Hematopoietic Function Research Articles

Enhancement of Immune Functions by Limosilactobacillus reuteri KBL346: In Vitro and In Vivo Studies.

Lactobacilli have been widely used as probiotics because of their benefits for intestinal health and physiological functions. Among a variety of Lactobacillus genera, Limosilactobacillus reuteri has been studied for its ability to exert anti-inflammatory functions and its role in controlling metabolic disorders, as well as the production of the antimicrobial compound reuterin. However, the effects and mechanisms of L. reuteri on enhancing immune responses in the immunosuppressed states have been relatively understudied. In this study, we isolated an immunomodulatory strain, namely, L. reuteri KBL346 (KBL346), from a fecal sample of a 3-month-old infant in Korea. We evaluated the immunostimulatory activity and hematopoietic function of KBL346 in macrophages and cyclophosphamide (CPA)-induced immunosuppressed mice. KBL346 increased the phagocytic activity against Candida albicans MYA-4788 in macrophages, and as biomarkers for this, increased secretions of nitric oxide (NO) and prostaglandin E2 (PGE2) were confirmed. Also, the secretions of innate cytokines (TNF-α, IL-1β, and IL-6) were increased. In CPA-induced immunosuppressed mice, KBL346 at a dosage of 1010 CFU/kg protected against spleen injury and suppressed levels of immune-associated parameters, including NK cell activity, T and B lymphocyte proliferation, CD4+ and CD8+ T cell abundance, cytokines, and immunoglobulins in vivo. The effects were comparable or superior to those in the Korean red ginseng positive control group. Furthermore, the safety assessment of KBL346 as a probiotic was conducted by evaluating its antibiotic resistance, hemolytic activity, cytotoxicity, and metabolic characteristics. This study demonstrated the efficacy and safety of KBL346, which could potentially be used as a supplement to enhance the immune system.

The bone-liver interaction modulates immune and hematopoietic function through Pinch-Cxcl12-Mbl2 pathway.

Mesenchymal stromal cells (MSCs) are used to treat infectious and immune diseases and disorders; however, its mechanism(s) remain incompletely defined. Here we find that bone marrow stromal cells (BMSCs) lacking Pinch1/2 proteins display dramatically reduced ability to suppress lipopolysaccharide (LPS)-induced acute lung injury and dextran sulfate sodium (DSS)-induced inflammatory bowel disease in mice. Prx1-Cre; Pinch1f/f; Pinch2-/- transgenic mice have severe defects in both immune and hematopoietic functions, resulting in premature death, which can be restored by intravenous injection of wild-type BMSCs. Single cell sequencing analyses reveal dramatic alterations in subpopulations of the BMSCs in Pinch mutant mice. Pinch loss in Prx1+ cells blocks differentiation and maturation of hematopoietic cells in the bone marrow and increases production of pro-inflammatory cytokines TNF-α and IL-1β in monocytes. We find that Pinch is critical for expression of Cxcl12 in BMSCs; reduced production of Cxcl12 protein from Pinch-deficient BMSCs reduces expression of the Mbl2 complement in hepatocytes, thus impairing the innate immunity and thereby contributing to infection and death. Administration of recombinant Mbl2 protein restores the lethality induced by Pinch loss in mice. Collectively, we demonstrate that the novel Pinch-Cxcl12-Mbl2 signaling pathway promotes the interactions between bone and liver to modulate immunity and hematopoiesis and may provide a useful therapeutic target for immune and infectious diseases.

Characterization of the effects of Chinese peony (Radix paeoniae Alba) and its active ingredients on the hematopoietic function of the Chinese mitten crab (Eriocheir sinensis)

Hematopoietic tissue (HPT) is crucial for the proliferation and release of newly produced hemocytes during infection in crustaceans. Very few studies have reported how herbs affected the hematopoietic function of crustaceans. Radix paeoniae Alba is a candidate immunopotentiator herb with the potential to be a blood-tonifying drug. In this study, the effects of Radix paeoniae Alba and its monomers (paeoniflorin, albiflorin, and paeonol) on HPT and hemocytes of Eriocheir sinensis were evaluated. After feeding them commercial diets containing different ingredients for three weeks, the HPT development was determined via morphological observation and hematopoietic tissue body index (HTBI) calculation. The count and classification of hemocytes were evaluated using the hemocytometer and flow cytometer. The expression of hematopoietic factor astakine, transglutaminase (TGase), and marker genes for different types of hemocytes (KPI and SOD) and undifferentiated HPT cells (PCNA) were detected using real-time quantitative PCR (qRT-PCR) assay. The results indicated that the HTBI in the Radix Paeoniae Alba, paeoniflorin, and albiflorin groups increased by 114.96%, 111.42%, and 112.50%, which were higher than the control group. The Radix Paeoniae Alba, paeoniflorin, albiflorin, and paeonol groups had higher proportions of ovoid lobules than the control group, especially in the Radix Paeoniae Alba group. The total hemocyte count (THC) of the crab in the Radix Paeoniae Alba, paeoniflorin, and paeonol groups were 8.12, 9.69, and 8.57×106 ind/mL, respectively, which were higher than the control group. Furthermore, all experimental groups showed a higher proportion of granulocytes than the control group, with increased astakine protein being simultaneously detected in the HPT. The qRT-PCR results showed a relatively higher KPI expression in all the experimental groups, and also higher SOD mRNA expression in the Radix Paeoniae Alba, paeoniflorin, and paeonol groups, as compared with the control group. Therefore, these results implied the potential effect of Radix Paeoniae Alba and its monomers on the hematopoietic function of E. sinensis. However, due to the complex ingredient composition in Radix Paeoniae Alba and their different activities in the hematopoietic process, further research needs to be conducted in the future.

Efficacy Evaluation of a Tetracyclic Triterpene Compound Targeting Multiple Apoptosis Proteins for the Development of a Multiple Myeloma Treatment

Introduction:Multiple myeloma (MM) is an incurable hematologic cancer that originates in plasma cells and occurs primarily in older patients over 60. This study screened for hit compounds for MM treatments targeting mitochondria and apoptosis proteins. Further, non-clinical testing, including molecular docking analysis, was performed. Methods: Based on our previous studies, candidate compounds targeting mitochondria were screened (www.medchemexpress.com). The killing effect of the compounds on MM cell lines (RPMI8226, IM9, and U266) was examined using a sulforhodamine B assay. Apoptosis mechanisms were investigated in vitro. Complex immunocompromised mice were established by injecting RPMI8226-RFP-FLuc cells through the tail vein. Efficacy of the KBB-N2 activity was assessed following intraperitoneal (15 or 30 mg/kg/day) and tail vein (7 mg/kg/day) infusions for three days a week at daily intervals. Toxicity, pharmacokinetics, and molecular docking analyses were performed according to the established protocols. Results: The hit compound, KBB-N2, a tetracyclic triterpene, was identified as an MM-targeted compound with cytotoxic effects. The primary mechanism of action of KBB-N2 in MM cells was inhibiting the activity of the mitochondrial electron transport (ETC) complex, resulting in the generation of excess intracellular reactive oxygen species (ROS), which led to tumor cell death by ROS-mediated apoptosis. In addition, exposure to KBB-N2 inhibited the activity of catalase, a powerful antioxidant enzyme in MM cells. Severe suppression of normal hematopoietic stem cell function was observed with lenalidomide administration at a concentration of 25 μM. However, this toxicity was not observed when KBB-N2 was administered at doses up to 60 μM. KBB-N2 activity involves PARP cleavage and caspase activation in the apoptotic pathway. Furthermore, KBB-N2 inhibited cell growth and exerted cytotoxicity. KBB-N2 induced apoptosis by activating the P53 apoptosis pathway in MM cells. In vivo, all untreated tumor-bearing mice showed rapid tumor growth and severe plasmacytomas, which led to death within seven weeks. Mice treated with KBB-N2 had significantly inhibited tumor growth and longer survival times. The molecular docking assay revealed the optimal docking conformation binding energy of KBB-N2 with docking scores (kcal/M) of -7.3 and -9.8 for ETC complexes I and III, respectively. Conclusions: The triterpene compound, KBB-N2, was identified as a hit compound and may act as a targeted agent for MM treatment. The antimyeloma activity of KBB-N2 involves interference with the ETC complex and leads to MM cell death.

Local Oxygen Dictates Hematopoietic Cell Growth and Function

Functional hematopoietic stem (HSC) and progenitor cells (HPC) are responsible for the replenishment of blood/immune cells, are a source of treatment for diseases, and when dysregulated are a source of hematological neoplasia. To identify factors that impact HSC/HPC functional competency, we performed a transcriptomic screen which revealed that human cord blood (CB) HSCs/HPCs capable of early engraftment in an NSG mouse model of transplantation exhibited enriched expression of gene programs that vary with changes in oxygen (O 2) availability compared to non-transplanted input cells. This is physiologically and clinically relevant as HSCs/HPCs and diseased hematopoietic cells such as acute myeloid leukemia (AML) are exposed to O 2 tensions ranging from <1% to 21% as they function in various anatomical sites or are harvested for clinical applications. Thus, there are translational and practical rationales for understanding the growth and functional properties that hematopoietic cells exhibit across the range of O 2 they experience physiologically or artificially, which we examined here. First we found significantly different frequencies of HSCs/HPCs and colony forming units (CFU) in human bone marrow (BM), mobilized peripheral blood (PB), and CB, which have varied O 2 tensions, suggesting O 2 availability may impact hematopoiesis. To directly examine this, human CB CD34+ cells were split and plated in CFU assays or expanded in growth stimulating conditions at 1% O 2, 3% O 2, 5% O 2, 14% O 2 (physiologic tensions), or 21% O 2 (processing tension) . HPCs expanded best and formed more differentiated CFUs in high physiologic O 2 tensions and extra physiologic O 2 tensions, while HSCs expanded better and more potent CFUs were formed in a mid-physiologic O 2 tension (5%). This suggests that O 2 dependent pathways are critical to HSC/HPC growth but there may be a “sweet spot” for early HSCs/HPCs that allows for proliferation while maintaining self-renewal and potency to maintain a pool of primitive cells, possibly mirroring in vivo O 2 responses. In in vivo functional HSC/HPC analyses, NSG mice transplanted with CB units expanded in physiologic O 2 exhibited significantly better early neutrophil recovery and long-term myeloid reconstitution compared to input, suggesting that growth and maintenance of HSC/ myeloid HPC functional competency is better balanced in physiologic O 2 compared to extra physiologic tensions. Platelet recovery and lymphoid reconstitution were significantly better in mice receiving uncultured control CB, suggesting expansion does not affect all HSC/HPC subpopulations equally. Mechanistically, cells grown in low O 2 tensions (1-3%) had higher frequencies of early HSCs/HPCs, significantly lower intracellular ROS levels, a significantly higher percentage of cells in cell cycle G0, and lower levels of phosphorylated γH2AX than those grown in higher O 2. These data suggest that while to some extent HSCs/HPCs require O 2 to proliferate, they also exhibit properties associated with exhaustion when maintained in higher O 2. We next examined the effects varying O 2 tensions have on diseased hematopoietic cells using primary mouse AML cells or oncogene transformed human CB CD34+ cell lines. AML cells proliferated significantly more rapidly and formed more CFUs in higher O 2 tensions, suggesting that O 2 sensing pathways may be a dependency in AML that could affect disease progression as local O 2 tensions become perturbed. AML cells grown in lower O 2 exhibited lower resting levels of phosphorylated γH2AX (measured by flow cytometry) and lower O 2 consumption rates (measured by Seahorse metabolic flux analyzer), suggesting they are less susceptible to cellular stress. In line with this, AML cells maintained in low O 2 exhibited significantly better proliferation and recovery after treatment with the chemotherapeutic cytarabine compared to those in higher O 2 tensions. Thus, diseased cells growing in lower O 2 niches may be less susceptible to treatment and therefore may be a source of chemoresistance or relapse. These data reveal important roles for O 2 in balancing hematopoietic cell growth with normal and diseased cell function. Manipulating O 2 tension or O 2 sensing pathways could be used to improve HSC/HPC expansion for transplantation or to treat disease. Transcriptomic studies of HSCs/HPCs and AML cells are ongoing to determine the O 2 sensing mechanisms that dictate cellular function.

Human DOCK11 Deficiency Causes Defective Erythropoiesis and Systemic Inflammation

Erythropoiesis involves significant changes in the cells, which are mediated by the plasma membrane and the actin cytoskeleton. The composition of the actin cytoskeleton and the interaction between its components are dynamically modified during erythropoiesis, however the precise role that different actin regulators play during erythroid differentiation is poorly understood. The dedicator of cytokinesis (DOCK) family member DOCK11 regulates actin cytoskeleton dynamics via its guanine nucleotide exchange factor (GEF) activity, resulting in activation of the small Rho GTPase CDC42. CDC42 has been implicated in regulating the early stages of erythroid development, as well as the terminal maturation steps involving enucleation. However, the role of human DOCK11 in hematopoietic cell function and human disease had not been defined. We analyzed a cohort of four patients from four unrelated families presenting with recurrent infections, early-onset severe immune dysregulation, systemic inflammation, as well as normocytic anemia and anisocytosis of unknown origin. Using whole-exome sequencing, we identified rare, hemizygous germline mutations in DOCK11 in these patients. Two mutations - an early stop-gain mutation and a To study the role of DOCK11 during erythropoiesis, we generated a dock11-knockout zebrafish model. We found that the dock11-knockout zebrafish embryos recapitulated the anemia and aberrant erythrocyte morphology observed in human DOCK11 deficiency. The anemia was amenable to rescue with constitutively active CDC42, suggesting that DOCK11 regulates erythrocyte numbers in a CDC42-dependent manner. As a next step we modeled human erythroid differentiation in vitro using an erythroid liquid culture system starting from purified CD34+ cells. ShRNA-mediated knockdown of DOCK11 in CD34 + cells revealed impairment of cell growth and differentiation during erythroid development. In line with moderate erythroid hypoplasia observed in the bone marrow of one of the patients, these data suggest an erythroid-intrinsic role of DOCK11 during erythropoiesis. As the patients with germline DOCK11 defects also presented with recurrent infections and systemic inflammation, we further assessed the role of DOCK11 in immune cells. In line with its role in actin dynamics, we found that human DOCK11 regulates T-cell morphology and migration, suggesting that defects in DOCK11 might impact the T cells' capability to fight infections. We further uncovered that the immune dysregulation observed in the patients involved aberrant T-cell activation and cytokine production. Mechanistically, using cells from DOCK11-deficient patients and Dock11-knockout mice, we were able to show that Dock11 regulates cytokine production at the transcriptional level by modulating the nuclear translocation of the T-cell transcription factor NFATc1, known to regulate the production of several cytokines. Collectively, we identified germline loss-of-function mutations affecting the actin regulator DOCK11 in a previously unknown disorder associating anemia and systematic inflammation. This work earmarks the DOCK11-CDC42 axis as an attractive future target for the treatment of a broader range of immune and hematological diseases

A Longitudinal Study to Assess Hematopoietic Stem Cell and Immune Cell Health in Astronauts Pre-, during, and Post-Mission

The International Space Station (ISS) allows research to be done in low earth orbit (LEO), offering a uniquely accelerating environment to model disease and study human health. Studies done in microgravity can simulate models of stress, inflammation, aging, immune dysfunction, and pre-malignant transformation of stem cells. The NASA Twins Study suggests that extended periods of spaceflight may affect hematopoietic stem cell and immune cell health and function through their multi-omics approach of one ground-based twin, Mark Kelly, and one on board the ISS, Scott Kelly (Garrett-Bakelman et al., Science, 2019). A recent study also suggests an adaptive response to immune cell activity related to pre- and post-flight long-term missions after transcriptomic analysis of astronaut blood samples (Stratis, et al., Frontiers in Immunology, 2023). These studies emphasize the importance of assessing astronaut hematopoietic stem cell and immune cell function to further understand the mechanisms of cellular and molecular changes associated with time spent in LEO. We designed a longitudinal study to collect (under an IRB-approved protocol) peripheral blood from Axiom Mission 2 (Ax-2) astronauts to assess their hematopoietic stem cell and immune cell health. Timepoints include Launch-45 days, Launch-2 days, inflight, Return+1 day, Return+50 days, and yearly for 5 years. Ax-2 was a 10-day mission which launched on May 21, 2023 and splashed down on May 31, 2023. This was a private crew-tended mission with a sample size of 4 astronauts. As part of the stem cell health and fitness assessment, we selected for CD34+ hematopoietic stem and progenitor cells (HSPCs) from the 4 private astronauts and performed functional survival and self-renewal assays from each timepoint. Preliminary results suggest that the self-renewal capacity of HSPCs decreases inflight, but upon Return+1 day, HSPCs regain their ability to clone themselves at a higher capacity than pre-flight. Furthermore, we collected HSPCs for whole genome sequencing, 10x Genomics single cell sequencing, and whole transcriptome sequencing, to analyze pre-, during, and post-flight genomic and transcriptomic changes. Analyses will be done to identify mutational burden and mutational signatures associated with each timepoint. These mutational signatures will be compared to previously identified C-to-T mutations to assess the pre-malignant status of HSPCs due to short-term spaceflight (Jiang et al., Cell Reports, 2021). In addition to assessing CD34+ HSPC health, we will also profile the immune landscape by sequencing in parallel the CD34- fraction and performing targeted FACS analysis based on initial sequencing results. We also seek to identify markers of inflammation and anti-viral response through analysis of cytokines from biobanked plasma. This investigation has the potential to provide valuable insight into stem cell and immune cell health and fitness associated with short-duration missions in LEO.

Long-Term Hematopoietic Dysfunction in Patients with Single Large-Scale Mitochondrial DNA Deletion Syndromes

Pearson syndrome (PS) is the only single large-scale mitochondrial DNA deletion syndrome (SLSMDs) presenting substantial hematologic manifestations, usually a severe though transient cytopenia of childhood. Recently, progression to myeloid malignancies has been described among PS patients. Older PS patients, as well as patients with SLSMDs presenting with Kearns-Sayre syndrome (KSS) or with progressive external ophthalmoplegia (PEO), do not display hematologic abnormalities. Since the underlying mitochondrial DNA deletion is similar in all SLSMDs, we hypothesized that bone marrow (BM) dysfunction is a common and persistent manifestation in these patients. We studied 16 patients with molecular-confirmed SLSMDs: 13 with PS, and three with KSS. The median age at diagnosis was 25 months (range 2-136 months). Seventy-five percent of patients presented with cytopenia in infancy, and 56% required blood products. Resolution of cytopenia occurred in 92% of this subgroup, at a median age of 24 months (range 12-39 months). BM studies were performed in all patients, and 56% of patients had multiple BM evaluations. All BM biopsies examined showed signs of marrow dysfunction, including in patients without a history of cytopenia. Median BM cellularity was 50% (range 30-80%) at a median age of 5 years. BM samples showed a paucity of precursor myeloid cells, signs of dyserythropoiesis, and abnormal or missing megakaryocytes. We report low hematopoietic stem and progenitor cell functionality in patients with SLSMDs: The median CD34+ count was 0.68% (range 0.27-1.92%, expected range 1-2%). BM colony forming unit (CFU) capacity was reduced in SLSMD patients: the median CFU in patients was 280 per 10 6 cells compared to 1090 per 10 6 cells in healthy controls (p=0.004). Conventional cytogenetic analysis was done on 10 patients (62%), and FISH analysis was done on 15 patients (93%). Monosomy 7 was identified in seven patients (44%): in one patient the monosomy 7 resolved in a follow-up BM assay. Another patient with signs of myelodysplasia and 30% of the BM cells having monosomy 7 was referred to an allogeneic bone marrow transplantation. To conclude, long-term BM dysfunction is evident in all patients with SLSMD and includes a risk of developing clonal evolution and monosomy 7. This data, combined with other recent publications, highlights the ongoing marrow failure and suggest that SLSMD syndromes should be included under inherited BM failure syndromes.

Circadian Rhythms Metabolically Regulate Bone Marrow Retained Primitive Hematopoietic Stem Cell Size and Function

Most primitive hematopoietic stem cells (pHSC; LSK SLAM CD34 - EPCR +) are bone marrow (BM) retained in a quiescent non motile state, and are the only chemotherapy resistant HSC which requires their rapid exit from homeostatic quiescence on demand to prevent lethal hematology failure. Circadian rhythms induce daily release of progenitor cells and low levels of rarely cycling pHSCs to replenish the blood and lymphatics with new maturing cells. Metabolically, pHSCs are characterized by their dependence on glycolysis, with low mitochondrial activity and membrane potential (MMP), associated with higher competitive repopulation potential. However, if their metabolism is influenced by circadian cues is currently unknown. We report here that pHSCs at night are more glycolytic with higher surface expression of the glucose transporter Glut1, and higher glucose uptake. Repeated administration of the glucose analog, 2-deoxyglucose (2-DG), during daylight mimicked the night phenotype and enhanced HSC engraftment potential. However, nocturnal administration of 2-DG did not further improve glucose uptake nor HSC engraftment potential, suggesting glycolytic saturation. To further understand these metabolic oscillations of glucose uptake, we investigated the circadian, transcriptional signatures by single pHSC RNA-seq analysis. We found higher expression of mitophagy-related genes at night. Genes regulating mitochondrial function, were also significantly down regulated at night leading to low MMP in pHSC. While mitochondrial ROS levels were higher, cytoplasmic ROS levels were significantly reduced in pHSC at night. Importantly, we further found at night, low-functioning pHSC mitochondria to also undergo higher levels of fission as assessed by increased expression of the active mitochondrial fission marker, phospho-Drp1 (S616). To better understand the molecular pathways mediating daily circadian metabolic reprogramming between glycolysis and mitochondrial activity, we monitored our gene set enrichment analysis. We found the Wnt/b-catennin and HIF-1a signaling pathways to be significantly increased at night in pHSC. Active Wnt signaling, leads to enhanced b-catenin localization to the nucleus which is enhanced by downstream effector of Wnt activation, FOXM1. We observed higher total protein expression of FOXM1 at night. While its nuclear and cytoplasmic localization were reduced, we observed significantly higher FOXM1 levels in the mitochondria. FOXM1 localization to the energy powerhouse has been shown to inhibit mitochondrial activity. Blocking mitochondrial recruitment of FOXM1 with a specific canonical Wnt inhibitor at night led to upregulation of MMP, reversing mitochondrial metabolic responses in pHSC. Finally, daily genetic changes between night Wnt, HIF-1a and glycolysis, versus daylight GSK3b, m-TOR signaling and enhanced mitochondrial activity in BM retained pHSC, also led to cell size changes. We documented reduced quiescent pHSC size at night associated with their increased BM maintenance and higher repopulation potential. While larger sized pHSCs in the morning during the time of blood replenishment, increased their migration and development potential, for their rapid exit from quiescence on demand. Our study reveals daily circadian cues dynamically determine Wnt-mediated FOXM1 bidirectional shuttling between the nucleus and the mitochondria, leading to metabolic switching of pHSCs to maintain their cellular fitness. Our data define key signaling pathways controlling pHSC quiescence and diverse circadian metabolic activities for accumulated ROS and old mitochondria clearance and turnover, keeping them ready for rapid activation in alarm situations.

Deciphering the Differential Impact of Thrombopoietin/MPL Signaling on Hematopoietic Stem Cell Function in Bone Marrow and Spleen

Introduction Thrombopoietin (TPO) and its receptor MPL are key regulators of hematopoietic stem cell (HSC) function. In humans, ablation of either TPO or MPL leads to bone marrow failure in the early years of life. In contrast, mice lacking TPO or MPL display marked thrombocytopenia and severely reduced HSC numbers but manage to maintain normal survival up to one year of age. In this study, we investigated whether these mice eventually develop marrow failure with longer follow up, with a specific focus on understanding the effects of TPO/MPL signaling on HSC regulation in different hematopoietic niches. Methods Marrow and spleen hematopoiesis in the TPO -/- mice and MPL -/- mice were examined during a 2-yr follow-up. Results At 2 yrs of age, TPO -/- and MPL -/- mice exhibited not only a severely decreased platelet count (TPO -/- vs. MPL -/- vs. control: 38 vs. 107 vs. 1255 x 10 9/L) but also a moderate reduction in neutrophil count compared to age-matched wild-type controls (TPO -/- vs. MPL -/- vs. control: 1.1 vs. 1.7vs.3.3 x 10 3/ul). Femur marrow cell count or spleen cell count was not significantly different between TPO -/- or MPL -/- mice and control mice. There was no significant difference in overall survival between the TPO -/- or MPL -/- mice and wild-type controls. Marrow Lin -cKit +Sca1 +CD150 +CD48 - HSC cell numbers were significantly decreased in young (4-6mo) TPO -/- and MPL -/- mice (~4-fold) compared to age-matched control mice. This HSC loss was further worsened in old (~24mo) TPO -/- (25-fold) and MPL -/- (12-fold) mice compared to control mice. In contrast, there was no significant difference in splenic HSC cell numbers between young TPO -/- or MPL -/- mice and control mice, with only a moderate decrease of HSCs in old TPO -/- and MPL -/- (~4-fold) mice compared to control mice. I n vivo using BrdU labeling revealed an increased proliferation of marrow HSCs in TPO -/- and MPL -/- mice compared to age-matched control mice. In contrast, there was no significant increase in splenic HSC proliferation in TPO -/- or MPL -/- mice. These findings suggest that TPO/MPL signaling has distinct effects on both the number and the function of HSCs in the marrow and spleen. Competitive repopulation assays revealed that HSCs from 2yr old TPO -/- or MPL -/- marrow failed to engraft the recipient mice at 12 weeks post-transplantation. We also conducted competitive repopulation assays in which spleen cells from old TPO -/- mice (CD45.2) were injected together with spleen cells from 2yr old wild-type competitor mice (CD45.1) into lethally irradiated wild-type recipients (CD45.1). At 12 days after transplantation, the TPO -/- spleen donor showed comparable engraftment in the Lin -cKit +Sca1 + stem/progenitor cell compartment, similar to the wild-type competitor spleen donor, suggesting that at least the short-term engraftment capacity of the TPO -/- spleen donor was not impaired. We are currently conducting further investigation to assess the long-term repopulation capacity of TPO -/- spleen HSCs. To investigate how TPO/MPL ablation impacts the hematopoietic niche in the marrow and spleen, we used quantitative flow cytometry and/or fluorescence imaging to examine these niche cells and their expression of niche factors. First, while there was no significant difference in the marrow vascular area (measured by in vivo VE-cadherin labeling) between TPO -/- or MPL -/- mice and control mice, we observed a significant increase in the splenic vascular area in TPO -/- and MPL -/- mice. Second, CXCL12 levels were significantly decreased in marrow endothelial cells (ECs; CD45 -CD31 +) from TPO -/- and MPL -/- mice compared to control mice; in contrast, CXCL12 levels were significantly increased in splenic ECs from TPO -/- and MPL -/- mice. Similar results were also observed with marrow and splenic perivascular stromal cells (CD45 -CD31 -Ter119 -Sca1 -). Therefore, TPO/MPL ablation enhanced both the quantity and quality of the splenic hematopoietic niche, which could contribute to the maintenance of splenic hematopoiesis in TPO -/- and MPL -/- mice. Last, consistent with findings from the TPO/MPL ablated mice, exogenous TPO treatment increased marrow HSC proliferation but did not alter splenic HSC proliferation. In addition, TPO treatment increased CXCL12 expression in marrow ECs, but decreased CXCL12 levels in splenic ECs. Taken together, our study revealed the complexity of TPO/MPL signaling in HSC regulation which differs between the marrow and spleen

Early Efficacy of IST in Combination with Hetrombopag and Rhtpo As First-Line Treatment for Naïve Severe Aplastic Anemia

BACKGROUND: Immunosuppressive therapy (IST) (anti-thymocyte immunoglobulin (ATG) and cyclosporine (CSA)) is the treatment of choice for patients with acquired severe aplastic anemia (SAA) who are ineligible for hematopoietic stem cell transplantation (HSCT), which results in a hematological response (HR) in about 60-70% of SAA patients. TPO is a hepatic-secreted hematopoietic growth factor that specifically regulates platelet production, induces hematopoietic stem cell differentiation to megakaryocytes by binding to the TPO receptor (MPL), stimulates megakaryocyte proliferation and differentiation, and promotes platelet production and release. Several previous studies have shown that IST combined with rhTPO or hetrombopag can improve the early hematologic response of SAA patients, especially the proportion of good hematologic response (good HR) in the early stage. RhTPO and TPORA have different binding sites with thrombopoietin receptor (c-MPL) and do not have competitive binding, which may produce synergistic effect. A retrospective study showed that eltrombopag combined with rhTPO increased the IST HR and improved the quality of hematologic remission in SAA with mild adverse effects. Therefore the combination of TPORA and rhTPO in SAA patients undergoing IST treatment may result in faster and higher HR rates. Clinical studies have confirmed that hetrombopag, act similarly to eltrombopag in SAA, but there are no clinical studies of hetrombopag in combination with rhTPO in SAA. OBJECTIVE: To evaluate the early-efficacy and safety profile of IST in combination with hetrombopag, and rhTPO in patients with primary, severe aplastic anemia. METHODS: This study was designed for patients with primary aplastic anemia major aged 14-70 years after January 2023 from a single center. This group were planned to be get treat of IST combination with rhTPO 15000U/d for 8 weeks from the first day of ATG and hetrombopag 15mg/d for at least 6 months. The primary endpoint was to observe the good response rate, blood product transfusion and safety profile of the study regimen for the treatment of primary SAA for 3 months. Efficacy Criteria：Complete response (CR): HGB>100 g/L, PLT> 100×10 9/L and ANC>1.5×10 9/L; ② Good partial response (GPR): HGB>80g/L, PLT> 50×10 9/L and ANC>1.0×10 9/L; ③ Partial response (PR): withdrawal from blood product transfusion dependence (HGB>70g/L, PLT> 20×10 9/L), hematologic tests improved and no longer met the SAA criteria, but blood count did not meet the GPR criteria. ④No response to treatment (NR): Continuous transfusion dependency was classified as no response. and/or blood count still met SAA criteria. We defined CR+GPR+PR as HR and CR+GPR as good HR. RESULTS: The trial group proposes to enroll 39 patients and currently includes 8 patients who can be evaluated for 3-month efficacy of IST treatment. And the control group includes two groups of IST combined rhTPO group and IST combined HPAG group in our previous center, and the cohort study of the results of the three groups. In this study, the HR rate of 8 patients can be assessed at present as 75% (6/8), and the rate of good HR was 50% (4/8). The HR of 33 patients in the control IST+rhTPO group was 66.67% (22/33), and the rate of good HR was 36.4% (12/33); the HR rate of 32 patients in the control IST+HPAG group was 46.9% (15/32), and the rate of good HR was 21.9%. The HR rate and good HR rate of the trial group were higher than those of the IST+rhTPO group and the IST+HPAG group (75%v66.67%v46.9%; 50%v36.4%v21.9%). None of the 8 patients enrolled in the trial group had any transformation of myelofibrosis, myelodysplastic syndrome and acute myeloid leukemia during the follow-up period. CONCLUSION: HR was obtained at 3 months in 6 of the 8 patients with severe remodeling IST+rhTPO+ hetrombopag in this study, with a good HR rate of 50%, which is the highest good efficacy response we know of in a prospective study of Chinese SAA patients to date. This confirms that rhTPO combined with hetrombopag can synergize the recovery of bone marrow hematopoietic function in SAA patients treated with IST, and its clinical effect is superior to that of various TPO-Ras or rhTPO that have been widely used. It is expected that more cases will be included in the later stages and that better results will be obtained. 【Keywords】severe aplastic anemia; recombinant human thrombopoietin; hetrombopag ; immunosuppression treatment.

The Association of SEPTIN6 Mutations with Pediatric Myelodysplasia

Septin family proteins belong to the RAS-like GTPase superclass and are considered as a structural element of the cytoskeleton. They are evolutionarily conserved and are important in cytokinesis, polarity, cell cycle, vesicle trafficking, and exocytosis. They are implicated in T and B cell lymphomas; and acute myeloid leukemias, where they frequently act as fusion partners with the Mixed-Lineage Leukemia (MLL) gene. Previously, we described two non-syndromic patients with severe neutropenia which progressed to myelodysplastic syndrome (MDS) (Renella et al. AJH, 2022, Mohamad et al. ASH, 2022). Next-generation sequencing revealed both patients (P1 and P2) to have novel X-linked germline mutations in codon 428 of the SEPTIN6 gene (P1: SEPTIN6 c.1282T>C; P2: SEPTIN6 c.1282T>A). One of these patients (P1) had an additional somatic stop-gain mutation (P1: SEPTIN6 c.43C>T) of unclear function. Both patient germline mutations were predicted to add 9 amino acids to the C-terminal end creating a novel mutated SEPTIN6 protein. Since SEPTIN6 is located on the X-chromosome and expression of these mutated proteins occurred in males, no wild-type protein was present in the patient cells. To further explore the biological relevance of these mutations, single base-pair gene editing was used to introduce the (P1: T>C) mutation into normal male donor CD34+ cells. Morphological studies demonstrated that SEPTIN6 edited cells had enlarged, multi-nucleated and dysplastic nuclei; and reduced clonogenic activity. To further assess the effect of the T>C mutation on hematopoietic stem cell (HSC) function, the SEPTIN6 edited cells were transplanted into immunocompromised NSGW mice. Analysis of peripheral blood demonstrated reduced chimerism in edited recipients (4.27% +/-3.4%) compared to controls (20.56% +/-6.2%, mean+/- SD, p= 0.0008) indicating a functional HSC defect upon mutation acquisition. Bulk mRNA sequencing demonstrated changes in ribosome, DNA replication, homologous recombination and mismatch repair pathways which imply DNA damage and cell cycle defects (Figure 1). To examine the potential dominant negative vs haploinsufficiency role of these mutations, we introduced the mutated SEPTIN6 (P1: SEPTIN6 c.1282T>C) cDNA into wild type human male CD34+ cells. Morphological studies demonstrated enlarged cells with multi-nucleated and dysplastic nuclei. Moreover, clonogenic assays demonstrated that cells containing both the mutated and wild type protein had reduced number of colonies (17.7+/- 5.5) compared to controls (127+/- 13.7, mean+/- SD, p <0.0001). This indicates that the mutated SEPTIN6 protein has a dominant negative effect over the wild type protein. We hypothesized that the somatic stop-gain mutation in the patient (P1) genome may mitigate the deleterious effects of the primary germline SEPTIN6 c.1282T>C mutation. To test this hypothesis, and determine the effect of each individual mutation, we created lentivirus vectors which contained a shmiR against the native SEPTIN6 mRNA and also contained (a) the germline P1: SEPTIN6 c.1282T>C mutation (M2 mutation) alone, or (b) the germline M2 mutation and the somatic stop-gain mutation (P1: M1+M2 mutation) in tandem, or (c) germline P2: SEPTIN6 c.1282T>A (M3 mutation). The wild type SEPTIN6 gene was reintroduced as a control. Clonogenic assays demonstrated that SEPTIN6 knockdown in human CD34+ cells caused no significant change in progenitor activity (Table 1). Furthermore, introduction of the M2 or the M3 germline mutants in SEPTIN6 knocked down cells caused a reduction in the total number of colonies, especially the number of GM colonies with no change in the number of BFU-E colonies (Table 1). This data validates our previous experiments performed with single base pair gene editing in human CD34+ cells. Interestingly, introduction of the stop-gain M1 mutation along with the M2 mutation (M1+M2) reversed the reduced clonogenic activity seen with expression of the M2 mutation (Table 1). This indicates that the M1 mutation by introducing an early stop codon creates a SEPTIN6 knockdown effect and rescues hematopoietic function from the deleterious M2 mutation. Overall, our data demonstrates the acquisition of de novo gain of function mutations in the SEPTIN6 gene in myelopoiesis and suggests deleterious effects on engrafting hematopoietic stem cells. These mutations are associated with the development of MDS in two pediatric patients.

IST Combined with Full-Dose Rhtpo in the Treatment of Severe Aplastic Anemia -a Prospective Single-Center Clinical Study

BACKGROUND: Immunosuppressive therapy (IST) (anti-thymocyte immunoglobulin (ATG) and cyclosporine (CSA)) is the treatment of choice for patients with acquired severe aplastic anemia (SAA) who are ineligible for hematopoietic stem cell transplantation (HSCT), which results in a hematological response (HR) in about 60-70% of SAA patients, while 1/3 of patients fail to achieve a hematological response. TPO is a hepatic-secreted hematopoietic growth factor that specifically regulates platelet production, induces hematopoietic stem cell differentiation to megakaryocytes by binding to the TPO receptor (MPL), stimulates megakaryocyte proliferation and differentiation, and promotes platelet production and release. Previous retrospective studies have demonstrated that IST combined with rhTPO improves the early hematologic response in SAA patients, but efficacy reported by centers varied depending on the dose and duration of rhTPO. There are no prospective studies of rhTPO application in SAA to confirm its efficacy. So this study was designed to observe the early feeicacy and safety of the SAA patients who were treated with IST combination with full-dose rhTPO. METHODS: This study was a single-center, single-arm, prospective cohort clinical study, enrolling 33 naive SAA patients aged between 14-70 years who diagnosed after April 2021 in our study center. The patients treated with IST combined with full-dose rhTPO (300U/kg.d) for 8 weeks starting from the first day of ATG. The primary endpoint of the observational study protocol good response rate, blood product transfusion volume and safety profile at 3 months of treatment of initial SAA. The patients were also evaluated for total hematologic response rate and adverse effects of drug application, clonal shift rate, and recurrence rate at 3 and 6 months after treatment. Efficacy Criteria：Complete response (CR): HGB>100 g/L, PLT> 100×10 9/L and ANC>1.5×10 9/L; ② Good partial response (GPR): HGB>80g/L, PLT> 50×10 9/L and ANC>1.0×10 9/L; ③ Partial response (PR): withdrawal from blood product transfusion dependence (HGB>70g/L, PLT> 20×10 9/L), hematologic tests improved and no longer met the SAA criteria, but blood count did not meet the GPR criteria. ④No response to treatment (NR): Continuous transfusion dependency was classified as no response. and/or blood count still met SAA criteria. We defined CR+GPR+PR as HR and CR+GPR as good HR. RESULTS: The rhTPO group enrolled 33 patients, and the control group was 99 patients who applied the caliper value of 0.2 to match the previous unapplied TPO treatment of SAA. At 3 months after IST treatment efficacy, the HR rate of 33 patients in the rhTPO group was 66.67% (22 /33 ), and the rate of good HR was 36.4% (12/33). The HR rate in the control group was 32.32% (32 /99 ), and the rate of good HR was 12.12% (12/99). The HR rate and the good HR rate between the two group were significantly statistical differences (66.67% v 32.32%; 36.4% v 12.12%), (P value=0.001; 0.002). At 6 months after IST treatment, the HR rate and good HR rate in the rhTPO group had a significantly higher than those of the control group (66.67% v 32.32%; 36.4% v 12.12%), (P value=0.001; 0.002). No disease transformation such as myelofibrosis, myelodysplastic syndrome and acute myeloid leukemia occurred in all enrolled patients during the follow-up period. CONCLUSION: 22 of the 33 SAA patients in this study achieved HR at 3 months after IST treatment, including 36.4% cases achived good HR , which is the highest good efficacy response in a prospective study of SAA patients in China known to us to date. This confirms that full-dose rhTPO, as a macromolecular peptide, can also promote the recovery of bone marrow hematopoietic function, significantly improved the rapidity and strength of HR in SAA patients. And its clinical effect is no less than that of the various TPO-Ras that have been widely used so far, and may even be higher. 【Keywords】severe aplastic anemia; recombinant human thrombopoietin; immunosuppression treatment.

15-Pgdh Inhibition Alternatively Activates Macrophages to Promote Hematopoietic Function during Aging

Aging results in the progressive decline of organ function and integrity across species. In the hematopoietic system aging is associated with diminished stem cell regenerative capacity and contributes to compromised tissue homeostasis and heightened vulnerability to age-related diseases including anemia, myelodysplastic syndromes, and hematological malignancies. Macrophages, integral components of the innate immune system, demonstrate remarkable functional plasticity and play diverse roles in tissue homeostasis, inflammation, and immunity. The balance between pro-inflammatory M1 and anti-inflammatory M2 macrophages significantly influences immune and inflammatory responses, and thus the progression of hematopoietic aging. We have previously demonstrated that the prostaglandin degrading enzyme 15-hydroxyprostaglandin dehydrogenase (15-PGDH) regulates hematopoietic stem cell (HSC) activity, that 15-PGDH inhibition (PGDHi) following transplantation results in a markedly accelerated recovery of neutrophils, platelets, and HSCs, and that 15-PGDH knockout animals display enhanced HSC function with age. Here we expand on our previous work and demonstrate in aged mice that (1) 15-PGDH expression and activity remains conserved (2) that PGDHi induces a gene expression signature consistent with alternatively activated M2 macrophages, (3) that prolonged PGDHi results in a significant increase in phenotypic HSCs in both the bone marrow and spleen and induces a gene signature consistent with enhanced HSC function, and (4) that PGDHi treated bone marrow (BM) displays a advantage during primary competitive repopulation studies and prevents age-associated myeloid bias during secondary transplants. Taken together we propose PGDHi represents an in vivo switch to alternatively activate macrophages, which has significant implications to not only prevent age-associated hematopoietic decline, but also as a novel strategy to treat age-associated hematologic disease.

Impact of Dkc1 Deficiency on BM Niche Function during Hematopoietic Stem Cell Transplantation in a Conditional Murine Model of X-Linked Dyskeratosis Congenita

Dyskeratosis congenita (DC) is a telomere biology disorder associated with bone marrow failure (BMF) and the triad of dystrophic nails, skin rashes, and leukoplakia. X-linked DC (XDC) is caused by mutations in DKC1, which encodes dyskerin, a nucleolar protein associated with rRNA processing and telomerase complex formation. Hematopoietic stem cell transplantation (HSCT) is currently the only definitive curative treatment for BMF in XDC patients, though many patients exhibit only partial restoration of hematopoietic function. Our previous studies demonstrated that molecular and cellular defects in BM niches contribute to poor donor HSC engraftment in murine models of other BMF syndromes, including Shwachman-Diamond Syndrome (SDS), a disease whose pathogenesis intersects with the role of dyskerin in ribosome biogenesis. These studies led us to hypothesize that BM niche dysfunction contribute to poor graft function in DC patients post-HSCT. Defining specific deficits would enable development of novel targeting strategies to improve HSCT outcomes for patients with XDC. Here, we report that in a novel murine model of XDC driven by conditional Dkc1 deletion, Dkc1 deficiency within mesenchymal BM niche cells causes significant impairment of healthy donor HSC engraftment after HSCT. We have identified defects in multiple pathways within Dkc1-deficient BM niche in response to myeloablative conditioning that may drive this reduction in HSC supportive capacity. To generate a murine model with conditional Dkc1 deletion in BM niche, we crossed Dkc1l/lmice with Mx1 Cre+mice, inducing Cre expression in hematopoietic cells and Mx1-inducible niche cells by polyinosinic-polycytidylic acid administration. At baseline, the resulting Mx1 CreDkc1 Exc mice exhibit mild hematopoietic abnormalities versus Dkc1 l/l controls, including variable thrombocytopenia, increased blood monocytes, and reduced BM lymphoid progenitors. To assess capacity of BM niches from Mx1 CreDkc1 Exc mice to efficiently engraft donor HSC, we transplanted Mx1 CreDkc1 Exc mice and Dkc1 l/l controls with healthy GFP + wild-type donor BM and assessed efficiency of donor HSC engraftment at 2 weeks following HSCT using competitive secondary HSCT. The Mx1 CreDkc1 Exc recipients exhibited significantly impaired efficiency of donor HSC engraftment compared with controls, as indicated by a 25-30% reduction of long-term GFP + cell reconstitution in mature blood lineages in secondary recipients (Figure 1). We next sought to identify molecular and cellular defects in BM niche that contribute to the engraftment deficits in Mx1 CreDkc1 Exc mice. Flow cytometry analysis of BM niche cells 48 hours after total body irradiation (TBI) showed Mx1 CreDkc1 Exc mice exhibited a 20% reduction of CD11b+F4/80+ osteomacs compared to Dkc1 l/l controls. In contrast to our published findings in a model of SDS, the frequencies of osteoprogenitors, mesenchymal stem cell, endothelial cells and megakaryocytes in BM niche were similar between irradiated Dkc1-deficient and control mice. We then performed multiplex ELISA and RNA-seq analyses on isolated BM niche cells from Mx1 CreDkc1 Exc versus control BM at 48 hours post-TBI. Irradiated Mx1 CreDkc1 Exc BM niche demonstrated lower expression of multiple chemokines, including CXCL1, CXCL12, CCL1, CCL11 and CCL12, known to be critical for recruiting HSC and many leukocyte subsets. Dkc1 deficiency in post-TBI BM niche cells also led to lower expression of genes that regulate production of IFN-γ and IL-1, resulting in reduced expression level of both cytokines that may drive decreased osteomacs seen in Dkc1-deficient BM niches. Moreover, Gene Ontology analysis indicates that after TBI, Dkc1 deficiency within BM niches resulted in downregulation of gene expression involved in cell-cell adhesion and extracellular matrix, both of which are critical for HSC enlodgement and engraftment after HSCT. Taken together, our results demonstrated that Dkc1 deficiency in BM niche cells that are not replaced by HSCT impairs the ability of these niches to engraft donor HSC efficiently. Disruptions of molecular and cellular pathways implicated in the niche dysfunction of this XDC model are distinct from our previous findings with SDS model. Ongoing and future studies are needed to define which of these pathways have the highest potential to serve as translational targets to improve HSCT success rate to treat BMF in patients with XDC.

The Function and Mechanism Research of NLRP6 in Bone Marrow Microenvironment in Myeloproliferative Neoplasms

Since the important role of inflammation in myeloproliferative neoplasms (MPN) progression, inflammasome is an important regulator of inflammation involved in the development of tumor. Here, we aimed to investigate the function of NLRP6 in bone marrow (BM) microenvironment in MPN in the hope of getting better treatment for MPN. We first analyzed that NLRP6 was highly expressed in BM mononuclear cells from individuals newly diagnosed with ET, PV, PMF patients (with or without JAK2 V617Fmutation). Similar results were obtained in BM cells and BM stromal cells of MPL W515L transduced C57BL/6 mice (C57BL/6 MPN). The results were validated that NLRP6 of the BM microenvironment was involved in the development of MPN. We thus hypothesized that the absence of NLRP6 in BM microenvironment affects the development of MPN. NLRP6 -/- mice and C57BL/6 mice MPN models were established by the same method. The experiments demonstrated that overall survival of NLRP6 -/- MPN mice was significantly prolonged compared to C57BL/6 MPN mice. Loss of NLRP6 in BM microenvironment interfered with hematopoiesis of BM, but it had no effect on the extramedullary hematopoiesis (EMH) in spleen. Pathological analysis showed that the destruction of spleen structure in NLRP6 -/- MPN mice and the infiltration of atypical megakaryocytes were less than C57BL/6 MPN mice. The tumor load in spleen was significantly reduced and the structure of spleen was intact, the hematopoietic function of the BM returned to normal and the tumor load in the BM was significantly reduced at 5 weeks in NLRP6 -/- MPN mice after sequential transplantation. It suggested that knockout of NLRP6 in the BM microenvironment significantly attenuated the tumorigenicity of malignant HSCs, delayed or even inhibited the development of MPN disease. The expression of inflammatory cytokines, abnormal megakaryocytes and the phenomenon of aggregation and reticular fibers in BM microenvironment were significantly reduced in NLRP6 -/- MPN group compared with the control group. After sequential transplantation, the destruction of BM structure was alleviated, megakaryocytes aggregation and heteromorphic megakaryocytes in BM were reduced, and reticular fibers were significantly diminished. Furthermore, the cytokines promoting disease progression in BM microenvironment were significantly reduced in NLRP6 -/- MPN group, suggesting that knockout of NLRP6 can improve the BM microenvironment and alleviate the progression of myelofibrosis. Finally, to gain better insight into the role of NLRP6 inflammasome in MPN pathogenesis, we sought to dissect the changes with scRNA-seq and pathway identification based on available data and bioinformatic pathway analysis in NLRP6 -/- mice. The results of scRNA-seq analysis showed that C18 population of malignant HSCs of recipients in primary transplantation was added, C16, C13 and C9 cell populations disappeared after deletion of NLRP6 in BM microenvironment, while C5, C6, C7, C10 and C12 cell populations increased significantly. Regulon of various subpopulations of malignant HSCs changed significantly, mainly involving cell development and differentiation, immune response, inflammatory BM microenvironment, vascular endothelial homeostasis and tumor related genes. Our results indicate that knockout of NLRP6 in the BM microenvironment may affect the progression of MPN by altering some malignant HSCs subpopulations and regulons, participating in immune, inflammatory, tumor metabolism and other signaling pathways to regulate the functional characteristics of tumor stem cells of MPN.Our data here provide new evidence for NLRP6 as potential target in the treatment of MPN.

Abstract 14659: Muscle Oxygenation Responses to Acute Exercise and Cellular Biomarkers of Vascular Health in Veterans With Chronic Kidney Disease

Introduction: Increased susceptibility of endothelial dysfunction and skeletal muscle impairments suggest peripheral limitations as an explanation for diminished aerobic capacity in chronic kidney disease (CKD) patients. However, data on peripheral limitations in CKD patients is limited. Hypothesis: We hypothesized CKD patients would experience reduced muscle oxygenation responses to acute exercise and cellular evidence of declines in vascular health compared to those without CKD. Methods: Sixteen male Veterans (CKD: n=9, age 74±6 yrs, eGFR, 41.7±10 mL/min/1.73 m 2 ; non-CKD controls (CON): n=7, age, 67±8 yrs, eGFR, 75±10.6 mL/min/1.73 m 2 ) completed treadmill cardiopulmonary exercise testing via the Modified Bruce protocol. Oxygenation responses were obtained from the medial gastrocnemius using near-infrared spectroscopy. Blood samples were collected at rest and analyzed for colony-forming units (CFU), flow-cytometry of mononuclear cells (MNCs) focusing on endothelial lineage cells and gene expression of MNCs. Effect sizes between groups were determined using Hedge’s g (g). Results: At peak exercise, oxygen consumption (VO 2peak 19±5 vs 25.8±5.2 ml/kg/min -1 , g=1.2), heart rate (HR peak 132±21 vs 160±9 bpm, g=1.6), oxygen extraction (Δ[HHb] 14.8±10.8 vs 24.9±6.8 a.u., g=1.0), and change in total hemoglobin concentration (Δ[tHb] 8.9±5.1 vs 22.5±12.8 a.u., g=1.4) were lower in CKD compared to CON. At an exercise intensity of 3.4 METs, Δ[HHb] and Δ[tHb] were 42% and 54% lower (8.0±7.8 vs 13.8±8.8 a.u., g=0.6 and 4.9±4.1 vs 10.7±4.5 a.u., g=1.2) in CKD vs CON, despite no differences in oxygen consumption or heart rate. CD34 + 184 expression was 47% lower in CKD vs CON (0.2±0.04 vs 0.38±0.48 %, g=0.5). CFU was significantly inversely correlated with body mass index and waist circumference (r=-0.67, p=0.034; r=-0.65, p=0.041). Conclusions: Preliminary data from this ongoing study suggest oxygen extraction and capillary blood volume expansion are impaired during exercise in CKD patients when compared to those without CKD. CD34 + 184 expression was lower in CKD patients versus those without CKD, indicating poor hematopoietic stem cell migratory function in the CKD group. In addition, an inverse association between CFU and body habitus was observed.

Metabolic Programming of Hematopoietic Stem Cell Function By Prenatal Folate

Hematopoietic stem cells (HSCs) are programmed by metabolic changes resulting in alterations to HSC function. Here, we discovered that varying prenatal folate status metabolically programs HSCs during development and affects adult HSC self-renewal and engraftment potential into adulthood. Folate status varies greatly in the global population based on nutritional intake, common genetic polymorphisms, and widespread supplementation. Folate-derived one-carbon metabolism (OCM) regulates cellular methylation, de novo nucleotide biosynthesis and mitochondrial metabolism, processes critical to HSC function and establishment. Despite immense therapeutic potential, there is limited understanding of mechanisms driving HSC developmental programming. We hypothesize that varying prenatal folate status affects risk for hematologic dysfunction by developmentally programming HSCs. To test how prenatal folate modulated HSC function, female mice were assigned to one of three experimental diets to model population-wide folate consumption: 0mg/kg (deficient, FD), 2mg/kg (control, FC) and 8mg/kg (supplemented, FS). Profiling of fetal liver (FL) hematopoietic cells revealed that FD diet caused expansion of long term (LT)-HSCs that was propagated across the hematopoietic hierarchy, including myeloid and lymphoid progenitors and mature myeloid cells. In contrast, FS decreased FL cell output. We next determined if these changes were driven by metabolic activity. FD fetal HSPCs exhibited higher mitochondrial membrane potential and lower glycolytic activity, whereas FS fetal HSPCs displayed both higher glycolytic activity and oxygen consumption despite impaired production of downstream hematopoietic cells. Together, these data suggest that HSPC metabolic programming initiates during fetal development and is folate-dose dependent. To determine if prenatal folate programs hematopoiesis into adulthood, we quantified bone marrow (BM) hematopoietic cells in adult offspring after weaning onto standard chow. Surprisingly, FD expanded lymphoid progenitors in adult offspring with no impact on mature lymphoid cell output. In contrast, FS diet expanded myeloid and lymphoid progenitors in the BM as well as downstream mature cells, including monocytes and B-cells. To determine if these effects were HSC cell-intrinsic, we performed competitive transplantation assays of LT-HSCs isolated from adult offspring. FD offspring HSCs showed worse engraftment by chimerism across all peripheral blood (PB) populations compared to FC. In comparison, FS offspring HSCs showed enhanced total cellular output by donor cells/uL across all PB mature lineages except platelets. Secondary whole BM transplantation revealed that FS HSCs retained enhanced total cellular output and superior engraftment compared to FC, whereas FD HSCs demonstrated complete exhaustion. These data indicate that adult HSC function is programmed by prenatal folate status. To investigate molecular alterations driving metabolic programming of adult hematopoiesis by prenatal folate, we performed single-cell RNA sequencing (Scseq) and metabolomics on adult offspring HSPCs. Scseq analysis revealed most differential gene expression (DE) in HSC clusters, with less DE in progenitors. Re-clustering of all HSCs revealed distinct heterogeneity within this population, where both FD and FS HSCs were identified as distinct clusters compared to FC. Metabolomic profiling further suggested that FD HSCs exhibited signs of mitochondrial dysfunction through, 1) accumulating glucose and lactate suggesting reliance on glycolysis and, 2) accumulating succinate and glutamine suggesting complex II impairment and inadequate utilization of mitochondrial metabolism. Surprisingly, the same changes associated with mitochondrial dysfunction in FD HSCs persisted downstream into lymphoid-biased multipotent progenitors. In contrast, limited metabolic changes were observed in FS HSCs, suggesting alternative pathways for sustained programming. These changes indicate that FD and FS offspring have distinct metabolic programming. Together, our data suggest that prenatal folate status programs hematopoietic stem cell function into adulthood and is driven by alterations to metabolic activity. Ongoing studies examine how prenatal folate status affects the adult hematological and immune response to challenge.

Ruxolitinib Promotes Clinical Responses in Large Granular Lymphocytic Leukemia Via Suppression of JAK/STAT-Dependent Inflammatory Cascades

Background: Large granular lymphocytic leukemias (LGL) are T or NK-cell lymphoproliferative disorders that are characterized by myelosuppression and frequent mutations in STAT3. Commonly used agents include low doses of methotrexate or cyclophosphamide. Among 5 patients (pts) with LGL treated on our phase II study of ruxolitinib in T-cell lymphoma (Moskowitz, et al. Blood 2021), 2 achieved partial response (PR) and 2 achieved stable disease, suggesting oncogenic dependence on JAK/STAT pathway activity in LGL. Methods: To further explore the efficacy and mechanism of JAK1/2 blockade in LGL, we expanded our phase II study to include 23 pts with relapsed/refractory LGL. This sample size was chosen to allow for narrowing of the 95% confidence interval to 54-87% (Wilson score method) if similar efficacy was observed. Pts received ruxolitinib 20mg PO BID continuously. Responses were assessed after cycles 2, 5, and every 3 cycles thereafter using the response assessment from the ECOG-5998 study for LGL. Furthermore, stable disease (SD) was defined as improvement in cytopenia(s) by 1 grade (without meeting criteria for PR) for a minimum of 6 months. Overall response rate (ORR) included complete response (CR) plus PR; clinical benefit rate (CBR) included ORR plus SD. In parallel, we performed single-cell RNA sequencing (scRNA-seq) on paired C1D1 and C1D8 peripheral blood mononuclear cells (PBMCs) from 7 patients (5 PR, 1 SD, and 1 non-responder) and plasma proteomic profiling on 5 patients (3 PR, 1 SD, and 1 POD). Results: Among 23 pts, median age was 58 (31-86), 13 (57%) were male, 19 (83%) were white, 1 (4%) was black. Median number of prior therapies was 2 (range 0-5). 12/22 (54%) had STAT3 mutations (of whom 1 also had a JAK1 mutation); 1 had a JAK2 mutation, 1 pt was missing sequencing. Baseline cytopenias included anemia (n=14, 61%), thrombocytopenia (n=12, 52%), and/or neutropenia (n=16, 70%). Among 20 pts evaluable for response (3 too early), there were 5 (25%) CRs, 6 (30%) PRs, and 4 (20%) SD. ORR and CBR were 55% and 75% respectively. Median event free survival (EFS) was not reached and was 68% (95% CI: 50-93) at 14 months ( Fig. 1A). STAT3 mutation status predicted for improved EFS (14-month EFS 100% vs 40%, p=0.007). After a median duration of treatment of 13.1 months, there were no unexpected toxicities. Severe adverse events included febrile neutropenia (n=3, 13%) and herpes zoster (n=1, 4%). To understand the mechanism by which ruxolitinib improved hematologic parameters in LGL, we performed unbiased proteomic analysis of peripheral blood plasma samples using the OLink 96 Inflammation and Immune Response Platforms. Ruxolitinib led to a decrease in several soluble mediators known to be driven by interferon-driven JAK/STAT activation, including CXCL9, CXCL10, and PD-L1, as well as TNF ( Fig. 1B). Several of these factors have been shown to suppress hematopoietic stem cell function, suggesting a JAK/STAT-dependent paracrine mechanism by which LGL leads to myelosuppression. ScRNA-seq of PBMCs identified LGL cells based on expression of cytotoxic genes, including NKG7, CST7, CCL5, GZMB and PRF1. Analysis of cell type proportions within the peripheral blood showed that patients achieving a PR to Ruxolitinib showed a decrease in the proportion of circulating myeloid cells. Gene set enrichment analysis (GSEA) of LGL cells revealed substantial downregulation of both JAK/STAT signaling and TNF signaling in LGL cells in PR but not SD patients, whereas persistently elevated TNF signaling was seen not only in LGL cells but also circulating myeloid and naïve T-cells in SD patients, suggesting that LGL cells might promote bone marrow suppression via paracrine effects on the bystander host immune system. Network analysis of LGL cells and non-malignant peripheral blood immune cells is ongoing. Conclusions: Ruxolitinib induced durable responses in pts with LGL and therefore may represent an option for pts requiring treatment. STAT3 mutation status was predictive of EFS and responses were associated with JAK/STAT pathway down-regulation, demonstrating on-target effects. Integrated immune profiling suggests that ruxolitinib may interrupt JAK/STAT-dependent paracrine inflammatory signals that promote bone marrow suppression. An ongoing phase II study of ruxolitinib in LGL will aid in clarifying optimal dosing and confirming both efficacy and mechanism of action (NCT05592015).

Preclinical Efficacy and Mechanism for a Novel BTK-Protacs Against Mantle Cell Lymphoma

Background: Mantle cell lymphoma (MCL) is a rare and aggressive form of B-cell non-Hodgkin lymphoma (NHL) that exhibits diverse clinical courses and biological contexts. Given the significance of Bruton tyrosine kinase (BTK) as a therapeutic target in MCL, this study aimed to design a range of BTK-PROTACs. The objective was to develop efficient BTK-PROTACs capable of degrading BTK proteins, inhibiting the proliferation of MCL cells, and overcoming resistance to both covalent and non-covalent BTK inhibitors in both in vivo and in vitro settings. Methods:A series of BTK-PROTACs were gradually screened by Western blot and MTS assays, and the best active drug was finally determined. Cell function experiments in vitro were applied to verify the screening results and therapeutic efficacy was tested in vivo in NSG mice. RNA sequencing identified differentially expressed genes and important signaling pathways that influenced by BTK-PROTACs. BTKi-resistant cell lines were constructed to evaluate the ability of BTK-PROTACs to degrade BTK protein and suppress the proliferation of MCL cells. R esults: BTK-PROTACs C23 demonstrated effective degradation of BTK protein and inhibition of tumor cell growth across various MCL cell lines. Additionally, C23 exhibited the ability to induce cell apoptosis, activate the ubiquitin pathway, and inhibit the B-cell receptor (BCR), NF-κB, and PI3K-AKT-mTOR signaling pathways. Importantly, C23 effectively degraded BTK in both Z138-BTK C481S and Z138-BTK L528W cells, suggesting its potential to overcome resistance to both covalent and non-covalent BTK inhibitors. In our in vivo experiments using MCL xenograft NSG mouse models, BTK-PROTACs C23 significantly impeded tumor proliferation in Z138-BTK WT, Z138-BTK C481S, and Z138-BTK L528W MCL models. Additionally, C23 induced the degradation of BTK in tumor tissues. Notably, HE staining revealed no significant adverse effects of BTK-PROTACs C23 on normal organs and tissues. Moreover, routine blood tests and liver function tests showed no impairment of hematopoietic or hepatic function caused by BTK-PROTACs C23. C onclusion: In this study, we successfully designed and synthesized BTK-PROTACs C23, which demonstrated a remarkable ability to degrade BTK protein and effectively suppress the proliferation of MCL cells both in vitro and in vivo. Importantly, BTK-PROTACs C23 showed promising potential to overcome resistance to both covalent and non-covalent BTK inhibitors.