Analysis Of Bone Marrow Cells Research Articles

The characteristics and impact on prognosis of cytopenia after anti-BCMA-CAR-T therapy in patients with relapsed and refractory multiple myeloma

Objective: To investigate the characteristics of cytopenia and its impact on prognosis in patients with relapsed and refractory multiple myeloma (RRMM) after B-cell maturation antigen (BCMA) chimeric antigen receptor T-cell (CAR-T) immunotherapy therapy. Methods: Clinical data of 36 RRMM patients received BCMA CAR-T therapy at the First Affiliated Hospital of Nanjing Medical University from April 2017 to March 2023 were retrospectively collected. Among them, there were 17 males and 19 females, with an age [M (Q1, Q3)] of 62 (53, 67) years. The follow-up deadline was August 31, 2023, and the follow-up time [M (Q1, Q3)] was 33 (10, 30) months. The characteristics of cytopenia at different time points before lymphodepleting chemotherapy and after CAR-T cell infusion in all patients were analyzed. Kaplan-Meier method was used to compare the differences in progression-free survival (PFS) and overall survival (OS) in patients with different clinical characteristics. Single-cell sequencing analysis was used to analyze the changes in hematopoietic stem cells in three patients after CAR-T cell therapy. Results: The incidence of cytopenia after BCMA CAR-T cell therapy in 36 RRMM patients reached 100%. The incidence of neutropenia peaked on the 7th and 28th day after cell infusion with a biphasic pattern of change.Patients with all grade neutropenia reached 61.1% (22/36) and grade 3 or higher reached 33.3% (12/36) on the 7th day, while patients with all grade neutropenia reached 67.9% (19/28) and grade 3 or higher reached 28.6% (8/28) on the 28th day (P<0.001),respectively. The occurrence rate of lymphopenia reached a peak on the day of CAR-T cell infusion [97.2% (35/36) patients showed lymphopenia, while 80.6% (29/36) patients showed grade 3 or higher lymphopenia] (P<0.001).The incidence of all grade of thrombocytopenia and severe thrombocytopenia (grade 3 or higher) peaked on the 14th day after cell infusion, with the rates of 69.4% (25/36) and 30.6% (11/36) respectively, which had a prolonged duration(P<0.001). Even after 12 months, 40% (8/20) of patients still experienced thrombocytopenia.The incidence of anemia peaked on the 7th and 14th day after cell infusion, with a rate of 100% (36/36) (P<0.001). 50% (10/20) of patients still had anemia even 12 months after cell infusion. Kaplan-Meier survival analysis showed that patients with thrombocytopenia < grade 3 had undefined OS, while patients with thrombocytopenia ≥grade 3 had shorter OS [17 (95%CI: 2-32) months, χ2=4.154, P=0.042], indicating a poorer prognosis. However, there was no statistically significant difference in the relationship between other cytopenia and survival (all P>0.05). Single-cell sequencing analysis of bone marrow cells revealed decreased proliferation, increased apoptosis, and cell cycle arrest of hematopoietic stem cells after CAR-T cell infusion. Conclusions: All patients experienced varying degrees of cytopenia after receiving BCMA CAR-T cell infusion, and patients with thrombocytopenia ≥grade 3 had shorter OS and poorer prognosis.

The Sensitization of Murine Hematopoietic Progenitors and Stem Cells to DNA Damaging Agents By Quizartinib Is Associated with the Transcriptional Downregulation of DNA Damage Repair Genes

Background: The FLT3 inhibitor quizartinib induces quiescence of murine hematopoietic progenitors, and this affords their protection from S phase specific chemotherapies. In this study we investigated whether quizartinib can also protect hematopoietic progenitors from the cytotoxic effects of DNA damaging chemotherapies and radiation. Methods: C57BL/6 mice were primed with 30 mg/kg of quizartinib or vehicle (5% cyclodextrin) by oral gavage, and treated with DNA damaging chemotherapies, either cyclophosphamide, carboplatin, cisplatin, temozolomide or mitoxantrone 6 hours later, or 5.5Gy total body irradiation (TBI) after 9 hours. This TBI dose represents half the 11 Gy split dose used for bone marrow conditioning of C57BL/6 mice prior to HSC transplantation. Bone marrow cells were analyzed by flow cytometry after 3 to 6 days to identify progenitor and stem cell populations. Results: In contrast to quizartinib's protection against the cytotoxic effects of 5-FU and gemcitabine, all 5 DNA-damaging chemotherapies caused an increased loss of total bone marrow cellularity when mice were primed with quizartinib compared to vehicle primed controls. Flow cytometry analysis revealed that the numbers of myeloid and multipotent progenitors, and short-term and long-term hematopoietic stem cells (HSCs), were markedly lower in the quizartinib-treated mice. DNA damage and the resultant cell death caused by ionizing radiation is cell cycle specific, particularly for cells traversing from G 1 to S phases of the cell cycle. The ability of quizartinib to induce quiescence in myeloid and multipotent progenitors suggested that quizartinib may protect these cells from radiation. To test this, mice were dosed with vehicle or 30 mg/kg of quizartinib and 9 hours later exposed mice to 5.5 Gy of TBI. Analysis of bone marrow cells at day 6 revealed that total bone marrow cellularity and the numbers of progenitor and stem cells were markedly reduced in quizartinib-dosed mice compared to vehicle controls. For subsequent experiments mice were treated with 3 Gy TBI because the combination of quizartinib and 5.5 Gy caused severe myeloablation. As part of a series of experiments examining the kinetics of quizartinib's sensitization of bone marrow cells we tested whether the sensitization was maintained when mice were dosed with quizartinib 24 hours prior to 3 Gy TBI. Analysis of bone marrow cells 6 days later revealed that quizartinib dosed mice showed a significantly greater loss of total bone marrow cells, progenitor cells and stem cells compared to vehicle dosed mice (Figure 1). To identify possible mechanisms by which quizartinib sensitizes hematopoietic progenitors to DNA damaging agents we investigated quantitative gene expression profiling of the HPC7 hematopoietic progenitor cell line. These cells provide a suitable in vitro model since quizartinib induces their quiescence and provides protection against 5-FU. HPC7 cells were treated with 100 µM quizartinib, or vehicle, for 18 hr before RNA was harvested and sent for Next-generation sequencing. We found that quiescence induction in HPC7 cells by quizartinib correlated with the transcriptional downregulation of a wide range of genes involved in DNA damage repair pathways (including Brca1/2, Rad51, Palb2, Chek1 and Nbn), thus providing a possible mechanism for the enhanced cytotoxic effects of DNA damaging agents. Conclusions: Quizartinib sensitizes hematopoietic progenitors and stem cells to DNA-damaging agents, an effect that correlates with the transcriptional downregulation of many DNA damage repair genes. The enhanced depletion of hematopoietic progenitors and stem cells by DNA damaging agents in the presence of quizartinib has important clinical implications. Administering concurrent DNA damaging chemotherapy or radiotherapy with quizartinib treatment should be avoided. However, this interaction could facilitate a reduction in radiation/chemotherapy doses administered for bone marrow conditioning prior to HSC transplantation. By reducing these doses, but not compromising the conditioning process, non-hematopoietic side effects could be minimized and may allow currently excluded patients to be considered for HSC transplantation. Disclosure: This study was partly funded by Daiichi Sankyo.

Epigenetic Silencing of CD9 Shapes a New Mechanism to Render Differentiation Block and Immune Evasion in Pediatric Acute Myeloid Leukemia

Acute myeloid leukemia (AML) is a heterogeneous hematologic malignancy characterized by clonal expansion of myeloid progenitor cells with impaired terminal differentiation. Despite intensive treatment regimes, the clinical outcomes remain suboptimal. Emerging evidence suggest myeloblasts could evolve multiple machineries to evade immune patrol and hinder immunotherapies. Here, we present a new mechanism involving the tetraspanin protein CD9 in orchestrating differentiation arrest and immune escape in pediatric AML. We first examined CD9 expression and its prognostic impact in patient cohorts of childhood leukemia. The expression of CD9 on blasts of AML patients (12.2%, n=82) was significantly lower than those of ALL patients (90.4%, n=219, P&amp;lt;0.001) or stem cells from normal bone marrow donors (48.4%, n=22, P=0.014). Among AML cases, the blasts of 32 patients (39%) were CD9+. The expression pattern of CD9 was highly associated with cytogenetic/genetic anomalies and FAB subtypes, for instance enrichment of CD9 in M4, M5 and M7 AML. The 5-year relapse-free survival rate of CD9- patients was significantly lower than CD9+ patients (34.1% vs. 61.2%, P=0.018), with its prognostic significance exhibited distinctively in M4/M5 AML. To elucidate the mechanism governing CD9 silencing, DNA and histone methylation status of CD9 were evaluated by bisulfite and ChIP sequencing for the classical histone marks H3K9/27me3, which indicated methylation may not contribute to CD9 repression. However, a marked decrease of H3K9/27Ac occupancy at the CD9 locus was observed in AML than in ALL cells (4.8-14.2-fold, P&amp;lt;0.05), and strongly correlated with CD9 repression ( r=0.585-0.719, P&amp;lt;0.01). Exposure of CD9- AML cell lines (n=8) or samples (n=9) to the histone deacetylase inhibitor panobinostat significantly elevated CD9 mRNA and protein expression (3.1-32.2-fold, P&amp;lt;0.05), restored activating histone acetylation marks (4.1-41.6-fold, P&amp;lt;0.05), and potently suppressed myeloblast proliferation ex vivo (median IC50: 21.4 nM). An epigenetic compound library screen confirmed histone hypoacetylation as the key mechanism driving CD9 silencing. Enforced CD9 expression in MV4-11 cells significantly suppressed proliferation ( P&amp;lt;0.01) and colony formation ( P=0.002). NOD/SCID mice transplanted with CD9+ cells exhibited a drastically reduced leukemic load in hematopoietic organs by 70.7-91.8% ( P&amp;lt;0.05), a significantly prolonged survival duration ( P&amp;lt;0.001), and a marked regression of extramedullary myeloid sarcoma when compared with animals receiving CD9- cells. Global transcriptome profiling of pediatric AML (n=31) revealed decreased stemness (NES: -1.7, P=0.01) and increased monocyte (NES: 1.8, P=0.034) gene signatures in CD9+ samples. Concordantly, we observed a profound up-regulation of CD9 (9.4-51.1-fold, P&amp;lt;0.01) preceding the appearance of lineage markers (CD14, CD36, CD86, CD115, CD300e, iCD68 and iTNFα) in PMA-mediated monocyte/macrophage differentiation but not ATRA-mediated neutrophil differentiation of myeloblasts. Importantly, the gain of CD9 in THP-1 cells and primary myeloblasts (n=6, P&amp;lt;0.05) consistently augmented the expression of specific monocytic markers, corroborating its cell-intrinsic function in AML differentiation. Single-cell transcriptomic analyses of bone marrow cells from MV4-11-transplanted mice detected a significant enrichment of differentially regulated genes functioning in antigen processing and presentation. Confirmatory overexpression of CD9 promoted basal and IFNγ-induced MHC-I/II expression ( P&amp;lt;0.01) through the JAK2/STAT5 axis. Inter-patient comparisons (n=31) revealed a higher MHC-I expression in CD9+ AML ( P&amp;lt;0.001). Interestingly, CD9 physically bound to MHC-I/II and formed an immune complex at the cell membrane as revealed by IP-MS, co-IP and confocal microscopy. In NSG mice, co-transplantation of human PBMCs mounted an effective immunity against CD9+ but not CD9- AML (MV4-11 and MOLM-13, P&amp;lt;0.05), concomitant with a robust bone marrow infiltration of cytotoxic T cells. Taken together, our data provided molecular, cellular and clinical evidence showing the plausible function of CD9 as a key driver intertwining monocytic differentiation and immune recognition in pediatric AML, and inspired a new combinatorial epigenetic/immunotherapy for this rare but aggressive malignancy.

Mecom mutation related to radioulnar synostosis with amegakaryocytic thrombocytopenia reduces HSPCs in mice.

Radioulnar synostosis with amegakaryocytic thrombocytopenia (RUSAT) is an inherited bone marrow failure syndrome characterized by congenital fusion of the forearm bones. RUSAT is largely caused by missense mutations that are clustered in a specific region of the MDS1 and EVI1 complex locus (MECOM). EVI1, a transcript variant encoded by MECOM, is a zinc finger transcription factor involved in hematopoietic stem cell maintenance that induce leukemic transformation when overexpressed. Mice with exonic deletions in Mecom show reduced hematopoietic stem and progenitor cells (HSPCs). However, the pathogenic roles of RUSAT-associated MECOM mutations in vivo have not yet been elucidated. To investigate the impact of the RUSAT-associated MECOM mutation on the phenotype, we generated knock-in mice harboring a point mutation (translated into EVI1 p.H752R and MDS1-EVI1 p.H942R), which corresponds to an EVI1 p.H751R and MDS1-EVI1 p.H939R mutation identified in a patient with RUSAT. Homozygous mutant mice died at embryonic day 10.5-11.5. Heterozygous mutant mice (Evi1KI/+ mice) grew normally without radioulnar synostosis. Male Evi1KI/+ mice aged 5-15 weeks exhibited lower body weight and those aged 16 weeks and older showed low platelet counts. Flow cytometric analysis of bone marrow cells revealed a decrease in HSPCs in Evi1KI/+ mice at 8-12 weeks. Moreover, Evi1KI/+ mice showed delayed leukocyte and platelet recovery after 5-fluorouracil-induced myelosuppression. These findings suggest that Evi1KI/+ mice recapitulate the bone marrow dysfunction in RUSAT, similar to that caused by loss-of-function Mecom alleles.

Hematopoietic Stem Cell Identification Postirradiation.

Radiation exposure is particularly damaging to cells of the hematopoietic system, inducing pancytopenia and bone marrow failure. The study of these processes, as well as the development of treatments to prevent hematopoietic damage or enhance recovery after radiation exposure, often require analysis of bone marrow cells early after irradiation. While flow cytometry methods are well characterized for identification and analysis of bone marrow populations in the nonirradiated setting, multiple complications arise when dealing with irradiated tissues. Among these complications is a radiation-induced loss of c-Kit, a central marker for conventional gating of primitive hematopoietic populations in mice. These include hematopoietic stem cells (HSCs), which are central to blood reconstitution and life-long bone marrow function, and are important targets of analysis in these studies. This chapter outlines techniques for HSC identification and analysis from mouse bone marrow postirradiation.

Short-Term Effect of SARS-CoV-2 Spike Protein Receptor-Binding Domain-Specific Antibody Induction on Neutrophil-Mediated Immune Response in Mice.

Vaccination protects against COVID-19 via the spike protein receptor-binding domain (RBD)-specific antibody formation, but it also affects the innate immunity. The effects of specific antibody induction on neutrophils that can cause severe respiratory inflammation are important, though not completely investigated. In the present study, using a mouse model mimicking SARS-CoV-2 virus particle inhalation, we investigated neutrophil phenotype and activity alterations in the presence of RBD-specific antibodies. Mice were immunized with RBD and a week after a strong antibody response establishment received 100 nm particles in the RBD solution. Control mice received injections of a phosphate buffer instead of RBD. We show that the application of 100 nm particles in the RBD solution elevates neutrophil recruitment to the blood and the airways of RBD-immunized mice rather than in control mice. Analysis of bone marrow cells of mice with induced RBD-specific antibodies revealed the increased population of CXCR2+CD101+ neutrophils. These neutrophils did not demonstrate an enhanced ability of neutrophil extracellular traps (NETs) formation compared to the neutrophils from control mice. Thus, the induction of RBD-specific antibodies stimulates the activation of mature neutrophils that react to RBD-coated particles without triggering excessive inflammation.

PB1912: MYELODYSPLASTIC SYNDROME WITH ISOLATED 5Q DELETION AND ASSOCIATED 5Q DELETION TO 13Q DELETION

Background: The 5q deletion is the classic chromosomal abnormality in myelodysplastic syndrome (MDS). Critical regions are 5q31 and 5q33, leading to gene haplodeficiency and other pathogeneic mechanisms. 13q deletion occurs in about 2% of MDS cases. This deletion involved band 13q14. Aims: Here we describe cytogenetic abnormalities in a Tunisian cohort of MDS patients and we shed the light on cases with 5q deletion. Methods: Cytogenetic analyses of bone marrow cells were performed according to standard procedures for 12 Tunisian patients presenting with MDS. Cytogenetic analysis was performed on lymphocytes and bone marrow cells. Cultured cells were treated with colchicine and hypotonic KCl solution. The pellet was fixed and washed in methanol-acetic acid (3:1). The cells were resuspended in fixative and dropped onto slides. Chromosome-banding analysis was performed using RHG and GTG banding techniques. Three karyotypes and 25 metaphases were analyzed according to the International System for Human Cytogenetic Nomenclature. Results: Cytogenetic abnormalities were detected in 9 cases and del5q was recorded in four patients (3 patients with refractory anemia (RA) and a fourth with RA and excess blasts-T (RAEB-T)). The del5q was isolated in two RA cases as a del (5) (q13 q33) in a new diagnosed and a treated patients. It was associated in two patients: a RA patient explored during the evolution, harboring a del (5) (q12 q33) associated to a del (13) (q12 q14) and the RAEB-T patient harbouring a del (5) (q21 q31) associated to a clonal hyperploidy. Summary/Conclusion: The diagnosis of MDS was based on cytomorphology and cytogenetics. However, nowadays, an increasing number of molecular aberrations have been discovered and included in the WHO revised new classification of myeloid neoplasms. The del13q described in MDS seems to be a marker of the evolution of the clonal cells harboring 5q deletion. However, the molecular basis and the implicated genes of the deletion of the long arm of chromosome 13 associated to the 5q deletion clone remain until now not well explored.

S116: CELLULAR AND MOLECULAR MECHANISMS OF EVI1-EXPRESSING MLL-REARRANGED ACUTE MYELOID LEUKEMIA

Background: Expression of a doxycycline (DOX)-inducible acute myeloid leukaemia (AML)-associated iMLL-AF9 fusion transgene in long-term haematopoietic stem cells (LT-HSC) can lead to an invasive and chemo-resistant disease expressing the transcription factor EVI1. High EVI1 expression has been suggested as marker of poor outcome in AML patients even without rearrangements of the EVI1 locus at 3q26. Aims: We addressed the association between EVI1 expression, the cellular origin and poor disease outcome in AML driven by the iMLL-AF9 fusion gene. Methods: The role of EVI1 expression was studied in iMLL-AF9 transgenic mice carrying an Evi1-IRES GFP reporter in vitro using flow cytometry, colony formation and RT-qPCR assays, ex vivo with high-resolution bone marrow (BM) imaging, and in vivo, by transplantation of enriched naïve Evi1+ iMLL-AF9 hematopoietic stem and progenitor cells (HSPC) into irradiated recipients on DOX. Haematopoiesis of symptomatic mice was analysed by flow cytometry and histology. For mechanistic studies, single cell and bulk RNA sequencing was performed on enriched HSPC or BM samples from diseased mice. Results: Analysis of BM cells from Evi1-IRES-GFP reporter mice revealed that not only the mostly quiescent LT-HSC but also fractions of the more proliferating multipotent progenitors (MPP1-3) express abundant Evi1 (“Evi1high”). Induction of the iMLL-AF9 fusion did not result in significant changes in numbers of Evi1+ cells nor levels of Evi1 mRNA expression in the LT-HSC and MPP1 compartments. However, in colony assays, Evi1high iMLL-AF9 cells retained a more immature phenotype and produced more colonies with an invasive morphology than Evi1low cells (n=11, p<0.05). While Evi1 expression did not influence disease induction upon transplantation of LT-HSC, recipients of Evi1+ MPP1 cells developed AML earlier than Evi1- MPP1 (n=11, 79 vs. 269d, p<0.05). Disease induced by Evi1+ cells presented with more extensive leukemic organ infiltration than Evi1- AML. Evi1 expression also correlated with in vitro Ara-C resistance. We also examined whether some exogenous factors may increase AML susceptibility by expanding the Evi1+ HSPC. Although a single injection of recombinant mouse thrombopoietin (TPO) only increased the number of LT-HSC and not of MPP1, the Evi1high cell fraction was enlarged in both compartments (LT-HSC: 23 vs. 50%; MPP1: 22 vs. 47%; n=29, p<0.0001) supported by high-resolution imaging. Interestingly, increased TPO induced HSPC cycling was confined to the Evi1high cell population (n=3, p<0.05). Transplantation of TPO-treated iMLL-AF9 LT-HSC or MPP1 resulted in a significantly faster induction of Evi1+ AML than controls (n=19, MPP1: 35 vs. 79d, p<0.001; LT-HSC: 41 vs. 90d, p<0.001). To better understand mechanisms of aggravated AML after TPO mediated expansion of Evi1+ HSCP we performed multiplexed single cell RNA sequencing of highly enriched HSPC cells in iMLL-AF9 and control mice. While we observed no changes of cellular cluster organisation 2 days after TPO injection, we found some differentially expressed genes in TPO-stimulated cycling iMLL-AF9 HSPC, including potential stemness regulators. Summary/Conclusion: Our results suggest that expansion of Evi1-expressing HSPC by exogenous factors can result in a more aggressive MLL-AF9-driven AML. Ongoing data exploration and validation may characterize aberrantly expressed genes in TPO-stimulated Evi1+ iMLL-AF9-expressing HSPC as potential therapeutic targets to impair stemness of AML cells.

Drug-induced bone marrow changes

Die zyto- und histomorphologische Untersuchung des Knochenmarks bei Blutbildungsstörungen muss immer auch die Möglichkeit medikamentös induzierter Veränderungen berücksichtigen. Diese können einzelne Differenzierungslinien oder das gesamte Knochenmark betreffen. Sie bestehen aus quantitativen Verschiebungen, d. h. einer Hypo- oder Hyperplasie und/oder Reifungsstörungen. Letztere umfassen eine mitunter extreme Linksverschiebung oder imitieren Vitaminmangelzustände und Atypien wie bei einem myelodysplastischen Syndrom (MDS). Auch die gesamte Hämatopoese kann betroffen sein, wobei im Extremfall das Bild einer aplastischen Anämie hervorgerufen wird. Das Spektrum infrage kommender Medikamente ist sehr breit und die Veränderungen in der Regel zu unspezifisch, um gezielt auf das schädigende Agens zurückzuschließen, was erst in Kenntnis der Medikamentenanamnese möglich wird. In der onkologischen Therapie eingesetzte zytotoxische Substanzen können mit einer durchschnittlichen Latenzzeit von 2–6 Jahren MDS auslösen, die allerdings bei Medikamenten, die in die DNA-Reparatur eingreifen, auch kürzer sein kann.

A deep learning method and device for bone marrow imaging cell detection.

BackgroundMorphological analysis of bone marrow cells is considered as the gold standard for the diagnosis of leukemia. However, due to the diverse morphology of bone marrow cells, extensive experience and patience are needed for morphological examination. automatic diagnosis system through the comprehensive application of image analysis and pattern recognition technology is urgently needed to reduce work intensity, error probability and improves work efficiency.MethodsIn this article, we establish a new morphological diagnosis system for bone marrow cell detection based on the deep learning object detection framework. The model is based on the Faster Region-Convolutional Neural Network (R-CNN), a classical object detection model. The system automatically detects bone marrow cells and determines their types. As specimens have severe long-tail distribution, i.e., the frequency of different types of cells varies dramatically, we proposed a general score ranking loss to solve such a problem. The general score ranking loss considers the ranking relationship between positive and negative samples and optimizes the positive sample with a higher classification probability value.ResultsWe verified this system with 70 bone marrow specimens of leukemia patients, which proved that it can realize intelligent recognition with high efficiency. The software is finally integrated into the microscope system to build an augmented reality system.ConclusionsClinical tests show that the response speed of the newly developed diagnostic system is faster than that of trained diagnostic experts.

Antigenotoxic and antimutagenic effects of lignin derivative BP-C2 against dioxidine and cyclophosphamide in vivo in murine cells

The study investigated antigenotoxic and antimutagenic activity of novel lignin-derived polyphenolic composition (BP-C2) with ammonium molybdate towards cyclophosphamide and dioxidine in the bone marrow, blood and liver cells of BALB/c mice. BP-C2 was given to mice via gavage at 60, 80 and 120 mg/kg once 1 h before single intraperitoneal injection of a genotoxic agent. 1.5 h and 3 h after dioxidine or cyclophosphamide injection, respectively, cellular suspensions were obtained from mice and assessed with the comet test and cytogenetic analysis of bone marrow cells. It was observed that antigenotoxic activity of BP-C2 against DNA damage induced by dioxidine, a prooxidant genotoxic agent, in the bone marrow, liver and blood cells of mice in vivo was more pronounced at 60 and 80 mg/kg than at 120 mg/kg. When cyclophosphamide was used to induce a DNA damage, the genoprotective effect of BP-C2 was observed in bone marrow, liver and blood cells at 60 mg/kg dose but the effect was not significant at 80 mg/kg. When co-administered with 120 mg/kg BP-C2, cyclophosphamide induced a higher level of DNA damage in liver cells, but its genotoxic effect in bone marrow and blood cells was the same as when it was administered alone. When assessing the effect of BP-C2 on chromosomal aberrations induced by cyclophosphamide and dioxidine in bone marrow cells, it was revealed that all three tested doses of BP-C2 significantly decreased the number of cells with chromosome abnormalities. Thus, BP-C2 has a pronounced antimutagenic and genoprotective effects.

Abrogation of a Histone Chaperone Pathway Mitigates Inflammation-Driven AML Progression

Background: Genetic heterogeneity makes clinical interventions challenging for acute myeloid leukemia (AML) patients. Identifying and targeting microenvironment-driven pathways that are active across AML genetic subtypes should allow the development of more broadly effective therapies. Previously, we have shown that AML microenvironment is rich in proinflammatory cytokine interleukin-1β (IL-1β) and significantly promotes the growth of AML progenitors while suppressing healthy progenitors. To elucidate this paradoxical effect, we performed transcriptome (RNA-seq) analysis from IL-1β-stimulated CD34+ AML and normal progenitors and found that ASF1B (anti-silencing function-1B) is one of the most differentially expressed genes. ASF1B is a histone chaperone, which recruits H3-H4 histones onto the replication fork during S-phase. This process is regulated by tousled-like kinase 1 and 2 (TLKs). TLKs and ASF1B are overexpressed in multiple solid tumors and associated with poor prognosis. However, their functional roles in hematopoiesis and inflammation-driven leukemia are unexplored. Here, we reveal a novel molecular mechanism that IL-1β promotes leukemia progression by activating the TLK-ASF1B pathway.Methods and Results: We first confirmed that IL-1β stimulation upregulates ASF1B expression at both mRNA and protein levels in FLT3-ITD, MLL-ENL, and NPM1 positive primary AML samples. ASF1B upregulation is abolished upon treatment with a p38MAPK inhibitor, further suggesting that ASF1B is downstream of IL-1β/p38 signaling. Next, we stably knocked down ASF1B in an AML cell line MOLM-14 using doxycycline-inducible shRNA. ASF1B-depleted AML cells exhibited reduced cell viability, inhibited growth, blocked cell cycle progression, and impaired colony formation ability. We xenografted shASF1B expressing AML cells into NSG mice and induced knockdown in vivo. Flow cytometry analysis of bone marrow cells 3 weeks post-engraftment showed 80 percent reduction in leukemia burden following ASF1B silencing compared to controls.To determine whether upregulation of ASF1B contributes to IL-1β-driven leukemic growth, we overexpressed ASF1B with MLL-ENL oncogene in a murine bone marrow transplantation model. We found that daily IL-1β exposure accelerated leukemia progression compared to vehicle-treated group (median survival = 64 vs. 85 days, p<0.05), and this effect was phenocopied by overexpression of ASF1B (median survival = 62 vs. 85 days, p<0.05). Conversely, heterozygous and complete Asf1b deletion in the MLL-ENL AML model delayed the leukemia progression compared to wildtype mice. Furthermore, Asf1b deletion attenuated IL-1β-mediated AML progression compared to wildtype controls (median survival = 63 vs. 47 days, p <0.01). Immunophenotyping of Asf1b-deficient mice using flow cytometry suggested that ASF1B is dispensable for normal hematopoiesis. Together, these data suggested that targeting of the ASF1B pathway may spare healthy cells. Additionally, we found that TLK2 which regulates ASF1B activation, is also upregulated in AML progenitors compared to healthy cells at the baseline levels and upon IL-1β stimulation. We next knocked down TLKs in human AML cells and observed a similar pattern with growth arrest, higher replication stress and DNA damage response. Finally, we generated Vav-Cre+ Tlk2 mice allowing Tlk2 deletion only in hematopoietic cells and found that Tlk2 deletion prolongs survival of leukemic mice in a dose dependent manner with and without IL-1β stimulation.Conclusions: We demonstrate that increased TLK-ASF1B expression promotes survival of AML cells. We also provide the first in vitro and in vivo evidence that the TLK-ASF1B pathway plays a critical role in potentiating IL-1β-dependent AML growth. Therefore, we establish TLK-ASF1B pathway as a novel route for therapeutic strategy to suppress inflammation-driven growth in various AML genetic subtypes. DisclosuresNo relevant conflicts of interest to declare.

279 ILT3 blockade reduces tumor blast cells in acute myeloid leukemia PBMC and inhibits tumor cell growth in a humanized AML tumor model

BackgroundImmunoglobulin-like transcript 3 (ILT3), an immune-inhibitory receptor expressed on myeloid cells, is highly expressed in acute myeloid leukemia (AML) with monocytic differentiation (M4 and M5 subtypes) and clinically is negatively correlated with overall survival. We examined the effects of a highly selective and potent chimeric anti-ILT3 mAb (c52B8) on tumor cell survival/growth and human immune cell modulation in AML PBMC and AML tumor cell lines in pre-clinical in vitro and in vivo models.MethodsILT3 mRNA and cell surface expression levels were measured by RNAseq and FACS, respectively. PBMC from AML patients with different levels of ILT3 cell surface expression were treated with c52B8 in vitro.A systemic AML model was generated by IV inoculation of NSG mice with luciferase-transfected MV-4-11 human AML cells, with or without human PBMC engraftment. Tumor bearing mice were treated weekly with c52B8, modified to contain the human Fc of either the IgG1 or IgG4 subclass. MV-4-11 growth in vivo was measured by bioluminescence imaging (BLI).CyTOF was performed to phenotype AML PBMC in in vitro culture and bone marrow samples from the systemic MV-4-11 model after the treatment.THP-1 cells/human T cell co-cultures stimulated with anti-CD3/CD28 antibodies were treated with c52B8. Human T cell activity was assessed by levels of IFNγ production in the culture using MSD ELISA.ResultsIn ILT3 high AML patient PBMC, c52B8 treatment in vitro decreased the frequency of tumor blasts, modulated Tregs, and increased monocytic myeloid cells. In the MV-4-11 model, weekly treatment with c52B8 significantly reduced tumor burden in vivo, regardless of the IgG subclass. Importantly, tumor cell growth inhibition by c52B8 occurred in the absence of human PBMC. Additional post-mortem CyTOF analysis of bone marrow cells from MV-4-11/human PBMC inoculated mice confirmed a reduction in MV-4-11 cells, identified by CD3-CD19- human myeloid cells following c52B8 treatment. In THP-1/human T cell co-cultures, c52B8 treatment reversed THP-1 induced T cell suppression, as measured by enhanced IFNγ production.ConclusionsAnti-ILT3 mAb (c52B8) is efficacious in blocking progression of AML with monocytic differentiation by directly influencing tumor cell growth and enhancing human T cell activity in pre-clinical models. These studies support a potential role for this compound in humans.Ethics ApprovalAll animal work was reviewed and approved by Merck IACUC before experiments were conducted.

AML-027: Menin Inhibition Decreases Bcl-2 and Bcl-xL and Enhances Venetoclax Activity in NPM1/FLT3-Mutated AML

<h3>Context:</h3> MLL1-menin interaction in NPM1-mutated (NPM1c) AML shares a common HOX gene signature and dependencies as that of MLL-rearrangements (MLL1-r) with menin. Menin inhibition has demonstrated anti-leukemia activity in both NPM1c and MLL-r AML. NPM1c frequently occurs in AML with FLT3-ITD/FLT3-TKD mutations. Co-inhibition of menin and FLT3 has shown enhanced anti-leukemia activity in MLL-r/FLT3- and NPM1c/FLT3-mutated AML. Targeting Bcl-2 has emerged as a promising therapeutic option for AML patients. However, despite the major improvement of combining the Bcl-2 inhibitor venetoclax with hypomethylating agents, most patients develop resistance and ultimately relapse. <h3>Objective:</h3> Investigate anti-leukemic activities, potential synergism, and mechanisms of menin-MLL1 inhibitor SDNX-50469, an equipotent surrogate of the clinical compound SNDX-5613 and venetoclax combination <i>in vivo</i> in an NPM1c/FLT3-ITD/TKD PDX model. <h3>Design:</h3> The PDX cell-engrafted NSG mice were treated with 0.05 or 0.1% SNDX-50469-spiked chow, venetoclax (50 mg/kg), or 0.1% SNDX-50469 plus venetoclax (one month). Engraftment and disease progression were assessed by flow cytometric measurement of circulating human CD45+ cells. The treatment effects on leukemia cell populations and protein levels were determined by CyTOF mass cytometry. <h3>Results:</h3> Menin inhibition exhibited strong anti-leukemia activity, which was enhanced by venetoclax, while venetoclax alone had minimal effects. The combination most effectively extended survival (143 d for 0.1% SNDX-50469 plus venetoclax, P=0.0003; 131 and 125 d for 0.1% or 0.05% SNDX-50469, respectively, both P=0.0001; 69 d for venetoclax, P=0.025; <i>vs</i> 61 d for controls). At the end of treatment, CyTOF analysis of bone marrow cells demonstrated that menin inhibition preferentially targeted CD34+CD38+ cells, while venetoclax targeted CD34+CD38- cells. Only the combination effectively eliminated bulk and CD34+CD38+/CD34+CD38-stem/progenitor cells. Menin inhibition increased the percentage of CD11b+ myeloid cells. Mechanistically, menin inhibition decreased Bcl-2 and Bcl-xL and concomitantly increased Bax and Bim, which seemingly enhanced venetoclax activity. However, we observed increased p-FLT3 in the surviving leukemia cells, particularly in the combination group, which may contribute to the regrowth of leukemia cells. <h3>Conclusions:</h3> Our study validated menin as a therapeutic target and demonstrated that its inhibition synergizes with venetoclax in NPM1/FLT3-mutated AML, which warrants clinical evaluation. Inhibition of FLT3 may further enhance menin and Bcl-2 co-targeting efficacy in NPM1- and FLT3-mutated AML.

Analysis of Radiation-Induced Changes in Cell Cycle and DNA Damage of Murine Hematopoietic Stem Cells by Multi-Color Flow Cytometry.

Exposure of bone marrow to genotoxic stress such as ionizing radiation (IR) results in a rapid decline of peripheral blood cells and stimulates entry of the normally quiescent hematopoietic stem cells (HSCs) into the cell cycle to reconstitute the hematopoietic system. While several protocols have employed flow cytometry analysis of bone marrow cells to study changes in specific cell populations with respect to cell cycle proliferation and/or expression of γ-H2AX, a marker of DNA damage, these parameters were examined in separate panels. Here, we describe a flow cytometry-based method specifically designed to examine cell cycle distribution using Ki-67 and FXCycle violet in combination with γ-H2AX in HSCs and hematopoietic progenitor cells (HPCs) within the same sample. This method is very useful, particularly in studies involving genotoxic stresses such as IR, which substantially reduce the absolute numbers of HSCs and HPCs available for staining. Additionally, we describe several important considerations for the analysis of markers of HSCs in irradiated versus unirradiated samples. Examples include the use of fluorescence minus one (FMO) controls, the gating strategy for markers whose expression is typically impacted by IR such as Sca1, tips for staining of intracellular antigens like Ki67, and ensuring the detection of signal from at least 500 events in each gate to ensure robustness of the results. © 2021 Wiley Periodicals LLC. Basic Protocol: Immunostaining protocol for bone marrow mononuclear cells using a multi-fluorophore panel.

Dysregulation of erythropoiesis and altered erythroblastic NMDA receptor-mediated calcium influx in Lrfn2-deficient mice.

LRFN2 encodes a synaptic adhesion-like molecule that physically interacts with N-methyl-D-aspartate (NMDA) receptor 1 and its scaffold proteins. Previous studies in humans and mice have demonstrated its genetic association with neurodevelopmental disorders such as learning deficiency and autism. In this study, we showed that Lrfn2-deficient (KO) mice exhibit abnormalities of erythropoietic systems due to altered NMDA receptor function. In mature Lrfn2 KO male mice, peripheral blood tests showed multilineage abnormalities, including normocytic erythrocythemia, and reduced platelet volume. Colony forming unit assay using bone marrow cells revealed decreases in the counts of erythrocyte progenitors (CFU-E) as well as granulocytes and monocyte progenitors (CFU-GM). Whole bone marrow cell staining showed that serum erythropoietin (EPO) level was decreased and EPO receptor-like immunoreactivity was increased. Flow cytometry analysis of bone marrow cells revealed increased early erythroblast count and increased transferrin receptor expression in late erythroblasts. Further, we found that late erythroblasts in Lrfn2 KO exhibited defective NMDA receptor-mediated calcium influx, which was inhibited by the NMDA receptor antagonist MK801. These results indicate that Lrfn2 has biphasic roles in hematopoiesis and is associated with the functional integrity of NMDA receptors in hematopoietic cells. Furthermore, taken together with previous studies that showed the involvement of NMDA receptors in hematopoiesis, the results of this study indicate that Lrfn2 may regulate erythropoiesis through its regulatory activity on NMDA receptors.

Activation of SIRT6 by DNA hypomethylating agents and clinical consequences on combination therapy in leukemia

The FDA-approved DNA hypomethylating agents (DHAs) like 5-azacytidine (5AC) and decitabine (DAC) demonstrate efficacy in the treatment of hematologic malignancies. Despite previous reports that showed histone acetylation changes upon using these agents, the exact mechanism underpinning these changes is unknown. In this study, we investigated the relative potency of the nucleoside analogs and non-nucleoside analogs DHAs on DNA methylation reversal using DNA pyrosequencing. Additionally, we screened their effect on the enzymatic activity of the histone deacetylase sirtuin family (SIRT1, SIRT2, SIRT3, SIRT5 and SIRT6) using both recombinant enzymes and nuclear lysates from leukemia cells. The nucleoside analogs (DAC, 5AC and zebularine) were the most potent DHAs and increased the enzymatic activity of SIRT6 without showing any significant increase in other sirtuin isoforms. ChIP-Seq analysis of bone marrow cells derived from six acute myeloid leukemia (AML) patients and treated with the nucleoside analog DAC induced genome-wide acetylation changes in H3K9, the physiological substrate for SIRT6. Data pooling from the six patients showed significant acetylation changes in 187 gene loci at different chromosomal regions including promoters, coding exons, introns and distal intergenic regions. Signaling pathway analysis showed that H3K9 acetylation changes are linked to AML-relevant signaling pathways like EGF/EGFR and Wnt/Hedgehog/Notch. To our knowledge, this is the first report to identify the nucleoside analogs DHAs as activators of SIRT6. Our findings provide a rationale against the combination of the nucleoside analogs DHAs with SIRT6 inhibitors or chemotherapeutic agents in AML due to the role of SIRT6 in maintaining genome integrity and DNA repair.

BCOR Mutation and TLS-ERG Expression in Acute Myeloid Leukemia with Monoclonal Immunoglobulinemia

Acute myeloid leukemia(AML) originates from the abnormal clonal proliferation of myeloblast which often combined with clinical symptoms. Cytogenetic and molecular abnormalities are frequent in AML patience. To date, the driver genes for leukemia remain largely undiscovered. Monoclonal immunoglobulinemia is a group of diseases caused by excessive proliferation of plasma cells or immunoglobulin-producing lymphoid plasma cells and B lymphocytes. It can develop into malignant plasma cell disease. Herein, we report a AML patient was concomitant with monoclonal immunoglobulinemia, the patient was also accompanied by BCOR mutation and TLS-ERG fusion gene. A 55-year-old married female was admitted into our hospital due to repeated edema for 3 weeks. On admission, peripheral blood counts: PLT142×10^9/L, HB77g/L↓, WBC35.2×10^9/L.Bone marrow examination showed the mononuclear cell system proliferated actively, and the primitive infantile monocytes accounted for 86%. Cell morphology suggested M5b(Figure1A ). Fusion gene screening in bone marrow revealed that TLS-ERG expression. Immunophenotype of bone marrow cell:Abnormal myeloid primitive cells accounted for 96.39% of the nuclear cells,expressCD33, CD13, CD123, CD34, CD9, MPO(Figure 1D). Karyotype analysis of bone marrow cells showed in Figure 1B. Thus, AML was diagnosed. Next-generation DNA sequencing technology showed that BCOR (51.7%),PLCG1(49.9%),DIS3(48.4%),BRAF(51.6%), JAK2(45.1%) ,JAK3(49.0%) were mutated. Meanwhile, we found that Peripheral blood immunofixation electrophoresis showed that Gamma region is seen with a monoclonal light chain lambda component((Figure 1C.).Then, the patient underwent one cycle of IA(Idabisine hydrochloride 10mg d1-4, cytarabine 0.075g q12h d1-7). Twenty-five after chemotherapy onset, bone marrow examination showed that primitive and immature monocytes accounted for 3%. Chromosome become normal. Minimal residual disease(MRD):0.01%. The disease reached complete remission(CR). Peripheral blood immunofixation electrophoresis turned negative. Fusion gene detection showed that TLS-ERG turned negative. BCOR mutation was not detected by Next-generation DNA sequencing. Mutations of PLCG1,DIS3,BRAF,JAK2,JAK3 still exist. Monoclonal immunoglobulinemia and AML are both clonal diseases, but originated from different clones. This case has both malignant clones of granulocyte stem cell and malignant clones of B line, so it is worthy of discussion. By comparing CR before and after we found that while the patient's M protein turned negative, the TLS-ERG fusion gene and BCOR gene mutation also disappeared. The TLS-ERG fusion gene is formed by the rearrangement of TLS and ERG genes on chromosomes 16 and 21. The current study holds that the expression of this fusion gene indicates rapid disease progression and poor prognosis. BCOR mutations can be found in AML and often coincide with DNMT3 gene mutations, suggesting it may affect the occurrence of leukemia through epigenetics. BCOR is a newly discovered corepressor of BCL-6, which can play a supporting role when BCOR combines with DNA; when BCOR is overexpressed, it can enhance the inhibition of BCL-6. BCL-6 is highly expressed in tumor cells,it encodes transcriptional repressors which are required for the formation of germinal center and may affect apoptosis. We thinked that the monoclonal immunoglobulinemia of this patient may caused by the BCOR abnormal expression which increased the inhibitory effect of BCL-6 and affect the apoptosis of B cells, and B cells continue to secrete immunoglobulin. BCOR mutations are associated with poor prognosis. The patient with TLS-ERG fusion gene which is a poor prognosis gene.However, the BCOR gene mutation site is a non-hot spot mutation which has few clinical studies. Whether the BCOR gene mutation results in the combination of the two diseases requires further study. Acknowledgment:The research was supported by fundings of the public technology research projects of Yiwu,China (2016-S-05), the key medical discipline of Yiwu,China(Hematology,2018-2020),and the academician workstation of the Fourth Affiliated Hospital of Zhejiang University School of Medicine. Correspondence to: Dr Jian Huang, Department of Hematology, The Fourth Affiliated Hospital of Zhejiang University School of Medicine. N1 Shangcheng Road. Yiwu, Zhejiang, Peoples R China. Email: househuang@zju.edu.cn Figure 1 Disclosures No relevant conflicts of interest to declare.

Glutathione Metabolism Contributes to the Pathobiology of Waldenstrom's Macroglobulinemia

Cancer cells have altered energy demand due to their increased proliferative capacity. In Waldenstrom Macroglobulinemia (WM), an indolent yet incurable B-cell lymphoma, the homeostasis of the bone marrow (BM) environment is disturbed due to the infiltration of the lymphoplamacytic cells that continuously produce monoclonal IgM. An alteration in energy demand could skew the balance of key proteins and metabolites towards a permissive niche for WM tumor cells growth, and unfavorably effect on the immune response against tumor cells. Therefore, the aim of our study was to identify how changes in certain metabolites and/or proteins could contribute to the pathobiology of WM and whether the cytokine and chemokine composition of the BM microenvironment play a role in such changes. WM patient's samples including BM plasma, peripheral blood serum and BM cells (n=101) as well as equivalent normal counterparts (n=86) were collected and used for metabolomics analysis. Comprehensive targeted metabolomics analysis was performed using Capillary Electrophoresis Time-of-Flight Mass spectrometry (CE-TOFMS), CE-triple quadrupole mass spectrometry (CE-qQqMS) and Liquid Chromatography (LC-TOFMS). Normal and WM peripheral blood serums samples were also used for untargeted proteomics analysis using a fully automated proteomic technology platform that includes an Agilent 1200 Series Quaternary HPLC system connected to a Q Exactive Plus mass spectrometer. Real-time PCR analysis was performed to detect the gene expression of the relevant metabolite transporters located on the cell membrane. BM cells from control and WM patients' samples were used for flow cytometry analysis. IHC was used to detect the proteins on the BM tissues. Principal Component Analysis (PCA) and Hierarchal Clustering Analysis (HCA) on both metabolomics and proteomics data identified two distinct clusters for disease and normal samples, indicating that there are differentially expressed proteins and metabolites in WM versus normal samples. Furthermore, pathway analysis showed that the majority of the altered metabolites were the members of the glutathione (GSH) metabolism pathway. This finding was further validated not only by data obtained from metabolomics analysis of BM cells and BM plasma, but also by proteomic data WM patients serum, implying that GSH metabolism is key to the biology of WM. Moreover, stimulation of WM cell lines by IL-6 and IL-21, cytokines involved in inducing WM cell proliferation and IgM secretion, resulted in increased gene expression of the transporters mediating uptake of metabolites required for GSH synthesis, including SLC7A11, 4F2HC and LAT1, indicating that cytokines in the WM BM could modulate GSH metabolism. In addition, IHC staining of the BM tissues as well as flow cytometry analysis of patients' lymphoplasmacytic cells identified glutathione peroxidase as one of the major proteins modulating GSH metabolism in WM. In summary, our data highlight a central role for GSH metabolism in WM disease biology and indicate that intervening with the metabolic processes could be a potential therapeutic strategy for patients with WM. Disclosures Ansell: AI Therapeutics: Other: research funding for clinical trials; Seattle Genetics: Other: research funding for clinical trials; Regeneron: Other: research funding for clinical trials; Affimed: Other: research funding for clinical trials; Bristol Myers Squibb: Other: research funding for clinical trials; Pfizer: Other: research funding for clinical trials; Merck: Other: research funding for clinical trials; Takeda: Other: research funding for clinical trials.

Liraglutide Attenuates Preestablished Atherosclerosis in Apolipoprotein E-Deficient Mice via Regulation of Immune Cell Phenotypes and Proinflammatory Mediators.

We have shown that the glucagon-like peptide-1 receptor agonist (GLP-1RA) liraglutide (Lir) inhibits development of early atherosclerosis in vivo by modulating immune cell function. We hypothesized that Lir could attenuate pre-established disease by modulating monocyte or macrophage phenotype to induce atheroprotective responses. Human atherosclerotic plaques obtained postendarterectomy and human peripheral blood macrophages were treated ex vivo with Lir. In parallel, apolipoprotein E-deficient (ApoE-/-) mice received a high-fat, high-cholesterol diet to induce atherosclerosis for 8 weeks, after which ApoE-/- mice received 300 μg/kg of Lir daily or vehicle control for a further 4 weeks to investigate the attenuation of atherosclerosis. Lir inhibited proinflammatory monocyte chemoattractant protein-1 secretion from human endarterectomy samples and monocyte chemoattractant protein-1, tumor necrosis factor-α, and interleukin (IL)-1β secretion from human macrophages after ex vivo treatment. An increase in CD206 mRNA and IL-10 secretion was also detected, which implies resolution of inflammation. Importantly, Lir significantly attenuated pre-established atherosclerosis in ApoE-/- mice in the whole aorta and aortic root. Proteomic analysis of ApoE-/- bone marrow cells showed that Lir upregulated the proinflammatory cathepsin protein family, which was abolished in differentiated macrophages. In addition, flow cytometry analysis of bone marrow cells induced a shift toward reduced proinflammatory and increased anti-inflammatory macrophages. We concluded that Lir attenuates pre-established atherosclerosis in vivo by altering proinflammatory mediators. This is the first study to describe a mechanism through which Lir attenuates atherosclerosis by increasing bone marrow proinflammatory protein expression, which is lost in differentiated bone marrow-derived macrophages. This study contributes to our understanding of the anti-inflammatory and cardioprotective role of GLP-1RAs. SIGNIFICANCE STATEMENT: It is critical to understand the mechanisms through which liraglutide (Lir) mediates a cardioprotective effect as many type 2 diabetic medications increase the risk of myocardial infarction and stroke. We have identified that Lir reduces proinflammatory immune cell populations and mediators from plaque-burdened murine aortas in vivo and augments proresolving bone marrow-derived macrophages in attenuation of atherosclerotic disease, which provides further insight into the atheroprotective effect of Lir.