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Cell membrane nanoparticles have attracted increasing interest in nanomedicine because they allow to exploit the complexity of cell membrane interactions for drug delivery. Several methods are used to obtain plasma membrane to generate cell membrane nanoparticles. Here, an optimized method combining nitrogen cavitation in isotonic buffer and sucrose gradient fractionation is presented. The method allows to obtain cell membrane fractions of high purity from both suspension and adherent cells. Comparison with other common methods for membrane extraction, where mechanical lysis using cell homogenizers is performed in isotonic or hypotonic buffers, shows that the optimized procedure yields high purity membrane in a robust and reproducible way. Procedures to mix the purified membrane with synthetic lipids to obtain cell membrane liposomes (CMLs) are presented and indications on how to optimize these steps are provided. CMLs made using crude membrane isolates or the purified membrane fractions show different uptake by cells. The CMLs made with the optimized procedure and liposomes of the same composition but without cell membrane components are thoroughly characterized and compared for their size, zeta potential, bilayer and mechanical properties to confirm membrane protein inclusion in the CMLs. Cell uptake studies confirm that the inclusion of membrane components modifies liposome interactions with cells., (© 2024 The Author(s). Small Methods published by Wiley‐VCH GmbH.)

Abstract: The interaction between menin and histone-lysine N-methyltransferase 2A (KMT2A) is a critical dependency for KMT2A- or nucleophosmin 1 (NPM1)-altered leukemias and an emerging opportunity for therapeutic development. JNJ-75276617 (bleximenib) is a novel, orally bioavailable, potent, and selective protein-protein interaction inhibitor of the binding between menin and KMT2A. In KMT2A-rearranged (KMT2A-r) and NPM1-mutant (NPM1c) acute myeloid leukemia (AML) cells, JNJ-75276617 inhibited the association of the menin-KMT2A complex with chromatin at target gene promoters, resulting in reduced expression of several menin-KMT2A target genes, including MEIS1 and FLT3. JNJ-75276617 displayed potent antiproliferative activity across several AML and acute lymphoblastic leukemia (ALL) cell lines and patient samples harboring KMT2A or NPM1 alterations in vitro. In xenograft models of AML and ALL, JNJ-75276617 reduced leukemic burden and provided a significant dose-dependent survival benefit accompanied by expression changes of menin-KMT2A target genes. JNJ-75276617 demonstrated synergistic effects with gilteritinib in vitro in AML cells harboring KMT2A-r. JNJ-75276617 further exhibited synergistic effects with venetoclax and azacitidine in AML cells bearing KMT2A-r in vitro, and significantly increased survival in mice. Interestingly, JNJ-75276617 showed potent antiproliferative activity in cell lines engineered with recently discovered mutations (MEN1M327I or MEN1T349M) that developed in patients refractory to the menin-KMT2A inhibitor revumenib. A cocrystal structure of menin in complex with JNJ-75276617 indicates a unique binding mode distinct from other menin-KMT2A inhibitors, including revumenib. JNJ-75276617 is being clinically investigated for acute leukemias harboring KMT2A or NPM1 alterations, as a monotherapy for relapsed/refractory acute leukemia (NCT04811560), or in combination with AML-directed therapies (NCT05453903)., (© 2024 American Society of Hematology. Published by Elsevier Inc. Licensed under Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International (CC BY-NC-ND 4.0), permitting only noncommercial, nonderivative use with attribution. All other rights reserved.)

Identification of specific and therapeutically actionable vulnerabilities, ideally present across multiple mutational backgrounds, is needed to improve acute myeloid leukemia (AML) patients' outcomes. We identify stearoyl-CoA desaturase (SCD), the key enzyme in fatty acid (FA) desaturation, as prognostic of patients' outcomes and, using the clinical-grade inhibitor SSI-4, show that SCD inhibition (SCDi) is a therapeutic vulnerability across multiple AML models in vitro and in vivo. Multiomic analysis demonstrates that SCDi causes lipotoxicity, which induces AML cell death via pleiotropic effects. Sensitivity to SCDi correlates with AML dependency on FA desaturation regardless of mutational profile and is modulated by FA biosynthesis activity. Finally, we show that lipotoxicity increases chemotherapy-induced DNA damage and standard chemotherapy further sensitizes AML cells to SCDi. Our work supports developing FA desaturase inhibitors in AML while stressing the importance of identifying predictive biomarkers of response and biologically validated combination therapies to realize their full therapeutic potential., (© 2024. The Author(s).)

Acute lymphoblastic leukemia (ALL), a complex malignancy, displays varying expression profiles of PIP4K2-related genes in adult patients. While PIP4K2A expression is elevated in ALL bone marrow cells compared to healthy bone marrow cells, PIP4K2B is downregulated, and PIP4K2C remains relatively unchanged. Despite the correlation between increased PIP4K2A expression and increased percentage of peripheral blood blasts, clinical outcomes do not strongly correlate with the expression of these genes. Here we investigated the therapeutic potential of three PIP4K2 inhibitors (THZ-P1-2, a131, and CC260) in ALL cell models. THZ-P1-2 emerges as the most effective inhibitor, inducing cell death and mitochondrial damage while reducing cell viability and metabolism significantly. Comparative analyses highlight the superior efficacy of THZ-P1-2 over a131 and CC260. Notably, THZ-P1-2 uniquely disrupts autophagic flux and inhibits the PI3K/AKT/mTOR pathway, indicating a distinct molecular mechanism. In summary, our findings elucidate the differential expression of PIP4K2-related genes in ALL and underscore the potential role of PIP4K2A in disease pathogenesis. The therapeutic promise of THZ-P1-2 in ALL treatment, along with its distinct effects on cell death mechanisms and signaling pathways, enriches our understanding of PIP4K2's involvement in ALL development and offers targeted therapy prospects., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Background and Aims: Previous studies have shown that skin autofluorescence (SAF), measured with an advanced glycation end product (AGE) reader, estimates the accumulation of AGEs in tissues. SAF is predictive of incident type 2 diabetes, cardiovascular disease (CVD), and CV mortality in the general population. Studies in diabetic mice have shown that activation of the receptor for AGEs in hematopoietic progenitor cells increases blood neutrophils and monocytes, impairing atherosclerosis regression. We asked whether SAF is associated with blood neutrophil and monocyte counts in the general population, and whether this was moderated by prediabetes, diabetes, and sex., Methods: We examined the associations between SAF and blood neutrophil/monocyte counts in participants of the Lifelines cohort (n = 58,923: n = 24,382 men, and n = 34,541 women), a prospective population-based cohort from the North of the Netherlands, employing multivariable regression analyses., Results: SAF positively associated with blood neutrophil and monocyte counts in the whole cohort. The positive association between SAF and monocyte, but not neutrophil, counts was moderated by prediabetes and diabetes. Positive associations between SAF and blood neutrophil and monocyte counts were moderated by male sex. Moreover, three-way interaction analyses revealed that the positive associations between SAF and neutrophil and monocyte counts were moderated by prediabetes, but not diabetes, in male sex., Conclusions: SAF is positively associated with blood neutrophil and monocyte counts in the general population, especially in men with prediabetes. This may contribute to the increased CV risk in men with prediabetes., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Sensing of the biophysical properties of membranes using molecular reporters has recently regained widespread attention. This was elicited by the development of new probes of exquisite optical properties and increased performance, combined with developments in fluorescence detection. Here, we report on fluorescence lifetime imaging of various rigid and flexible fluorescent dyes to probe the biophysical properties of synthetic and biological membranes at steady state as well as upon the action of external membrane-modifying agents. We tested the solvatochromic dyes Nile red and 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N-(7-nitro-2-1,3-benzoxadiazol-4-yl) (ammonium salt) (NBD), the viscosity sensor Bodipy C 12 , the flipper dye FliptR, as well as the dyes 3,3'-dioctadecyloxacarbocyanine perchlorate (DiO), Bodipy C 16 , lissamine-rhodamine, and Atto647, which are dyes with no previous reported environmental sensitivity. The performance of the fluorescent probes, many of which are commercially available, was benchmarked with well-known environmental reporters, with Nile red and Bodipy C 12 being specific reporters of medium hydration and viscosity, respectively. We show that some widely used ordinary dyes with no previous report of sensing capabilities can exhibit competing performance compared to highly sensitive commercially available or custom-based solvatochromic dyes, molecular rotors, or flipper in a wide range of biophysics experiments. Compared to other methods, fluorescence lifetime imaging is a minimally invasive and nondestructive method with optical resolution. It enables biophysical mapping at steady state or assessment of the changes induced by membrane-active molecules at subcellular level in both synthetic and biological membranes when intensity measurements fail to do so. The results have important consequences for the specific choice of the sensor and take into consideration factors such as probe sensitivity, response to environmental changes, ease and speed of data analysis, and the probe's intracellular distribution, as well as potential side effects induced by labeling and imaging., Competing Interests: Declaration of interests G.Y. is an employee of Antikor Biopharma (Stevenage, UK)., (Copyright © 2024 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

Mass cytometry by time-of-flight (CyTOF) is an emerging technology allowing for in-depth characterization of cellular heterogeneity in cancer and other diseases. Unfortunately, high-dimensional analyses of CyTOF data remain quite demanding. Here, we deploy a bioinformatics framework that tackles two fundamental problems in CyTOF analyses namely (1) automated annotation of cell populations guided by a reference dataset and (2) systematic utilization of single-cell data for effective patient stratification. By applying this framework on several publicly available datasets, we demonstrate that the Scaffold approach achieves good trade-off between sensitivity and specificity for automated cell type annotation. Additionally, a case study focusing on a cohort of 43 leukemia patients reported salient interactions between signaling proteins that are sufficient to predict short-term survival at time of diagnosis using the XGBoost algorithm. Our work introduces an automated and versatile analysis framework for CyTOF data with many applications in future precision medicine projects., Competing Interests: The authors declare no competing interests., (© 2024 The Authors.)

Despite advancements in utilizing genetic markers to enhance acute myeloid leukaemia (AML) outcome prediction, significant disease heterogeneity persists, hindering clinical management. To refine survival predictions, we assessed the transcriptome of non-acute promyelocytic leukaemia chemotherapy-treated AML patients from five cohorts (n = 975). This led to the identification of a 4-gene prognostic index (4-PI) comprising CYP2E1, DHCR7, IL2RA and SQLE. The 4-PI effectively stratified patients into risk categories, with the high 4-PI group exhibiting TP53 mutations and cholesterol biosynthesis signatures. Single-cell RNA sequencing revealed enrichment for leukaemia stem cell signatures in high 4-PI cells. Validation across three cohorts (n = 671), including one with childhood AML, demonstrated the reproducibility and clinical utility of the 4-PI, even using cost-effective techniques like real-time quantitative polymerase chain reaction. Comparative analysis with 56 established prognostic indexes revealed the superior performance of the 4-PI, highlighting its potential to enhance AML risk stratification. Finally, the 4-PI demonstrated to be potential marker to reclassified patients from the intermediate ELN2017 category to the adverse category. In conclusion, the 4-PI emerges as a robust and straightforward prognostic tool to improve survival prediction in AML patients., (© 2024 British Society for Haematology and John Wiley & Sons Ltd.)

Background: VISTA is a well-known immune checkpoint in T cell biology, but its role in innate immunity is less established. Here, we investigated the role of VISTA on anticancer macrophage immunity, with a focus on phagocytosis, macrophage polarization and concomitant T cell activation., Methods: Macrophages, differentiated from VISTA overexpressed THP-1 cells and cord blood CD34 + cell-derived monocytes, were used in phagocytosis assay using B lymphoma target cells opsonized with Rituximab. PBMC-derived macrophages were used to assess the correlation between phagocytosis and VISTA expression. qRT-PCR, flow cytometry, and enzyme-linked immunosorbent assay were performed to analyze the impact of VISTA on other checkpoints and M1/M2-like macrophage biology. Additionally, flow cytometry was used to assess the frequency of CD14 + monocytes expressing VISTA in PBMCs from 65 lymphoma patients and 37 healthy donors., Results: Ectopic expression of VISTA in the monocytic model cell line THP-1 or in primary monocytes triggered differentiation towards the macrophage lineage, with a marked increase in M2-like macrophage-related gene expression and decrease in M1-like macrophage-related gene expression. VISTA expression in THP-1 and monocyte-derived macrophages strongly downregulated expression of SIRPα, a prominent 'don't eat me' signal, and augmented phagocytic activity of macrophages against cancer cells. Intriguingly, expression of VISTA's extracellular domain alone sufficed to trigger phagocytosis in ∼ 50% of cell lines, with those cell lines also directly binding to recombinant human VISTA, indicating ligand-dependent and -independent mechanisms. Endogenous VISTA expression was predominantly higher in M2-like macrophages compared to M0- or M1-like macrophages, with a positive correlation observed between VISTA expression in M2c macrophages and their phagocytic activity. VISTA-expressing macrophages demonstrated a unique cytokine profile, characterized by reduced IL-1β and elevated IL-10 secretion. Furthermore, VISTA interacted with MHC-I and downregulated its surface expression, leading to diminished T cell activation. Notably, VISTA surface expression was identified in monocytes from all lymphoma patients but was less prevalent in healthy donors., Conclusions: Collectively, VISTA expression associates with and drives M2-like activation of macrophages with a high phagocytic capacity yet a decrease in antigen presentation capability to T cells. Therefore, VISTA is a negative immune checkpoint regulator in macrophage-mediated immune suppression., (© 2024. The Author(s).)

Amanita phalloides poisonings account for the majority of fatal mushroom poisonings. Recently, we identified hematotoxicity as a relevant aspect of Amanita poisonings. In this study, we investigated the effects of the main toxins of Amanita phalloides , α- and β-amanitin, on hematopoietic cell viability in vitro. Hematopoietic cell lines were exposed to α-amanitin or β-amanitin for up to 72 h with or without the pan-caspase inhibitor Z-VAD(OH)-FMK, antidotes N-acetylcysteine, silibinin, and benzylpenicillin, and organic anion-transporting polypeptide 1B3 (OATP1B3) inhibitors rifampicin and cyclosporin. Cell viability was established by trypan blue exclusion, annexin V staining, and a MTS assay. Caspase-3/7 activity was determined with Caspase-Glo assay, and cleaved caspase-3 was quantified by Western analysis. Cell number and colony-forming units were quantified after exposure to α-amanitin in primary CD34+ hematopoietic stem cells. In all cell lines, α-amanitin concentration-dependently decreased viability and mitochondrial activity. β-Amanitin was less toxic, but still significantly reduced viability. α-Amanitin increased caspase-3/7 activity by 2.8-fold and cleaved caspase-3 by 2.3-fold. Z-VAD(OH)-FMK significantly reduced α-amanitin-induced toxicity. In CD34+ stem cells, α-amanitin decreased the number of colonies and cells. The antidotes and OATP1B3 inhibitors did not reverse α-amanitin-induced toxicity. In conclusion, α-amanitin induces apoptosis in hematopoietic cells via a caspase-dependent mechanism.

Abstract: Cysteine is a nonessential amino acid required for protein synthesis, the generation of the antioxidant glutathione, and for synthesizing the nonproteinogenic amino acid taurine. Here, we highlight the broad sensitivity of leukemic stem and progenitor cells to cysteine depletion. By CRISPR/CRISPR-associated protein 9-mediated knockout of cystathionine-γ-lyase, the cystathionine-to-cysteine converting enzyme, and by metabolite supplementation studies upstream of cysteine, we functionally prove that cysteine is not synthesized from methionine in acute myeloid leukemia (AML) cells. Therefore, although perhaps nutritionally nonessential, cysteine must be imported for survival of these specific cell types. Depletion of cyst(e)ine increased reactive oxygen species (ROS) levels, and cell death was induced predominantly as a consequence of glutathione deprivation. nicotinamide adenine dinucleotide phosphate hydrogen oxidase inhibition strongly rescued viability after cysteine depletion, highlighting this as an important source of ROS in AML. ROS-induced cell death was mediated via ferroptosis, and inhibition of glutathione peroxidase 4 (GPX4), which functions in reducing lipid peroxides, was also highly toxic. We therefore propose that GPX4 is likely key in mediating the antioxidant activity of glutathione. In line, inhibition of the ROS scavenger thioredoxin reductase with auranofin also impaired cell viability, whereby we find that oxidative phosphorylation-driven AML subtypes, in particular, are highly dependent on thioredoxin-mediated protection against ferroptosis. Although inhibition of the cystine-glutamine antiporter by sulfasalazine was ineffective as a monotherapy, its combination with L-buthionine-sulfoximine (BSO) further improved AML ferroptosis induction. We propose the combination of either sulfasalazine or antioxidant machinery inhibitors along with ROS inducers such as BSO or chemotherapy for further preclinical testing., (© 2024 by The American Society of Hematology. Licensed under Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International (CC BY-NC-ND 4.0), permitting only noncommercial, nonderivative use with attribution. All other rights reserved.)

In recent years, the therapeutic (re)activation of innate anticancer immunity has gained prominence, with therapeutic blocking of the interaction of Signal Regulatory Protein (SIRP)-α with its ligand CD47 yielding complete responses in refractory and relapsed B cell lymphoma patients. SIRP-α has as crucial inhibitory role on phagocytes, with e.g., its aberrant activation enabling the escape of cancer cells from immune surveillance. SIRP-α belongs to a family of paired receptors comprised of not only immune-inhibitory, but also putative immune-stimulatory receptors. Here, we report that an as yet uninvestigated SIRP family member, SIRP-beta 2 (SIRP-ß2), is strongly expressed under normal physiological conditions in macrophages and granulocytes at protein level. Endogenous expression of SIRP-ß2 on granulocytes correlated with trogocytosis of cancer cells. Further, ectopic expression of SIRP-ß2 stimulated macrophage adhesion, differentiation and cancer cell phagocytosis as well as potentiated macrophage-mediated activation of T cell Receptor-specific T cell activation. SIRP-ß2 recruited the immune activating adaptor protein DAP12 to positively regulate innate immunity, with the charged lysine 202 of SIRP-ß2 being responsible for interaction with DAP12. Mutation of lysine 202 to leucine lead to a complete loss of the increased adhesion and phagocytosis. In conclusion, SIRP-ß2 is a novel positive regulator of innate anticancer immunity and a potential costimulatory target for innate immunotherapy., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Visser, Nelemans, He, Lourens, Corrales, Huls, Wiersma, Schuringa and Bremer.)

High metabolic flexibility is pivotal for the persistence and therapy resistance of acute myeloid leukemia (AML). In 20-30% of AML patients, activating mutations of FLT3, specifically FLT3-ITD, are key therapeutic targets. Here, we investigated the influence of FLT3-ITD on AML metabolism. Nuclear Magnetic Resonance (NMR) profiling showed enhanced reshuffling of pyruvate towards the tricarboxylic acid (TCA) cycle, suggesting an increased activity of the pyruvate dehydrogenase complex (PDC). Consistently, FLT3-ITD-positive cells expressed high levels of PDP1, an activator of the PDC. Combining endogenous tagging of PDP1 with genome-wide CRISPR screens revealed that FLT3-ITD induces PDP1 expression through the RAS signaling axis. PDP1 knockdown resulted in reduced cellular respiration thereby impairing the proliferation of only FLT3-ITD cells. These cells continued to depend on PDP1, even in hypoxic conditions, and unlike FLT3-ITD-negative cells, they exhibited a rapid, PDP1-dependent revival of their respiratory capacity during reoxygenation. Moreover, we show that PDP1 modifies the response to FLT3 inhibition. Upon incubation with the FLT3 tyrosine kinase inhibitor quizartinib (AC220), PDP1 persisted or was upregulated, resulting in a further shift of glucose/pyruvate metabolism towards the TCA cycle. Overexpression of PDP1 enhanced, while PDP1 depletion diminished AC220 resistance in cell lines and peripheral blasts from an AC220-resistant AML patient in vivo. In conclusion, FLT3-ITD assures the expression of PDP1, a pivotal metabolic regulator that enhances oxidative glucose metabolism and drug resistance. Hence, PDP1 emerges as a potentially targetable vulnerability in the management of AML., (© 2023. The Author(s).)

Phosphatidylinositol-5-phosphate 4-kinase type 2 (PIP4K2) protein family members (PIP4K2A, PIP4K2B, and PIP4K2C) participate in the generation of PIP 4 , 5 P 2 , which acts as a secondary messenger in signal transduction, a substrate for metabolic processes, and has structural functions. In patients with acute myeloid leukemia (AML), high PIP4K2A and PIP4K2C levels are independent markers of a worse prognosis. Recently, our research group reported that THZ-P1-2 (PIP4K2 pan-inhibitor) exhibits anti-leukemic activity by disrupting mitochondrial homeostasis and autophagy in AML models. In the present study, we characterized the expression of PIP4K2 in the myeloid compartment of hematopoietic cells, as well as in AML cell lines and clinical samples with different genetic abnormalities. In ex vivo assays, PIP4K2 expression levels were related to sensitivity and resistance to several antileukemia drugs and highlighted the association between high PIP4K2A levels and resistance to venetoclax. The combination of THZ-P1-2 and venetoclax showed potentiating effects in reducing viability and inducing apoptosis in AML cells. A combined treatment differentially modulated multiple genes, including TAp73 , BCL2 , MCL1 , and BCL2A1 . In summary, our study identified the correlation between the expression of PIP4K2 and the response to antineoplastic agents in ex vivo assays in AML and exposed vulnerabilities that may be exploited in combined therapies, which could result in better therapeutic responses.

Competing Interests: The authors have no conflicts of interest to disclose.

Dependency on mitochondrial oxidative phosphorylation (OxPhos) is a potential weakness for leukemic stem cells (LSC) that can be exploited for therapeutic purposes. Fatty acid oxidation (FAO) is a crucial OxPhos-fueling catabolic pathway for some acute myeloid leukemia (AML) cells, particularly chemotherapy-resistant AML cells. Here, we identified cold sensitivity at 4°C (cold killing challenge; CKC4), commonly used for sample storage, as a novel vulnerability that selectively kills AML LSCs with active FAO-supported OxPhos while sparing normal hematopoietic stem cells. Cell death of OxPhos-positive leukemic cells was induced by membrane permeabilization at 4°C; by sharp contrast, leukemic cells relying on glycolysis were resistant. Forcing glycolytic cells to activate OxPhos metabolism sensitized them to CKC4. Lipidomic and proteomic analyses showed that OxPhos shapes the composition of the plasma membrane and introduces variation of 22 lipid subfamilies between cold-sensitive and cold-resistant cells. Together, these findings indicate that steady-state energy metabolism at body temperature predetermines the sensitivity of AML LSCs to cold temperature, suggesting that cold sensitivity could be a potential OxPhos biomarker. These results could have important implications for designing experiments for AML research to avoid cell storage at 4°C., Significance: Mitochondrial metabolism fueled by FAO alters the membrane composition and introduces membrane fragility upon cold exposure in OxPhos-driven AML and in LSCs. See related commentary by Jones, p. 2441., (©2023 The Authors; Published by the American Association for Cancer Research.)

Knowledge about evolution of clonal hematopoiesis, which may drive malignant progression, is crucial for clinical decision-making. We investigated the landscape of clonal evolution by error-corrected sequencing on 7,045 sequential samples from 3,359 individuals in the prospective population-based Lifelines cohort, with a special focus on cytosis and cytopenia. Spliceosome (SRSF2/U2AF1/SF3B1) and JAK2 mutated clones show highest growth rates over a median 3.6-year period, while clone sizes for DNMT3A and TP53 increase only marginally, independent of cytosis or cytopenia. Nevertheless, large differences are observed between individuals carrying the same mutation, indicative of modulation by non-mutation-related factors. Clonal expansion is not dependent on classical cancer risk factors (e.g., smoking). Risk for incident myeloid malignancy diagnosis is highest for JAK2, spliceosome, or TP53 mutations and absent for DNMT3A, and it is mostly preceded by cytosis or cytopenia. The results provide important insight into high-risk evolutionary patterns to guide monitoring of "CHIP" and "CCUS.", Competing Interests: Declaration of interests The authors declare to have no competing interests., (Copyright © 2023 Elsevier Inc. All rights reserved.)

It is increasingly becoming clear that cancers are a symbiosis of diverse cell types and tumor clones. Combined single-cell RNA sequencing, flow cytometry, and immunohistochemistry studies of the innate immune compartment in the bone marrow of patients with acute myeloid leukemia (AML) reveal a shift toward a tumor-supportive M2-polarized macrophage landscape with an altered transcriptional program, with enhanced fatty acid oxidation and NAD + generation. Functionally, these AML-associated macrophages display decreased phagocytic activity and intra-bone marrow coinjection of M2 macrophages together with leukemic blasts strongly enhances in vivo transformation potential. A 2-day in vitro exposure to M2 macrophages results in the accumulation of CALR low leukemic blast cells, which are now protected against phagocytosis. Moreover, M2-exposed "trained" leukemic blasts display increased mitochondrial metabolism, in part mediated via mitochondrial transfer. Our study provides insight into the mechanisms by which the immune landscape contributes to aggressive leukemia development and provides alternatives for targeting strategies aimed at the tumor microenvironment.

Inadequate mobilization of peripheral blood progenitor cells (PBPCs) is a limiting factor to proceeding with autologous hematopoietic cell transplantation (auto-HCT). To assess the impact of clonal hematopoiesis (CH) on mobilization failure of PBPC for auto-HCT, we investigated the characteristics of poor mobilizers (with a total PBPC collection <2 × 106 CD34+ cells per kg) in a consecutive single-center cohort of 776 patients. Targeted error-corrected next-generation sequencing of 28 genes was performed in a nested case-control cohort of 90 poor mobilizers and 89 matched controls. CH was detected in 48 out of 179 patients (27%), with most patients carrying a single mutation. The presence of CH (detected at variant allele frequency [VAF] ≥ 1%) did not associate with poor mobilization potential (31% vs 22% in controls, odds ratio, 1.55; 95% confidence interval, 0.76-3.23; P = .238). PPM1D mutations were detected more often in poor mobilizers (P = .005). In addition, TP53 mutations in this cohort were detected exclusively in patients with poor mobilization potential (P = .06). The incidence of therapy-related myeloid neoplasms (t-MN) was higher among patients with mobilization failure (P = .014). Although poor mobilizers experienced worse overall survival (P = .019), this was not affected by the presence of CH. We conclude that CH at low VAF (1%-10%) is common at the time of stem cell mobilization. TP53 mutations and PPM1D mutations are associated with poor mobilization potential and their role in subsequent development of t-MN in these individuals should be established., (© 2023 by The American Society of Hematology. Licensed under Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International (CC BY-NC-ND 4.0), permitting only noncommercial, nonderivative use with attribution. All other rights reserved.)

Previous studies have reported an association between ABO type blood group and cardiovascular (CV) events and outcomes. The precise mechanisms underpinning this striking observation remain unknown, although differences in von Willebrand factor (VWF) plasma levels have been proposed as an explanation. Recently, galectin-3 was identified as an endogenous ligand of VWF and red blood cells (RBCs) and, therefore, we aimed to explore the role of galectin-3 in different blood groups. Two in vitro assays were used to assess the binding capacity of galectin-3 to RBCs and VWF in different blood groups. Additionally, plasma levels of galectin-3 were measured in different blood groups in the Ludwigshafen Risk and Cardiovascular Health (LURIC) study (2571 patients hospitalized for coronary angiography) and validated in a community-based cohort of the Prevention of Renal and Vascular End-stage Disease (PREVEND) study (3552 participants). To determine the prognostic value of galectin-3 in different blood groups, logistic regression and cox regression models were used with all-cause mortality as the primary outcome. First, we demonstrated that galectin-3 has a higher binding capacity for RBCs and VWF in non-O blood groups, compared to blood group O. Additionally, LURIC patients with non-O blood groups had substantially lower plasma levels of galectin-3 (15.0, 14.9, and 14.0 μg/L in blood groups A, B, and AB, respectively, compared to 17.1 μg/L in blood group O, p < 0.0001). Finally, the independent prognostic value of galectin-3 for all-cause mortality showed a non-significant trend towards higher mortality in non-O blood groups. Although plasma galectin-3 levels are lower in non-O blood groups, the prognostic value of galectin-3 is also present in subjects with a non-O blood group. We conclude that physical interaction between galectin-3 and blood group epitopes may modulate galectin-3, which may affect its performance as a biomarker and its biological activity.