Hematopoietic stem and progenitor cells (HSPC) are regulated by interactions with stromal cells in the bone marrow (BM) cavity, which can be segregated into two spatially defined central marrow (CM) and endosteal (Endo) compartments. However, the importance of this spatial compartmentalization for BM responses to complex conditions like inflammation remains largely unknown. Here, we extensively validate a combination of scRNA-seq profiling and matching flow cytometry isolation that reproducibly identifies 7 key CM and Endo populations and accurately surveys both niche locations. We demonstrate that inflammatory perturbations exert specific effects on different cellular compartments, with type I interferon responses causing leptin receptor-expressing mesenchymal stromal cells to abandon their normal stromal functions and instead adopt an inflammatory phenotype associated with overproduction of chemokines that modulate local monocyte dynamics in the surrounding microenvironment. Our results provide a comprehensive method for molecular and functional stromal characterization and highlight the importance of altered stomal cell activity in regulating hematopoietic responses to inflammatory challenges.

Circulating tumor cells (CTC) represent a high-risk biomarker in newly diagnosed multiple myeloma (NDMM); however, their prognostic value among transplant-eligible (TE) patients receiving daratumumab with bortezomib/lenalidomide/dexamethasone (D-VRd) remains unknown. Here, we analyzed CTC in the phase 3 PERSEUS trial (EMN017/NCT03710603). TE patients with NDMM were randomized (1:1) to D-VRd with daratumumab/lenalidomide maintenance (D-VRd group) or VRd with lenalidomide maintenance (VRd group), both with transplant. A subset of 451/709 patients from PERSEUS (D-VRd, 231/355; VRd, 220/354) had screening blood samples collected for CTC analysis by flow cytometry (median follow-up, 47.6 months). CTC were detected in 370/451 (82%) patients (median limit of detection, 0.0004%). CTC were prognostic of progression-free survival (PFS), independently of other factors, as a continuous (HR, 1.36 [95% CI, 1.15-1.60]; P<0.001) and categorical variable (≥0.175% CTC-high, optimal threshold). D-VRd improved PFS versus VRd in CTC-low patients (4-year rates: 88% vs 74%; HR, 0.42 [95% CI, 0.25-0.70]; P=0.0013). Regardless of study treatment, minimal residual disease (MRD)-negativity rates were lower in CTC-high versus CTC-low patients (10-5: 52.2% vs 66.2%; 10-6: 34.8% vs 52.4%). D-VRd significantly increased MRD-negativity rates versus VRd among CTC-high (10-5: 69.4% vs 33.3%; 10-6: 47.2% vs 21.2%; both P<0.05) and CTC-low patients (10-5: 74.4% vs 57.8%; 10-6: 65.6% vs 38.5%; both P<0.001), with similar observations for sustained MRD negativity. CTC levels are an independent prognostic factor in TE-NDMM patients treated with standard-of-care frontline quadruplet. D-VRd improved overall and sustained MRD-negativity rates in CTC-high and CTC-low patients, and improved PFS for CTC-low with a positive trend in CTC-high patients. NCT03710603.

Chronic graft-versus-host-disease (cGvHD) is the primary non-relapse limitation to a successful hematopoietic cell transplantation (HCT) and is largely treated as a single biological entity. We hypothesized that there exist different biological subtypes of cGvHD. Using the ABLE network database, derived from the largest pediatric cGvHD cohort worldwide, we applied clustering analysis to subtype cGvHD patients from the ABLE1.0 and 2.0 studies (51 cGvHD patients, 158 non-cGvHD patients). We found three distinct cGvHD subtypes: cGvHD-1 was characterized by an effector memory (TEM), cytotoxic NK cell, and early precursor B cell predominant pattern; cGvHD-2 was phosphatidylcholine, cytokine, and plasma cells predominant; and cGvHD-3 had more naïve CD4+ T cell (TN) and naïve Treg, had later onset, and the only subtype with measurable TREC. We partially replicated these subtypes using metabolomic data from a separate pediatric cohort of the COG trial ASCT0031 (33 cGvHD patients, 39 non-cGvHD patients). Further, cGvHD-1 was associated with serotherapy (predominantly ATG) exposure, and cGvHD-3 was associated with receiving peripheral blood stem cells (PBSC) from donors, total body irradiation (TBI), and no previous acute GvHD. cGvHD-2 was associated with liver involvement and cGvHD-2 and -3 with de novo cGvHD. Overall, none of the subtypes closely associated with organ involvement. Contrasting each subtype against non-cGvHD patients, the 3 subtypes shared common markers, all of which were used in our previous cGvHD diagnostic classifier. These findings suggest the presence of distinct biological subtypes of cGvHD that may help guide therapeutic strategies.

The primary objective in multiply-relapsed hairy cell leukemia and variant (HCL/HCLv) was to determine if pentostatin-rituximab (DCFR) and bendamustine-rituximab (BR) each have overall response rate (ORR) exceeding that historically achieved by rituximab alone (~40%) in favor of 65%. Prospective data was unreported for either regimen. Fifty-six patients received six 28-day cycles of rituximab (375 mg/m² days 1, 15) with either bendamustine (90 mg/m² days 1, 2) or pentostatin (4 mg/m² days 1, 15). Eligibility required ≥2 purine analogs, or one plus rituximab for initial response <1 year. Complete remission (CR) and minimal residual disease (MRD) were studied, and progression-free survival (PFS) with hazard ratios (HR) determined. Although patients were assigned to either regimen through randomization for increasing homogeneity of the two treatment groups, the DCFR arm had fewer prior purine analogs (p=0.021) and lower baseline marrow HCL/HCLv infiltration (p=0.013). ORRs for DCFR and BR were 93% and 86%, 95% confidence intervals (95%CI) 83-102% and 73-99%, each exceeding the primary objective (40% in favor of 65%, p<0.0001 for each). Rates for CR and MRD-free CR and median PFS (141 vs 50 months, HR 0.63, 95%CI 0.32-1.25) numerically favored DCFR but that arm was significantly enriched with lower prior purine analogs and marrow infiltration each of which was associated post hoc with better response. Post hoc subgroup analysis particularly for the 41 patients with classic HCL suggested any superiority of DCFR vs BR might apply to the more favorable patients. DCFR and BR were highly effective in multiply relapsed HCL/HCLv. Possible DCFR superiority was hypothesis-generating, given uneven baseline risks and trial design. NCT01059786.

Aged hematopoietic stem cells (HSCs) expand in clusters over time, while reducing their regenerative capacity and their ability to preserve the homeostasis of the hematopoietic system. The expression of Notch ligands in the bone marrow (BM) niche is essential for hematopoiesis. However, the impact of Notch signaling for adult HSC function and its involvement in HSC aging remains controversial. Here we show that Notch activation in young HSCs is not homogeneous, and it is triggered by sinusoidal expression of the Notch ligand Jagged2 (Jag2). Sinusoidal Jag2 deletion in young mice recapitulates the decrease in Notch activity observed in aged HSCs and alters HSC divisional symmetry and fate priming, promoting myeloid-biased HSCs (My-HSCs) expansion. Mechanistically, our data reveals that upon decreasing sinusoidal Jag2 expression, HSCs themselves upregulate Jag2, which cis-inhibits Notch signaling, resulting in the expansion of My-HSCs and in reduced hematopoietic regeneration. Collectively, these findings identify the crosstalk between BM niche-driven and HSC intrinsic features in regulating HSC fate priming and regenerative potential and reveal an extrinsic Notch trans-activation to intrinsic cis-inhibition switch underlying HSC aging.

Blood platelets are crucial in hemostasis, thrombosis and thrombo-inflammation. Current evidence highlights the considerable heterogeneity within subjects in platelet structure, age, and activation properties. This heterogeneity has major implications for the diverse functions of platelets in physiology and pathophysiology extending to therapeutic targeting in hemostasis and cancer. In this review we provide a general concept of heterogeneity or diversity of platelet populations with emphasis on the diagnostic and advanced methodologies to assess and study differences between platelets. We describe conventional and novel approaches to address clinical and in research questions addressing platelet heterogeneity and discuss strengths and limitations of the available techniques.

Pirtobrutinib, a non-covalent, reversible Bruton tyrosine kinase inhibitor (BTKi), demonstrated efficacy in patients with chronic lymphocytic leukemia (CLL), resistant to covalent BTKi (cBTKi). Genomic correlations with response and resistance to pirtobrutinib were analyzed in relapsed/refractory (R/R) CLL patients pretreated with cBTKi and enrolled in the phase 1/2 BRUIN trial. DNA sequencing was performed on PBMCs at baseline, on treatment, and at progressive disease (PD). Common alterations at baseline included mutations in BTK (43%), TP53 (38%), SF3B1 (25%), NOTCH1 (23%), ATM (19%), XPO1 (11%), PLCG2 (9%), BCL2 (8%) and 17p deletion (28%). Common baseline BTK mutations included C481S (85%), C481R (10%), C481F (6%) and C481Y (4%). At PD, 60/88 patients (68%) acquired ≥1 mutation, including 44% with acquired BTK mutations and 24% with other acquired mutations. A total of 55 acquired BTK mutations were detected in 39 patients, including gatekeeper mutations (T474I/F/S/L/Y/P, 26%), kinase-impaired L528W (16%), C481F/R/Y (5%), V416L (2%), and A428D (1%) and others proximal to the ATP-binding pocket, D539G/H/A (1%) and Y545N (1%). Decrease or complete clearance of BTK C481x was observed at PD in 36/43 patients (84%). Using a more sensitive assay, 37% (18/49) of acquired BTK mutations were detected at baseline at low allele frequency. Using the highly sensitive assay at progression, a similar frequency of acquired BTK mutations (39%) was detected, and all patients had detectable acquired mutations. This study highlights the complex clonal dynamics of BTK mutations in R/R CLL patients undergoing pirtobrutinib treatment, and the extent of resistance without an obvious genomic driver. NCT03740529.

Serine-threonine kinase 10 (STK10) is a member of Ste20 family of serine/threonine kinases and regulates lymphocyte adhesion. Quantitative phosphoproteomic assay showed increased STK10 phosphorylation in activated platelet. However, its role in platelet function remains unclear. In our study, we investigated the expression and role of STK10 in platelet function. We first showed STK10 expression in human and mouse platelets. By establishing megakaryocyte/platelet-specific STK10 knockout mice, we found that deletion of platelet STK10 impaired hemostasis and arterial thrombosis. Consistently, platelet aggregation, a-granule release, aIIbb3 activation, procoagulant activity, spreading and clot retraction were all reduced after deletion of STK10. Quantitative phosphoproteomic assays revealed several dysregulated phosphoproteins, which were enriched in platelet activation and focal adhesion. By using immunoprecipitation coupled to mass spectrometry and protein phosphorylation profiles screening approaches, we identified that STK10 interacts with integrin-linked protein kinase (ILK) and deletion of STK10 significantly reduced ILK phosphorylation (Ser343). Following in vitro phosphorylation assay demonstrated that STK10 directly phosphorylated ILK at Ser343. Additionally, inhibition of calcium, PKC or PI3K inhibited STK10 phosphorylation in activated platelets. Moreover, deletion of platelet STK10 reduced platelet-neutrophil interactions, neutrophil accumulation and neutrophil extracellular traps formation, ameliorated thromboinflammation, as well as increased the survival of sepsis mice. Furthermore, an increase of the activation of platelet STK10 and ILK was observed in sepsis mice and patients. In conclusion, our study identifies a novel regulatory role of STK10 in platelet function, arterial thrombosis and thromboinflammation, implying that it might be a potential target for the treatment of thrombotic or cardiovascular diseases.

Antiphospholipid antibodies targeting b2-glycoprotein I (b2GPI) are a hallmark of antiphospholipid syndrome (APS), associated with an increased risk of thrombosis and pregnancy morbidity. Among these, antibodies targeting Domain I (DI) are common in individuals at higher risk; however, their epitopes and prevalence among APS phenotypes remain unclear. Here, we employ a large collection of 29 structurally and functionally validated b2GPI variants to provide new insights into the molecular mechanisms of autoantibody recognition in APS. Using the prototypic human-derived monoclonal anti-DI antibody MBB2, we identified positively charged residue R39 as the key driver for MBB2 binding, followed by residues R43, N56, and T57. Structural analyses revealed that, while R39 is solvent-exposed, R43 is not, as it is caged by residues N56 and T57. The narrow epitope footprint explains why MBB2 exhibits a modest affinity for soluble b2GPI. The cage structure accounts for the epitope being conformational rather than linear. Mutational analyses of IgG anti-b2GPI antibodies from 52 triple-positive APS patients, 37 with a history of thrombosis and 15 non-vascular obstetric patients, confirmed significant reactivity against DI and showed signatures of two conformational epitopes: one similar to MBB2 (epitope I), in which the presence of R39 is essential, and another that does not require R39 (epitope II). While less frequent than epitope-II in our cohort, epitope-I reactivity was notably enriched in thrombotic obstetric patients. Varying epitope specificities for DI may therefore aid in identifying different APS phenotypes and predicting clinical outcomes.

MLL rearrangements (MLLr) are the most common cause of congenital and infant leukemias. MLLr arise prior to birth and can transform fetal/neonatal progenitors with the help of only a few additional cooperating mutations. Despite the low threshold for transformation, infant leukemias are rare, and congenital leukemias, which arise before birth, are even less common. These observations raise the question of whether mechanisms exist to suppress leukemic transformation during fetal life, thereby protecting the developing fetus from malignancy during a period of rapid hematopoietic progenitor expansion. To test this possibility, we used a mouse model of temporally controlled MLL::ENL expression to show that fetal MLL::ENL exposure establishes a heritable, leukemia-resistant state within hematopoietic progenitors that persists after birth. When we induced MLL::ENL expression prior to birth and transplanted hematopoietic stem and progenitor cells, very few recipient mice developed acute myeloid leukemia (AML) despite robust engraftment. When we induced MLL::ENL expression shortly after birth, all recipient mice developed a highly penetrant AML. Fetal MLL::ENL expression imposed a negative selective pressure on hematopoietic progenitors before birth followed by loss of self-renewal gene expression and enhanced myeloid differentiation after birth that precluded transformation. These changes did not occur when MLL::ENL expression initiated shortly after birth. The fetal barrier to transformation was enforced by the histone methyltransferase MLL3, and it could be overcome by cooperating mutations, such as NrasG12D. Heritable fetal protection against leukemic transformation may contribute to the low incidence of congenital and infant leukemias in humans.

Calprotectin, a calcium- and zinc-binding protein composed of the subunits S100A8 and S100A9, has been extensively studied as a biomarker of gastrointestinal (GI) inflammation through fecal and serum analyses. However, its role in intestinal tissue remains poorly understood due to limited availability of biopsies. In this study, we analyzed S100A8 and S100A9 mRNA expression in 579 intestinal biopsies from allogeneic stem cell transplant (ASCT) patients and observed a strong association with acute GI graft-versus-host disease (aGI-GvHD) (p<0.001). Neutrophil infiltration correlated with the severity of aGI-GvHD (p<0.001), and calprotectin expression was strongly linked to Toll-like receptor 4 (TLR4) (p<0.001) and TLR2 (p<0.001) expression. TLR4 and aGI-GvHD were associated with elevated calprotectin mRNA levels (p<0.001). When patients received broad-spectrum antibiotics at disease onset, expression of calprotectin was suppressed (S100A8, p=0.001; S100A9, p=0.01). Gastrointestinal site-specific differences in calprotectin expression were identified: during severe aGI-GvHD, levels increased up to 30-fold in the small intestine and up to 5-fold in the large intestine with respect to mild/no aGI-GVHD, while under homeostasis, the large intestine exhibited higher baseline calprotectin (p=0.001). The high clinical relevance is evident from the observation that calprotectin expression was prognostic for transplant-related mortality (TRM). Our study suggests that (a) calprotectin is a potential biopsy biomarker in aGI-GvHD, (b) calprotectin expression and neutrophil infiltration possibly indicate translocation of microbiota, which (c) may be modulated by antibiotics.

Immune thrombocytopenia (ITP) is characterized by overproduction of anti-platelet autoantibodies. While B-cell depletion therapies show promise in ITP, their high relapse rates suggest a potential de novo breakdown of tolerance during an early stage of B cell development. Here, we investigated how central B-cell tolerance mechanisms affect autoantibody production in ITP. Paired single-cell RNA/B cell receptor (BCR) sequencing and bulk BCR sequencing revealed reduced V-J genomic distances in immunoglobulin kappa-chain (IGK) genes within bone marrow and peripheral B cells from ITP patients, along with decreased expression of recombination activating gene (RAG) in the immature B cells, suggesting insufficient receptor editing. Single-cell antibody cloning demonstrated increased autoreactive and polyreactive naïve B cells in ITP, indicating defective central B-cell tolerance. Through in vivo study, we established a causal link between receptor editing defects and anti-platelet antibody production, validating the immature B cell stage as the key phase of dysregulation. These findings suggest that insufficient receptor editing of immature B cells triggers central B-cell tolerance deficiency and autoantibody accumulation in ITP.