Differentiation Of Hematopoietic Progenitor Cells Research Articles

Effects of PM2.5 exposure on hematopoiesis and coupled immune disorder in adult male mice

Fine particulate matter, namely PM2.5, increases the risk of morbidity and mortality in various systems, including the hematopoietic and immune systems. However, there is limited experimental evidence supporting the association between PM2.5 exposure and hematopoietic outcomes as most studies focus on epidemiological data. In this study, adult male mice were exposed to PM2.5 for a long time to investigate hematopoietic toxicity. There were significant increases in red blood cells (RBC), hemoglobin (HGB), and white blood cells (WBC) in peripheral blood after 5-month real-environmental PM2.5 exposure, indicating elevated circulatory inflammation and potential hematopoietic abnormality. Moreover, we observed abnormal proliferation and differentiation of hematopoietic stem and progenitor cells (HSPCs), along with altered mRNA levels of hematopoietic genes and increased proinflammatory factors in the bone marrow (BM). Transcriptomic analysis of BM cells suggested that PM2.5 exposure activated immune responses associated with lymphocytes. Then, PM2.5 exposure via intratracheal instillation was performed for 8 weeks to verify the toxic effect. The results of the complete blood count were similar to those of real-environmental exposure, with drastic changes in the number and function of HSPCs, as well as mRNA levels of the hematopoietic genes and increased inflammatory factors in the BM being detected. Furthermore, lymphocyte subsets changed significantly in the BM and spleen, confirming immune disorder following PM2.5 exposure. In conclusion, PM2.5 interfered with the process of BM hematopoiesis by triggering inflammation and leading to immune disorder. Our study provided experimental evidence for the hematopoietic toxicity of PM2.5 and highlighted the significance of reducing air pollution.

Molecular mechanisms promoting long-term cytopenia after BCMA CAR-T therapy in multiple myeloma

AbstractHematologic toxicity is a common side effect of chimeric antigen receptor T-cell (CAR-T) therapies and is particularly severe among patients with relapsed or refractory multiple myeloma (MM). In this study, we analyzed a cohort of 48 patients who were treated with B-cell maturation agent (BCMA) CAR-Ts to characterize the kinetics of cytopenia, identify predictive factors, and determine potential mechanism that underlie these toxicities. The overall incidence of cytopenia was 95.7%, and grade >3 thrombocytopenia and neutropenia, 1 month after infusion, was observed in 57% and 53% of the patients, respectively, and was still present after 1 year in 4 and 3 patients, respectively. The presence of cytopenia at baseline and high peak inflammatory markers were highly correlated with cytopenia that persisted up to 3 months. To determine potential mechanisms that underpin cytopenias, we evaluated the paracrine effect of BCMA CAR-Ts on hematopoietic stem and progenitor cell (HSPC) differentiation using an ex vivo myeloid differentiation model. Phenotypic analysis showed that supernatants from activated CAR-Ts (spCARs) halted HSPC differentiation, thereby promoting more immature phenotypes with a reduction in the expression of granulocytic, monocytic, and erythroid markers, which could be prevented with a combination of interferon gamma, tumor necrosis factor α/β, transforming growth factor β, interleukin-6 (IL-6) and IL-17 inhibitors. Single-cell RNA sequencing demonstrated upregulation of transcription factors associated with the early stages of hematopoietic differentiation in the presence of spCAR (GATA2, RUNX1, CEBPA) and a decrease in the activity of key regulons involved in neutrophil and monocytic maturation (ID2 and MAFB). Our results suggest that CAR-T activation negatively influences hematopoietic differentiation through paracrine effects, thereby inducing HSPCs maturation arrest. Moreover, our study contributes to the understanding of severe cytopenia observed after CAR-T therapy in MM and provides potential treatments to prevent or decrease its severity.

FLI1 is associated with regulation of DNA methylation and megakaryocytic differentiation in FPDMM caused by a RUNX1 transactivation domain mutation

Familial platelet disorder with associated myeloid malignancies (FPDMM) is an autosomal dominant disease caused by heterozygous germline mutations in RUNX1. It is characterized by thrombocytopenia, platelet dysfunction, and a predisposition to hematological malignancies. Although FPDMM is a precursor for diseases involving abnormal DNA methylation, the DNA methylation status in FPDMM remains unknown, largely due to a lack of animal models and challenges in obtaining patient-derived samples. Here, using genome editing techniques, we established two lines of human induced pluripotent stem cells (iPSCs) with different FPDMM-mimicking heterozygous RUNX1 mutations. These iPSCs showed defective differentiation of hematopoietic progenitor cells (HPCs) and megakaryocytes (Mks), consistent with FPDMM. The FPDMM-mimicking HPCs showed DNA methylation patterns distinct from those of wild-type HPCs, with hypermethylated regions showing the enrichment of ETS transcription factor (TF) motifs. We found that the expression of FLI1, an ETS family member, was significantly downregulated in FPDMM-mimicking HPCs with a RUNX1 transactivation domain (TAD) mutation. We demonstrated that FLI1 promoted binding-site-directed DNA demethylation, and that overexpression of FLI1 restored their megakaryocytic differentiation efficiency and hypermethylation status. These findings suggest that FLI1 plays a crucial role in regulating DNA methylation and correcting defective megakaryocytic differentiation in FPDMM-mimicking HPCs with a RUNX1 TAD mutation.

CRISPR/Cas9-mediated gene disruption determines the roles of MITF and CITED2 in human mast cell differentiation

The differentiation of hematopoietic stem and progenitor cells into the various cell lineages is regulated by cell-intrinsic factors and the progenitors' microenvironment. Experiments in mice have allowed the identification of transcriptional modulators that control mast cell differentiation. However, a comprehensive approach that allows efficient disruption of individual transcriptional modulators in primary human hematopoietic progenitors coupled with a mast cell formation readout has not been described. Here, we report a simple electroporation- and ribonucleoprotein-based knockout system that allows the identification of genes that are required and dispensable for human mast cell differentiation. We show that the transcription factor MITF is upregulated in human mast cell progenitors and reveal that the loss of MITF results in the suppressed formation of mast cells. By contrast, CITED2, another transcriptional modulator that is upregulated along the mast cell differentiation trajectory, was dispensable for human mast cell differentiation. Taken together, we report a CRISPR/Cas9-based framework that serves to identify genes involved in regulating the formation of human mast cells, and the results uncover the role of two transcriptional modulators in controlling human mast cell differentiation.

RIO-kinase 2 is essential for hematopoiesis.

Regulation of protein synthesis is a key factor in hematopoietic stem cell maintenance and differentiation. Rio-kinase 2 (RIOK2) is a ribosome biogenesis factor that has recently been described an important regulator of human blood cell development. Additionally, we have previously identified RIOK2 as a regulator of protein synthesis and a potential target for the treatment of acute myeloid leukemia (AML). However, its functional relevance in several organ systems, including normal hematopoiesis, is not well understood. Here, we investigate the consequences of RIOK2 loss on normal hematopoiesis using two different conditional knockout mouse models. Using competitive and non-competitive bone marrow transplantations, we demonstrate that RIOK2 is essential for the differentiation of hematopoietic stem and progenitor cells (HSPCs) as well as for the maintenance of fully differentiated blood cells in vivo as well as in vitro. Loss of RIOK2 leads to rapid death in full-body knockout mice as well as mice with RIOK2 loss specific to the hematopoietic system. Taken together, our results indicate that regulation of protein synthesis and ribosome biogenesis by RIOK2 is essential for the function of the hematopoietic system.

Interleukin 3 Inhibits Glutamate-Cytotoxicity in Neuroblastoma Cell Line.

Interleukin 3 (IL-3) is a well-known pleiotropic cytokine that regulates the proliferation and differentiation of hematopoietic progenitor cells, triggering classical signaling pathways such as JAK/STAT, Ras/MAPK, and PI3K/Akt to carry out its functions. Interestingly, the IL-3 receptor is also expressed in non-hematopoietic cells, playing a crucial role in cell survival. Our previous research demonstrated the expression of the IL-3 receptor in neuron cells and its protective role in neurodegeneration. Glutamate, a principal neurotransmitter in the central nervous system, can induce cellular stress and lead to neurotoxicity when its extracellular concentrations surpass normal levels. This excessive glutamate presence is frequently observed in various neurological diseases. In this study, we uncover the protective role of IL-3 as an inhibitor of glutamate-induced cell death, analyzing the cytokine's signaling pathways during its protective effect. Specifically, we examined the relevance of JAK/STAT, Ras/MAPK, and PI3 K signaling pathways in the molecular mechanism triggered by IL-3. Our results show that the inhibition of JAK, ERK, and PI3 K signaling pathways, using pharmacological inhibitors, effectively blocked IL-3's protective role against glutamate-induced cell death. Additionally, our findings suggest that Bcl-2 and Bax proteins may be involved in the molecular mechanism triggered by IL-3. Our investigation into IL-3's ability to protect neuronal cells from glutamate-induced damage offers a promising therapeutic avenue with potential clinical implications for several neurological diseases characterized by glutamate neurotoxicity.

Towards an integrated understanding of inflammatory pathway influence on hematopoietic stem and progenitor cell differentiation.

Recent research highlights that inflammatory signaling pathways such as pattern recognition receptor (PRR) signaling and inflammatory cytokine signaling play an important role in both on-demand hematopoiesis as well as steady-state hematopoiesis. Knockout studies have demonstrated the necessity of several distinct pathways in these processes, but often lack information about the contribution of specific cell types to the phenotypes in question. Transplantation studies have increased the resolution to the level of specific cell types by testing the necessity of inflammatory pathways specifically in donor hematopoietic stem and progenitor cells (HSPCs) or in recipient niche cells. Here, we argue that for an integrated understanding of how these processes occur in vivo and to inform the development of therapies that modulate hematopoietic responses, we need studies that knockout inflammatory signaling receptors in a cell-specific manner and compare the phenotypes caused by knockout in individual niche cells versus HSPCs.

Potential marker subset of blood-circulating cytokines on hematopoietic progenitor-to-Th1 pathway in COVID-19.

In this study, we analyzed a relatively large subset of proteins, including 109 kinds of blood-circulating cytokines, and precisely described a cytokine storm in the expression level and the range of fluctuations during hospitalization for COVID-19. Of the proteins analyzed in COVID-19, approximately 70% were detected with Bonferroni-corrected significant differences in comparison with disease severity, clinical outcome, long-term hospitalization, and disease progression and recovery. Specifically, IP-10, sTNF-R1, sTNF-R2, sCD30, sCD163, HGF, SCYB16, IL-16, MIG, SDF-1, and fractalkine were found to be major components of the COVID-19 cytokine storm. Moreover, the 11 cytokines (i.e., SDF-1, SCYB16, sCD30, IL-11, IL-18, IL-8, IFN-γ, TNF-α, sTNF-R2, M-CSF, and I-309) were associated with the infection, mortality, disease progression and recovery, and long-term hospitalization. Increased expression of these cytokines could be explained in sequential pathways from hematopoietic progenitor cell differentiation to Th1-derived hyperinflammation in COVID-19, which might also develop a novel strategy for COVID-19 therapy with recombinant interleukins and anti-chemokine drugs.

The Effect of Diet Composition on the Post-operative Outcomes of Roux-en-Y Gastric Bypass in Mice.

Roux-en-Y gastric bypass (RYGB) leads to the improvement of many obesity-associated conditions. The degree to which post-operative macronutrient composition contributes to metabolic improvement after RYGB is understudied. A mouse model of RYGB was used to examine the effects of diet on the post-operative outcomes of RYGB. Obese mice underwent either Sham or RYGB surgery and were administered either chow or HFD and then monitored for an additional 8weeks. After RYGB, reductions to body weight, fat mass, and lean mass were similar regardless of diet. RYGB and HFD were independently detrimental to bone mineral density and plasma vitamin D levels. Independent of surgery, HFD accelerated hematopoietic stem and progenitor cell proliferation and differentiation and exhibited greater myeloid lineage commitment. Independent of diet, systemic iron deficiency was present after RYGB. In both Sham and RYGB groups, HFD increased energy expenditure. RYGB increased fecal energy loss, and HFD after RYGB increased fecal lipid content. RYGB lowered fasting glucose and liver glycogen levels but HFD had an opposing effect. Indices of insulin sensitivity improved independent of diet. HFD impaired improvements to dyslipidemia, NAFLD, and fibrosis. Post-operative diet plays a significant role in determining the degree to which RYGB reverses obesity-induced metabolic abnormalities such as hyperglycemia, dyslipidemia, and NAFLD. Diet composition may be targeted in order to assist in the treatment of post-RYGB bone mineral density loss and vitamin D deficiency as well as to reverse myeloid lineage commitment. HFD after RYGB continues to pose a significant multidimensional health risk.

The Mechanism of Setd2 in Mesenchymal Stromal Cell in MDS-Associated Ineffective Hematopoiety

Myelodysplastic syndrome (MDS) is a heterogeneous, clonal, hematopoietic stem progenitor cell malignant disease. Bone marrow microenvironment participates in the occurrence and development of MDS and plays a key role. Histone methyltransferase Setd2 is the only enzyme that can catalyze the methylation of mammalian histone H3K36Me3. Previous studies have confirmed that Setd2 is involved in the the transformation of MDS to leukemia, and the expression of S100A9 decreases after Setd2 knockout. This study is based on NHD13 mice with mesenchymal stem cell (MSC) targeted knockout of Setd2. Through experimental methods such as flow cytometry, peripheral blood routine, Wright staining, HE staining, and Gomori staining, it finds that Setd2 targeted knockout mice undergo faster leukemia transformation compared to NHD13 mice; Peripheral blood white blood cells increase, while hemoglobin and platelets decrease; The differentiation of hematopoietic stem progenitor cells in bone marrow is abnormal, with abnormal hematopoiesis and increased blast cells; Spleen fibrosis aggravates and megakaryocyte infiltrates; and Extramedullary hematopoiesis in liver. Through flow cytometry,β- Galactosidase staining and other experimental methods, we finds that compared with NHD13 mice, the MSC morphology of knockout mice is irregular, and the support function for hematopoietic cells is weakened. This study demonstrates that MSC targeted knockout of Setd2 has a promoting effect on the progression of MDS. We used mass spectrometry to identify proteins that are significantly upregulated in the targeted knockout mouse bone marrow supernatant compared to the NHD13 mouse bone marrow supernatant, and and select the protein Uba5 related to hematopoiesis from them. The expression of Uba5 in the bone marrow supernatant of knockout mice is significantly higher than that in the NHD13 group through WB validation. It is speculated that the hematopoietic changes in Setd2 targeted knockout mice may be related to the downstream target protein Uba5 of Setd2. This study takes the change of bone marrow microenvironment as an entry point to explore the possible mechanism of MDS ineffective hematopoiesis, hoping to provide microenvironment regulation therapy for some MDS patients with genes that hematopoietic stem cell transplantation that cannot cure(such as TP53, NRAS, KRAS), and provide new ideas for MDS treatment. There are still many limitations in this study, further clinical validation and vivo and vitro rescue experiments on downstream target genes and proteins are still needed in the future.

Roles for Heme Synthesis in the Maintenance of Hematopoietic Stem and Progenitor Cells

Heme is required not only in red blood cells for oxygen transport but also in non-erythroid cells for the proper function of hemoproteins. Cytochrome c is one of the hemoproteins and inevitable for mitochondrial oxidative phosphorylation, suggesting that heme possesses important roles for ATP synthesis and mitochondria maintenance. Heme also can skew the differentiation of hematopoietic stem and progenitor cells (HSPCs) by altering the functions of transcription factors. Recent CRISPR Cas9 dropout screens repeatedly showed that some of the heme synthases, such as CPOX, might be inevitable for the survival and/or proliferation of HSPCs. However, still little is known about the roles for heme synthesis in the maintenance (survival, proliferation and differentiation) of HSPCs. To reveal the roles for heme synthesis in HSPCs in vivo, we developed polyI:C inducible hematopoietic cell specific Cpox knock-out mice ( Cpox f/f; Mx1-Cre). PolyI:C treated Cpox f/f; Mx1-Cre mice (KO mice) showed significant increase in the numbers of hematopoietic stem cells (HSCs) and multipotent stem and progenitor cells (MPP2s and MPP3s) but decrease in the numbers of common myeloid progenitors (CMPs) and some of their progenies (i.e., Gr1 positive cells and platelets). In contrast, its effects on erythroid and lymphoid lineages were limited. This result suggests that heme synthesis inhibition in HSPCs (HSCs, MPP2 and MPP3s) can cause their differentiation arrest. On the other hand, competitive transplantation analysis of LSKs (lineage -sca1 +c-kit +) or HSCs revealed that myelopoiesis were markedly decreased compared to control, suggesting that heme synthesis in HSPCs is important for the proper reconstitution of hematopoiesis. These results indicate that cell intrinsic heme synthesis is inevitable for the maintenance of HSPCs and their lineage specific output in vivo. In depth, we revealed that mitochondrial respiratory activity of Cpox KO HSCs were increased compared to control HSCs, suggesting that alterations of mitochondrial status and/or functions by the inhibition of heme synthesis might contribute to the aberrance in Cpox KO HSPCs. Comprehensive gene expression analysis is now ongoing to support this notion. To further reveal the roles for heme synthesis in human HSPCs, we treated human leukemia cell lines (MV4-11, KG1a and U937) with heme synthesis inhibitors (SA or NMPP). Concentration-dependent cell growth inhibitory effect and cell death were observed with increased apoptosis factors (cleaved caspase 3 and cleaved PARP), suggesting that heme synthesis inhibition triggered apoptosis. We also revealed that CPOX inhibition under doxycycline inducible CRISPR Cas9 system caused cell growth inhibitory effect and cell death in these cells. Second mitochondria-derived activator of caspases (Smac) and cytochrome c were released to cytoplasm, and the inhibitor of apoptosis protein (XIAP) was decreased by heme synthesis inhibitions. Therefore, caspase-3 activation by accumulation of cytoplasmic cytochrome c and Smac might be the inducer of this apoptosis pathway. Comprehensive transcriptomic analysis (RNA-seq) revealed that heme synthesis inhibition induced the release of cytochrome c and Smac from mitochondria into cytoplasm by the transcriptomic upregulation of Oxidative Stress Induced Growth Inhibitor 1 (Osgin1) that is the known target of NRF2. Collectively, this study demonstrated the significance of heme synthesis in the maintenance of HSPCs especially in vivo (mouse) and in vitro (human). Dysfunctions of heme synthesis in HSPCs can cause their aberrance by the alterations in their mitochondrial status which can cause apoptosis and/or differentiation arrest. Considering the metabolic differences (i.e., demands for mitochondrial oxidative phosphorylation and subsequent ATP synthesis) between normal HSPCs and leukemia or unperturbed hematopoiesis and perturbed hematopoiesis (i.e., infections and hematopoietic stem cell transplantations), further analysis to reveal the roles for heme synthesis in the maintenance of HSPCs will pave the way to establish optical approaches in these clinical settings.

N-3 Polyunsaturated Fatty Acid (N-3 PUFA) Derived Lipid Mediators Increase Hematopoiesis during Zebrafish Embryogenesis

Polyunsaturated fatty acids (PUFAs), which include n-3 PUFAs and n-6 PUFAs, are essential fatty acids that we can only get from diet. The richest source of PUFAs is fish oil. N-3 PUFAs have been used in the prevention and treatment of coronary artery disease, diabetes, hypertension, arthritis, cancer, and other inflammatory and autoimmune disorders in humans. Lipid mediators derived from n-3 PUFAs are implicated in mediating the resolution of inflammation. However, their effect on hematopoietic stem and progenitor cell (HSPC) proliferation and differentiation and on hematopoiesis has not been studied. Therefore, in this project, we investigated the pro-hematopoietic effects of n-3 PUFA-derived lipid mediators. We exposed wild-type zebrafish embryos to different n-3 PUFA-derived lipid mediators. After fixation, we performed whole mount in situ hybridization (WISH) using antisense RNA probes such as c-myb (hematopoietic stem cell marker), gata1 (erythroid progenitor cell marker), hbbe1 (erythrocyte marker), l-plastin (macrophage and neutrophil cell marker ), mpx (neutrophil cell marker), lyz (neutrophil cell marker), and rag1 (immature lymphocyte marker); reverse transcription-quantitative polymerase chain reaction (RT-qPCR); and fluorescence-activated cell sorting (FACS) to understand the effect of lipid mediator exposure on the proliferation of HSPCs, erythroid progenitor cells, erythrocytes, granulocytic cells, neutrophils, macrophages, and T lymphocytes in different development stages during vertebrate hematopoiesis. In addition, we also used o-dianisidine staining to study red blood cell (RBC) formation and transgenic zebrafish to observe different blood lineages including HSPCs, neutrophils, macrophages, erythroid progenitors, and erythrocytes with fluorescent microscopy. We identified two novel lipid mediators which promote HSPC proliferation, enhance erythroid progenitor cell and erythrocyte production, and increase neutrophil cell number. These two molecules could be applied as novel therapeutics for blood disorders and blood cancers such as anemia and leukemia in humans. These two compounds can be intensely investigated to become candidate supplements to enhance HSPC amplification before HSPC transplantation for leukemia patients.

Avatrombopag in Combination with Immunosuppressive Therapy for Hepatitis-Associated Aplastic Anemia

Hepatitis-associated aplastic anemia (HAAA) is an uncommon but distinct variant of aplastic anemia (AA), in which an episode of seronegative acute hepatitis precedes the onset of pancytopenia. Previous studies indicated that about only 20% of HAAA patients can get their hematologic partially restored after 3 months treatment with standard immunosuppressive therapy (IST). Recent studies have shown that adding thrombopoietin receptor agonist (TPO-RA) eltrombopag to standard IST can lead to a more, faster, and better hematologic responses. Nevertheless, eltrombopag is often associated with hepatic adverse effects, i.e., elevated transaminases and bilirubin, which maybe limit its use in patients with HAAA who have just experienced severe liver damage. Avatrombopag is also a TPO-RA, which was approved for the treatment of thrombocytopenia in patients with chronic liver disease (CLD) and patients with chronic immune thrombocytopenia (ITP) by the U.S. Food and Drug Administration (FDA) and European Medicines Agency (EMA). The pre-clinical studies indicated that avatrombopag can promote the hematopoietic and megakaryocyte progenitor cells proliferation and differentiation by the dose-dependence. These in vitro effects are similar to those of other TPO-RAs, including Eltrombopag. Theoretically, on the basis of adequate IST, avatrombopag can also promote hematopoietic recovery in patients with aplastic anemia just as Eltrombopag works. If this is true, then avatrombopag should be particularly suitable for HAAA due to its favorable hepatic adverse effect profile. Nevertheless, avatrombopag added to standard IST as first-line treatment for aplastic anemia could increase the rapidity and strength of hematologic response is still unknown. Herein, we retrospectively reviewed 8 consecutive cases of patients with hepatitis-associated aplastic anemia who were treated with avatrombopag plus IST regimen. From September 2021 to December 2022, eight patients with hepatitis-associated aplastic anemia treated with avatrombopag in combination with standard IST were enrolled in this study. Among them, seven patients were diagnosed as VSAA and one SAA. The median age was 17 (9-20) years old and the median absolute neutrophil count was 0.09 (0.02-0.39) ×10 9/L. The median time from onset of hepatitis to hematopoietic failure was 2 (0.5-5.5) months, and at the time of beginning of IST all the eight patients had their liver biochemical testing returns to normal. The karyocytes were normal in all patients. One patient presented positive PNH clone at the diagnosis of HAAA, with PNH clone size of 4.7%. Avatrombopag was incorporated into day 1 of standard immunosuppressive therapy with p-ATG and cyclosporine for all the eight HAAA patients. The dose of avatrombopag was 40 mg/d at least 6 months. Dosage adjusting of avatrombopag was according to patient's platelet counts and adverse events. Encouragingly, four of the eight patients (50.0%) achieved hematologic response (HR) with two (20.0%) getting complete response (CR) at 3 months after IST and avatrombopag. Between 3 and 6 months after IST, the other two patient entered their hematologic responded. Then, six of eight (75.0%) patients got HR at 6 months, including 3 CR, 2 good partial hematologic response (GPR), and 1 partial response (PR). Unfortunately, one patient who was diagnosed as very severe HAAA, was without getting hematologic response and died at 118 days after treatment due to gastrointestinal infection. We evaluate the side effects of avatrombopag and graded according to the Common Terminology Criteria for Adverse Events (CTCAE) version 5.0. No hepatic adverse events were observed in all the eight patients, that indicated avatrombopag may not increase the hepatic burden and is safe for HAAA. The median follow-up time was 371(118-665) days. Except for one patient who died within 6 months, six cases achieved hematologic response, with 4 CR and 2 GPR. There is no clonal evolution, i.e., MDS, AML, and PNH, was observed. In conclusion, avatrombopag may be used as a safe and effective TPO-RA, added to IST for the treatment of hepatitis-associated aplastic anemia, which maybe improve the efficacy and the strength of hematologic response in HAAA.

Real-time quantification of proteins secreted of conditioned media from mesenchymal stromal cells (MSC) in co-culture with hematopoietic progenitor cells

The interaction of mesenchymal stromal cells (MSC) in co-cultures with hematopoietic progenitor cells (HPC) in ex-vivo models induce the secretion of pro-inflammatory protein factors and influences HPC cell proliferation and differentiation processes. Different studies have shown the quantification of these biomarkers in other conditioned media using an ELISA and Luminex™ type immunoassay format. In this paper, we developed real-time quantification of biomarkers in conditioned media of these cells in co-cultures from bone marrow, umbilical cord blood, and Wharton's jelly sources using surface plasmon resonance imaging (SPRi). We show the detection and quantification of CCL2, CCL5, bFGF, IL-6, IL-8 and VEGF proteins with high sensitivity and specificity. SPRi can be used with a real-time analysis methodology and could be considered a tool for biomarker assays.

A Phase 2 Study of Canakinumab in Patients with Lower-Risk Myelodysplastic Syndromes or Chronic Myelomonocytic Leukemia

Background Myelodysplastic syndromes (MDS) lie on a continuum of myeloid neoplastic progression between clonal hematopoiesis (CH) and acute myeloid leukemia (AML). Interleukin 1 beta (IL-1β)-induced inflammasome activation is a mechanism of cancer initiation and progression. Given that IL-1β upregulation induces aberrant myeloid differentiation of hematopoietic stem and progenitor cells (HSPCs), we hypothesize that IL-1β plays a critical role in MDS pathogenesis, and targeting IL-1β signaling will overcome aberrant MDS HSPCs' myeloid skewing. Here, we report final results of a phase 2 study evaluating the safety and efficacy of canakinumab, an IL-1β inhibitor, in patients with lower-risk MDS or chronic myelomonocytic leukemia (CMML). Methods We conducted a single center, open-label phase 2 trial at The University of Texas MD Anderson Cancer Center (Houston, TX, USA). Patients aged ≥ 18 with IPSS-R ≤ 3.5 MDS/CMML who have previously been treated were included in the study. Patients received 300 mg of canakinumab on day 1 of a 4-week cycle. The primary objective was to determine the clinical activity of canakinumab in MDS/CMML patients. Response was assessed using the modified International Working Group 2006 criteria for MDS (Cheson et al. 2006). Secondary objectives were to evaluate the safety profile including rate of transfusion independence (TI), duration of response (DoR), progression-free survival (PFS), and overall survival (OS). All patients who received 1 dose of study drug were included in the analyses. This study is registered with ClinicalTrials.gov (NCT04239157). Findings Between August 2020 and May 2023, 27 patients were enrolled in the phase 2 portion of this study, and 2 patients failed screening. The median age was 74 years with 15 (60%) male patients; 20 patients (80%) were post-hypomethylating agent failure with a median number of prior lines of therapy of 2 (1-5). Transfusion dependency was observed in 24 patients (96%) prior to canakinumab initiation. Fourteen (56%) patients showed normal karyotype. IPSS-R stratification revealed an intermediate-2 risk in 12 (48%) patients and a high risk in one (4%). The most common mutations were SF3B1 (40%), TET2 (32%), DNMT3A (28%), and RUNX1 (24%). IPSS -M risk score was calculated for 24 cases showing one (4%) very low (VL), four (16%) low (L), 14 (16%) moderate low (ML), eight (32%) moderate high (MH), five (20%) high (H), and two (8%) very high (VH) risk categories. Canakinumab was well tolerated and no drug-related toxicities were observed. One death due to sepsis, which was deemed not treatment-related, occurred on the study drug. Out of 23 evaluable patients, the overall response rate was 17·4%, with erythroid and platelet hematological improvement (HI-E and HI-P, respectively) confirmed in 3 (13.0%) patients and 1 (4.3%) patient, respectively. Thirteen patients had stable disease (56·5%) and 6 (26·1%) progressed during therapy, 1 of which transformed to AML (Fig. 1). TI was achieved in 3 patients (median DoR 8·53 months (95% CI 0·41-16·1) and 2 of them maintained TI for over 12 months. With a median follow-up time of 22·6 months (95% CI 15·0-29·4), median OS was 17·3 months (95% CI 14·3-not estimable). We performed separate univariate analyses to evaluate any associations between the IPSS-M and OS/PFS. The median OS in patients with higher-risk MDS by IPSS-M (MH, H, VH) was 15·0 months vs 29·4 months in the lower-risk disease by IPSS-M (VL, L, ML) group (p=0·12). Interestingly, statistically significant findings were observed with 1-year PFS when stratifying patients into higher vs lower risk MDS by IPSS-M (64·3% vs 100·0%, respectively; p=0·022, Fig. 2). Conclusion In this cohort of MDS patients who had experienced multiple lines of prior therapy and exhibited high-molecular-complexity, canakinumab showed limited efficacy (HI 17.3%). Nevertheless, canakinumab showed a good safety profile and yielded sustained long-term responses in patients with single somatic driver mutation in TET2 or DNMT3A. This suggests that clonal complexity, and therefore disease burden, may be a determining factor in response to canakinumab. Therefore, we have amended the protocol of a phase 1/2 clinical trial evaluating the safety and activity of canakinumab in clonal cytopenia of unknown significance (CCUS) and low-molecular complexity MDS patients. Our results will clarify the role of IL-1β signaling in MDS initiation and progression.

BGS-2456 Is a Novel Potent Covalent Inhibitor of FLT3 That Highly Discriminates Against KIT and Is Not Toxic Toward Normal Hematopoiesis in Vitro

Introduction: Class III receptor tyrosine kinases include FLT3, KIT, PDGFRA/B and CSF1R. Activating mutations in FLT3 represent the most common genetic anomalies in AML (30%). While there are numerous examples of KIT TKIs that do not inhibit FLT3 (imatinib, avapritinib, dasatinib), to date, all clinically active FLT3 TKIs (quizartinib, gilteritinib, midostaurin, sorafenib) fail to spare KIT inhibition. Preclinical studies suggest that coinhibition of FLT3 and KIT can result in non-specific myelosuppression, which can limit dose intensification and the ability to safely achieve maintenance dosing. A potent FLT3 TKI that is non-myelosuppressive may enable prolonged treatment in conjunction with chemotherapy and thereby substantially improve clinical outcomes, similar to BCR::ABL1 TKIs in Ph+ ALL (Slayton et al, JCO 2018). FF-10101 is a recently described covalent inhibitor to irreversibly bind FLT3. In vitro and in vivo studies have revealed that FF-10101 exhibits substantial activity against FLT3 and resistant mutants F691L and D835V/Y. However, it does not effectively discriminate against KIT inhibition (Yamara et al, Blood 2018). In clinical experience, a majority of responses were associated with persistent myelosuppression (Levis et al, JCO 2021 39:15_suppl, 7008). Further clinical development of FF-10101 has been abandoned. Here we describe a novel covalent inhibitor (BGS-2456) that potently targets FLT3 with outstanding selectivity against KIT. Results: To confirm that BGS-2456 is a covalent FLT3 inhibitor, we tested various Cys→Ser substitutions in FLT3-ITD. Of these, only substitution of C695 resulted in loss of activity, suggesting that this residue is critical for covalent bond formation with BGS-2456, as has also been demonstrated for FF-10101 (Yamara et al, Blood 2018). Additionally, the compound without the covalent warhead (BGS-2457) was found to be unimpacted by the C695S substitution and substantially less potent than BGS-2456 (Figure). To characterize in vitro properties of BGS-2456 and whether it achieves selectively against FLT3 relative to KIT, we performed cell viability assays of the patient-derived FLT-ITD-positive AML Molm14 cell line as well as the gastrointestinal stromal tumor cell line GIST-T1, which contains an activating juxtamembrane mutation in KIT. We compared EC50 ratios with other FLT3 TKIs and found that BGS-2456 is highly potent, with an EC50 value of ~0.2nM. Moreover, BGS-2456 displayed remarkable selectivity toward FLT3, >10-fold greater than all other TKIs tested. Kinome profiling confirmed FLT3 as the kinase that was most highly inhibited by BGS-2456 among the 300 kinases assessed, with BMX being a distant second (Figure). To assess whether the selectivity of BGS-2456 results in less myelosuppression in vitro, we performed CFU assays of healthy bone marrow. Compared to FF-10101 and gilteritinib, BGS-2456 exhibited the least amount of hematologic toxicity, facilitating in vitro proliferation and differentiation of normal hematopoietic progenitor cells even at 100x EC50 concentration against Molm14 cells. Finally, we assessed BGS-2456's ability to retain activity against the known clinically problematic drug-resistant FLT3-ITD mutants D835Y and F691L. Encouragingly, BGS-2456 retained activity against both D835Y and F691L mutants in the low nanomolar range. Conclusion: This is the first description of a potent and exquisitely specific FLT3 inhibitor that spares KIT inhibition and displays no myelosuppression in vitro at >100x EC50 concentration. The potent inhibitory effects of BGS-2456 on both D835Y and F691L mutants support its promise as a best-in-class TKI for the treatment of FLT3-mutant AML. Efforts to molecularly dissect the basis of the high degree of selectivity of BGS-2456 are ongoing.

Quantity and Functional Exhaustion of Mucosal-Associated Invariant T Cells in Acute Myeloid Leukemia: Friends or Foes?

Abstract Introduction Acute Myeloid Leukemia (AML) is a hematological malignancy affecting hematopoietic progenitor cell differentiation, proliferation, and cell cycle progression, possibly involving disturbances in host immune function. Comprehensive understanding of the immune system composition and function in AML patients is crucial for developing novel immunotherapeutic strategies. Mucosa-Associated Invariant T cells (MAITs), as a subset of unconventional T cells, bridge innate and adaptive immunity and have been predominantly studied in the context of mucosa-related cancers. However, their role in AML remains largely unexplored. Investigating the association between MAITs and AML is of significant importance for understanding the immune escape mechanisms in AML, developing novel immunotherapeutic approaches, and predicting patient prognosis. Methods This study aimed to delineate the longitudinal immune profile of MAITs during AML pathogenesis and treatment, which may contribute to understanding immunological changes of MAITs in AML and predicting patient prognosis. In this prospective study, we collected peripheral blood or bone marrow samples from 131 AML patients, including 99 newly diagnosed cases, 18 in remission, and 14 in relapse, as well as 69 healthy controls. We performed multi-parameter flow cytometry for phenotypic and functional analysis of MAITs and explored their relationship with disease features, progression, and prognosis. Results We initially demonstrated that MAITs reflected immunological defects in AML patients. MAITs showed signs of depletion in both frequency and absolute count at initial diagnosis (p<0.0001) (Fig. 1A), especially in the CD8 + MAITs subset. The frequency of MAITs reflected AML cell genetics, tumor burden, disease status, and treatment responsiveness (Fig. 1B). AML patients exhibited reduced frequency of effector memory MAITs (p<0.0001) and increased frequency of terminal differentiated MAITs (p<0.0001) in peripheral blood (Fig. 1C), indicating altered reactivity of MAIT cells to antigen stimulation. MAITs displayed a state of high activation and even exhaustion, as evidenced by upregulated PD-1 expression. Impaired secretion of Th1-type cytokines and increased secretion of Th17-type cytokines (Fig. 1D), granzyme B, and perforin were observed in MAITs, while MAITs also shifted towards secreting tumor-promoting cytokines such as IL-8. Lower frequency of MAITs was correlated with inferior Overall Survival (OS) and Progression-Free Survival (PFS) (Fig. 1E). Multivariate analysis identified MAITs frequency <2.12% as an independent prognostic factor affecting OS. Conclusion Our study highlights the role of MAITs in immunological defects in Acute Myeloid Leukemia and reveals their functional impairment and enhanced tumor-promoting effects. The frequency of MAITs in peripheral blood reflects the genetic features, tumor burden, disease status, and treatment responsiveness of AML patients. Lower frequency of MAITs is associated with inferior OS and PFS, serving as a prognostic indicator for AML.

Effect and Mechanism of Specnuezhenide on Chemotherapy-induced Myelosuppression.

This study aimed to investigate the therapeutic effect of Specnuezhenide on myelosuppression induced by chemotherapy and clarify its mechanism. In this study, we measured peripheral blood cells, thymus index, spleen index, bone marrow nucleated cells (BMNCs), and the number of cell colonies counted in vitro by hematopoietic progenitor cells (HPCs) to determine the effect of SPN on cyclophosphamide (CTX)-induced myelosuppression. The alterations in the expression of relevant proteins, the cell cycle, and cytokines associated with hematopoietic cells were examined to better understand how it works. In the cyclophosphamide-induced mouse model, our study discovered that SPN can increase the number of peripheral blood cells and BMNCs after treatment, increase the thymus index and decrease the spleen index, and promote the proliferation and differentiation of HPCs. SPN can improve the production of cultured colonies in vitro, reduce the level of hematopoietic factors in vivo, regulate the proportion of G0/G1 phase cells, and promote the normal growth and development of cells. SPN can increase the expression levels of key proteins MEK and p-ERK in the MAPK signaling pathway, which may be one of the important mechanisms for improving myelosuppression. SPN can enhance the hematological and immunological functions of myelosuppressionmice, and it is hypothesized that SPN is extremely helpful to the hematopoietic and immune functions of tumor patients following chemotherapy. SPN might be used to treat myelosuppression. Additionally, high doses of SPN have a stronger therapeutic effect than low levels of SPN.

HCMV UL8 interaction with β-catenin and DVL2 regulates viral reactivation in CD34+ hematopoietic progenitor cells.

CD34+ hematopoietic progenitor cells (HPCs) are an important cellular reservoir for latent human cytomegalovirus (HCMV). Several HCMV genes are expressed during latency that are involved with the maintenance of the viral genome in CD34+ HPC. However, little is known about the process of viral reactivation in these cells. Here, we describe a viral protein, pUL8, and its interaction and stabilization with members of the Wnt/β-catenin pathway as an important component of viral reactivation. We further define that pUL8 and β-catenin interact with DVL2 via a PDZ-binding domain, and loss of UL8 interaction with β-catenin-DVL2 restricts viral reactivation. Our findings will be instrumental in understanding the molecular processes involved in HCMV reactivation in order to design new antiviral therapeutics.

The NCOR-HDAC3 co-repressive complex modulates the leukemogenic potential of the transcription factor ERG

The ERG (ETS-related gene) transcription factor is linked to various types of cancer, including leukemia. However, the specific ERG domains and co-factors contributing to leukemogenesis are poorly understood. Drug targeting a transcription factor such as ERG is challenging. Our study reveals the critical role of a conserved amino acid, proline, at position 199, located at the 3’ end of the PNT (pointed) domain, in ERG’s ability to induce leukemia. P199 is necessary for ERG to promote self-renewal, prevent myeloid differentiation in hematopoietic progenitor cells, and initiate leukemia in mouse models. Here we show that P199 facilitates ERG’s interaction with the NCoR-HDAC3 co-repressor complex. Inhibiting HDAC3 reduces the growth of ERG-dependent leukemic and prostate cancer cells, indicating that the interaction between ERG and the NCoR-HDAC3 co-repressor complex is crucial for its oncogenic activity. Thus, targeting this interaction may offer a potential therapeutic intervention.