Apoptosis In CD34 Research Articles

CD34+ orbital fibroblasts contribute to the pathogenesis of thyroid eye disease via miR-182-5p.

CD34+ orbital fibroblasts (OFs) play a pathogenic role in thyroid eye disease (TED). Several micro (mi)RNAs have been shown to promote TED progression. This study aims to explore the regulatory effect of miRNAs on CD34+ OFs, to find potential therapeutic target. In this case-control study, orbital connective tissues (OCTs) and OFs were obtained from 25 TED patients and 24 healthy donors. MiRNA-seq was performed to examine differential expression of miRNAs in OCTs, and miR-182-5p was selected for subsequent experiments. MiR-182-5p was detected both in CD34+ and CD34- OFs. The upstream regulators of miR-182-5p were studied. Downstream targets of miR-182-5p were analyzed. The functionality of miR-182-5p and its target genes in CD34+ OFs was evaluated. MiR-182-5p was highly expressed in TED OCTs and their derived CD34+ OFs. TED OCTs displayed increased expression of interluekin (IL)-6, IL-17A, CD34, and phosphorylated STAT3 at Ser727 and Tyr705. Activation of IL-6/STAT3 signaling promoted the expression of miR-182-5p in CD34+ OFs. MiR-182-5p enhanced wound repair ability, proliferation, and RANTES expression while inhibiting apoptosis in CD34+ OFs. CD34+ OFs transfected with miR-182-5p were susceptable to TGF-β-initiated myofibroblast differentiation. Luciferase reporter and pull-down assays revealed Smad7 as the downstream target of miR-182-5p, which modulated the proliferation, migration, fibrosis, and apoptosis of CD34+ OFs. IL-6/STAT3/miR-182-5p pathway led to activation of CD34+ OFs. MiR-182-5p promoted the proliferation, migration, fibrosis, and anti-apoptosis of CD34+ OFs via targeting Smad7. Our findings suggest that miR-182-5p may potentially serve as a therapeutic target for TED.

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.

Skewed fate and hematopoiesis of CD34+ HSPCs in umbilical cord blood amid the COVID-19 pandemic.

Umbilical cord blood (UCB) is an irreplaceable source for hematopoietic stem progenitor cells (HSPCs). However, the effects of SARS-CoV-2 infection and COVID-19 vaccination on UCB phenotype, specifically the HSPCs therein, are currently unknown. We thus evaluated any effects of SARS-CoV-2 infection and/or COVID-19 vaccination from the mother on the fate and functionalities of HSPCs in the UCB. The numbers and frequencies of HSPCs in the UCB decreased significantly in donors with previous SARS-CoV-2 infection and more so with COVID-19 vaccination via the induction of apoptosis, likely mediated by IFN-γ-dependent pathways. Two independent hematopoiesis assays, a colony forming unit assay and a mouse humanization assay, revealed skewed hematopoiesis of HSPCs obtained from donors delivered from mothers with SARS-CoV-2 infection history. These results indicate that SARS-CoV-2 infection and COVID-19 vaccination impair the functionalities and survivability of HSPCs in the UCB, which would make unprecedented concerns on the future of HSPC-based therapies.

Preclinical Studies and Phase II Trial of Venetoclax in Combination with Chidamide and Azacitidine in Relapsed/Refractory Acute Myeloid Leukemia

Background: The treatment for relapsed and refractory acute myeloid leukemia (R/R AML) remains challenging and leukemia stem cells (LSCs) play a causal role in R/R AML. Recent progress in genomics and molecular biology has uncovered a spectrum of mutations in AML suggesting that translational research combined with targeted therapy for LSCs has greater guiding significance to improve outcome in R/R AML. Therefore, we used a high-throughput screening platform to identify a novel and effective therapeutic combination, which is venetoclax, chidamide, and azacitidine (VCA). Next, to assess the efficacy of VCA, we performed preclinical studies on LSCs-like cell lines and patient-derived xenograft (PDX) mouse model. In addition, a subsequent phase II study was conducted to test the safety and preliminary efficacy of this combination. Methods:Preclinical: We performed an automated drug screen to evaluate the drug sensitivity in CD34+CD38- KG-1α and Kasumi-1 cells simultaneously. Ultimately select the optimal combination therapy of venetoclax, chidamide, and azacitidine (VCA) by using a high-throughput platform. To validate the efficacy of the combination in vivo, we used primary CD34+ AML PDX model. Mice were treated with single, dual, or triple of venetoclax (VEN, 30 mg/kg, oral gavage), chidamide (CS055, 7.5 mg/kg, oral gavage), and azacitidine (5-AZA, 2 mg/kg, intraperitoneal injection) every 3 days for 12 days consecutively. Phase II: In a multi-center, single-arm, open-labeled phase II study, patients aged ≥18 years with R/R AML were treated with VEN at 200 mg orally daily for 14 days, CS055 at 30 mg orally twice per week for 14 days, and 5-AZA at 100 mg by continuous hypodermic injection (IH) daily for 7 days for up to two 28-day cycles. The primary objective was to assess the safety and efficacy of VCA. The primary efficacy measurement was overall response rate (ORR), which was the combined complete remission (CR), CR with incomplete hematologic recovery (CRi) and partial remission (PR). ResultsPreclinical: VCA exhibited synergistic effects on attenuating cell viability and inducing apoptosis in CD34+CD38- KG-1α and Kasumi-1 cells. Importantly, triple therapy intensively reduced leukemia burden and improved prognosis in a primary CD34+ AML derived PDX model without significant adverse effects versus single or double combination. Mechanistically, co-exposure to three agents led to the inhibition of anti-apoptotic BCL-2 family proteins (including BCL2, BCL-XL and MCL-1), associating with enhanced ubiquitination. Moreover, CS055-mediated HDAC inhibition increased F-box and WD repeat domain-containing protein 7 (FBW7) acetylation, thus resulting in FBW7 stabilization, which further enhanced the MCL-1 protein degradation. Phase II: As of July 30th, 2022, 29 patients were enrolled in the study. Patients had a median age of 51 years (range: 35-64). The median time on study was 31 (range: 4-not reached [NR]) days. 25 patients (86.2%) had confirmed responses. The ORR was 75% (12/16: 2 CR, 7 CRi and 3PR) with one complete and 88.9% (8/9: 5 CR, 2 CRi and 1 PR) with two completes. The most common non-hematologic adverse events (AE) were nausea (75%) and vomiting (25%), and no grade III or IV non-hematologic AE was observed. There was no tumor lysis syndrome (TLS) or treatment-discontinuation due to therapy. Conclusions: This novel combination strategy of VEN, CS055 and 5-AZA has shown synergy in AML LSCs-like cells and PDX model. The phase II data suggests that VCA as a chemo-free therapy exhibits promising efficacy and is safe and well-tolerated in patients with R/R AML.

PHF6 Maintains Acute Myeloid Leukemia Via Regulating NF-Κappab Signaling Pathway

Acute myeloid leukemia (AML) is a major hematopoietic malignancy characterized by the uncontrolled expansion of the immature myeloid cells in the hematopoietic system. Current chemotherapy regimens remain ineffective as assessed by low remission and 5-year-survival rate. Many efforts have been dedicated to decipher the molecular events underlying AML transformation. PHF6 is a conserved epigenetic regulator and has been found to be frequently altered in hematologic malignancies. Previous studies demonstrated that PHF6 plays a suppressive role in T-ALL leukemogenesis. However, the functional role(s) of PHF6 in AML initiation and progression remains unknown. Here in this study, we analyzed the mRNA expression of PHF6 in blood cells from healthy donors and leukemia cells from AML patients, and found that the PHF6 expression was significantly increased in AML cells when compared with that of normal PMNs (www.Bloodspot.com) (p < 0.05). Significantly, the average survival time of AML patients with high PHF6 expression was much shorter than that of AML patients with low PHF6 expression (p = 0.022). Overexpressing of PHF6 (PHF6 OE) in Kasumi-1 or K562 leukemia cell lines showed increased growth of Kasumi-1 and K562 cells, decreased apoptosis of Kasumi-1 cells. Additionally, we knocked down (KD) PHF6 expression in AML cells and CD34+ cord blood cells, and found that PHF6 KD significantly decreased the growth of Kasumi-1 and promoted cell apoptosis, while had little effects on apoptosis of CD34+ cord blood cells. These results suggested that PHF6 is required for the growth of myeloid leukemia cells but is dispensable for normal blood cells. To further evaluate the functional role of Phf6 in leukemogenesis in vivo, we generated Phf6 KO+RUNX1-ETO9a (RE9a) and Phf6 KO+MLL-AF9 (MA9) AML mice. We found the survival time of Phf6 KO+RE9a mice was significantly longer than that of WT Phf6+RE9a mice. The Phf6 KO+RE9a mice showed much milder disease phenotypes than WT Phf6+RE9a mice, including lower counts of GFP+ leukemia cells in the PB and BM, higher body weights, lower spleen and liver weights, and lower degree of extramedullary infiltration in spleen, liver, lung and brain. Extreme limiting dilution transplantation assays demonstrated a marked decrease in leukemia stem cell (LSC) activity in Phf6 KO+RE9a cells when compared with Phf6 WT+RE9a cells. Consistent with RE9a-induced AML mouse model, PHF6 deficiency impaired the self-renewal of LSCs and delayed the progression of MA9-induced AML, supporting a general oncogenic role of Phf6 in sustaining myeloid leukemia. To investigate the underlying molecular mechanisms of Phf6 in regulating AML development, we performed RNAseq to analyze the transcriptome programing changes associated with Phf6 deficiency in isogenic Phf6 WT+RE9a or Phf6 KO+RE9aAML cells. Gene set enrichment analysis (GSEA) showed up-regulation of apoptosis and down-regulation of NF-κB-regulated gene expressions in Phf6 KO+RE9a cells, suggesting that Phf6 depletion might promote AML cell apoptosis by blocking the NF-κB signaling. Immunocytochemistry studies showed that PHF6 depletion partially inhibited the nuclear translocation of p50. Co-immunoprecipitation experiments revealed that PHF6 directly bound to p50 in myeloid leukemia cells, suggesting that PHF6 might activate NF-κB signaling pathways by binding with p50 and maintaining the nuclear translocation of p50. To confirm this, we treated PHF6 OE AML cells with BAY11-7082 (a selective NF-κB inhibitor), and found BAY11-7082 significantly inhibited the growth of PHF6 OE Kasumi-1 cells in vitro. In summary, we revealed that PHF6 promoted RE9a- or MA9-induced AML progression in mice by activating NF-κB signaling pathway, and promoting P50 nuclear translocation. Our study thus defined a unique role for Phf6 in regulating NF-κB signaling pathway in AMLs, and potentially can be used as a therapeutic target for treating myeloid leukemia patients.

CDK4/CDK6 Inhibitors Synergize with Midostaurin, Avapritinib, and Nintedanib in Inducing Growth Inhibition in KIT D816V+ Neoplastic Mast Cells.

Simple SummaryAdvanced systemic mastocytosis (AdvSM) is a rare malignant disease with a poor prognosis due to the drug resistance of neoplastic mast cells. We found that drugs targeting the cell cycle regulators CDK4 and CDK6 profoundly suppress the growth and survival of neoplastic mast cells. Furthermore, these drugs can overcome resistance against KIT D816V-targeting drugs, including midostaurin, in neoplastic mast cells. Finally, the CDK4/CDK6 inhibitors applied induced apoptosis in CD34+/CD38− stem cells in AdvSM. Based on these results, we believe that CDK4/CDK6 inhibition may be a new and interesting therapeutic approach with curative potential for AdvSM. Whether combinations of KIT D816-targeting drugs and CDK4/CDK6 inhibitors can induce long-term remission in patients with AdvSM remains to be determined in clinical trials.In most patients with advanced systemic mastocytosis (AdvSM), neoplastic mast cells (MC) express KIT D816V. However, despite their disease-modifying potential, KIT D816V-targeting drugs, including midostaurin and avapritinib, may not produce long-term remissions in all patients. Cyclin-dependent kinase (CDK) 4 and CDK6 are promising targets in oncology. We found that shRNA-mediated knockdown of CDK4 and CDK6 results in growth arrest in the KIT D816V+ MC line HMC-1.2. The CDK4/CDK6 inhibitors palbociclib, ribociclib, and abemaciclib suppressed the proliferation in primary neoplastic MC as well as in all HMC-1 and ROSA cell subclones that were examined. Abemaciclib was also found to block growth in the drug-resistant MC line MCPV-1, whereas no effects were seen with palbociclib and ribociclib. Anti-proliferative drug effects on MC were accompanied by cell cycle arrest. Furthermore, CDK4/CDK6 inhibitors were found to synergize with the KIT-targeting drugs midostaurin, avapritinib, and nintedanib in inducing growth inhibition and apoptosis in neoplastic MCs. Finally, we found that CDK4/CDK6 inhibitors induce apoptosis in CD34+/CD38− stem cells in AdvSM. Together, CDK4/CDK6 inhibition is a potent approach to suppress the growth of neoplastic cells in AdvSM. Whether CDK4/CDK6 inhibitors can improve clinical outcomes in patients with AdvSM remains to be determined in clinical trials.

Chidamide and apatinib are therapeutically synergistic in acute myeloid leukemia stem and progenitor cells

BackgroundLeukemia stem cells (LSCs) are responsible for the initiation and perpetuation of acute myeloid leukemia (AML), and also represent leukemia relapse reservoirs with limited therapeutic approaches. Thus, additional treatment strategies are medical unmet needs to eliminate LSCs.MethodsCell counting kit-8 and Annexin-V-FITC/PI assays were used to examine the interaction of chidamide and apatinib on LSC-like cell lines (CD34+CD38− KG1α and Kasumi-1 cells) and primary CD34+ AML cells. AML patient-derived xenografts were established to investigate the in vivo efficacy of the combined regimen. RNA sequencing, Glutamine uptake assay, oxygen consumption assay, and western blotting were employed to explore the molecule mechanism for the cytotoxicity of chidamide with or without apatinib against LSC-like cell lines and/or primary CD34+ AML cells.ResultsIn this study, chidamide and apatinib were synergisitc to diminish cell viability and induce apoptosis in CD34+CD38− KG1α and Kasumi-1 cells and in CD34+ primary AML cells. Importantly, chidamide combined with apatinib had more powerful in reducing leukemia burden and improving prognosis than single drug alone in an AML PDX model without significant adverse effects. Chidamide cytotoxicity was associated with decreasing glutamine uptake. The therapeutic synergy of chidamide and apatinib correlated with reprogramming of energy metabolic pathways. In addition, inactivating the VEGFR function and reducing the anti-apoptotic ability of the Bcl2 family contributed to the synergism of chidamide and apatinib in CD34+CD38− KG1α cells and CD34+ primary AML cells.ConclusionChidamide in combination with apatinib might be a promising therapeutic strategy to get rid of the population of AML stem and progenitor cells, and thus provide a potentially curative option in the treatment of patients with AML, although further clinical evaluations are required to substantiate the conclusion.

An Unbalanced Monocyte Macrophage Polarization in the Bone Marrow Microenvironment of Patients with Poor Graft Function after Allogeneic Hematopoietic Stem Cell Transplantation

BackgroundPoor graft function (PGF) remains a life-threatening complication following allogeneic hematopoietic stem cell transplantation (allo-HSCT), and the underlying mechanisms have not yet been elucidated. Considerable evidence from murine studies has demonstrated that effective hematopoiesis depends on the specific bone marrow (BM) microenvironment, where hematopoietic stem cells (HSCs) reside. In this regard, we previously reported that PGF patients had impaired BM endosteal cells and endothelial progenitor cells, as well as dysregulated T cell responses in the BM microenvironment, which may be involved in the occurrence of PGF (BBMT 2013; BMT2016; Oncotarget 2016; BBMT 2016; Blood 2016; J Transl Med2017). Macrophage is one of the important components of BM immune microenvironment. Meanwhile, Murine studies suggest that macrophages regulate the retention of HSCs by regulating osteoblastic cell activity and nestin-expressing cells. Increasing evidence shows that BM resident macrophages are indispensable for HSCs function and BM erythroid output. However, little is known about the quantity and function of BM macrophages and whether the unbalanced BM macrophages directly interact with HSCs in PGF patients post allo-HSCT.AimsTo compare the number and function of BM macrophages between patients with PGF and GGF after allo-HSCT. Moreover, to investigate whether the unbalanced BM macrophages directly interact with HSCs in PGF patients.MethodsThis prospective nested case-control study enrolled 30 patients with PGF, 60 matched patients with good graft function (GGF), defined as persistent successful engraftment after allotransplant, and 30 healthy donors (HD). Standard monocyte subsets, defined by cluster of differentiation CD14 and CD16, were quantified by flow cytometry. In addition, based on the phenotype of polarized macrophages in vitro, classically activated inflammatory macrophage (M1:CD68+CCR2+) and alternatively activated anti-inflammatory macrophage (M2:CX3CR1+CD163+/CD206+) from BM samples were analyzed by flow cytometry. BM CD14+ monocytes were isolated from BM mononuclear cell (BMMNCs) and were cultured with 100 ng/ml recombinant human M-CSF for 10 days to obtain macrophages. The functions of BM derived macrophages were evaluated by migration assay and phagocytosis assay. CD34+ cells from healthy donors were co-cultured with BM derived macrophages from PGF patients, and the levels of reactive oxygen species (ROS) and apoptosis in CD34+ cells were analyzed by flow cytometry after 5 days co-culture.ResultsAlterations in standard monocyte subsets (classical, intermediate and non-classical) were found when comparing subjects among PGF, GGF and normal controls. In particular, elevated intermediate and non-classical monocyte subsets were observed in PGF patients when compared with those in GGF patients. Moreover, PGF patients displayed an imbalanced M1/M2 ratio compared with the GGF and normal controls, attributable to a reduction in M2 and an increase in M1. Dysfunctional BM derived macrophages, which were characterized by impaired proliferation, migration, phagocytosis and higher level of apoptosis, were revealed in patients with PGF. BM CD34+ cells from healthy donors were co-cultured respectively with BM derived macrophages from patients with PGF and normal controls. Apoptosis and intracellular levels of ROS were markedly increased in CD34+ bone marrow cells which were co-cultured with PGF patients' BM derived macrophages compared with normal controls.Summary/ConclusionIn summary, the current study demonstrated the abnormal number and impaired function of BM macrophages and unbalanced BM macrophages directly interact with HSCs in PGF patients. We speculate that reduction of anti-inflammatory M2 monocytes while increase of pro-inflammatory M1 monocytes in PGF patient BM have a negative impact on HSCs in BM microenvironment. Thus, our results indicate that unbalanced BM M1/M2 and dysfunctional BM macrophages may play an important role in the pathogenesis of PGF following allo-HSCT. Therefore, it would be of value to investigate the appropriate approach to balance the M1/M2 and enhance function of BM macrophages, which may be a promising therapeutic approach for PGF patients. DisclosuresNo relevant conflicts of interest to declare.

Up-regulation of miR-155 potentiates CD34+ CML stem/progenitor cells to escape from the growth-inhibitory effects of TGF-ß1 and BMP signaling.

microRNAs (miRNAs or miRs) play key roles in different stages of chronic myeloid leukemia (CML) pathogenesis. The present study aimed to demonstrate whether miR-155 enables CD34+ CML cells to escape from the growth-inhibitory effects of TGF-β1 and bone morphogenetic protein (BMP) signaling. Among differentially expressed miRNAs in CD34+ CML cells, miR-155 was highly up-regulated. QRT-PCR revealed an inverse correlation between miR-155 and two key members of the TGF-β pathway-TGF-βR2 and SMAD5. Results showed that SMAD5 is not only up-regulated through BMPs treatment, but recombinant TGF-β1 can also induce SMAD5 in CML cells. We also demonstrated that TGF-β1-mediated phosphorylation of SMAD1/5 was abolished by pre-treatment with the blocking TGF-βR2 antibody, suggesting a possible involvement of TGF-βR2. Additionally, overexpression of miR-155 significantly promoted the proliferation rate of CD34+ CML cells. Results showed that siRNA-mediated knockdown of SMAD5 had a promoting effect on CD34+ CML cell proliferation, suggesting that SMAD5 knock-down recapitulates the proliferative effects of miR-155. Importantly, TGF-β1 and BMP2/4 treatment had inhibitory effects on cell proliferation; however, miR-155 overexpression enabled CD34+ CML cells to evade the anti-proliferative effects of TGF-β1 and BMPs. Consistently, down-regulation of miR-155 augmented the promoting effects of TGF-β1 and BMP signaling on inducing apoptosis in CD34+ CML stem cells. Our findings demonstrated that targeting of SMAD5 and TGF-βR2 links miR-155 to TGF-β signaling in CML. Overexpression of miR-155 enables CD34+ CML cells to evade growth-inhibitory effects of the TGF-β1 and BMP signaling, providing new perspectives for miR-155 as a therapeutic target for CML.

A drug targeting 5-lipoxygenase enhances the activity of a JAK2 inhibitor in CD34+ bone marrow cells from patients with JAK2V617F-positive polycythemia vera in vitro.

Janus kinase 2 (JAK2) inhibitors, the first targeted treatments for myeloproliferative neoplasms (MPNs), provide substantial benefits, including a marked reduction in splenomegaly and MPN-associated symptoms. However, these drugs rarely induce molecular remission in patients with MPNs. Zileuton, a 5-lipoxygenase (5-LO) inhibitor, has been demonstrated to selectively deplete hematopoietic stem cells (HSCs) expressing a JAK2 point mutation (JAK2V617F) in mouse models of JAK2V617F-induced polycythemia vera (PV). To determine the potential activity of 5-LO inhibitors in combination with JAK inhibitors against human PV HSCs, the present study first analyzed 5-LO expression in CD34+ bone marrow cells from patients with JAK2V617F-positive PV using western blotting and reverse transcription-quantitative PCR, and then examined the effect of zileuton combined with ruxolitinib on colony formation using a colony formation assay. Furthermore, cell cycle and apoptosis in CD34+ cells from patients with PV and healthy volunteers were determined by flow cytometry. In the present study, 5-LO expression was upregulated in CD34+ cells from patients with PV compared with in CD34+ cells from healthy volunteers. Higher levels of leukotriene B4, a product of the 5-LO signaling pathway, were detected in patients with PV compared with in healthy volunteers. Zileuton treatment suppressed the colony formation of CD34+ cells from patients with PV in a dose-dependent manner. Furthermore, zileuton and ruxolitinib exerted their anticancer effects by suppressing hematopoietic colony formation, inducing apoptosis and arresting the cell cycle of human CD34+ cells from patients with PV. The combination of these two drugs exerted a more beneficial effect than either agent alone. Based on these data, zileuton enhanced the antitumor activity of low-dose ruxolitinib in hematopoietic progenitor cells from patients with PV, providing conceptual validation for further clinical applications of combination treatment with ruxolitinib and zileuton for patients with PV.

Human Cytomegalovirus UL7, miR-US5-1, and miR-UL112-3p Inactivation of FOXO3a Protects CD34+ Hematopoietic Progenitor Cells from Apoptosis.

Human cytomegalovirus (HCMV) infection of myeloid lineage cells, such as CD34+ hematopoietic progenitor cells (HPCs) or monocytes, results in the upregulation of antiapoptotic cellular proteins that protect the newly infected cells from programmed cell death. The mechanisms used by HCMV to regulate proapoptotic cellular proteins upon infection of CD34+ HPCs have not been fully explored. Here, we show that HCMV utilizes pUL7, a secreted protein that signals through the FLT3 receptor, and miR-US5-1 and miR-UL112-3p to reduce the abundance and activity of the proapoptotic transcription factor FOXO3a at early times after infection of CD34+ HPCs. Regulation of FOXO3a by pUL7, miR-US5-1, and miR-UL112 results in reduced expression of the proapoptotic BCL2L11 transcript and protection of CD34+ HPCs from virus-induced apoptosis. These data highlight the importance of both viral proteins and microRNAs (miRNAs) in protecting CD34+ HPCs from apoptosis at early times postinfection, allowing for the establishment of latency and maintenance of viral genome-containing cells.IMPORTANCE Human cytomegalovirus (HCMV) causes serious disease in immunocompromised individuals and is a significant problem during transplantation. The virus can establish a latent infection in CD34+ hematopoietic progenitor cells (HPCs) and periodically reactivate to cause disease in the absence of an intact immune system. What viral gene products are required for successful establishment of latency is still not fully understood. Here, we show that both a viral protein and viral miRNAs are required to prevent apoptosis after infection of CD34+ HPCs. HCMV pUL7 and miRNAs miR-US5-1 and miR-UL112-3p act to limit the expression and activation of the transcription factor FOXO3a, which in turn reduces expression of proapoptotic gene BCL2L11 and prevents virus-induced apoptosis in CD34+ HPCs.

Protective effect of microRNA-134-3p on multiple sclerosis through inhibiting PRSS57 and promotion of CD34+ cell proliferation in rats.

MicroRNAs (miRs) have been extensively studied for their involvement in multiple sclerosis (MS). We investigated the involvement of miR-134-3p on MS. The MS rat model was established, and positive expression of interleukin-17 (IL-17) was detected using the immunohistochemical method while the expression of miR-134-3p and serine protease 57 (PRSS57) was determined by means of reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and Western blot analysis. Second, the miR-134-3p overexpression or short hairpin RNA against PRSS57 was introduced into the CD34+ cells to investigate the levels of proliferation and apoptosis-related genes by RT-qPCR and Western blot analysis. In addition, analysis of the targeting relations of miR-134-3p and PRSS57 was conducted using online software and dual-luciferase reporter gene assay. Furthermore, neuronal functions, inflammatory response, proliferation, and apoptosis of CD34+ cells were assayed by flow cytometry, enzyme-linked immunosorbent assay, and methyl thiazolyl tetrazolium. IL-17 and PRSS57 expression increased while miR-134-3p expression decreased in the spinal cord from MS rats. miR-134-3p could target PRSS57. miR-134-3p overexpression or PRSS57 silencing enhanced mitochondrial activity of neurons, mitochondrial membrane potential content, CD34+ cell proliferation, while decreasing Cyt C content, inflammatory response, and cell apoptosis. Collectively, overexpression of miR-134-3p promotes CD34+ cell proliferation via inhibition of PRSS57 in MS, which may serve as a promising target for MS intervention.

ERK Activity in Immature Leukemic Cells Drives Clonal Selection during Induction Therapy for Acute Myeloid Leukemia

Selection of resistant clones following intensive chemotherapy is a common obstacle for cure in many cancers, particularly in acute myeloid leukemia (AML). In AML, clone-specific sensitivity to chemotherapy varies even within the same patient. Multiple mutations and genetic aberrations are associated with clones surviving chemotherapy. The current study explored the role of activated signaling pathways in chemoresistance as a function of cell maturation, reflected by CD34 expression. In-vitro, Kasumi-1 leukemic cell line, sorted by CD34 expression, showed increased apoptosis only in the CD34− subpopulation after exposure to cytosine arabinoside (Ara-C) or daunorubicin. The resistant CD34+ subset demonstrated higher expression of ERK1/2 and BCL-2 proteins than CD34− cells. MEK1/2 inhibition elevated Ara-C ability to induce apoptosis in CD34+ cells, suggesting that MEK1/2-ERK1/2 is surviving signaling, which correlates to cell maturation levels and plays a role in chemoresistance. Deep sequencing of sorted CD34+/− populations, both derived from the same patient samples, demonstrated various subclonal distribution of NPM1, DNMT3A and FLT3-ITD mutations. Interestingly, in these samples, p-ERK levels and apoptosis rates following chemotherapy exposure significantly differed between CD34+/− populations. Hence, clones may be selected due to their ability to escape apoptosis rather than a direct effect of chemotherapy on a specific mutated clone.

Decellularized Aortic Scaffold Alleviates H2O2-Induced Inflammation and Apoptosis in CD34+ Progenitor Cells While Driving Neovasculogenesis.

Bone marrow-derived stem/progenitor cells have been utilized for cardiac or vascular repair after ischemic injury, but they are subject to apoptosis and immune rejection in the ischemic site. Multiple scaffolds were used as delivery tools to transplant stem/progenitor cells; however, these scaffolds did not show intrinsically antiapoptotic or anti-inflammatory properties. Decellularized aortic scaffolds that facilitate cell delivery and tissue repair were prepared by removing cells of patient-derived aortic tissues. Scanning electron microscopy (SEM) showed cells attached well to the scaffold after culturing for 5 days. Live/dead staining showed most seeded cells survived at day 7 on a decellularized aortic scaffold. Ki67 staining demonstrated that decellularized aortic scaffold promoted proliferation of bone marrow-derived CD34+ progenitor cells. Apoptosis of CD34+ progenitor cells induced by H2O2 at high concentration was significantly alleviated in the presence of decellularized aortic scaffolds, demonstrating a protective effect against oxidative stress-induced apoptosis. Furthermore, decellularized aortic scaffolds significantly reduced the expression of proinflammatory cytokines (IL-8, GM-CSF, MIP-1β, GRO-α, Entoxin, and GRO) concurrently with an increase in anti-inflammatory cytokines (IL-2 and TGF-β) released from CD34+ progenitor cells when exposed to H2O2 at low concentration. Finally, neovascularization was observed by H&E and immunohistochemical staining 14 days after the decellularized aortic scaffolds were subcutaneously implanted in nude mice. This preclinical study demonstrates that the use of a decellularized aortic scaffold possessing antiapoptotic and anti-inflammatory properties may represent a promising strategy for cardiovascular repair after ischemic injury.

Protective effects of cytokine combinations against the apoptotic activity of glucocorticoids on CD34+ hematopoietic stem/progenitor cells.

Haematopoietic stem cells can self-renew and produce progenitor cells, which have a high proliferation capacity. Chemotherapeutic drugs are toxic to normal cells as well as cancer cells, and glucocorticoids (GCs), which are essential drugs for many chemotherapeutic protocols, efficiently induce apoptosis not only in malignant cells but also in normal haematopoietic cells. Studies have shown that haematopoietic cytokines can prevent the apoptosis induced by chemotherapy and decrease the toxic effects of these drugs. However, the apoptosis induction mechanism of GCs in CD34+ haematopoietic cells and the anti-apoptotic effects of cytokines have not been well elucidated. In this study, we investigated the apoptotic effects of GCs on CD34+, a haematopoietic stem/progenitor cell (HSPC) population, and demonstrated the protective effects of haematopoietic cytokines. We used a cytokine cocktail containing early-acting cytokines, namely, interleukin-3 (IL-3), thrombopoietin, stem cell factor and flt3/flk2 ligand, and dexamethasone and prednisolone were used as GCs. Apoptotic mechanisms were assessed by immunohistochemical staining and quantified using H-scoring. Dexamethasone and prednisolone induced apoptosis in CD34+ HSPCs. GC treatment caused a significant increase in apoptotic Fas, caspase-3, cytochrome c and Bax, but a significant decrease in anti-apoptotic Bcl-2. Furthermore, as expected, cytokines caused a significant decrease in all apoptotic markers and a significant increase in Bcl-2. Thus, our findings suggest that CD34+ HSPCs are an extremely sensitive target for GCs and that cytokines protect these cells from GC-induced apoptosis.

The Novel Small Molecule Inhibitor KS99 Targets AML and Inhibits Stemness Markers STAT3 and ALDH

Introduction: Signal transducer and activator of transcription 3 (STAT3) has been shown to be activated in leukemic stem cells (LSCs), especially in Pre-LSCs, and activation has been associated with poor prognosis in multiple cancers including AML. In AML, sorted CD34+/CD38- and aldehyde dehydrogenase-positive (ALDH+) cells have significantly phosphorylated STAT3 when compared to healthy controls. The Isatin analog, KS99 has shown selective anti-cancer activity against Multiple Myeloma (MM) which may, in part, be mediated by inhibition of BTK activation. Here we demonstrate that KS99 selectively targets AML CD34+/CD38- and ALDH+ cells and inhibits STAT3 phosphorylation while sparing normal hematopoietic stem and progenitor cells (HSPCs).Experimental Design: The pro-apoptotic activity of KS99 for primary human AML cells (n=21) and human AML cell lines (n=9) was evaluated using Annexin V and cell proliferation (MTS) assays. Colony forming assays were carried out to demonstrate the specific LSC-targeted effect of KS99. Flow cytometry was used to detect STAT3 phosphorylation and apoptosis in CD34+/CD38- LSCs and ALDH activity in a series of human AMLs. Next, to assess the preclinical efficacy, the human AML cell line U937 was injected intravenously (IV) into NSG mice. Mice with an established leukemic burden were randomized into treatment groups and treated through an intraperitoneal (IP) injection with either vehicle control or KS99 (2.5mg/kg) every other day x8. Two days after the final treatment, leukemic burden (hCD45+) was quantitated in the bone marrow.Results: AML cell lines exhibited dose-dependent sensitivity to KS99 in the nanomolar range (IC50: 150-400nM) as did most primary AML cells (Figure: A). Interestingly, poor prognosis AML subsets, AML with MDS related changes (MDS-RCs), were more sensitive than De Novo AML cases (p= 0.0077) with or without a NMP1 mutation (p= 0.012, p=0.045, respectively). Within De Novo AML, NMP1 wild-type cases were more sensitive than NMP1 mutant cases (p= 0.02) (Figure: B). Furthermore, KS99 treatment selectively reduced the clonogenicity of human AML patient cells vs. normal cord blood mononuclear cells (p<0.001) (Figure: C). KS99 induced apoptosis in the CD34+/CD38- gated cell population of primary AML, leading to reduced blast-like and stem-like cells, but not other cells. It also showed a reduction in ALDH+ cells in an Aldefluor assay. Likewise, KS99 treatment showed reduced pSTAT3 in viable CD34+/CD38- cells (Figure: D). Inhibition of pBTK, BCL-2, and pSTAT3 were seen in Western blot experiments. Animal studies showed 76 % reduction of hCD45+ U937 cells in bone marrow of KS99-treated mice when compared to bone marrow isolated from vehicle-treated animals (p<0.05) (Figure: E).Conclusion: KS99 selectively targets LSCs from poor prognosis AML patients while sparing normal HPSCs. This effect may be mediated via targeting of STAT3 signaling and ALDH, which are critical for proliferating LSCs, but not normal HPSCs. Further studies will aim to demonstrate mechanisms of activity and preclinical efficacy in patient-derived AML xenograft mouse models. [Display omitted] DisclosuresDovat:Elf Zone, Inc.: Membership on an entity's Board of Directors or advisory committees.

Disulfram/Copper Complex May Enhance Its Cytotoxic Effect on CD34+CD38- KG1-Alpha Cells By Down Regulating the Expression of HIF1-Alpha in the Co-Cultured MSCs

Backgroud We had reported that Disulfram/copper complex (DS/ Cu) had a potent and selective anti-leukemia property in vitro against leukemia stem-like cells (e.g., CD34+/CD38- KG1α and Kasumi-1 cells and primary CD34+ cells isolated from AML patients) as well as was highly effective in vivo in CD34+/CD38- leukemic cell-derived xenograft mouse models. Here, we report the in vitro activity of DS/Cu against CD34+/CD38- leukemia stem-like cells sorted from KG1α AML cell line by affecting the co-cultured AML bone marrow mesenchymal stromal cells. Methods KG1α cells were cultured and stained with hCD34-APC, hCD38- PE, and hCD123-PE-Cy7 to determine the percentage of cells expressing CD34, CD38, and CD123. To isolate CD38- cells, CD38 microbeads were used for the depletion of CD38+ cells. First, we examined the cytotoxic effect of DS/Cu on CD34+CD38- KG1α cells and MSCs using the Cell Counting Kit-8 (CCK-8) and monitor expression of HIF-1α expression by western blot. Next, the sorted CD34+CD38- KG1α cells were co-cultured with primary AML bone marrow MSCs isolated from a de novo AML patient in the hypoxic condition induced by Cobalt Chloride (CoCl2) for 24 hr. The co-cultured cells were treated with DS/CU with/without YC1(HIF-1α inhibitor)、IDA(idarubicin) and then separated. CCK8 assay, fow cytometry were used to determine the cell proliferation and apoptosis, respectively. Finally, we used western bolting to explore the underlying mechanism of the cytotoxicity of DS/CU in the AML cells and the sensitizated effect in AML MSCs. Results First, we examined the cytotoxic effect of DS/Cu on CD34+CD38- KG1α cells and MSCs using the Cell Counting Kit-8 (CCK-8). As shown in Figure 1, DS/Cu (DS 1umol/L,Cu 0.5umol/l) administrated alone was unable to induce apoptosis in MSCs (P>0.05 vs untreated control; see below Figure 1). However, DS in combination with 0.5 μM Cu for 24 hrs resulted in signifcantly increased apoptosis in CD34+CD38- KG1α cells.( P<0.001 vs untreated control; see Figure 1) .The co-cultured cells were treated with DS/CU、IDA with/without YC1 and then separated. CCK8 assay, fow cytometry were used to determine the cell apoptosis, respectively. Treatment with DS/CU resulted in signifcantly increased apoptosis in the co-cultured CD34+/CD38- KG1α cells (Figure 2; P<0.05, DS/Cu vs either untreated control). Comparable phenomena were observed in co-cultured CD34+/CD38- KG1α cells treated with idarubicin. However, while treatment with YC1 and DS/CU had no signifcant effect on cell apoptosis (P>0.05 vs untreated control). Taken together, these results indicated that DS/CU was active against co-cultured CD34+/CD38- KG1α cells and the effect can be reversed by HIF1-α inhibitor(see Figure 2).We examined the cytotoxic effect of DS/Cu on CD34+/CD38- KG1α cells in different culture systems using the CCK-8. As shown in Figure 4, while treatment with DS/CU had significant inhibited proliferation on cell proliferation of the co-cultured CD34+/CD38- KG1α cells (P<0.05 vs un-co-cultured control). However, while treatment with DS/CU and CoCl2 (HIF-1α promoter) had no signifcant effect on cell proliferation of the co-cultured CD34+/CD38- KG1α cells (P>0.05 vs un-co-cultured control,see Figure 3).CD34+CD38- KG1α cells and MSCs separated from the co-cultured system were treated with DS/CU with/without YC1 and Cocl2, followed by Western blot analysis to monitor expression of HIF-1α. As shown at the figure 4, the expressions of HIF-1α and SDF-1α between the two cells were clearly down-regulated treated with DS/CU alone. And the expressions of HIF-1α and SDF-1α were up-regulated treated with DS/CU and CoCl2. However, when co-treated with YC1, the down-regulation of HIF-1α was reverse. DS/Cu down redulated the expressions of HIF1-α、SDF1-α、CXCR4 and VLA4 in the co-cultured CD34+CD38- KG1α cells and these effect could be reserved by HIF1-α inhibitor.Conclusion DS/Cu preferentially induces apoptosis of CD34+CD38- KG1α cells, but not MSCs. DS/Cu exhibited cytotoxicity in CD34+/CD38- KG1α cells, and MSCs enhances this effect. The mechanism may be related to the HIF1α/SDF1/CXCR4 and VLA4 pathway. DS/CU may enhance its inhibitory effect on CD34+CD38- KG1α cells by down regulating the expression of HIF1-α in MSCS. DisclosuresNo relevant conflicts of interest to declare.

Характеристика костномозгового клеточного трасплантата больных хронической сердечной недостаточностью до и после кратковременной экспозиции с эритропоэтином

Аутологичные костномозговые стволовые клетки являются альтернативным способом терапии пациентов с сердечной недостаточностью. Цель работы - изучение фенотипа и функциональных свойств костномозговых мононуклеарных клеток (КМ-МНК) больных хронической сердечной недостаточностью (ХСН) до и после кратковременной экспозиции с эритропоэтином in vitro . Методика. КМ-МНК выделяли на градиенте плотности фиколл/верографин (r = 1,077 г/л). Фенотип КМ-МНК, клеточный цикл и апоптоз CD34+ клеток определяли до и после экспозиции с эритропоэтином на проточном цитометре. Пролиферативный потенциал КМ-МНК до и после экспозиции с эритропоэтином оценивали в спонтанном и стимулирующем тесте. Пролиферацию, миграцию и ангиогенный потенциал клеток EA.hy 929 изучали в тесте «раневого дефекта» монослоя клеток и на матригеле под влиянием 30% кондиционных сред от КМ-МНК. Результаты. Показано, что КМ-МНК представляют собой смесь гемопоэтических стволовых клеток (ГСК), эндотелиальных прогениторных клеток (ЭПК) на разных этапах созревания и дифференцировки, и мезенхимных стволовых клеток (МСК). Под действием эритропоэтина увеличивается количество CD34+ клеток в G0G1 фазе клеточного цикла, CD45+/EpoR+, CD31-/CD184+, CD31+/CD184+ и CD34+/CD184-, и уменьшается количество CD34+/CD133+, CD34+/EpoR-. Кондиционная среда от КМ-МНК способствует пролиферации, миграции и формированию сосудисто-подобных структур клетками EA.hy 929. Заключение. Полученные результаты свидетельствуют, что кратковременная экспозиция КМ-МНК больных ХСН задерживает CD34+ клетки в стадии покоя, увеличивает пул ЭПК, экспрессирующих «хоуминг» рецептор, а кондиционная среда от КМ-МНК стимулирует пролиферацию, миграцию и ангиогенный потенциал EA.hy 929, что следует учитывать при выборе методов усиления «приживаемости» клеточного трансплантата. Autologous bone marrow stem cells are an alternative therapy for patients with heart failure. The aim of this work was to study the phenotype and functional properties of bone marrow mononuclear cells (BM-MNCs) from patients with chronic heart failure (CHF) before and after a short-term in vitro exposure to erythropoietin. Methods. BM-MNCs were isolated using density gradient. The BM-MNC phenotype, cell cycle, and apoptosis of CD34+ cells were evaluated before and after exposure to erythropoietin with a flow cytometer. Proliferation of BM-MNCs before and after the erythropoietin exposure was evaluated in a spontaneous and stimulating test. Proliferation, migration, and angiogenic potential of EA.hy 929 cells were studied in the wound closure test and in the tubule formation test under the influence of 30% conditioned medium from BM-MNCs. Results. BM-MNCs represented a mixture of hematopoietic stem cells (HSCs), endothelial progenitor cells (EPCs) at different stages of maturation and differentiation, and mesenchymal stem cells (MSCs). Erythropoietin increased the number of CD34+ cells in the G0/G1 cell cycle phase, CD45+/EpoR+, CD31-/CD184+, CD31+/CD184+, and CD34+/CD184-, and decreased the number of CD34+/CD133+ and CD34+/EpoR. The erythropoietin exposure of BM-MNCs reduced their proliferative capacity. The BM-MNCs-conditioned medium promoted EA.hy 929 cell proliferation, migration, and formation of vascular-like structures. Conclusion. A short-term exposure of BM-MNCs delayed the resting stage of CD34+ cells, increased the pool of EPCs expressing the homing receptor while the BM-MNC conditioned medium stimulated EA.hy 929 proliferation, migration and tubule formation, which should be taken into account when selecting methods to enhance survival of cellular grafts.

The pan-Bcl2 Inhibitor AT101 Activates the Intrinsic Apoptotic Pathway and Causes DNA Damage in Acute Myeloid Leukemia Stem-Like Cells.

Leukemia stem cells (LSCs) are considered to be the cause of treatment failure and relapse in acute myeloid leukemia (AML). Overexpression of the Bcl-2 family of anti-apoptotic proteins such as Bcl-2, Bcl-xl, and Mcl-1 accounts for survival and self-renewal of LSCs. AT101 binds to the BH3 motif of all Bcl-2 family anti-apoptotic proteins and demonstrates anti-tumor activity in multiple types of tumor. Thus, we hypothesized that this agent might have the potential to deplete LSCs. The present study aims to investigate if and by what mechanism AT101 is able to target AML stem-like cells. As LSCs and hematopoietic stem cells (HSCs) are enriched in CD34+CD38- populations, CD34+CD38- cells from KG1α and Kasumi-1 cell lines as well as CD34+ blasts from AML patients were used as LSC models, while CD34+ cells from healthy donors were used as normal hematopoietic cells. Cell proliferation and apoptosis were assessed by a cell counting kit-8 (CCK8) assay and an Annexin V/PI assay using flow cytometry, respectively. Colony-forming units experiments were performed to monitor the stemness features of AML cells. Western blot and quantitative real-time polymerase chain reaction (qPCR) analysis were performed to examine the levels of proteins and mRNAs related to either the intrinsic apoptotic pathway or DNA damage response. AT101 inhibited proliferation and induced apoptosis in CD34+CD38- KG1α and Kasumi-1 cells in a dose- and time-dependent manner. Exposure to AT101 for 24h resulted in apoptosis in primary CD34+ AML blasts (EC50[concentration needed for a 50% maximal effect] =2.45-76.00μmol/L), while it only had a modest effect on normal CD34+ hematopoietic cells. Mechanistically, AT101 activated the intrinsic apoptotic pathway by inhibition of Bcl-2 anti-apoptotic proteins, reflected by a decrease in mitochondrial membrane potential. Moreover, AT101 caused DNA damage (e.g., increased γH2AX phosphorylation), which might also contribute to its anti-leukemic effects. Interestingly, the ex vivo efficacy of AT101 in primary AML samples significantly correlated to hyperleukocytosis and FLT3-ITD mutations. AT101 was also effective against CD34+ blasts isolated from elderly patients and patients who did not achieve complete remission after induction therapy. AT101 effectively eliminates LSCs in vitro through the induction of DNA damage and activation of the intrinsic apoptotic pathway. AT101 is effective towards leukemic cells from patients with adverse prognostic factors, suggesting that AT101 could have the potential as an alternative salvage therapy for the treatment of relapsed and refractory AML.

Novel Use of All-Trans-Retinoic Acid in A Model of Lipopolysaccharide-Immunosuppression to Decrease the Generation of Myeloid-Derived Suppressor Cells by Reducing the Proliferation of CD34+ Precursor Cells.

All-trans-retinoic acid (ATRA) is a derivative of vitamin A with antiproliferative properties. Endotoxin shock and subsequent immunosuppression (IS) by lipopolysaccharide (LPS) stimulates myelopoiesis with expansion of myeloid-derived suppressor cells (MDSC). Since we have previously shown that ATRA reverses the IS state by decreasing functional MDSC, our aim was to investigate if ATRA was able to modulate MDSC generation by regulating myelopoiesis in murine hematopoietic organs. We found that ATRA administration in vivo and in vitro decreased the number of CD34+ precursor cells that were increased in IS mice. When we studied the cellular mechanisms involved, we did not find any differences in apoptosis of CD34+ precursors or in the differentiation of these cells to their mature counterparts. Surprisingly, ATRA decreased precursor proliferation, in vitro and in vivo, as assessed by a reduction in the size and number of colony forming units generated from CD34+ cells and by a decreased incorporation of H-thymidine. Moreover, ATRA administration to IS mice decreased the number of MDSC in the spleen, with a restoration of T lymphocyte proliferation and a restitution of the histological architecture. Our results indicate, for the first time, a new use of ATRA to abolish LPS-induced myelopoiesis, affecting the proliferation of precursor cells, and in consequence, decreasing MDSC generation, having a direct impact on the improvement of immune competence. Administration of ATRA could overcome the immunosuppressive state generated by sepsis that often leads to opportunistic life-threatening infections. Therefore, ATRA could be considered a complementary treatment to enhance immune responses.