Mature Neutrophils Research Articles

Type 1 diabetes mellitus: Roles of neutrophils in the pathogenesis.

Circulating neutrophil counts are reduced both in healthy autoantibody-positive individuals and in patients with type 1 diabetes, which may be related on cell-specific autoimmunity. This paper was written to give an update on roles of neutrophils in the pathogenesis of type 1 diabetes mellitus. Different research search engines like PubMed Central, Scopus, Web of Science, Researchgate, Google Scholar etc were utilised for writing this paper. A drop in blood neutrophil counts in type 1 diabetes may be caused by decreased neutrophil generation and maturation, tissue maintenance, consumption, or peripheral damage. Neutrophil count variations between studies may be explained by results from various stages of diabetes or by ethnic groups. Neutrophils can induce type 1 diabetes by colonizing pancreatic islets and interacting with other immune cells, according to exciting findings that shed new light on their role in the pathogenesis of the disease. Knowing more about the function of neutrophils in the pathogenesis of type 1 diabetes will help in early diagnosis, treatment, and even prevention of the disease.

The methylome of buccal epithelial cells is influenced by age, sex, and physiological properties.

Epigenetic modifications, particularly DNA methylation, have emerged as regulators of gene expression and are implicated in various biological processes and disease states. Understanding the factors influencing the epigenome is essential for unraveling its complexity. In this study, we aimed to identify how the methylome of buccal epithelial cells, a noninvasive and easily accessible tissue, is associated with demographic and health-related variables commonly used in clinical settings, such as age, sex, blood immune composition, hemoglobin levels, and others. We developed a model to assess the association of multiple factors with the human methylome and identify the genomic loci significantly impacted by each trait. We demonstrated that DNA methylation variation is accurately modeled by several factors. We confirmed the well-known impact of age and sex and unveiled novel clinical factors associated with DNA methylation, such as blood neutrophils, hemoglobin, red blood cell distribution width, high-density lipoprotein cholesterol, and urea. Genomic regions significantly associated with these traits were enriched in relevant transcription factors, drugs, and diseases. Among our findings, we showed that neutrophil-impacted loci were involved in neutrophil functionality and maturation. Similarly, hemoglobin-influenced sites were associated with several diseases, including aplastic anemia, and the genomic loci affected by urea were related to congenital anomalies of the kidney and urinary tract. Our findings contribute to a better understanding of the human methylome plasticity and provide insights into novel factors shaping DNA methylation patterns, highlighting their potential clinical implications as biomarkers and the importance of considering these physiological traits in future medical epigenomic investigations.NEW & NOTEWORTHY We have developed a quantitative model to assess how the human methylome is associated with several factors and to identify the genomic loci significantly impacted by each trait. We reported novel health-related factors driving DNA methylation patterns and new site-specific regulations that further elucidate methylome dynamics. Our study contributes to a better understanding of the plasticity of the human methylome and unveils novel physiological traits with a potential role in future medical epigenomic investigations.

APOE‐4 dependent dysfunctional neutrophil‐microglia crosstalk accelerates Alzheimer’s disease

AbstractBackgroundAPOE4 is the strongest genetic risk factor for late‐onset Alzheimer’s disease (AD). The role of human APOE variants in AD has been studied extensively in the regulation of brain cells but not in peripheral immunity. APOE is also expressed in neutrophils and controls their activation. Moreover, neutrophils have been shown to play a negative role in AD mice via the induction of neutrophil extracellular traps (NETs)MethodHere we aimed to dissect the impact of APOE4‐neutrophils on AD pathology, using Ly6g‐CRE mice crossed to APOE‐KI(E3 and E4)fl/fl:APP/PS1.ResultWe identified a cell intrinsic role of APOE3 in controlling neutrophil maturation and degranulation, which is exacerbated in APOE4‐KI neutrophils and reduced in APOE4‐KO neutrophils. Moreover, APOE4 neutrophils accumulated in the brains of AD mice and in AD patients, associated with microglia at sites of plaque pathology. Here we show that genetic deletion of APOE4 in neutrophils suppressed their activation and reduced their recruitment to the brains of APP/PS1 mice. RNAseq analysis showed increased proportion of disease associated microglia (DAM/MGnD) in APP/PS1: Ly6g‐CRE:APOE4fl/fl mice, associated with reduced plaque pathology. Furthermore, we show impaired induction of MGnD signature in AD brains of APOE4 carriers. Our findings show that APOE4‐induced inflammatory neutrophils are recruited to the diseased brain and impair MGnD response leading to accelerated AD pathology.ConclusionTaken together, these findings identify a cell‐intrinsic role of APOE4 in the induction of hyperactivated neutrophils recruited to the brain, which may provide new molecular targets to modulate and restore functional microglia in AD.

Absence of Neutropenia in Patient with an Exon 2 Nonsense Mutation in ELANE: Clinical Evidence to Support Gene Therapy Efforts for Severe Congenital Neutropenia

Introduction Severe congenital neutropenia (SCN) is an inherited bone marrow failure condition most often caused by heterozygous mutations in the ELANE gene. While granulocyte colony stimulating factor (G-CSF) is effective in increasing neutrophil counts in the majority of patients, approximately 15% of patients do not respond, necessitating the creation of alternative treatment strategies. Due to its association with significant, life-threatening infections and heightened risk of progression to myelodysplastic syndrome and/or acute myeloid leukemia, SCN has been the focus of recent gene therapy efforts by multiple groups (Nasri et al., 2020; Rao et al., 2021). Pathogenic variants in ELANE most often cause missense mutations or in-frame indels which lead to the production and expression of mutant neutrophil elastase (NE) proteins. Frameshift mutations resulting in out-of-frame stop codons or nonsense mutations are also seen in the third through fifth (final) exons of ELANE due to their ability to circumvent nonsense-mediated decay (NMD) and generate mutant NE. In contrast, frameshift and nonsense mutations in the first and second exons have not been shown to cause neutropenia, most likely because NMD prevents the translation of mutant NE (Rao et al., 2021; Horwitz, 2021). Collectively, these observations suggest that the pathogenicity of ELANE mutations may rely on gain-of-function alterations in mutant NE rather than loss-of-function alterations in normal NE. Because the absence of one copy of ELANE does not appear to be deleterious, one theory posits that therapeutic inactivation of mutant copies of ELANE may have corrective potential (Horwitz et al., 2019; Nasri et al., 2020; Rao et al., 2021). Thus, the focus of several gene editing efforts has been CRISPR/Cas9-mediated knockout in early exons of ELANE in hematopoietic stem and progenitor cells (HSPCs) of patients with ELANE-mutated SCN for allogeneic stem cell transplant (Nasri et al., 2020; Rao et al., 2021). With this study, we sought to understand whether an inherited ELANE mutation analogous to those utilized in gene therapy would cause neutropenia. Case Presentation Here, we present a six-month-old Caucasian male with an ELANE mutation whose clinical findings support this model. The patient was born at full term to a 35-year-old G6P2 mother. The patient's constellation of complex symptoms, including polymicrogyria, corpus callosum dysgenesis, hypotonia, seizures, and feeding difficulties prompted clinical trio whole genome sequencing (WGS) at approximately two months of age. The patient was incidentally found to have a de novo nonsense variant in exon 2 of the ELANE gene (NM_001972.2:c.151G>T, p.[G51*]), predicted to result in protein truncation or NMD. WGS also identified variants of uncertain significance in CHRNB2 and HFE. Importantly, although the ELANE variant was classified as likely pathogenic in the clinical genetics report, the patient has not had clinically significant neutropenia. Absolute neutrophil counts have ranged from 1.27 - 10.95 K cells/μL, with mean and median values of 4.09 and 2.95 K cells/μL, respectively (n=10). The patient has not had a significant infectious history, nor has he required any treatment for hematologic concerns. Discussion The absence of a neutropenia pathology in this patient, at least in his first six months of life, provides evidence in support of gene therapy efforts which seek to ameliorate pathogenic ELANE mutations with gene editing to produce frameshifts in early exons. Most likely, in this patient, NMD of mutant ELANE mRNA prevented its translation, thus allowing for ample production and maturation of neutrophils. A similar pattern was observed in another report which examined patients with whole gene deletions of ELANE (Horwitz et al., 2019), but this is the first report of a nonsense variant analogous to those proposed to be introduced by gene or base editing for gene therapy of SCN. Thus, the present case adds to a growing body of evidence which suggests that ELANE knockout may have therapeutic potential for SCN.

Severe Chronic Idiopathic Neutropenia Managed with Chronic Filgrastim: A Case Report

Myelodysplastic syndrome (MDS) represents a heterogeneous group of hematological disorders characterized by ineffective hematopoiesis and abnormal blood cell production. Chronic neutropenia is one such MDS and is classified into mild (Absolute Neutrophil Count (ANC): 1,000 to 1,500/µL), moderate (ANC: 500 to 1,000/µL), or severe (ANC: below 500/µL), with the latter posing the highest risk of infection-related complications. In an attempt to alleviate neutropenia, patients with MDS are often treated with colony stimulating factors (CSFs), such as filgrastim to promote growth and maturation of neutrophils. The target ANC is 1,500 to 2,000/µL for adequate protection against most infections. However, CSF treatments have the potential to stimulate the transformation of MDS into acute myeloid leukemia (AML) so CSF is often used with caution. Presented below is a case of severe chronic idiopathic neutropenia responsive to chronic filgrastim that has yet to show evidence of transformation. A 79 year-old female with a past medical history of distant tobacco use, hypertension, acute cerebrovascular accident, and Hashimoto's thyroiditis presents to the clinic for routine follow-up of her now stable chronic idiopathic neutropenia being managed with weekly filgrastim injections for three weeks with one week off in between cycles. She had originally presented to medical attention in 2004 complaining of chronic mouth sores and ulcers with leukopenia and a severely decreased ANC of <500/µL. Continued monitoring with more than 3 complete blood counts (CBC) with a differential consistently noted a decreased ANC in the setting of persisting oral mucosal erosion. Leukocytes were not noted to be dysplastic, quantitative IgG, IgA, IgM levels were noted to be within normal limits, anti-nuclear antibodies/ anti-neutrophil antibodies were noted to be negative argued against an etiology of large granular lymphocytic leukemia, immunodeficiency, and/or autoimmune disorders, respectively. A trial of glucocorticoids did not significantly increase her ANC which led to her ultimately receiving a bone marrow biopsy (BMB) in 2008. A mildly hypercellular marrow with trilineage hematopoiesis was found, with maturation of all lines, negative flow cytometry, and myelodysplasia. Comforted by the lack of MDS pathologically, the patient was started on filgrastim with significant improvement. Over the course of years the frequency and duration of filgrastim was optimized to her current regimen of weekly filgrastim injections for 3 weeks followed by one week off which allowed her to maintain an adequate ANC and avoid oral ulcers to this day. Despite the feared complication of kickstarting the transformation of MDS into AML with CSF therapy, patients such as the one mentioned here can benefit greatly from judicious use of CSF when blood work can be monitored for complications. The use of G-CSF in severe chronic neutropenia to raise levels from below 500/µL to the ideal target ANC is 1,500 to 2,000 cells per microliter (µL) for adequate protection is in line with our current understanding that even long-term use of G-CSF does not appear to increase the risk of AML (Dale et al., 2022). G-CSF should continue to be considered in the management of severe chronic neutropenia as the risk for transformation may be insignificant compared to the clinical benefit of its use when properly monitored.

Base Edited HSPCs Are Shielded from Targeted CD33 Therapy but Preserve CD33 Expression

Targeted therapy for AML remains challenging as there is broad overlap between targetable surface proteins on transformed cells and healthy hematopoietic stem and progenitor cells (HSPC). CD33 is expressed in about 90% of patients with AML but also on healthy myeloid cells with strong expression on progenitor cells and only weak expression on mature neutrophils. Given the higher expression on leukemic blasts compared to their healthy counterparts, CD33 is an attractive target in AML. However, targeting CD33 e.g. with the antibody-drug conjugate (ADC) gemtuzumab ozogamicin often leads to prolonged cytopenia due to suppression of normal myelopoiesis. Transplantation of genetically engineered CD33 knock-out allogenic HSPCs protects reconstituting hematopoietic cells from targeted immunotherapy administered for disease control after transplantation. Early clinical trials explore this option but the long-term effect of loss-of-function of CD33 remains unknown. We recently demonstrated that single amino acid substitutions engineered into CD45, CD117 or CD123 protect HSPCs from targeted therapies against these antigens while preserving their function. Here, we describe the identification of CD33 shielding amino acid substitutions that can be engineered by base editing and preserve CD33 expression on HSPCs. We identified binding sites of two CD33 antibodies by alanine scanning of the entire extracellular domain. The most favorable amino acid substitutions at key residues were analyzed computationally and binding reduction was subsequently validated by flow cytometry. 23 individual amino acid substitutions at 3 positions were tested. Eleven retained similar expression, purity and biophysical properties as wildtype CD33. Those variants are shielded from antibody 1 even at high concentrations when assessed by biolayer interferometry. In addition, three variants are shielded from antibody 2 binding. We used the ABE8e_SpRY base editor to engineer HSPCs and tiled 21 sgRNAs centered around the intended edit. Five days after electroporation, HSPCs were evaluated for editing efficiency (Sanger sequencing) and binding to antibody 1 (flow cytometry). sgRNA4 showed high editing efficiency of about 80% as well as loss of binding to antibody 1 (Figure A) while binding to the control antibody that binds a different epitope was maintained. Importantly, Sanger sequencing did not show off-target editing of the closely related SIGLEC22P pseudogene. Edited (sgRNA4) and non-edited control (NTC) HSPCs formed comparable numbers of myeloid and erythroid colonies (Figure B). Furthermore, in vitro differentiation showed no difference in frequency of CD14, CD15, CD19, CD33, CD41 and GlyA positive cells indicating that the edited HSPCs did not display major functional alterations. In summary, CD33 variants that can be engineered into HSPCs with high editing efficiency by base editing protected from binding to antibody 1 but preserved CD33 expression. These edited HSPCs could be therapeutically useful, in particular for continued immunotherapy after allogenic HSC transplantation. Similar to HSPCs devoid of CD33 these engineered cells could enable tumor-selective immunotherapy using ADCs or CAR T cells but with preserved CD33 expression and function. Furthermore, since base editing is suitable for multiplexing, they may in the future be multiplexed to protect CD33 and other targets for combination immunotherapy.

In Vivo CRISPR Screening Platform for Studying Myeloid Inflammation and Clonal Dynamics

Previous experimental approaches using CRISPR to study factors governing hematopoiesis in vivo have been hampered by heterogeneity in cellular expansion during hematopoietic reconstitution. To control for such ‘cellular jackpotting’, we developed an in vivo hematopoietic stem and progenitor (HSPC)-based CRISPR screening system with unique molecular identifiers (UMIs) and applied these techniques to study myeloid inflammation and clonal expansion of Tet2-mutated HSPCs. Transplantation of Cas9-expressing Lin -Sca1 +c-Kit + (LSK) cells transduced with high-diversity UMI-labeled guide-RNAs (gRNAs) enabled sufficient gRNA representation at a genome-wide level after lentiviral integration of one gRNA per cell and controlled for 10 log-fold differences in cellular expansion during hematopoietic reconstitution. Simultaneous comparison of barcoded CRISPR screening with non-barcoded screening revealed that barcoding decreases false-positive biological processes related to cell division while improving sensitivity for detecting biological processes related to transcriptional events and cellular differentiation. To study inflammatory mediators, we applied this CRISPR screening tool towards studying the recruitment of CD101 +Ly6G +CD11b + mature neutrophils during an inflammatory insult, which revealed the majority of genes involved in human congenital neutropenia (Figure A). To demonstrate the reproducibility of this screening system, we performed a targeted transcription factor (TF)-based CRISPR screen, which revealed concordance between the genome-wide screen and the targeted screen. Next, we also applied this screening system to studying dynamics of Tet2-mutated clonal hematopoiesis. To study master regulators of clonal expansion in Tet2 KO, we performed a UMI-based CRISPR screen using chromatic and transcription factors, which revealed Tet3 and Bcor, genes previously identified to cooperate with Tet2 KO to promote myeloproliferation (Figure B). Validation of additional novel genetic hits is currently ongoing. We hypothesize that this screening platform will be useful for dissecting many aspects of hematopoiesis including inflammatory mediators and clonal dynamics of HSPCs.

A Multiomic Single-Cell Atlas of Human Myelopoiesis Reveals Cellular and Molecular Drivers of Immunomodulatory Drug-Induced Neutropenia

Neutrophils are crucial innate immune cells which protect the host by killing infectious pathogens. Single cell RNA-sequencing analyses have previously described transcriptional heterogeneity of neutrophils and precursors, however the genome regulatory events underlying the transcriptional changes are less well characterised. Understanding this is important as neutropenia can be caused by dysregulation of the genome regulatory events that orchestrate neutrophil maturation. A good example is the use of immunomodulatory drugs (IMiDs, lenalidomide [LEN] & pomalidomide [POM]) for the treatment of multiple myeloma (MM) which are now standard-of-care, but their use is frequently complicated by neutropenia because of neutrophil maturation impairment. This is due to Cereblon (CRBN)-driven substrate ubiquitination and Ikaros degradation, causing PU.1 downregulation. However, restoration of Ikaros levels cannot fully alleviate the IMiD-induced myeloid differentiation block suggesting other mechanisms might also contribute. Our aim was to build a multiomic atlas of human myelopoiesis and use this to interrogate the molecular drivers of IMiD-induced neutropenia. We developed an ex vivo myeloid cell differentiation assay using human mobilized peripheral blood CD34 + cells from healthy donors and optimised methods for single cell multiomic (combined RNA-seq & ATAC-seq) analysis. This platform was validated orthogonally (immunophenotype, morphology & functional) and employed to construct a multi-omic single-cell landscape of normal human neutrophil differentiation and it's perturbation by IMiDs. Single cell transcriptomes from >110,000 cells from untreated and IMiD-treated samples were analyzed, capturing the full spectrum of human myelopoiesis spanning from early myeloid progenitors through to neutrophil populations (band, segmented, mature) (Fig.1A-left). Ex vivo differentiated neutrophils retained transcriptional signatures reflective of effector functions (granule biogenesis, chemotactic activity, respiratory burst) and mapped to previously described human neutrophil populations in vivo. Ex vivo generated neutrophils showed expected functional properties of phagocytosis and neutrophil extracellular trap formation upon stimulation. Exposure to IMiDs caused a maturation arrest with a 40-50% (p<0.001) decrease in abundance of differentiating metamyelocytes, and neutrophil populations. Crucially, this maturation arrest was due to an accumulation of transcriptionally distinct myeloid precursors that differentiated though an aberrant pathway (population 6, Fig 1A). Furthermore, immature erythroid clusters representing 10% of live cells accumulated specifically in IMiD-treated samples (p<0.001) in absence of erythropoiesis-stimulating cytokines. Simultaneous profiling of gene expression and chromatin accessibility in individual cells confirmed the presence of distinct phenotypes for untreated and IMiD-treated myeloid cells (Fig.1A) enabling the characterisation of the genome landscape of myelopoiesis, which has previously proven challenging. As expected, we observed dysregulation of Ikaros targets including PU.1 upon IMiD treatment (Fig. 1B-frame). The ability to study distinct and aberrant cellular populations at transcriptional and genome regulation level in parallel allowed us to identify several novel regulators of normal and perturbed neutrophil development. For example, we observed perturbed activation patterns of the transcription factors (TFs) zinc finger protein (ZNF467) and interferon regulatory factor 5 (IRF5) and the expression of their downstream targets as illustrated in Fig.1B. ZNF467 orchestrates a transcriptional program supporting terminal neutrophil differentiation in untreated cells (Fig.1B-top right), whilst the regulatory repertoire of IRF5, a hallmark of neutrophil activation is confined primarily to treatment-specific immature populations (Fig.1B-bottom right). We present a multiomic atlas of normal myelopoiesis which we believe will provide an important resource for the study or normal and perturbed neutrophil development. We use this platform to study IMiD-induced neutropenia and identify two aberrant cellular progenitor populations (aberrant myeloid precursors & erythroid progenitors) with distinct molecular properties as the likely cause of myelosuppression.

Granulocyte Colony-Stimulating Factor Use in Patients with Hemophagocytic Lymphohistiocytosis: A Single Center Experience

Background: Hemophagocytic lymphohistiocytosis (HLH) is a rare, life-threatening syndrome caused by activation of T cells that results in extreme inflammation. HLH is commonly associated with underlying conditions such as infection, autoimmune disease, or malignancy. HLH management includes treatment directed at the specific trigger, suppression of maladaptive cytokines, and inhibition of T cell and macrophage proliferation. Granulocyte colony-stimulating factor (GCSF) is a myeloid growth factor that stimulates the production, maturation, and activation of neutrophils. GCSF may theoretically promote production of inflammatory cytokines and enhance hemophagocytosis, thereby intensifying HLH. By contrast, GCSF could play a vital role in the treatment of granulocytopenia caused by HLH and thus limit the development of infections. There is a paucity of data examining the role of GCSF in patients with HLH. Current literature is limited to case reports of HLH exacerbated by GCSF administration. Practice patterns of GCSF use in HLH are highly variable, both within and between institutions. Aim: To compare the clinical course and overall survival of patients with HLH who received GCSF treatment and those who did not. Methods: We performed a retrospective, single-center cohort study of patients admitted with a diagnosis code of HLH from July 1997 through February 2023. Patients at least 18 years old with active HLH were included. Each case was reviewed to confirm the diagnosis of HLH based on available diagnostic evaluation and the final discharge diagnoses as documented by the primary treating team. In cases indeterminate for HLH, a second independent reviewer performed additional evaluation to reach consensus. The primary outcome was overall survival as determined by the Kaplan Meier method with statistical comparison using the log-rank test. Other examined outcomes included: incidence of infection, length of hospital stay (LOS), incidence of intensive care unit (ICU) admissions, length of ICU stay, incidence of 30-day re-admissions due to HLH, and time to first change in HLH-directed therapy. Results: Database analysis revealed 130 patients with a diagnosis code of HLH, of which 68 patients met inclusion criteria. The study cohort comprised 26 patients that received GCSF treatment and 42 patients that did not. The median follow-up time was 3.1 months (interquartile range [IQR] 0.6-29.4). The most common HLH triggers were infection (30.8% GCSF group vs 45.2% control, p=0.31) and malignancy (65.5% GCSF group vs 19% control, p<0.001). A median of 10 doses (IQR 6-16) of GCSF were administered per patient in the GCSF group, and the median time between diagnosis and the first administered dose was 6.5 days (IQR 2.25-18.75). Median overall survival in the GCSF group was 122.6 months compared to 1.5 months in the control group (p=0.02, Figure 1). There was a higher but statistically insignificant difference in infection rate in the GCSF arm (92.3%) compared to the control arm (71.4%, p=0.06). The median LOS was higher in the GCSF group (38 days vs 15 days, p<0.001). Rates of ICU admission (53.4% vs 62%, p=0.69), intubation and mechanical ventilation (34.6% vs 33.3%, p=1.0), and initiation of renal replacement therapy (23.1% vs 16.7%, p=0.74) were similar across the GCSF and control groups, respectively. Two patients in each group were re-admitted for HLH within 30 days of initial discharge. The median time to change in HLH-directed therapy was not reached in either group, and there was no significant difference in incidence of therapy change between groups at 1, 3, or 6 months (Figure 2). Conclusions: In this single center, retrospective cohort study of patients with HLH, treatment with GCSF was not associated with inferior overall survival. While over-representation of a malignancy-associated trigger in the GCSF group may confound interpretation of this result, it nevertheless reflects an important clinical finding that contradicts previously published case report-level evidence. This study highlights the need for further prospective investigation of GCSF administration in HLH.

Isocitrate Dehydrogenase 2 Mutation Allows Myeloid Differentiation but Impairs Bone Marrow Macrophage Polarization and Function Via Metabolic Dysregulation

Isocitrate dehydrogenase 2 (IDH2) mutations, while less common than other mutations associated with myelodysplastic syndromes (MDS), are also found in clonal hematopoiesis and are associated with increased risk of transformation to leukemia. IDH2 mutations lead to metabolic dysfunction and DNA hypermethylation due to the accumulation of D-2-hydroxyglutarate (2HG), an oncometabolite derived from alpha-ketoglutarate (aKG), which disrupts hematopoiesis. However, the mechanisms that induce clonal progression and transformation remain incompletely understood. Furthermore, the intrinsic and extrinsic impact of IDH mutations on mature myeloid cell functions and the bone marrow microenvironment remains to be elucidated. Previous literature suggested that hypermethylation via this mutant leads to incomplete differentiation and failed mature myeloid cell generation. Our lab previously demonstrated that efferocytic defects in bone marrow macrophages can skew hematopoietic stem and progenitor cells via increased inflammatory mediators. Based on these data, we hypothesized that IDH2 mutations prevent complete polarization down pro-efferocytic phenotypes in macrophages due to blocking of metabolic pathways required for mature macrophage function. Congenic CD45.1+ mice were transplanted with retrovirally infected hematopoietic stem cells containing human WT or R172K IDH2 mutant with an mCherry reporter and showed macrocytic anemia, thrombocytopenia, and elevated reticulocytes by 16 weeks post-transplant. In contrast to prior literature, IDH2 mutants gave rise to mature reporter positive monocytes, macrophages, and neutrophils in the bone marrow and peripheral blood. As expected, we observed myeloid skewed hematopoiesis, a trademark of MDS. When cultured ex vivo, there was loss of efferocytic capacity in IDH2 mutant bone marrow macrophages when fed apoptotic neutrophils. We then generated a genetic murine model using the Vav1 Cre IDH2 R140Q floxed mutant. The genetic model allowed us to study disease progression in the absence of radiation induced stress, and to observe mature myeloid populations while clinically defined MDS was absent. We performed ATACseq and bulk RNAseq analysis on bone marrow macrophages isolated from 3-month-old and 6-month-old mice, which demonstrated a significant decrease in global chromosomal accessibility; however, this had limited impact on the transcriptional program. Based on existing literature, the majority of macrophages in the bone marrow are in a poised state and have not yet activated genes associated with pro-efferocytic/pro-resolution or pro-inflammatory phenotypes. Since aKG is accumulated in macrophages during polarization toward reparative and pro-efferocytic phenotypes and is necessary for metabolic reprogramming for oxidative phosphorylation, we hypothesized that, in the presence of IDH2 mutations, which drain aKG from the system, pro-efferocytic polarized macrophages would be metabolically stunned. To test this theory, we isolated bone marrow macrophages from IDH2 mutants and cultured them to generate pro-efferocytic polarized macrophages. We then tested their efferocytic potential and metabolic capacity by seahorse assay. Continual efferocytosis, a hallmark of pro-efferocytic macrophages, was impaired in IDH2 mutant macrophages as early as 2-3 months of age. Consistent with this finding, mito stress testing by seahorse demonstrated that IDH2 mutant macrophages had severely impaired maximal respiratory capacity compared to WT macrophages. Treatment with global hypomethylating agent azacytidine or a cell permeable aKG analog were unable to restore respiratory capacity or efferocytic activity in the IDH2 mutant. These data strongly suggest that the efferocytic defect in IDH2 mutant macrophages is due to metabolic rather than epigenetic reprogramming. In summary, we identify defects in efferocytic function and oxidative phosphorylation in IDH2 mutant macrophages. This represents a novel mechanism that could alter the bone marrow microenvironment and impact progression of MDS and leukemic transformation. Furthermore, our data suggest that the metabolic dysregulation induced by IDH mutations may play a greater role than epigenetic impairment in IDH2 mutant mature myeloid cells, which could explain why approaches targeting hypermethylation have shown limited efficacy in clinical settings.

309. Optimized hematopoietic stem cell source for the ex vivo production of mature human neutrophils as a potential cell-based therapy for neutropenia

Abstract Background Neutrophils or granulocytes are the most common innate immune cell and are critical for eliminating invasive pathogens. Patients with low neutrophil counts face an increased risk of fatal opportunistic infections. Allogeneic granulocyte transfusions offer a solution to supplement endogenous neutrophil counts but are challenging due to insufficient preservation methods and donation feasibility. Here, we compare the capacity of three different hematopoietic stem cell (HSC) sources; 1) cord blood (CB), 2) live adult bone marrow (LABM), and 3) cadaveric adult bone marrow (CABM), to expand and generate an ex vivo neutrophil source. Methods HSCs were expanded ex vivo with or without UM729 then differentiated and evaluated for neutrophil yield and purity. Ex vivo differentiated neutrophils were characterized by flow cytometry for activated phenotype surface markers. Finally, the functional capacity of ex vivo neutrophils, including phagocytosis, ectodomain shedding, and reactive oxygen species (ROS) production, was measured in response to the fungal pathogen Candida albicans. Results CB-derived HSCs were found to be superior in expansion capacity compared to LABM- or CABM-derived HSCs by 3-fold at day six. The use of UM729 reduced the purity of neutrophil differentiation by 44% for both live and deceased BM marrow-derived HSCs. UM729 had no observable effect on the purity of neutrophil differentiation for CB-derived HSCs. Additionally, due to enhanced Fc gamma receptor I expression, ex vivo neutrophils from all sources were ten times more sensitive to recognizing IgG-opsonized C. albicans than primary neutrophils. Ex vivo neutrophils were capable of phagocytosis, ROS production, and ectodomain shedding to a similar capacity as primary donor neutrophils. Conclusion We find that cadaveric bone marrow-derived HSCs are a viable source for HSC-generated neutrophils. That said, CB-derived HSC were superior in expansion capacity. We also highlight the unique opsonin-activating features of ex vivo generated neutrophils which warrant further study in granulocyte transfusion animal models of systemic infection. These data underscore the significance of neutrophil cellular therapeutics as a novel modality for preventing infection in high-risk neutropenic patients. Disclosures Nathan E. Jeffries, BA, Safi Biotherapeutics: Salary Michael Mansour, MD, PhD, Thermofisher: Grant/Research Support

Abstract 167: Neutrophil Maturity Following Resuscitation From Cardiac Arrest

Introduction: Immature neutrophils mobilized from bone marrow in response to injury do not express CD10 and are characterized as pro-inflammatory. Maturity status of neutrophils has not yet been described in patients after the global ischemic-reperfusion injury following cardiac arrest (CA). Hypothesis: Immature neutrophils predominate the systemic cellular response after CA and are associated with mortality. Goal: To characterize neutrophils and their associations with clinical parameters in patients following resuscitation from CA. Methods: After informed consent, 100 subjects were enrolled. Blood samples were collected at 6, 12, 24, 48, 72, and 168 hours after return of spontaneous circulation (ROSC). White blood cells (WBC) were isolated and stained for flow cytometry. Data analysis was performed with FlowJo and GraphPad Prism; a P-value of <0.05 was deemed statistically significant. Results: In our cohort of 51 non-survivors and 49 survivors, there were no significant differences in WBC count at any time point. At 72 hours after ROSC, we found a higher absolute number (6.8 vs. 5.0; p=0.018) and percentage ( 87.1 vs. 80.3; p=0.030) of neutrophils in the WBC population in non-survivors compared to survivors. Analysis of neutrophil CD10 subpopulations revealed a higher number of immature CD10 - neutrophils in non-survivors at 72 hours after ROSC (6.1 vs. 4.4; p=0.007) compared to survivors. There was no significant difference between non-survivors and survivors in mature CD10 + neutrophil count or temporal changes. The CD10 - /CD10 + neutrophil ratio was significantly higher in non-survivors at 24 hours (4.7 vs. 3.1; p=0.036) and 48 hours (11.5 vs. 6.6; p=0.040) after ROSC. Conclusions: Neutrophils are important to inflammation resolution after injury; however certain subpopulations of immune cells may be involved in and perpetuate tissue damage after CA. The levels and temporal changes of pro-inflammatory, immature (CD10 - ) neutrophils in non-survivors point to the need for further research including functional studies. The identification of neutrophil marker targets that may impact the balance of pro- and anti-inflammatory immune cells and their related signaling pathways could offer opportunities to improve outcomes after cardiac arrest.

Abstract 428: Clinical and Molecular Characterization of Post-Resuscitation Phenotypes: Cardiac vs. Neurorespiratory Etiology

Background: Establishment of clinical and molecular phenotypes is needed to individualize post-resuscitation cardiac arrest (CA) care. Leukocyte subpopulations may characterize ongoing organ injury, and strongly correlate with outcomes. We analyzed patients determined to have a cardiac (COR) vs. neurorespiratory (NR) etiology of CA. Hypothesis: Clinical and molecular phenotypes differ according to the etiology of CA. Goals: Prospectively characterize clinical and molecular phenotypes of patients with cardiac or neurorespiratory etiology of CA. Methods: After IRB approval and informed consent, we collected blood samples at 24, 48, and 72 hours after return of spontaneous circulation. We excluded patients <18 years of age, moribund, Hgb <7mg/dL, or whose CA etiology could not be determined. Flow cytometry was performed on fresh blood; outcomes were established by 6-month telephone call. Results: Among patients with COR (n=61) vs. NR (n=42) etiology of CA, COR patients were older (61±14 vs 54 ±14, P=0.021), more often had shockable rhythm (74% vs. 7.1%, P<0.001), had higher initial blood glucose (227±89 vs 195±95, P=0.046), higher incidence of moderate or severe left ventricular dysfunction on presentation (67% vs 33.5%, P=0.004), lower EEG suppression ratio at 6 hours [22 (4,84) vs. 88 (66,93), P<0.001), lower NSE levels [30 (19,140) vs 104 (33,268), P=0.034], higher in-hospital survival (64% vs. 33%, P=0.002), less neurological-etiology death (25% vs 55%, P<0.001) and more CPC 1-2 at 6 months (49% vs 12%, P<0.001). There were no differences in total number of leukocytes, but flow cytometry revealed leukocyte subsets, characterized by variable expression of CD4, CD8, CD10, CD14, CD16, CD54, CXCR1 and other surface proteins, indicating a trend toward enhanced neutrophil maturation and a preserved pool of circulating T cells in COR vs. NR patients. Conclusions: Separating CA survivors into a cardiac vs. neurorespiratory etiology reveals different clinical risk profiles and inflammatory responses during the acute phase after resuscitation. Further research should evaluate how specific interventions affect patients with dissimilar risk profiles, and how various therapies modulate the immuno-inflammatory response.

UK-5099, a mitochondrial pyruvate carrier inhibitor, recovers impaired neutrophil maturation caused by AK2 deficiency in human pluripotent stem cell models

Reticular dysgenesis (RD) is a rare genetic disease caused by gene mutations in the ATP:AMP phosphotransferase, adenylate kinase 2 (AK2). Patients with RD suffer from severe combined immunodeficiency with neutrophil maturation arrest. Although hematopoietic stem cell transplantation can be a curative option, it is invasive, and complications of agranulocytosis-induced infection worsen the prognosis. Here, we report that the use of UK-5099, an inhibitor of the mitochondrial pyruvate carrier (MPC), on hemo-angiogenic progenitor cells (HAPCs) derived from AK2-deficient induced pluripotent stem cells improved neutrophil maturation. Reactive oxygen species (ROS) levels in AK2-deficient HAPCs remained unchanged throughout all experiments, implying that UK-5099 improved the phenotype without affecting ROS levels. Overall, our results suggest that the MPC is a potential therapeutic target for the treatment of neutrophil maturation defects in RD.

CRISPR/Cas9 Strategy for Correcting ELANE Mutations and Restoring Neutrophil Differentiation in Severe Congenital Neutropenia: Insights from Patient-Derived iPSCs

Introduction The ELANE gene, encoding neutrophil elastase (NE), has been identified as the primary causative gene for more than half of patients with severe congenital neutropenia (SCN). For patients refractory to standard therapy (G-CSF), the only available option is an allogeneic hematopoietic stem cell transplant. Therefore, there is a great clinical interest in the development of novel gene therapies, including CRISPR/Cas9, which has shown promise for the correction of ELANE mutations and the restoration of neutrophil function. Here, we introduce a gene editing strategy using CRISPR/Cas9 to repair ELANE gene mutations in patient-derived induced pluripotent stem cells (iPSCs), aiming to restore proper neutrophil development and function. Methods We utilized a targeted NGS panel composed of 725 genes related to hematological disorders to sequence 282 pediatric patients suffering from chronic neutropenia. Patient-derived epithelial cells collected from urine were reprogrammed into iPSCs using the EBV-derived oriP/EBNA-1 system carrying transcription factors. CRISPR-Cas9 technology was employed to repair mutations in patient-derived iPSCs. The efficiency of transfection and gene editing was subsequently assessed by analyzing the targeted region by Sanger sequencing. Control iPSCs (healthy donors) and iPSCs from patients were differentiated into neutrophils with specific cytokine cocktail (BMP4, CHIR99021, VEGF, SCF, bFGF, SB431542, SCF, IL-3, TPO, Flt-3, G-CSF). Cells were harvested at day 25 and analyzed for surface markers resembling the distinct myeloid progenitors. Flow cytometry antibody panel (CD14, CD45, CD49d, Siglec-8, CD101, CD11b, CD35, CD16) and gating strategy allowed to identify myeloid populations such as proNeu1, preNeu, immature-Neu and mature Neu representing myeloblasts, promyelocytes, myelocytes/metamyelocytes and band/segmented neutrophils, respectively. The morphology of myeloid precursors and mature neutrophils as well as the localization of neutrophil elastase in control iPSCs were evaluated by confocal microscopy. Results From a sequenced cohort of 282 patients, causative mutations were identified in 49 individuals, including 17 with ELANE mutations, and 9 novel variants were discovered. For this study, we selected two patients exhibiting different phenotypes for further investigation. One patient carries the NM_001972.3:c.[163T>C];[=], NP_001963:p.[(Cys55Arg)];[(=)] ELANE mutation and presents severe neutropenia (ANC=100/µL), currently undergoing G-CSF treatment. The second patient carries the NM_001972.3:c.[597+5G>A];[=], NP_001963:p.[(Val190_Phe199del)];[(=)] ELANE mutation which demonstrates in the fluctuating blood neutrophil counts characteristic for cyclic neutropenia. iPSCs were generated from both patients. Alkaline phosphatase and immunocytochemical staining confirmed that the reprogrammed cells express pluripotent stem cell markers and can differentiate into the three germ layers. Genetic defect repair was observed in 13.3% of tested cell colonies from the patient with the c.163T>C Cys55Arg ELANE mutation and in 30% of sequenced colonies from the patient with the c.597+5G>A V190_F199del ELANE mutation. Molecular cytogenetics (SNP-array) showed no significant genomic rearrangements or off-targets in iPSCs selected for differentiation. iPSC cells from healthy donors, derived from c.597+5G>A V190_F199del ELANE patient and the repaired clone were successfully differentiated into neutrophils (CD16+ CD35+) and their myeloid precursors (CD49d+ CD11b+). The percentage of immature and mature neutrophils (CD16+ and CD35+) derived from control or corrected iPSC was significantly higher compared to patient-derived neutrophils (Figure 1). Conclusions Our study demonstrates the successful use of CRISPR/Cas9 to repair ELANE gene mutations in patient-derived iPSCs, aiming to restore proper neutrophil development. Patient-derived iPSCs exhibit impaired differentiation at the myelocytes/metamyelocytes stage, as evidenced by a notably lower population of immature and mature neutrophils when compared to CRISPR/Cas9 repaired and healthy donor iPSCs. This observation aligns with the bone marrow phenotype observed in patients with SCN. Our findings indicate that iPSCs generated from patients' epithelial cells serve as a valuable cellular model for further research.

Celmod Induced Disruption of Granulopoiesis Continuum Identified Using Humanized Cereblon Mice

Introduction: CRBN mediated degradation of Ikaros by Pomalidomide (Pom) and Mezigdomide (Mezi) results in robust antiproliferative activity against myeloma cells, with concomitant activation of immune cells such as T and NK cells. However, degradation of Ikaros has been shown to mediate maturation arrest of neutrophils leading to neutropenia. Unlike chemotherapy drugs, CELMoD driven neutropenia is an on-target adverse effect. Although the next generation cereblon E3 ligase modulators are potent and highly selective, neutrophil biased adverse effects remain persistent. Here, we sought to define the impact of Ikaros degradation through a longitudinal assessment of granulopoiesis in the bone marrow of humanized CRBN mice treated with Pom and Mezi. Methods: We have performed multi-parametric studies leveraging a humanized CRBN (hCRBN) C57BL/6 mouse model. Driven by the endogenous mouse CRBN promoter, human CRBN was inserted into the mouse CRBN gene, preventing expression of mCRBN. These hCRBN mice are capable of specific degradation of CELMoD mediated substrates, thus enabling an in vivo model to understand pathophysiological mechanisms. Homozygous hCRBN mice were treated daily with Pom and Mezi via oral gavage until days 7, 14 and 21 when mice were euthanized. Peripheral blood and bone marrow were collected for flow cytometric and molecular analyses. Critical population classes such as multipotent progenitors, lineage committed progenitors, pre-myelocytes, immature neutrophils and mature neutrophils were defined by immunophenotypes, and pharmacodynamic parameters such as Aiolos/Ikaros degradation were evaluated. All data is represented as fold change over median of vehicle control. Results: The hCRBN mouse model was able to recapitulate Pom and Mezi driven clinical neutropenia. Peripheral absolute neutrophil count gradually decreased with increased dosing (QD7, QD14 and QD21) in mice treated with Pom (26%, 45% and 74%) and Mezi (35%, 57% and 70%). Treatment with Pom and Mezi resulted in disruption of early hematopoietic multi-progenitor populations leading to a widespread effect at multiple stages of haematopoiesis. Intriguingly, in bone marrow these effects were both temporally differential for multi-potent progenitors, and across various stages of neutrophil maturation. Myeloid biased, MPP3 population increased to ~55% and ~103% at QD7, followed by a steady decline, renormalizing to baseline at QD14 and QD21 for Pom and Mezi respectively. The first myeloid lineage committed progenitors, CMP population, reduced to 21% (Pom) and 42% (Mezi) at QD7 stabilizing at ~17-21% below baseline by QD21. In contrast GMP population, descendants from CMP, increased to ~50% (Pom) and ~117% (Mezi) at QD7. For Pom treatment (but not with Mezi), GMP proportions renormalized to baseline at QD21. Pre-myelocytes, one of the earliest neutrophil maturation stages, shrunk to ~24-30% at QD21 without any differences between Pom and Mezi. On the other hand, immature neutrophils steadily diminished to 51% (Pom) and 70% (Mezi) at QD21. This effect was more pronounced in mice treated with Mezi. Mature neutrophils in bone marrow, part of a highly dynamic reservoir that extravasates into blood, increased to 10-18% at all the time points for both Pom and Mezi. Overall, impairment of granulopoiesis is more profound in hCRBN mice treated with Mezi than Pom, owing to the more potent Ikaros degradation. Mechanistically, degradation of neo-substrates such as Ikaros affect the myelopoiesis and neutrophil maturation but are likely not the sole drivers. Addition of corticosteroids such as dexamethasone have been shown to mitigate IMiD/CELMoD mediated neutropenia in the clinical setting. Mezi treated animals who were co-administered dexamethasone demonstrated partially enhanced early progenitor populations compared to single agent Mezi. To investigate patterns of Ikaros transcriptional control, specific populations were flow sorted in parallel, for focused molecular analyses such as RNAseq using the SMARTseq platform and will be included in the presentation. Conclusion: These studies delineate previously unreported effects of CELMoDs on a multifaceted continuum of myelopoiesis. These insights will help rationally design dose and schedule considerations for CELMoD treatment regimens alone, or in combination with other therapeutic agents to alleviate CELMoD induced neutropenia.

Severe Congenital Neutropenia-Type 5: Impaired T Cell Proliferation, Aberrant Th1 Cytokine Production, Abnormal Megakaryocytes, and Impaired Platelet Granule Formation in a Patient with VPS45 Deficiency Caused By Uniparental Isodisomy

Introduction VPS45 is essential in early endosome formation with rabenosyn-5 and syntaxin 16. VPS45 deficiency is one of the inborn errors of immunity characterized by severe congenital neutropenia and myelofibrosis. Impaired production and function of neutrophils, increased cell death, and abnormal hematopoiesis have been reported, whereas there are several undetermined pathophysiologies, such as T and/or NK lymphopenia or myelofibrosis. Here, we experienced a girl with VPS45 deficiency presenting with neutropenia at birth, progressive T and NK lymphopenia, hepatosplenomegaly, and myelofibrosis in infancy. We examined the pathophysiology of VPS45 deficiency focusing on hematologic disorder, T cell function, and myelofibrosis. Methods We sequentially evaluated peripheral blood and bone marrow cell counts and morphologic features. Genetic analysis was performed using DNA from peripheral blood. We evaluated T cell proliferation using the CFDA-SE cell tracer kit with the stimulation of PHA-L or anti-CD3/CD28 beads. Serum cytokine analysis was performed by beads array. Protein expression was examined in platelets from the patient and the healthy control by western blot analysis. Results Case: The patient was born to nonconsanguineous Japanese parents. She received a blood examination for mild respiratory distress at birth. This examination demonstrated neutropenia, the emergence of myeloid cells, and nucleated red blood cells in the peripheral blood, whereas mature neutrophils were confirmed in the bone marrow. Genetic analysis demonstrated homozygous pathogenic variants of p.E238K in VPS45. The same heterozygous variant was confirmed in her father, but not in her mother. SNP array analysis demonstrated that she inherited chromosome 1 only from her father and developed VPS45 deficiency by paternal uniparental isodisomy. At 7 months, she demonstrated T and NK lymphocytopenia, macrothrombocytopenia, and hepatosplenomegaly. Bone marrow examination demonstrated the presence of blast cells, micro or separated megakaryocytes, and pseudo-Pelger-Huet anomaly. Electronic microscopy of peripheral blood demonstrated abnormal granules in neutrophils and macro-thrombocytes with reduced alpha granules and open channel enlargement. Elevated interferon-gamma, IL-12, and IL-23 levels, representing T helper 1 (Th1) activation were found. We initiated ruxolitinib at 7 months to suppress aberrant Th1 activation and extra-medullar hematopoiesis which could be associated with massive hepatosplenomegaly. Ruxolitinib was effective for hepatosplenomegaly and reduced Th1 cytokine levels without significant complications. She received hematopoietic cell transplantation with bone marrow from an HLA-matched unrelated donor 30 days after the initiation of ruxolitinib. T cell function: With anti-CD3/CD28 beads stimulation, the patient's CD8-positive cells were significantly less proliferative. Either CD4-positive or CD8-positive cells did not proliferate with PHA-L stimulation. Platelet analysis: Western blot analysis of platelet demonstrated a reduced amount of alpha granule proteins (von Willebrand factor and PF4), not of p-selectin. Discussion VPS45 deficiency was characterized by severe congenital neutropenia and myelofibrosis. Furthermore, T cell deficiency, especially CD8-positive T lymphopenia was observed in some patients with VPS45 deficiency. Our study suggested that T cell lymphopenia would be associated with impaired TCR signaling and mitogen response, although the detailed mechanism remains to be elucidated. The impaired platelet alpha granule formation confirmed by electron microscopy and western blot analysis in our patients was compatible with the previous VPS45 deficiency patient. A similar manifestation is observed in gray platelet syndrome which is also associated with affected endosomal transport. Affected endosomal transport would be responsible for myelofibrosis. We hypothesized that massive hepatosplenomegaly was attributable to the hyper-activated Th1 cells and extra-medullar hematopoiesis associated with JAK1 and JAK2 signaling. Indeed, JAK1/ JAK2 inhibitor, ruxolitinib improved hepatosplenomegaly in this patient. Conclusion VPS45 deficiency could broadly affect not only neutropenia and myelofibrosis but T cell, NK cell, megakaryocyte, and platelet function.

Real-World Performance of 2022 Revised World Health Organization (WHO) Criteria for Mixed Phenotype Acute Leukemia (MPAL) B/Myeloid Defined Only By Isolated MPO Expression (isoMPO)

Mixed phenotype acute leukemia (MPAL) is biologically and immunophenotypically heterogeneous, and optimal diagnostic criteria remain unclear. The two current pathologic classifications [2022 World Health Organization (WHO) 5 th Ed. and International Consensus Classification (ICC)] group MPAL by cell lineages and, when present, cytogenetic drivers. In both, myeloperoxidase (MPO) is a key determinant of myeloid lineage. However, the diagnosis of MPAL has been inconsistently applied to cases of acute leukemia expressing MPO alone to define myeloid lineage. To address this, the 2022 WHO revised prior cell lineage definitions to emphasize antigen intensity (≥50%) relative to a positive control population, attempting to standardize lineage assignment across flow cytometer settings and pathologist interpreters. Distinction between pediatric B-lymphoblastic leukemia (B-ALL) and MPAL B/Myeloid with isolated MPO is crucial for therapeutic decision-making. For example, on Children's Oncology Group (COG) regimens, patients with MPAL B/Myeloid receive an anthracycline during induction and intensified chemotherapy post-induction, whereas standard-risk B-ALL patients do not. We hypothesized that the 2022 WHO criteria applied to MPO expression in children with acute leukemia would identify leukemic subtypes (B-ALL vs. MPAL B/Myeloid with isolated MPO) with differences in clinical characteristics and/or outcome. We applied 2022 WHO criteria to flow cytometry data from a multicenter pediatric cohort of leukemias originally classified as MPAL B/Myeloid or B-ALL (Oberley AJCP 2017; Raikar Blood 2018; Oberley Leukemia 2020). Maximal antigen intensity on the leukemic population was measured in 0.5 log increments and normalized to maximal intensity of a control (neutrophil) population. MPO expression was scored as none, low (<50% intensity of mature neutrophils, the typical level in B-ALL with isolated MPO expression) or high (≥50% intensity of neutrophils, meeting 2022 WHO criteria for myeloid lineage assignment). Since the current study focused on MPAL B/Myeloid cases with isolated MPO expression, cases of MPAL T/Myeloid and of MPAL B/Myeloid with myeloid lineage assignment based on monocytic markers were excluded. Clinicopathologic data were compared with MPO expression scores by univariate and multivariate analyses. Primary endpoint was categorical difference in presenting features. Secondary endpoints were end of induction minimal residual disease (EOI MRD) ≥0.01%, event-free survival (EFS) and overall survival (OS). All analyses were two-sided; p<0.05 was the threshold for statistical significance. Among all patients at diagnosis (n=176), median age was 6.14 years (range 0.3-21), 68% were <10 years old, 59% were male, 63% were Hispanic, 76% had initial white blood cell (WBC) count <50K/µL, and 81% were CNS1. MPO was absent, low (<50%) or high (≥50%) in 48%, 29% and 23% of the cohort, respectively. Prevalence of favorable cytogenetics ( ETV6:: RUNX1 or double trisomy) was inversely proportional to MPO intensity [no MPO (50%), low MPO (27%), high MPO (17%); p<0.001). MPO expression (low or high) was more prevalent in patients >10 years of age (p<0.001). There was no difference in MPO by presenting WBC count (p=0.40). Among patients treated with ALL induction with known EOI MRD results (n=166), MPO intensity was not significantly associated with EOI MRD positivity, EFS or OS (multivariate p>0.40 for all three endpoints after accounting for presenting features and initial therapy). Our study demonstrates that 2022 WHO criteria for interpreting MPO intensity can be consistently applied to flow cytometry data from a variety of laboratories. Based on our analysis, only 44% of previous MPAL B/Myeloid cases with isolated MPO were now classified as MPAL. However, the clinical significance of this distinction remains unclear. After accounting for differences in presenting features, MPO expression was not additionally predictive of MRD positivity, EFS or OS. Further investigation is required to support the proposed threshold of 50% MPO intensity of a neutrophil control population as clinically relevant to assign myeloid lineage. Prospective standardized application of 2022 WHO cell lineage assignment criteria across centers will facilitate detailed understanding of the biological underpinnings and optimal therapeutic strategies for this heterogeneous disease.

Loss of EFL1 Which Causes a Shwachman-Diamond Syndrome Reduces Cell Proliferation and Alters Transcriptional Profiling during Neutrophil Differentiation

Introduction. Shwachman-Diamond syndrome (SDS) is an inherited bone marrow failure syndrome characterized by neutropenia, exocrine pancreatic insufficiency, and skeletal abnormalities. Patients with SDS are predisposed to develop myeloid malignancy. Almost all individuals with SDS harbor biallelic mutations in SBDS. The partner of SBDS is EFL1, a ribosomal protein with GTPase activity. More than a dozen patients have been found with SDS due to biallelic mutations in EFL1. EFL1 serves to release EIF6, facilitating the assembly of the 80S ribosome from 60S and 40S ribosome subunits. Our lab has generated both sbds and efl1 null zebrafish, which phenocopy much of the human syndrome. We recently reported that Sbds and Efl1 depletion leads to upregulation of tp53 and cdkn1a ( p21) and accumulation of Eif6 in SDS zebrafish models, which is hypothesized to cause bone marrow failure. However, the mechanism(s) of EFL1's loss-of-function leading to neutropenia is not known. Methods. We used CRISPR/Cas9 editing to generate mutations in exon 17 of Efl1 of 32Dcl3, an IL-3-dependent murine myeloid cell line that can be differentiated to mature neutrophils. We analyzed the effects of Efl1 mutations on cell proliferation, survival, and differentiation with G-CSF. Immunoblotting and qPCR-based gene expression analysis were performed to identify affected mechanistic pathways. Results. Two clones were isolated and were sequenced to verify biallelic deleterious mutations. A clone with compound heterozygous inframe deletion and frameshift mutations showed trace expression of EFL1 (EFL1-KD) and the other with compound heterozygous frameshift mutations showed no expression (EFL1-KO). They presented a myeloblast-like morphology indistinguishable from parental cells. They proliferated significantly less than parental cells, although there was no increased apoptosis. Both EFL1-KD and KO cells showed significantly increased protein expression of EIF6 and transcriptional expression of Trp53 and Cdkn1a. EFL1 was downregulated during differentiation of parental 32Dcl3 cells to neutrophils, whereas EIF6 levels did not change. Withdrawal and replenishment of IL-3 from culturing media did not alter EFL1 protein expression. When EFL1-KD and KO were cultured in differentiating media, they did not proliferate and underwent greater apoptosis, particularly after 3 days of differentiation compared to parental 32Dcl3. The surviving EFL1-KD and KO matured into neutrophils after 7 days. Gene profiling for neutrophil differentiation identified equivalent expression of key transcriptional factors ( Cebpa, Cebpe, and Spi1) of EFL1-KD and KO in the maintenance media, except for Gfi1 which was two-fold and three-fold high in EFL1-KD and KO compared to parental cells, respectively. G-CSF induced significantly higher expression of Cebpa and lower expression of Cebpe in EFL1-KO from those in parental cells, respectively. Based on the observation that somatic genetic rescue by EIF6 mutations occurs in human SDS patients and animal models, we transfected 32Dcl3 cells with siRNA against Eif6. In parental 32Dcl3 cells, reduction of EIF6 caused slow cell growth; however, transfection of siRNA to Eif6 partially rescued proliferation of EFL1-KD and KO in both maintenance and differentiating media. Conclusions. Our results suggested that imbalance of late maturing factors of ribosome associated with SDS may cause decreased proliferation in myeloid-committed cells, thereby leading to neutropenia in SDS. Newly established Efl1-deficient cells recapitulated key observation from SDS patients, with an increased proapoptotic profile in hematopoietic cells. Because EFL1 has only been recognized recently as a rare cause of SDS, we do not know if EFL1 deficiency serves as a leukemia predisposition syndrome. We speculate that the aberrant normal neutrophil differentiation may form the foundation for myeloid neoplasia in SDS.

The Mechanism of Therapy Resistance By Lineage Plasticity in AML and How to Overcome It

Acute myeloid leukemia (AML) is an aggressive blood cancer driven predominantly by mutations in epigenetic regulators. This characteristic allows AML to thrive in a hostile and rapidly changing environment where it needs to adapt to stressors both extrinsic (chemotherapy) and intrinsic (replicative and oxidative stress) to survive. Our study is the first to demonstrate the mechanism of how AML uses epigenetic reprogramming to adjust to these stressors via lineage plasticity. Venetoclax and Azacitidine (Ven/Aza) co-treatment is increasingly the front-line therapy for AML, functioning through BH3 inhibition and DNA hypomethylation. However, most patients will relapse with a highly aggressive refractory AML. Some studies suggest that monocytic character of the AML is responsible for relapse, but more recent evidence demonstrates that Ven/Aza treatment itself can induce a lineage switch to monocytic character. A potent selection mechanism facilitated by epigenetic dysregulation generating plastic clones is likely driving resistance, though the mechanism remains unclear and represents a significant barrier for AML treatment. Using a combination of single cell transcriptomics with surface immunophenotype sequencing in multiple human xenograft and primary patient samples, we discovered that this plasticity is widespread and mediated by epigenetic reprogramming of the tumor in direct response to the therapy. Broadly, we found that AML responds to Ven/Aza by transitioning into a reversible state of cell cycle arrest with transcriptional character similar to hematopoietic stem cells. This transient state protects AML from Azacitidine through replication arrest and protects from Venetoclax by upregulation of anti-apoptotic transcriptional programs. These protections operate simultaneously with reprogramming events driven by a switch from MYC-driven transcriptional programs to a PU.1-driven program. Reprogramming is both facilitated by and dependent on repression of the Chromatin Assembly Factor 1 (CAF1) complex, a chromatin-bound master epigenetic regulator responsible for maintaining lineage fidelity through transcriptional regulation via its role as a nucleosome assembly factor. Repressing CAF1 facilitates reprogramming where the output is an altered cell population that is distinct from the initial tumor both transcriptionally and by ATACseq. AML can repeatedly return to this plastic state, regardless of the number of cycles of Ven/Aza, and introducing other cytotoxic compounds does not remove this plastic stem-like population but instead initiates the iterative process of reprogramming anew until a different resistant population emerges. To find therapeutic vulnerabilities, we generated several PDX models of Ven/Aza resistance so that we could track the stages of acquired resistance in real time. When single cell data from these experiments were projected onto a reference map of normal hematopoiesis, we found that Ven/Aza resistance stems from a plastic population of cells that actively samples the niche by generating short-lived multi-lineage hybrid differentiated products. While the predominating output of this process was monocytic, we found that the induced plastic stem-like population was durably retained even after Ven/Aza withdrawal. To undermine this process, we took advantage of the plastic state to drive differentiation through an external stimulus. We expected this strategy to be particularly effective because CAF1 was repressed (CAF1 repression facilitates differentiation in every metazoan model) and, based on our single cell data, these plastic populations of AML cells express pro-differentiation surface receptors. In particular, Ven/Aza induced the expression of CSF3R (the G-CSF receptor) which when stimulated, drives neutrophil maturation through the JAK/STAT pathway. While G-CSF as a single agent or in sequence with Ven/Aza was unable to achieve maturation, when used concurrently with Ven/Aza (or recapitulated by CAF1 depletion using shRNA) we were able to drive neutrophil differentiation in vitro and fully resolve primary mouse and PDX tumors in vivo, even when using Ven/Aza resistant tumors as starting material. Together, these data demonstrate the possibility that Ven/Aza resistance can be completely subverted by taking advantage of the plastic state induced by the therapy to stimulate differentiation and resolve the disease.