukemic cells were transplanted into non-irradiated neutrophil reporter (Ly6G-tdTomato; Catchup) hosts. Neutrophils could not completely mature, suggesting impaired granulopoiesis. Single-cell transcriptomics of AML-exposed neutrophils revealed higher inflammation signatures and expression of CD14, an inflammatory marker. To address the factors contributing to this biology, an ex vivo cytokine screen was performed on marrow neutrophils and identified that NFκB signaling drove CD14 expression. AML-exposed neutrophils displayed widespread chromatin remodeling, and de novo motif discovery predicted increased binding sites for CCAAT-enhancer-binding proteins (C/EBPs) and Interferon regulatory factors (IRFs). Moreover, AML-exposed neutrophils inhibited T-cell proliferation, highlighting their immune-suppressive capability. Finally, similar biology of immature, inflammatory neutrophils was found in AML patients, again indicating dysregulated granulopoiesis. Collectively, these data show that AML-associated inflammation alters neutrophil granulopoiesis, impairs neutrophil function, and drives immunosuppression, thus contributing to patient susceptibility to infection.

Hematologists have a significant role in the diagnosis and management of light chain (AL) amyloidosis and are also frequently involved in diagnosing transthyretin (ATTR) and other rarer types of amyloidosis. Recent advances in diagnostic techniques and therapies are dramatically improving patient prognosis. Radionuclide imaging methods are emerging as a highly specific and non-invasive way to diagnose, quantify and monitor organ involvement representing a major advance in amyloidosis management.

Advanced mycosis fungoides (MF) and Sézary syndrome (SS) have a poor prognosis with overall survival <5 years. Studies have found the current clinical staging (IA-IVB) inadequate for risk stratification. Developing a prognostic index in MF/SS will identify patients with poor outcomes and may allow better management decisions and improved survival.PROCLIPI (Prospective Cutaneous Lymphoma International Prognostic Index) Study was launched in 2015 at 46 international expert MF/SS Centers, prospectively collecting pre-defined datasets in newly diagnosed MF/SS patients to determine a cutaneous lymphoma IPI (CLIPI).552 advanced stage MF/SS patients were recruited. The 5-year overall survival (OS) was IIB=50.0%, IIIA=64.8%, IIIB=43.9%, IVA1=50.8%, IVA2=25.9%, IVB=36.9%. Factors at diagnosis associated with a significantly worse survival were N3 status; p<0.001, age>60yrs; p<0.001, raised serum lactate dehydrogenase; p=0.005 and large-cell transformation in skin; p=0.006. Modelling these 4 independent risk-factors into a CLIPI found there was a statistically significant worse OS in high-versus low-risk p<0.001, high-versus intermediate-risk p=0.002 as well as intermediate-versus low-risk p=0.010. 5 Year OS were 63.3%, 44.7% and 18.3% in the low-, intermediate- and high-risk PROCLIPI cohort respectively.In advanced stage MF/SS there was a low 5-year survival rate and increasing stage was not associated with worsening survival. The use of CLIPI to stratify patients into low, intermediate, and high-risk prognostic groups has the potential to improve patient outcomes by helping guide optimal treatment selection. CPMS ID 17662 (PROCLIPI), RRK4970, ClinicalTrials.Gov ID: NCT02848274.

Deficiency of (f)actor VIII causes hemophilia A and excess FVIII function increases venous thromboembolic risk. The phenotypic consequences of aberrant FVIII function underscore the importance of understanding mechanisms that downregulate (a)ctivated FVIII to inform disease pathology and therapeutic drug design. Spontaneous A2-domain dissociation and activated protein C (APC) proteolysis are established mechanisms of FVIIIa inactivation. However, we know very little about how FVIIIa binding interactions with FIXa and FX impact FVIIIa inactivation in vivo. Here we investigate this using recombinant FVIIIa variants to probe A2-domain dissociation (FVIIIa-D519V,E665V) and APC cleavage (FVIIIa-R336Q,R562Q) or both (FVIIIa-R336Q,R562Q/D519V,E665V) in biochemical assays and in HA mouse injury models. We found that FIXa binding to FVIIIa stabilized the A2 domain and increased the contribution of APC to FVIIIa inactivation. Additional studies using individual APC cleavage site variants (FVIIIa-R336Q and FVIIIa-R562Q) demonstrated that FIXa and FX can protect FVIIIa from APC cleavage at Arg562 and Arg336, respectively, in a manner that is incomplete in vivo. Data also demonstrate that APC inactivation of FVIIIa exceeds FVIII suggesting differential APC recognition of FVIIIa relative to FVIII. Hemostatic studies of FVIII variants with altered inactivation demonstrated that both A2-domain dissociation and APC cleavage contribute to in vivo FVIIIa regulation. Specifically, stabilizing the A2-domain, inhibiting APC cleavage, or both improved potency 2.4, 4.8, and >10-fold, respectively, over wild type FVIII in a mouse hemostatic assay. Data support that both mechanisms of FVIIIa inactivation and FIXa interactions could be leveraged to enhance FVIII function for therapeutic benefit.

Tumor inflammation-associated neurotoxicity (TIAN) was recently proposed as a unique complication of immunotherapy in brain tumor patients. Here, we report a first comprehensive characterization of TIAN in CNS lymphoma (CNSL) patients treated with CD19-directed chimeric antigen receptor T-cells (CD19-CAR). TIAN occurred in 10/56 (17.9%) CNSL with clinical onset at a median 3.5 days (range: 1-9) after CD19-CAR infusion. It was less frequently associated with cytokine release syndrome (60% vs 100%, p = 0.009) than immune effector cell-associated neurotoxicity syndrome (ICANS). Although symptoms were usually transient and fully reversible, TIAN was associated with a fatal outcome in one patient. Larger CNS tumor volume at baseline allowed the identification of patients at risk for TIAN (AUC: 0.847, p = 0.002). Maximizing Youden J statistics, a discriminatory tumor volume threshold >3.4cm3 was determined, which carried 87.5% sensitivity and 80.5% specificity. TIAN correlated with higher overall response rates to CD19-CAR (90% vs 52%, p = 0.036) and improved progression-free survival (Hazard ratio: 0.22; 95%-Confidence interval: 0.07-0.61, p = 0.006) on multivariate Cox proportional hazard regression. Post-mortem histopathological evaluation of a TIAN lesion revealed a dense macrophage population with central necrosis and peripheral reactive gliosis, accompanied by loss of white matter and intracytoplasmic myelin in foamy macrophages. Collectively, our work supports TIAN as a localized on-tumor, on-target neurotoxicity syndrome, closely related to pre-existing CNSL lesions and distinct from ICANS. CNS tumor volume at baseline may allow to identify patients at risk and may guide management.

Hematopoietic stem cells (HSCs) are responsible for sustaining hematopoietic system throughout life, and their functional decline contributes to hematological disorders and organismal aging. Understanding the molecular mechanisms that govern HSC function is critical for developing interventions for treating and preventing aging-related diseases. Here, we show that DCAF8, a substrate recognition component of Cullin-RING E3 ubiquitin ligases, is highly expressed in HSCs and undergoes a progressive decline with age. Loss of DCAF8 in mice results in impaired function in HSCs, characterized by increased number yet decreased self-renewal capacity, which associates with cellular senescence and elevated DNA damage. Mechanistically, DCAF8 mediates the degradation of DOCK11, a guanine nucleotide exchange factor for CDC42. In the absence of DCAF8, DOCK11 accumulates, leading to elevated CDC42 activity and consequential loss of polarity of HSCs. Knocking out Dock11 mitigates the senescence, DNA damage, and self-renewal defects of Dcaf8-/- HSCs. This study highlights a critical role of DCAF8 in preventing HSC senescence via the DOCK11-CDC42 axis and suggests potential therapeutic targets for preventing functional decline in HSCs.

The megakaryocytic (MK) specific immunoreceptor G6b-B plays an essential role in MK development. Since germline loss-of-function mutations of G6b-B in man and its deletion in mouse models leads to thrombocytopenia and a myelofibrosis-like clinical phenotype (MF-MPIG6B), we explored the role of G6b-B in patients with MF due to a myeloproliferative neoplasm (MPN) with thrombocytopenia (MPN-MF-T). We demonstrated that MKs generated from mononuclear cells (MNCs) from a patient with MF-MPIG6B as well as patients with MPN-MF-T, failed to express GATA1 and G6B and possessed a protein pattern expression characteristic of MKs primed for inflammation rather than platelet production. MNCs from MPN-MF-T patients also generated fewer MK biased hematopoietic stem cells (HSCs) and greater numbers of small cytoplasmic immature MKs (CD41+CD42-G6B-) as compared to MNCs from non-thrombocytopenic MPN-MF patients (MPN-MF-NT). Plasma levels of TGFβ1 and YKL-40 which were shown to arrest normal MK maturation were elevated in the MF-MPIG6B patient. Although TGFβ1 plasma levels were similarly elevated in MPN-MF-T and MPN-MF-NT patients, TNFα and YKL-40 levels were upregulated to a greater extent in MPN-MF-T than MPN-MF-NT patients. Moreover, we identified a reciprocal positive regulatory loop involving TGFβ1 and YKL-40 in MF MKs. These findings indicate that impaired MK maturation, and reduced G6B expression lead to the predominance of pro-inflammatory MKs which produce factors that further arrest MK development in MF-MPIG6B and MPN-MF-T patients. NCT03895112.