Apoptosis Of Hematopoietic Cells Research Articles

Effects of PTPN6 Gene Knockdown in SKM-1 Cells on Apoptosis, Erythroid Differentiation and Inflammations.

Objective: Protein tyrosine phosphatase non-receptor type 6 (PTPN6) is a cytoplasmic phosphatase that acts as a key regulatory protein in cell signaling to control inflammation and cell death. In order to investigate the role of PTPN6 in hematologic tumor myelodysplastic syndrome (MDS), this study infected SKM-1 cell line (MDS cell line) with packaged H_PTPN6-shRNA lentivirus to obtain H_PTPN6-shRNA SKM-1 stable strain. The effect of PTPN6 knockdown on apoptosis, erythroid differentiation, and inflammations in SKM-1 cell line was examined. Methods: The stable knockdown SKM-1 cell line was validated using qPCR and Western blot assays. The proliferation activity, apoptosi, erythroid differentiation, and inflammatory cytokines in SKM-1 cells were assessed before and after transfection. Results: qPCR confirmed that the expression level of H_PTPN6-shRNA in SKM-1 cells was significantly reduced, and Western blot showed that the protein expression level of H_PTPN6-shRNA in SKM-1 cells was also significantly reduced. The CCK-8 cell viability assay confirmed that stable gene knockdown did not affect cell viability. Flow cytometry revealed that the apoptosis rate of cells in the PTPN6 knockdown group was 0.8%, lower than the 2.7% observed in the empty plasmid group; the expression rate of the erythroid differentiation marker CD235a was 13.2%, lower than the 25.0% observed in the empty plasmid group. The expression levels of the proinflammatory factors IL-6 and IL-8 increased, and the expression levels of the inhibitor factor IL-4 decreased. Conclusions: The PTPN6 gene was successfully knocked down using lentivirus-mediated transduction, and the constructed cell line was validated using PCR and Western blot. The CCK-8 cell viability assay confirmed that stable gene knockdown did not affect cell proliferation viability. Flow cytometry analysis of apoptosis and erythroid differentiation indicated that PTPN6 knockdown inhibits apoptosis and erythroid differentiation in SKM-1 cells and also alters the level of inflammations in the bone marrow microenvironment. It suggests that the PTPN6 gene acts as a tumor suppressor in myelodysplastic syndrome cells, influencing hematopoietic cell apoptosis, erythroid differentiation, and inflammations. This provides a reliable experimental basis for further in-depth studies on the mechanism of PTPN6 in MDS and related pharmacological research.

The Role of SIRT1 in Leukemia.

Leukemia is a type of hematological malignancy (HM) caused by uncontrolled proliferation, apoptosis, and differentiation of hematopoietic stem cells (HSCs). Leukemia cells proliferate greatly in the bone marrow (BM), infiltrate other tissues and organs, and affect the normal hematopoietic function. Although the emergence of new targeted agents and immune agents has improved the prognosis of patients, due to the complex pathogenic factors and heterogeneity of leukemia, there are still some patients with poor prognosis. Recent studies have shown that silent information regulator 1 (SIRT1) is involved in the proliferation, apoptosis, metabolism, and senescence of leukemia cells. As a double-edged sword in leukemia cells, SIRT1 can both promote and inhibit the growth of leukemia cells. Since its mechanism of action has not been elucidated, it is urgent to explore the regulatory mechanism of SIRT1 in leukemia. In this review, we discussed the mechanisms of SIRT1 in different aspects of leukemia, providing a theoretical basis for the treatment of patients with leukemia.

Chinese herbal formula, modified Guilu Erxian glue, alleviates apoptosis of hematopoietic stem cells by regulating SLAM-SAP signal pathway in aplastic anemia mice model

Ethnopharmacological relevanceGuilu Erxian Glue (GEG) and Danggui Buxue Tang (DBT) are traditional Chinese herbal formulas. According to the theory of traditional Chinese medicine, the combination of those two formulas (Modified Guilu Erxian Glue, MGEG) has the effects of tonifying the kidney and producing blood, was usually used to treat bone marrow failure diseases, including aplastic anemia (AA). Aim of the studyT lymphocytes play a crucial role in the disease pathogenesis and progression of AA. Our preliminary results confirmed that GEG can improve the damage of hematopoietic stem cells in mice, while DBT can reduce the proliferation and differentiation of T lymphocytes and inhibit the production of IFN-γ. We hypothesized that the combination of those two herbal formulas could inhibit immune attack and restore hematopoietic function through multiple mechanisms. In this study, we aim to study the curative effect of MGEG on regulating the expression of Signal lymphocyte activating molecule (SLAM), an activation-related molecule in T lymphocytes, thereby suppressing the immune function of T cells and decelerating the damage to hematopoietic stem cells. Materials and methodsHigh-performance liquid chromatography-electrospray ionization/mass spectrometry system was used to identify the components of the MGEG formulation. Induction of aplastic anemia mouse model by injecting allogeneic lymphocyte suspension into BABL/c mice after ionizing radiation. Cyclosporine A (CsA) was used as a positive control drug. Flow cytometry was used to detect the number and apoptosis rate of hematopoietic stem cells in the bone marrow. Enzyme-linked immunosorbent assay was performed to measure the levels of IFN-γ and TNF-α. Immunofluorescence staining was used to assess the expression of T-bet and SLAM-SAP. Western Blot was conducted to examine the expression of activation-related molecules in T lymphocytes and proteins related to the Fas signal pathway. Hematoxylin-eosin staining was performed to observe pathological changes in the bone marrow tissue. Wright-Giemsa staining was utilized to evaluate alterations in the cellular composition and basic structure of the bone marrow cells (BMCs). Transmission electron microscopy was employed to observe changes in the structure and morphology of hematopoietic stem cells. The hematology analyzer was used to detect peripheral blood parameters. ResultsTwenty-three different components were identified in MGEG. After MGEG treatment, the expression levels of Fyn and SLAM-SAP binding were increased in AA mice, while the expression levels of T-bet were decreased and the secretion of IFN-γ was reduced significantly. Additionally, MGEG also could downregulate the protein levels of Fas, caspase-3, and cleaved caspase-3 in AA mice. ConclusionMGEG could attenuate the production of IFN-γ by promoting the SLAM-SAP signal pathway to regulate the generation and distribution of T-bet in T cells. Additionally, it suppresses apoptosis of HSCs through intervention in the Fas-dependent pathway, thereby mitigating immune-mediated damage to HSCs.

In Fanconi anemia, impaired accumulation of bone marrow neutrophils during emergency granulopoiesis induces hematopoietic stem cell stress

Fanconi Anemia (FA) is an inherited disorder of DNA-repair due to mutation in one of 20+ interrelated genes that repair intra-strand DNA crosslinks and rescue collapsed or stalled replication forks. The most common hematologic abnormality in FA is anemia, but progression to bone marrow failure (BMF), clonal hematopoiesis, or acute myeloid leukemia (AML) may also occur. In prior studies, we found that Fanconi DNA-repair is required for successful emergency granulopoiesis; the process for rapid neutrophil production during the innate immune response. Specifically, Fancc-/- mice did not develop neutrophilia in response to emergency granulopoiesis stimuli, but instead exhibited apoptosis of bone marrow hematopoietic stem cells (HSCs) and differentiating neutrophils. Repeated emergency granulopoiesis challenges induced BMF in most Fancc-/- mice, with AML in survivors. In contrast, we found equivalent neutrophilia during emergency granulopoiesis in Fancc-/-Tp53+/- mice and wild type (WT) mice, without BMF. Since termination of emergency granulopoiesis is triggered by accumulation of bone marrow neutrophils, we hypothesize neutrophilia protects Fancc-/-Tp53+/- bone marrow from the stress of a sustained inflammation that is experienced by Fancc-/- mice. In the current work, we found that blocking neutrophil accumulation during emergency granulopoiesis led to BMF in Fancc-/-Tp53+/- mice, consistent with this hypothesis. Blocking neutrophilia during emergency granulopoiesis in Fancc-/-Tp53+/- mice (but not WT) impaired cell cycle checkpoint activity, also found in Fancc-/- mice. Mechanisms for loss of cell cycle checkpoints during infections challenges may define molecular markers of FA progression, or suggest therapeutic targets for bone marrow protection in this disorder.

Establishment of human hematopoietic organoids for evaluation of hematopoietic injury and regeneration effect

BackgroundHuman hematopoietic organoids have a wide application value for modeling human bone marrow diseases, such as acute hematopoietic radiation injury. However, the manufacturing of human hematopoietic organoids is an unaddressed challenge because of the complexity of hematopoietic tissues.MethodsTo manufacture hematopoietic organoids, we obtained CD34+ hematopoietic stem and progenitor cells (HSPCs) from human embryonic stem cells (hESCs) using stepwise induction and immunomagnetic bead-sorting. We then mixed these CD34+ HSPCs with niche-related cells in Gelatin-methacryloyl (GelMA) to form a three-dimensional (3D) hematopoietic organoid. Additionally, we investigated the effects of radiation damage and response to granulocyte colony-stimulating factor (G-CSF) in hematopoietic organoids.ResultsThe GelMA hydrogel maintained the undifferentiated state of hESCs-derived HSPCs by reducing intracellular reactive oxygen species (ROS) levels. The established hematopoietic organoids in GelMA with niche-related cells were composed of HSPCs and multilineage blood cells and demonstrated the adherence of hematopoietic cells to niche cells. Notably, these hematopoietic organoids exhibited radiation-induced hematopoietic cell injury effect, including increased intracellular ROS levels, γ-H2AX positive cell percentages, and hematopoietic cell apoptosis percentages. Moreover, G-CSF supplementation in the culture medium significantly improved the survival of HSPCs and enhanced myeloid cell regeneration in these hematopoietic organoids after radiation.ConclusionsThese findings substantiate the successful manufacture of a preliminary 3D hematopoietic organoid from hESCs-derived HSPCs, which was utilized for modeling hematopoietic radiation injury and assessing the radiation-mitigating effects of G-CSF in vitro. Our study provides opportunities to further aid in the standard and scalable production of hematopoietic organoids for disease modeling and drug testing.

Abstract 5014: Analytical validation of a complete system for detection and characterization of genomic aberrations in circulating tumor DNA

Abstract Cell-free DNA (cfDNA) fragments are readily detected at a low concentration in the human blood stream. In healthy patients cfDNA is primarily derived from apoptosis of hematopoietic cells. In cancer patients circulating DNA derived from tumor cells (ctDNA) undergoing apoptosis, shedding, and necrosis contribute to total cfDNA. Next generation sequencing (NGS) of cfDNA isolated from patient plasma can be used to fully characterize cancer driven changes in the tumor DNA including the relative contribution of cfDNA, single nucleotide variants (SNVs), small insertions and deletions (Indels), larger deletions, regions of amplified DNA, and structural rearrangements. Frontage has developed a complete lab system for analysis of circulating cfDNA including automated sample extraction, preparation and sequencing of libraries targeting exon sequences of 293 oncology-related genes, and a bioinformatic reporting system. The Frontage sample extraction process uses a post-extraction size selection step to remove high molecular weight DNA derived from lysed blood cells in the plasma sample to increase the sensitivity of detection of ctDNA. The NGS platform couples hybridization-based selection of genomic DNA fragments derived from protein-coding exons with deep sequencing to ~4,000X gross coverage on the NextSeq 2000. The bioinformatic pipeline integrates unique molecular barcode-based generation of consensus reads for each template molecule, somatic variant calling with GATK Mutect2 and FreeBayes, and removal of false positive variants by comparison to a 60 samples healthy patient data set. Analytical validation was performed for each component of the system. For verification of the sample extraction process, cfDNA was extracted in duplicate from four lots of pooled K2 EDTA plasma of healthy patients as well as individual K2 EDTA plasma of colorectal cancer (CRC) patients in four independent extraction runs. The mean yields of cfDNA from healthy samples ranged from 2.3 - 4.2 nanograms per ml plasma and all 32 of the isolated cfDNA aliquots from healthy patient plasma showed a prominent peak around 167 nt with minimal contamination from high molecular weight DNA. For validation of the NGS and bioinformatics platform a set of 8 different reference cfDNAs were processed through four independent runs of library preparation, sequencing, and analysis. For each of the validation runs, greater than 97% of 208 SNVs and Indels with expected allele frequencies (AF) between 2-3% were detected by the platform with higher levels of accuracy for variants at above 3% AF. Two or less false positive variants per run were detected out of 278,000 nucleotides of verified non-mutated sequence that overlapped with the 293 gene panel. Four known DNA amplification events were detected in each of the validation runs. Data from characterization of CRC and other clinical samples with the system will be presented. Citation Format: Ericca Stamper, Weiming Shen, Aman Pruthi, Casey Nagel, Marcin Piechota, Katarzyna Tomala, Qi Wei, David Willoughby. Analytical validation of a complete system for detection and characterization of genomic aberrations in circulating tumor DNA [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5014.

Landscape of FLT3 Variations Associated with Structural and Functional Impact on Acute Myeloid Leukemia: A Computational Study.

Acute myeloid leukemia (AML) is hallmarked by the clonal proliferation of myeloid blasts. Mutations that result in the constitutive activation of the fms-like tyrosine kinase 3 (FLT3) gene, coding for a class III receptor tyrosine kinase, are significantly associated with this heterogeneous hematologic malignancy. The fms-related tyrosine kinase 3 ligand binds to the extracellular domain of the FLT3 receptor, inducing homodimer formation in the plasma membrane, leading to autophosphorylation and activation of apoptosis, proliferation, and differentiation of hematopoietic cells in bone marrow. In the present study, we evaluated the association of FLT3 as a significant biomarker for AML and tried to comprehend the effects of specific variations on the FLT3 protein's structure and function. We also examined the effects of I836 variants on binding affinity to sorafenib using molecular docking. We integrated multiple bioinformatics tools, databases, and resources such as OncoDB, UniProt, COSMIC, UALCAN, PyMOL, ProSA, Missense3D, InterProScan, SIFT, PolyPhen, and PredictSNP to annotate the structural, functional, and phenotypic impact of the known variations associated with FLT3. Twenty-nine FLT3 variants were analyzed using in silico approaches such as DynaMut, CUPSAT, AutoDock, and Discovery Studio for their impact on protein stability, flexibility, function, and binding affinity. The OncoDB and UALCAN portals confirmed the association of FLT3 gene expression and its mutational status with AML. A computational structural analysis of the deleterious variants of FLT3 revealed I863F mutants as destabilizers of the protein structure, possibly leading to functional changes. Many single-nucleotide variations in FLT3 have an impact on its structure and function. Thus, the annotation of FLT3 SNVs and the prediction of their deleterious pathogenic impact will facilitate an insight into the tumorigenesis process and guide experimental studies and clinical implications.

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.

Fluacrypyrim Protects Hematopoietic Stem and Progenitor Cells against Irradiation via Apoptosis Prevention.

Ionizing radiation (IR)-induced hematopoietic injury has become a global concern in the past decade. The underlying cause of this condition is a compromised hematopoietic reserve, and this kind of hematopoietic injury could result in infection or bleeding, in addition to lethal mishaps. Therefore, developing an effective treatment for this condition is imperative. Fluacrypyrim (FAPM) is a recognized effective inhibitor of STAT3, which exhibits anti-inflammation and anti-tumor effects in hematopoietic disorders. In this context, the present study aimed to determine whether FAPM could serve as a curative agent in hematopoietic-acute radiation syndrome (H-ARS) after total body irradiation (TBI). The results revealed that the peritoneally injection of FAPM could effectively promote mice survival after lethal dose irradiation. In addition, promising recovery of peripheral blood, bone marrow (BM) cell counts, hematopoietic stem cell (HSC) cellularity, BM colony-forming ability, and HSC reconstituting ability upon FAPM treatment after sublethal dose irradiation was noted. Furthermore, FAPM could reduce IR-induced apoptosis in hematopoietic stem and progenitor cells (HSPCs) both in vitro and in vivo. Specifically, FAPM could downregulate the expressions of p53-PUMA pathway target genes, such as Puma, Bax, and Noxa. These results suggested that FAPM played a protective role in IR-induced hematopoietic damage and that the possible underlying mechanism was the modulation of apoptotic activities in HSCs.

Unraveling Hematotoxicity of α-Amanitin in Cultured Hematopoietic Cells.

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

MITOL deficiency triggers hematopoietic stem cell apoptosis via ER stress response.

Hematopoietic stem cell (HSC) divisional fate and function are determined by cellular metabolism, yet the contribution of specific cellular organelles and metabolic pathways to blood maintenance and stress-induced responses in the bone marrow remains poorly understood. The outer mitochondrial membrane-localized E3 ubiquitin ligase MITOL/MARCHF5 (encoded by the Mitol gene) is known to regulate mitochondrial and endoplasmic reticulum (ER) interaction and to promote cell survival. Here, we investigated the functional involvement of MITOL in HSC maintenance by generating MX1-cre inducible Mitol knockout mice. MITOL deletion in the bone marrow resulted in HSC exhaustion and impairment of bone marrow reconstitution capability in vivo. Interestingly, MITOL loss did not induce major mitochondrial dysfunction in hematopoietic stem and progenitor cells. In contrast, MITOL deletion induced prolonged ER stress in HSCs, which triggered cellular apoptosis regulated by IRE1α. In line, dampening of ER stress signaling by IRE1α inihibitor KIRA6 partially rescued apoptosis of long-term-reconstituting HSC. In summary, our observations indicate that MITOL is a principal regulator of hematopoietic homeostasis and protects blood stem cells from cell death through its function in ER stress signaling.

Combining Allogeneic Gamma Delta T Cells with a Phosphoantigen Prodrug Promotes Apoptosis of Acute Myeloid Leukemia Stem and Progenitor Cells

Despite recent therapeutic advances, patients with relapsed/refractory acute myeloid leukemia (r/r AML) have limited treatment options and poor prognosis. Leukemic stem cells (LSCs) are considered as relapse-initiating cells and the “culprit” of treatment resistance. Allogeneic and autologous gamma delta (γδ) T cell-based therapies have been explored for a long time and proven to be safe in patients through multiple clinical trials. However, quite a few AML blasts obtained from r/r AML patients show resistance to γδ T cell-mediated cytotoxicity. Novel strategies targeted LSCs may improve efficacy of γδ T cell-mediated r/r AML therapy. Given that γδ T cells exert antitumor capacity via multiple pathways, to identify the way matters most in the resistance of γδ T cell-mediated cytotoxicity, we examined the median fluorescence intensity (MFI) of the ligands expressed in tumor cells by flow cytometry, including butyrophilin 3A (BTN3A), butyrophilin 2A1 (BTN2A1), the human MHC class I chain-related genes (MICA and MICB), the unique-long 16 binding proteins (ULBP1, ULBP2/5/6, ULBP3), Fas, the receptor of TNF-related apoptosis-inducing ligand (TRAIL) DR5, CD112 and CD155. The cytotoxicity activity of γδ T cells was measured by flow cytometry-based killing assay or bright-lite luciferase assay system after coculturing with AML cell lines and blasts at an appropriate effector to target (E:T) ratio. Then we employed a Support Vector Machine model to predict the killing efficiency and identified that BTN3A and BTN2A1 play the predominant role in γδ T cell-mediated cytotoxicity based on the Shapley additive explanation. BTN3A and BTN2A1 are the key ligands in the γδ TCR-phosphoantigen (pAg) recognition activity. PAgs, which are produced in tumor cells function as “molecular glues” to promote heteromeric association of BTN3A1 and BTN2A1 (Yuan L et al., BioRxiv 2022). To promote the γδ T cell-mediated cytotoxicity targeting r/r AML, here we synthesized a pAg prodrug, which show great plasma stability and membrane permeability. We anticipate this small molecule would lead pAg accumulation in tumor cells and cause increased γδ T cell activation. To identify the killing efficacy of pAg prodrug-induced γδ T cell activation, we select KG-1α (a human leukemia stem cell-like cell) as target cell. Zoledronate (ZOL), which is employed to stimulate γδ T cells in multiple γδ T cell-based clinical trials, is used in our killing assay as compared group. As expected, combining γδ T cells with pAg prodrug or ZOL both significantly improved the percent of specific killing when the E:T ratio is 1:1. The pAg prodrug showed more potent activity than ZOL group with EC50 value of 0.1 nM vs 17.93μM. And the specific killing percent of pAg prodrug group is 83.26±2.1 % (1 nM), comparing with negative control 15.17±3.4 % (only γδT cells). Futhermore, at relatively low E:T ratio (1:4), γδ T cells with pAg prodrug remain excellent cytotoxicity when targeting MV411 cell and OCI-AML3 cell, which are widely used in r/r AML research. After coculturing with primary leukemia stem and progenitor cells (CD34 +) obtained from r/r AML patients for 6~8 h, the apoptosis percentage of CD34 + leukemia cells was 42.34% comparing with negative control (only γδT cells) 2.53%. Then we evaluated the in vivo efficacy of combining γδT cells and pAg prodrug in NSG mice bearing MV411 cells. Compared with only γδT cells-treated group, combination with pAg prodrug presented better tumor clearing. To elucidate the pathway of pAg prodrug-mediated killing, we tested mitochondria apoptosis related events. After treated by γδ T cells with pAg prodrug, KG-1α cells showed loss of mitochondrial outer membrane potential and increasingly actived caspase 3/7. Meanwhile, pAg prodrug directly activates effector γδ T cells with increased secretion of CD107a and IFN-γ. Considering the possibility of toxicity towards normal hematopoietic stem and progenitor cells, we conducted colony-forming units (CFUs) assay. After treatment with γδT cells and pAg prodrug, the number of CFUs of CD34 + cells from cord blood was not effected compared with the CD34 + cells only group. Overall, we explored the predominant factor in γδ T cell-mediated killing and synthesized a pAg prodrug to improve the killing efficacy. We demonstrated that combining γδ T cells with pAg prodrug has effectively promoted apoptosis of AML stem and progenitor cells sparing normal hematopoietic stem and progenitor cells.

Aplastic anemia from past to the present: A bibliometric analysis with research trends and global productivity during 1980 to 2022.

There is no bibliometric study in the literature on Aplastic Anemia (AA). In the present study, the purpose was to summarize the intellectual structure of the subject, uncover the global productivity in this respect, and identify the latest research trends by performing a bibliometric analysis of the articles published on AA. For this purpose, outputs for different research components of scientific outputs (i.e., countries, institutions, journals, and authors) were analyzed. A total of 3221 articles on Aplastic Anemia published between 1980 and 2022 were analyzed by using various statistical methods and bibliometric approaches. The Spearman Correlation Coefficient was used for correlation analysis and bibliometric network visualization maps were used to identify trending topics, citation analysis, and international collaborations. The top 3 contributing countries to the literature were the USA in this respect (800, 24.8%), China (514, 15.9%), and Japan (442, 13.7%). The top 3 most active institutions were the National Institutes of Health USA (n = 177), National Heart Lung Blood Institute (n = 153), and Udice French Research Universities (n = 136). The top 3 most productive journals were the British Journal of Haematology (n = 239), Blood (n = 181), and Bone Marrow Transplantation (n = 137). The most prolific author was Neal Stuart Young (n = 130). Specific keywords that were most frequently used in articles were severe aplastic anemia, immunosuppressive therapy, pediatrics/children, anti-thymocyte globulin, cyclosporine, hematopoietic stem cell transplantation, myelodysplastic syndromes, Paroxysmal Nocturnal Hemoglobinuria, hepatitis-associated aplastic anemia, allogeneic stem cell transplantation, haploidentical hematopoietic stem cell transplantation, pancytopenia, eltrombopag, fludarabine, Graft-Versus-Host Disease, survival, apoptosis, cytokines, and cyclophosphamide. It was determined that the trend topics in recent years were eltrombopag, COVID-19, Treg, Th17, thrombopoietin receptor agonists, haploidentical hematopoietic stem cell transplantation, haploidentical donor/transplantation, and posttransplantation cyclophosphamide. In the formation of the AA literature, it was determined that the research leadership belonged to the USA, China, Japan, European countries (United Kingdom, Italy, Germany, France, Switzerland), India, and South Korea, which have large economies.

The Molecular and Genetic Mechanisms of Inherited Bone Marrow Failure Syndromes: The Role of Inflammatory Cytokines in Their Pathogenesis.

Inherited bone marrow failure syndromes (IBMFSs) include Fanconi anemia, Diamond-Blackfan anemia, Shwachman-Diamond syndrome, dyskeratosis congenita, severe congenital neutropenia, and other rare entities such as GATA2 deficiency and SAMD9/9L mutations. The IBMFS monogenic disorders were first recognized by their phenotype. Exome sequencing has validated their classification, with clusters of gene mutations affecting DNA damage response (Fanconi anemia), ribosome structure (Diamond-Blackfan anemia), ribosome assembly (Shwachman-Diamond syndrome), or telomere maintenance/stability (dyskeratosis congenita). The pathogenetic mechanisms of IBMFSs remain to be characterized fully, but an overarching hypothesis states that different stresses elicit TP53-dependent growth arrest and apoptosis of hematopoietic stem, progenitor, and precursor cells. Here, we review the IBMFSs and propose a role for pro-inflammatory cytokines, such as TGF-β, IL-1β, and IFN-α, in mediating the cytopenias. We suggest a pathogenic role for cytokines in the transformation to myeloid neoplasia and hypothesize a role for anti-inflammatory therapies.

Upregulation of S100A6 and its relation with CD34+ cells apoptosis in high-risk myelodysplastic syndromes patients

ObjectivesMyelodysplastic syndromes (MDS) are a group of myeloid malignancies characterized by peripheral blood cytopenia and hematopoietic dysplasia that often progress to acute myeloid leukemia (AML). Increased apoptosis of normal hematopoietic cells and decreased apoptosis of malignant clonal hematopoietic cells in patients with MDS is some of the mechanisms leading to ineffective hematopoietic cells in the bone marrow. S100 calcium-binding protein A6 (S100A6) is upregulated in many malignancies. The overexpression of S100A6 in these malignancies has been associated with proliferation, migration, and invasion phenotypes in cancer cells, and we aimed to investigate the expression of S100A6 in CD34+ cells and the relationship between S100A6 expression and apoptosis of CD34+ cells in high-risk patients with MDS. MethodsWe measured S100A6 mRNA expression in bone marrow (BM) CD34+ cells from high-risk patients with MDS using RT-PCR. Next, we examined S100A6 expression in CD34+ cells using flow cytometry. We also analyzed the correlation between CD34+ cell apoptosis and S100A6 expression in high-risk patients with MDS. ResultsOur data showed increased S100A6 mRNA expression in CD34+ cells in patients with MDS (1.05 ± 0.69 vs. 0.17 ± 0.12; P＜0.01). The expression of S100A6 in BM CD34+ cells also increased (58.40 ± 13.18 vs. 45.83 ± 15.01). The expression of S100A6 in CD34+ cells and apoptosis of CD34+ cells were negatively correlated in patients (r = −0.75; P < 0.01). ConclusionsCollectively, S100A6 may be a potential marker of CD34+ cells in high-risk patients with MDS and may participate in the pathological behaviors of CD34+ cells, such as evasion of apoptosis. Thus, S100A6 may be a potential target for eliminating minimal residual disease.

Mitigating the Risk of t-MNs Development: TP53 or Not TP53?

Therapy-related myeloid neoplasms (t-MNs) are severe consequences of cytotoxic therapies used to treat solid tumors, nonmyeloid hematological cancers, or autoimmune disorders.1 They consist of acute myeloid leukemia (AML) or myelodysplastic syndromes (MDS) and associate to poor prognostic features. The majority of t-MNs are believed to arise from pre-existing mutant hematopoietic stem cell (HSC) clones which do not undergo apoptosis in response to cytotoxic therapies and are thus favored under this selective pressure. Albeit numerous evidence demonstrated that the clonal hematopoiesis of indeterminate potential (CHIP) constitutes the soil for t-MNs development, it remains to be fully elucidated how individual therapies shape the selection and the evolution of specific mutant HSC clones. Understanding this aspect could enable modulating therapeutic regimens based on their effects on pre-existing mutant HSCs and thus reducing the risk of t-MNs development. In a recent Blood article, Sperling and colleagues provided interesting clinical and experimental evidence to tackle this aspect.2 The researchers first performed a mutational analysis of 416 patients diagnosed with t-MNs, including 40% of AML cases and 60% of MDS patients. This revealed the predominance of mutations in genes involved in DNA damage responses such as TP53 and PPM1D, which were followed by common CHIP-associated mutations such as TET2, DNMT3A, ASL1, and SRF2. PPM1D and TP53 mutations occurred more frequently in t-MNs compared to AML-MDS cases without prior exposure to chemoradiation therapies. TP53 mutations, moreover, significantly associated to prior exposure to lenalidomide, hence suggesting that this drug could directly promote the development of t-MNs by selecting TP53 mutant HSCs. To investigate this hypothesis, the authors performed competitive transplant assays using a murine model deficient for Trp53 and sensitive to thalidomide analogs such as lenalidomide and pomalidomide. This revealed an outgrowth of Trp53 knock-out HSCs over wild-type cells, which occurred only following exposure to lenalidomide but not to pomalidomide. To explain these findings on a mechanistic level, the authors built on their previous findings, reasoning that lenalidomide selective toxicity on wild-type HSCs could derive from its exclusive ability to degrade casein kinase alpha (CK1α), a phenomenon that triggers p53-mediated apoptosis.3 Supporting this hypothesis, HSCs haploinsufficient for Csnk1a (the gene coding for CK1α) were out-competed by wild-type cells in competitive transplant assays when exposed to lenalidomide. However, they did not show a competitive disadvantage upon treatment with pomalidomide or iberdomide, 2 thalidomide analogs which do not degrade CK1α. These data suggest an interesting model linking lenalidomide and t-MNs bearing TP53 mutations. In this model (Figure 1), CK1α degradation, selectively induced by lenalidomide but not by other thalidomide analogs, induces p53-mediated apoptosis in wild-type cells but not in TP53 mutant HSCs, hence positively selecting them. Although the researchers provided some experimental evidence excluding the possibility that lenalidomide could select for HSCs bearing other CHIP-associated mutations (ie, Dnmt3A, Tet2, Asxl, Pmp1d, Ezh2), it remains to be investigated whether this holds true for other thalidomide analogs. Further investigating these aspects will be important to define whether the careful choice of thalidomide analogs in chemotherapy regimens may allow to mitigate the risk of developing t-MNs.Figure 1.: By degrading CK1α, lenalidomide induces p53-dependent apoptosis in wild-type hematopoietic stem cells but not in TP53 mutant clones, hence favoring their positive selection and, ultimately, the development of t-MNs. CK1α = casein kinase alpha; t-MNs = therapy-related myeloid neoplasms.As the patients analyzed in Sperling’s study included multiple myeloma cases, the vast majority of which were exposed to both lenalidomide and proteasome inhibitors, one may argue that the expansion of TP53 mutant HSC clones may result from the combined exposure of both drugs. The clinical and experimental evidence provided by the authors, however, do not support this possibility. On one side, the multivariate analysis the authors performed to adjust for the confounding effects of multiple exposure confirmed the significant association of TP53 mutation and lenalidomide exposure. On the other side, in vitro long-term competitive experiments using an immortalized murine hematopoietic stem/progenitors line showed that proteasome inhibitors such as bortezomib or carfilzomib conferred only a mild proliferative advantage to Trp53 mutant cells in contrast to the pronounced proliferative advantage provided by lenalidomide exposure. To conclude, the study by Sperling and colleagues provided an interesting molecular and functional characterization of the interplay linking patients genotype and specific cytotoxic drugs in driving the selection of premalignant clones. This has important consequences as it suggest at least 2 strategies to possibly reduce the risk of developing t-MNs, namely screening patients undergoing lenalidomide-based therapies for TP53 mutant CHIP and/or adapting chemotherapy regimens by choosing the appropriate thalidomide analog. DISCLOSURES The author has no conflicts of interest to disclose.

Apoptosis of Hematopoietic Stem Cells Contributes to Bone Marrow Suppression Following Chimeric Antigen Receptor T Cell Therapy

Chimeric antigen receptor (CAR) T cell (CAR-T) therapy represents a revolutionary treatment for patients with relapsed/refractory hematologic malignancies. However, its use can result in significant toxicities, including cytokine release syndrome (CRS), a potentially life-threatening clinical syndrome resulting from the release of proinflammatory cytokines upon T cell activation. In addition, patients who develop CRS often experience prolonged cytopenias, and those with the most severe CRS also have the longest delays in full marrow recovery. Although an association between CRS and delayed bone marrow recovery has been established, the precise mechanism underlying this phenomenon remains unknown. This study was conducted to test our hypothesis that delayed bone marrow recovery following CAR-T therapy is caused by elevation of proinflammatory cytokines, leading to apoptosis and depletion of hematopoietic stem and progenitor cells (HSPCs). SCID-beige mice bearing intraperitoneal CD19+ Raji cell tumors were treated with injection of human CD19.28z CAR T cells. Bone marrow was then harvested for analysis by flow cytometry, and HSPCs were isolated for whole-transcriptome analysis by RNA sequencing. Complete blood counts and serum cytokine levels were measured as well. A second model was developed in which SCID-beige mice were treated with murine IFN-γ (mIFN-γ), murine IL-6 (mIL-6), or both. Bone marrow was harvested, and flow cytometry assays were conducted to evaluate the degree of apoptosis and proliferation on specific HSPC populations. SCID-beige mice bearing intraperitoneal Raji cell tumors that were treated with CAR-T therapy developed CRS, with elevations of several proinflammatory cytokines, including profound elevation of human IFN-γ. Gene set enrichment analysis of RNA sequencing data revealed that genes associated with apoptosis were significantly upregulated in HSPCs from mice that developed CRS. Endothelial protein C receptor (EPCR)-negative HSCs, a subset of HSCs that is poised for terminal differentiation, was found to be specifically decreased in mice that were treated with CAR T cells. Furthermore, HSPCs were found to have increased levels of apoptosis upon treatment with mIFN-γ and mIL-6, whereas short-term HSCs and multipotent progenitors exhibited increases in proliferation with mIFN-γ treatment alone. The results from this study provide evidence that the elevation of proinflammatory cytokines following CAR-T therapy impacts the bone marrow through a combined mechanism: pluripotent HSCs that are exposed to elevated levels of IFN-γ and IL-6 undergo increased cell death, while more committed progenitor cells become more proliferative in response to elevated IFN-γ. These combined effects lead to depleted stores of repopulating HSCs and ultimately cytopenias. © 2023 American Society for Transplantation and Cellular Therapy. Published by Elsevier Inc.

Biomimetic Prussian blue nanozymes with enhanced bone marrow-targeting for treatment of radiation-induced hematopoietic injury

There is an urgent medical need to develop effective therapies that can ameliorate damage to the radiation-exposed hematopoietic system. Nanozymes with robust antioxidant properties have a therapeutic potential for mitigating radiation-induced hematopoietic injury. However, enhancing nanozyme recruitment to injured tissues in vivo while maintaining their catalytic activity remains a great challenge. Herein, we present the design and preparation of a biomimetic nanoparticle, a mesenchymal stem cell membrane camouflaged Prussian blue nanozyme (PB@MSCM), which exhibits biocompatible surface properties and demonstrates enhanced injury site-targeting towards the irradiated murine bone marrow niche. Notably, the constructed PB@MSCM possessed redox enzyme-mimic catalytic activity and could scavenge overproduced reactive oxygen species in the irradiated bone marrow cells, both in vitro and ex vivo. More importantly, the administration of PB@MSCM significantly mitigated hematopoietic cell apoptosis and accelerated the regeneration of hematopoietic stem and progenitor cells. Our findings provide a new targeted strategy to improve nanozyme therapy in vivo and mitigate radiation-induced hematopoietic injury.

Evaluation of Unrestricted Somatic Stem Cells Therapy in an Ex-Vivo and in-Vivo Acquired Severe Aplastic Anemia Model

Background: In acquired severe aplastic anemia (aSAA), oligoclonal expansion of dysregulated CD8+ cytotoxic T cells, abnormal function of CD4+ T helper cells, along with elevated production of IFN-γ and TNF-α have been associated with the apoptosis of hematopoietic stem and progenitor cells (HSPC) (Young, N Engl J Med, 2018). Liao and Cairo et al (Cell Transplantation, 2014) have demonstrated that cord blood-derived unrestricted somatic stem cells (USSCs) could significantly reverse the abnormal/inverted ratio of CD4:CD8 T cells, increases the number of FOXP3A positive Treg cells and decrease the level of IFN-γ in an animal model of recessive dystrophic epidermolysis bullosa (RDEB). Alternative therapies are in great need for patients with aSAA as immunosuppressive therapy (IST) overall responses are sub-optimal. Objectives:To determine the effect of USSCs on HSPC survival, differentiation and phosphorylation pathways in an ex-vivo culture of aSAA with human CD34+ cells.To determine the effect of USSCs administration on blood cell count recovery, bone marrow (BM) cellularity and survival in an in-vivo inflammatory cytokine mediated mice model of aSAA. Design/Methods: Human HSPC were cultured with SCF, FLT3 and human TPO. HSPCs alone, with and without IFN-γ and TNF-α served as controls. USSCs (transwell and direct) were added to the culture. HSPC were harvested and assayed for their survival at day 7 and 14. Annexin-V+/PI+ were referred to as late apoptotic cells. Each experimental condition was set up in triplicate. The cells were cultured at 37°C with 5% CO2. Multi-lineage differentiation capacity was assessed with a selective colony forming units (CFU) assay and compared between experimental groups. Signaling pathways were determined using phosphor-flow cytometric analysis of pSTAT1. For the immune mediated mouse model of aSAA, 8-week-old C57BL/6 mice were irradiated with 6.5Gy TBI. Spleen lymphocytes were collected from FVB donors, and injected 4 hours post-radiation into B6 recipient mice via the retro-orbital vein, at 1-1.5 × 106 cells/recipient in 100μL PBS. Each experimental group (PBS control and USSCs) included 20 mice per group. Mice were continuously monitored for their survival. Peripheral blood (PB) was analyzed using a VETSCAN® HM5 Analyzer. Mean ± SEM of the blood counts was compared between experimental groups. Mice femur bones were extracted and cleaned of soft tissue, fixed and sectioned. Paraffin sections were stained with Hematoxylin and Eosin (H&E) to assess morphology. Results: The myelosuppressive effect of IFN-γ and TNF-α was significantly rescued (4-fold increase) by the addition of USSCs (transwell) at day 7. After 28 days direct co-culture with USSCs increased CD34+ survival as well (p<0.001) from 5 to 35 cells/µl. A significantly higher percent of viable cells (73% versus 63%) was observed among the USSCs treated cells (P<0.05). Phosphor-flow cytometric analysis demonstrated an almost 50% decrease in pSTAT1 in the presence of USSCs (p<0.05). Based on CFU analysis, CD34+ cells cultured in the presence of USSCs were able to produce more CFU (52, SD±3.7) at day 7 (p<0.01) compared to IFN-γ and TNF-α treated cells (16, SD±2.8). Also, USSCs were associated with more BFU (p<0.05) at day 14. In our immune mediated aplastic anemia mice model, treatment with USSCs was associated with substantial prolonged mice survival (p=0.02) (Figure 1a). Evaluation of PB counts demonstrated increased hemoglobin levels at day 14 in the USSCs treated mice (p<0.05) 5.6g/dL (SD±0.6), compared to PBS treated mice 2.1g/dL (SD±0.1). BM evaluation of USSCs treated mice versus PBS control displayed higher cellularity in the USSCs treated sections (Figure 1b). Conclusions: Cellular therapy, such as USSCs, may be instrumental in the treatment of aSAA, specifically by increasing BFU-E and eventually hemoglobin levels. Future studies will need to be done to investigate the exact mechanisms of action and the clinical efficacy of this novel cellular therapy. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Apoptosis of Hematopoietic Stem Cells Contributes to Bone Marrow Suppression Following CAR-T Therapy

Apoptosis of Hematopoietic Stem Cells Contributes to Bone Marrow Suppression Following CAR-T Therapy