Myelodysplastic syndrome (MDS) represents a heterogeneous group of myeloid neoplasms, with approximately two-thirds identified as lower-risk MDS (LR-MDS). LR-MDS with thrombocytopenia is associated with poor prognosis, a high risk of life-threatening hemorrhage, and limited treatment options. We explored a prospective single-arm, single-center study on the combination of very-low-dose decitabine (VLD-DAC, 3.5 mg/m2/day from days 1 to 5 of a 28-day cycle) and recombinant human thrombopoietin (rhTPO, 1.5 million units/day from days 1 to 14) for treating LR-MDS with thrombocytopenia (platelet count < 50 × 109/L). The primary endpoint was the hematologic improvement-platelet (HI-P) rate. Among 20 patients, 19 completed at least two treatment cycles. Of these, 63

Progressive aging of bone marrow hematopoietic stem cells (HSCs) underlies clonal hematopoiesis and age-associated hematologic disorders. Defining early molecular events driving HSC functional decline is essential for rejuvenation strategies. Here, we identify P-selectin (Selp) as a surface marker that stratifies HSCs into conserved functional and transcriptional states during organismal aging in humans and mice. P-selectin expression increases early during aging and remains elevated in old HSCs. Selphigh-HSCs exhibit increased DNA damage and bias towards megakaryocytic/myeloid lineage fate, whereas Selplow-HSCs maintain metabolic integrity, enhanced antioxidant capacity, and reduced myeloid skewing. Transcriptomic analysis revealed that Selphigh-HSCs adopt megakaryocytic-primed, pro-inflammatory, and oxidative stress programs, while Selplow-HSCs retain lymphoid-associated and redox-balanced signatures consistent with a more preserved stem-cell state. Further ATAC-seq analysis demonstrates distinct chromatin landscapes with Selphigh-HSCs being enriched for inflammatory and platelet-related regulatory elements and CTCF motifs, but Selplow-HSCs displaying accessible ETS-driven networks linked to metabolic fitness and stem cell resilience. Together, these findings uncover conserved heterogeneity during blood stem cell aging and establish P-selectin as an early biomarker and potential therapeutic target to mitigate age-associated hematopoietic decline. This study identifies P-selectin (Selp) as an evolutionarily conserved surface marker that captures functional and transcriptomic heterogeneity within the elderly hematopoietic stem cell (HSC) compartment. Early aged HSC pools are stratified by Selp expression levels into functionally divergent populations, driven by separate transcriptional programs and chromatin landscapes. P-selectin captures distinct functional and transcriptomic states in aged blood stem cells.

One of the primary causes of tissue functional degeneration during aging is the functional decline of somatic stem cells. Research on the regulatory mechanisms of hematopoietic stem cells (HSCs) during the aging process is gaining significant attention. Nupr1, as a transcriptional cofactor, is strongly induced by various cellular stresses. Among hematopoietic cells, HSCs exhibit markedly high expression of Nupr1.Previous work revealed that HSCs with higher mitochondrial mass (MM) survive more effectively in the aged bone marrow microenvironment (BME) and display elevated Nupr1 expression compared with those with lower MM. Notably, Nupr1 expression is significantly upregulated in HSCs from 9-month-old mice. Nupr1 knockdown experiments showed that both young and aged HSCs exhibited reduced mitochondrial membrane potential. Additionally, Nupr1 knockdown also drove both young and aged HSCs to exit quiescence and enter the cell cycle. Intriguingly, Nupr1 knockdown elicited opposed effects on DNA double-strand break sites (DSBs) and apoptosis in young and aged HSCs. In young HSCs, reduced Nupr1 expression decreased DSB formation and apoptosis, whereas in aged HSCs, it increased both parameters. Functional assays utilizing Nupr1-knockdown HSC transplants further revealed contrasting outcomes. In young mice, Nupr1 knockdown resulted in lower reconstitution compared with controls, whereas aged mice exhibited enhanced reconstitution.These findings suggest that Nupr1 may play a pivotal role in DNA damage repair, modulation of cell metabolism, and the enhancement of HSC resilience under various forms of cellular stress. This likely endows HSCs with a survival advantage within the aging BME. Therefore, therapeutic targeting of Nupr1 and its downstream effectors in the early stages of aging could potentially mitigate or even reverse the functional deterioration of aged HSCs.

T cell large granular lymphocytic leukemia (T-LGLL) is a clonal lymphoproliferative disorder, originated from mature effector memory CD8+ T cells. It is a challenge to define the leukemic T cell clones due to the lack of definite markers. Here, we decipher the heterogeneity of CD8+ T cells using cellular indexing of transcriptomes and epitopes by sequencing (CITE-seq) and T cell receptor (TCR) profiling in T-LGLL patients. A CD8+ terminal effector subset is identified, marked by reduced KLRG1 expression. Remarkably, high fidelity of leukemic clonality was specially limited in KLRG1- large granular lymphocytes (LGLs), not seen in KLRG1+ LGLs in T-LGLL patients or in KLRG1- LGLs in healthy controls. KLRG1- leukemic LGLs show upregulated PI3K signaling with enhanced cytotoxicity and exhaustion, persisting after conventional treatment. In a pilot trial of linperlisib (a PI3Kδ inhibitor) for refractory cases, 7 of 8 participants quickly respond with satisfactory safety. This study is registered at ClinicalTrials.gov (NCT05676710).

While Azacitidine combined with HAG (HHT, low-dose cytarabine, G-CSF) regimen has shown promise in treating older and unfit patients with acute myeloid leukemia (AML), its efficacy in younger patients remains understudied. This study evaluates the effectiveness and safety of Azacitidine combined with the HAG regimen in a broader patient cohort, including newly diagnosed and relapsed/refractory AML patients. This single-center, prospective cohort included patients with acute myeloid leukemia admitted to our center from June 2019 to Oct 2022 for induction chemotherapy with the HAGA regimen (Azacitidine combined with HAG). We focused on patients' remission rate, MRD (minimal residual disease) conversion and safety. 71 patients with newly diagnosed or R/R AML were enrolled in this study, with a median follow-up time of 20.5 months. After two cycles of HAGA, patients received 3 cycles intermediate-dose Cytarabine and 1 cycle HAGA regimen as consolidation therapies. The CRc (composite complete remission) rate of HAGA regimen as induction chemotherapy for the overall cohort was 85.9% (61/71), which included 71.8% (51/71) CR and 14.1% (10/71) CRi. The CRc with MRD-negative rate was 76.1%. Median OS (overall survival) and DFS (disease free survival) were not yet reached, and the estimated 24-month OS rate was 65.4% (95%CI: 48.4-78.0%), the estimated 24-month DFS rate 66.0% (95%CI: 48.1-79.0%). Only one patient died in induction. The HAGA regimen can be a new option in addition to intense chemotherapy for newly diagnosed or R/R AML, and with high safety.

BACKGROUND:Immunosuppressive treatments induce and maintain remissions in a majority of patients with acquired pure red cell aplasia (aPRCA); however, salvage therapy for non-responders remains unsatisfactory. METHODS:In this prospective, open-label, clinical trial (NCT04423367), patients with refractory/relapsed aPRCA were recruited and received subcutaneous bortezomib at 1.3 mg/m2 in combination with dexamethasone 40 mg weekly(BD regimen) for 12 weeks. The primary endpoint was the overall response rate at 12 weeks. RESULTS:In total, 18 patients were enrolled, including 10 (55.6%) with primary aPRCA and 8 (44.4%) with plasma cell dyscrasia-associated aPRCA. The overall response rate and complete response rate were 61.1% and 55.6%, respectively. Intensive short-term BD regimen led to rapid response, with a median time to response of 40 days (range 21-77 days). Extended maintenance was unnecessary after the 12-week treatment. At a median follow-up of 16.5 (6-51) months, the median treatment-free survival (TFS) was 7 (2-48) months. There was no significant difference in the overall hematological response rate between primary and secondary aPRCA (60% vs. 62.5%, p = 0.914). Adverse events were recorded in 5 patients, including infection, deep venous thrombosis, hyperglycemia, constipation, and liver injury. CONCLUSION:The BD regimen may serve as an effective second-line therapy for refractory/recurrent/intolerant aPRCA patients, with manageable adverse events and possibly longer TFS.

Hematopoiesis is a finely tuned process that generates all blood cell types through self-renewal and differentiation, which is crucial for maintaining homeostasis. Acute infections can prompt a hematopoietic response known as emergency myelopoiesis. In this study, using a Candida albicans (C. albicans) infection model, we demonstrated for the first time that disruption of Fhl2 led to increased fungal burden, heightened inflammatory response and reduced survival rates. Impaired myeloid hematopoiesis and immune cell production were evident, as proved by the decreased numbers of hematopoietic stem and progenitor cells (HSPCs) and granulocytes in the bone marrow of Fhl2-deficient mice. In conclusion, FHL2 regulated emergency myelopoiesis in response to C. albicans, affecting the host’s defense against pathogens.

Topic: 20. Lymphoma Biology & Translational Research Background: T-cell large granular lymphocytic leukemia (T-LGLL) is a chronic lymphoproliferative disease of mature T cells, characterized by monoclonal T-cell large granular lymphocytosis, often clinically manifest as bone marrow failure and accompany with autoimmune symptoms. Immunosuppressive therapies are effective, but the dominant clone may persist even in responders. The pathogenesis of T-LGLL has not been fully elucidated. In this study, single-cell RNA sequencing and V(D)J profiling were performed to discern clonotypes and gene expression patterns of T lymphocytes from T-LGLL patients. Aims: To identify the biomarkers of leukemic large granular lymphocytes (LGLs) and explore novel agent for treating T-LGLL. Methods: Peripheral blood samples were collected from five T-LGLL patients, three T-cell clones of uncertain significance (T-CUS) patients and three healthy donors. single-cell RNA library was performed on the 10x Genomics Chromium Single Cell V(D)J and 5’ Gene Expression platform, and sequencing obtained on the Illumina Platform. Barcode assignment, alignment, unique molecular index counting and T cell receptor sequence assembly were performed using Cell Ranger 6.1.0. Immune cells were clustered and annotated, then transcriptome characteristics of each group of cells were analyzed, T cell receptor (TCR) clonetype and clonesize were identified. Results: By analyzing the characteristics of transcriptome and immune library of CD8 T cells from all samples, terminal effector T cells were divided into three clusters (which named CD8 TE1, CD8 TE2 and CD8 TE3). Almost all of TE3 cluster, which characterized by decreased clonetype diversity, were from T-LGLL patients. To identify whether TE3 cluster was pathogenic, we mapped the transcriptome on previous sequencing of T-LGLLs (Nat commun(2022)13:1981 and Nat commun (2022)13:1982) and verified TE3 cluster was the leukemic subpopulation. Compared with non-leukemic effector T cells, this leukemic subpopulation demonstrated higher toxicity and increased proliferative function. Interestingly, upregulated exhausted genes were also observed in this cluster. Furthermore, cell killing, TCR activation, interferon γ signaling pathway and cytokine production were upregulated in leukemic cluster. Remarkablely, the leukemic effector T cells demonstrated a lower level of killer cell lectinlike receptor G1 (KLRG1). We also verified the significantly decreased expression of surface KLRG1 on CD3+CD8+CD57+ cells in other five T-LGLL patients. Moreover, up-regulated PI3K-AKT pathway was observed in this KLRG1- leukemic subpopulation. Proliferation and cytotoxicity of leukemic lymphocytes from patients were significantly decreased by PI3K inhibitor. Summary/Conclusion: Large granular lymphocytes is a typical KLRG1- clonal T effector cluster, characterized by increased cytotoxicity and exhaustion. PI3K inhibitor, which could restrain proliferation and effector function of leukemic lymphocytes, is a promising agent for T-LGLL. Keywords: LGL

Aplastic anemia (AA) is an auto-activated T cell-mediated bone marrow failure, characterized by pancytopenia and hypocellular marrow.1 With the worldwide spread of the Omicron variant of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus, patients with AA are at high risk for infection due to disease-related neutropenia and enduring immunosuppressive therapy (IST).2 Furthermore, AA patients often do not receive the SARS-CoV-2 vaccination due to an ineffective protective specific antibody response to viral antigens and potential immune system activation that may worsen underlying marrow failure. Prior studies have evaluated the clinical manifestation of SARS-CoV-2 infection in severe or very severe AA (SAA/VSAA) patients through case reports or case series,3-8 but the severity and long-term prognosis of SARS-CoV-2 infection in AA is still not clear. To better explore whether patients with AA experienced more severe symptoms of infection or demonstrated higher relapse rate after infection, we prospectively collected 175 AA patients enrolled in National Longitudinal Cohort of Hematological Diseases (NICHE, NCT04645199) and described the clinical features and outcomes of AA after infecting Omicron SARS-CoV-2 between December 2022 and January 2023. This study demonstrated a low risk of relapse in AA after Omicron infection. In this study, SARS-CoV-2 illness severity was evaluated by the National Institutes of Health COVID-19 Treatment Guidelines. Severe illness was defined as individuals who had SpO2 < 94% on room air at sea level, a ratio of arterial partial pressure of oxygen to fraction of inspired oxygen <300 mmHg, a respiratory rate > 30 breaths/min, or lung infiltrates >50%. Hematological responses were evaluated based on established criteria.9 Robust response was defined as not only meeting standard response criteria,9 but fulfilling more than 50 × 109/L of platelet and 100 g/L of hemoglobin (Hb). A progressive and substantial decline in blood counts requiring reinitiation of high-dose cyclosporine or androgen or thrombopoietin receptor agonists was defined as relapse. Paired t-tests were used to compare the hematological indices before and after Omicron infection, including Hb, platelet, neutrophil, and lymphocyte counts. The study engaged a total of 175 patients, comprising 90 males and 85 females, with a median age of 33 years (range, 10–72). Among these patients, 29.7% had SAA/VSAA, 14.3% had transfusion-dependent AA, and 56.0% had transfusion-independent AA. Forty-six patients (26.3%) had been vaccinated against SARS-CoV-2, with a median interval of 18 (1–55) months between the last vaccine dose and Omicron infection. At the onset of infection, 64.0% (105/164) of AA patients achieved robust response, 20.1% (33/164) had inferior response, and 15.9% (26/164) were no response. Among the whole population, 136 patients received IST for at least 6 months before infection, with 36 (20.6%) of them accepting either antithymocyte or antilymphocyte globulin. As presented in Table S1, the most common initial symptoms were fever (78.9%), sore throat or cough (60.6%), headache (49.1%), fatigue (44.6%), and myalgia (40.0%). Approximately 47% of patients experienced symptoms beyond 2 weeks. A total of 11 patients developed pneumonia and required oxygen supplementation, and one of them needed admission to intensive care unit for invasive ventilation. Among these 11 patients, the median age was 34 years (range, 15–58), with four of them having SAA/VSAA and four being male. Furthermore, one of them had asthma as a complication, and one had a gastrointestinal tumor for whom the last chemotherapy was over 3 years ago. Four of these 11 patients had been vaccinated against SARS-CoV-2, with a median interval of 15 months (range, 12–16) between the last dose and infection. Nine individuals received IST before infection, with seven of them receiving IST for more than 6 months. At the onset of Omicron infection, three patients achieved robust response with a median lymphocyte of 1.45 × 109/L (range 0.9–3.61 × 109/L). All 11 patients recovered from Omicron infection following supportive therapies, including oxygen supplementation, antipyretic drugs, and a short course of broad-spectrum antibiotics. Only one case required steroids and intravenous immunoglobulin infusion to alleviate symptoms. To investigate the impact of Omicron infection on hematological profiles in AA patients, we analyzed the successive hematological indices during both the infection and convalescence periods (Figure 1A). During the 2-week infectious period, we observed a progressive decline in hematological profiles compared to preinfection levels, including Hb (112.4 vs. 107.4 g/L, p = .043), platelet (84.0 vs. 61.2 × 109/L, p < .0001), and neutrophil (2.11 vs. 1.73 × 109/L, p = .014). Nevertheless, no significant alteration was observed in lymphocyte count. During convalescence period of 2–4 weeks after infection, Hb (112.4 vs. 105.3 g/L, p < .001), platelet (84.0 vs. 69.7 × 109/L, p < .001), and neutrophil (2.01 vs. 1.70 × 109/L, p = .0084) were still significantly lower than those prior to the infection. However, the platelet count increased than that during 2-week infectious period (36 vs. 42 × 109/L, p = .018). Interestingly, the lymphocyte count increased from the preinfection level (1.75 vs. 1.87 × 109/L, p = .004). Within 4–8 weeks following the infection, the hematological parameters including Hb (112.4 vs. 109.5 g/L, p = .213) and platelet (84.0 vs. 82.1 × 109/L, p = .933), showed a spontaneous return to their preinfection levels and remained stable thereafter. The neutrophil returned to the preinfection level after 8 weeks (2.11 vs. 1.95 × 109/L, p = .489). To evaluate the extent of the decline in hematological indices, a meaningful decrease was defined as a reduction in Hb of more than 30 g/L, in neutrophil count of more than 0.5 × 109/L, or in platelet count of more than 30 × 109/L, compared to preinfection levels. As shown in Figure 1B, less than 5% of patients experienced the meaningful reduction in Hb, but a meaningful decrease in neutrophil was observed over 30% of patients within 8 weeks after infection. The proportion of patients with a meaningful platelet decrease gradually reduced within 8 weeks, but 5.93% of patients still had a meaningful decrease in platelet count. Moreover, after at least 8 weeks of infection, 76 AA patients had successive blood indices and only three individuals (4.3%) experienced relapse, including one complicated with paroxysmal nocturnal hemoglobinuria. The patients were classified based on whether they received IST for at least 6 months before Omicron infection into long-course and short-course IST groups. The long-course group had significant declines in platelet (92.4 vs. 72.3 × 109/L, p < .001) and neutrophil (2.17 vs. 1.75 × 109/L, p = .012) after 2 weeks of infection. Meanwhile, the short-course group had a significant decrease in platelet (52.8 vs. 24.2 × 109/L, p = .005). Remarkable pancytopenia was observed in long-course group within 2–4 weeks following infection, including reduced level of Hb (114.5 vs. 107.5 g/L, p < .001), platelet (92.4 vs. 77.7 × 109/L, p < .001), and neutrophil (2.17 vs. 1.90 × 109/L, p = .039) as compared to counts before infection. However, no aggravated pancytopenia was observed in short-course IST group. Following a minimum of 8 weeks from infection, both groups exhibited a return of all hematological parameters to their prior levels (Figure 1C). To explore the relationship between response status and postinfection pancytopenia, patients were divided into robust group and inferior group based on their preinfection response status. The former was comprising those who achieved robust response, and the inferior group was consisting of patients who only achieved inferior response or no response. Within first 2 weeks of infection, notable declines of platelet (110.8 vs. 81.1 × 109/L, p < .001) and neutrophil (2.42 vs. 1.88 × 109/L, p = .008) were observed in robust group. Following 2–4 weeks of infection, Hb (124.5 vs. 119.9 g/L, p = .001), platelet (110.8 vs. 95.4 × 109/L, p < .001), and neutrophil (2.42 vs. 2.01 × 109/L, p = .002) were still significantly decreased in robust group. Hematological profiles returned to their baseline levels after a minimum of 8 weeks from the onset of infection (Figure 1D). Nevertheless, no significant pancytopenia was observed in the inferior group. This prospective cohort from NICHE provides detailed clinical data in the largest cohort of Omicron-infected AA patients. The initial symptoms were fever, dizziness or headache, muscle or body aches, and sore throat, which were consistent with those reported by the general population during the 2022 Omicron waves in Tianjin.10 The percentage of patients with severe/critical illness in AA patients was similar to that of the general population (0.6% vs. 0.5%, p > .05).10 However, it was significantly lower than that of patients with hematological malignancies11 (0.6% vs. 8.9%, p < .05), possibly due to fewer complications in the AA population. Our study found that Omicron infection did not result in a high proportion (less than 5%) of relapse in AA patients. Nevertheless, aggravated cytopenia was observed within 4 weeks of infection, which may be due to the aberrant immune response triggered by virus.12 It's worth noting that the lymphocyte count significantly increased during 2–4 weeks after infection, which may reflect an activated immune response. This abnormal immune response may not be sustained, as the hematological parameters spontaneously returned to the preinfection levels for more than 4 or 8 weeks. Furthermore, patients receiving a long course of IST were more likely to experience temporally aggravated pancytopenia compared to the short-course group. Additionally, patients with a robust response may have a fragile balance of immune condition, which can result in a significant decrease of hematological indices. In conclusion, infection of Omicron in AA did not result in higher mortality. Moreover, our data highlight true relapse after Omicron infection is rare in AA, but decrease on hematological parameters is common during acute infection and early convalescence. Lele Zhang and Jingyu Zhao designed the study, analyzed data, interpreted data, and wrote manuscript. Ruonan Li, Hong Pan, Zhen Gao, Weiwang Li, and Liwei Fang collected the clinical data and recruited patients. Jun Shi conceptualized the study, interpreted the data, and supervised the project. All authors critically edited and reviewed the manuscript. All authors have access to the raw data used for this study and they all approved the final manuscript. Authors declare that they have no competing interests. Data openly available in a public repository that issues datasets with DOIs. TABLE S1. Demographic and clinical characteristics of 175 AA patients infected with the SARS-CoV omicron. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.

Background: The CR rate of standard “3 + 7” regimens for de novo acute myeloid leukemia range from 65% to 85%.Chemotherapy combine with targeted drugs like BCL2 inhibitors increase CR rate to 80~90%. Azacitidine(Aza) is commonly used to treat older patients with newly diagnosed AML，The complete remission (CR) rates of Aza combined regimens varied with different drugs. HAG(HHT, low-dose cytarabine, G-CSF) is a so called priming regimen and always used for elderly/unfit AML patients. The efficacy and safety of Aza combined with HAG(HAGA) in adult AML patients are not sure. We prospective enrolled our cohort of patients with AML treated with HAGA regimen. Aims: To explore the efficacy and safety of HAGA regimen in adult AML patient. Methods: We prospectively enrolled 69 patients from June 2019 to Oct 2022 from our centre presenting with AML and fit for chemotherapies. After 2 cycle of HAGA regimens, patients received 2 cycles of moderate doses of cytarabine and 2 cycles of HAGA as consolidation chemotherapy or underwent allo-HSCT. The primary endpoints were 2-year OS rate and 2-year DFS rate. The secondary endpoints were rate of composite complete response (CR+CRi) and rate of MRD negative composite complete response within 2 cycles. Results: Among the 69 AML patients who received HAGA induction therapy, 58 were newly diagnosed (including 22.4% in the ELN2022 high risk group,25.9% in the median risk group), and 9 were relapsed/refractory,1 secondary AML. The median follow-up time was 25.5 months (range:2.5-43.5 months). After the first cycle of HAGA, the composite CR rate were 82.6%(57/69), including 84.5% (49/58) in newly diagnosed patients, 66.7%(10/15) in ELN2022 high risk group and 80% (8/10) in relapsed/refractory patients. The rate of MRD negative CR rate is 47.8%(33/69), including 48.3% (28/58) in newly diagnosed patients, 40%(6/15) in ELN2022 high risk group and 50% (5/10) in R/R patients. 59 patients received a secondary HAGA reinduction/consolidation regimen and were evaluated for efficacy. After the 2th HAGA, the composite CR rate increase to 91.5%(54/59),the MRD-negative composite CR rate rise up to 81.4%(48/59). Especially in high-risk patients, after 2 cycles of HAGA, the composite CR rate was 90.9%(10/11), the MRD-negative composite CR rate was 72.7%(8/11). The overall 2-year OS rate was 66.3%, median OS was 31.7 months (95%CI: 27.6-35.9 months). And the 2-year DFS rate was 57.8%,median EFS was 26.3 months(95%CI: 21.6-31.0 months). As expected, Grade ≥3 toxicities were predominantly hematologic. The median duration of neutropenia was 11 (0-42) days in the first cycle and 6 (0-42) days in the second cycle. Only 1 patient died during induction therapy. Summary/Conclusion: Azacitidine combined with HAG regimen as induction therapy can achieve a high CR rate and MRD negative rate, provide patients with transplantation opportunities and better survival outcomes, and is expected to become the first-line treatment option for adult AML. Keywords: AML, Chemotherapy, Azacitidine

Acute myeloid leukemia (AML) is a heterogeneous hematological malignancy with dismal prognosis. Identification of better biomarkers remained a priority to improve established stratification and guide therapeutic decisions. Therefore, we extracted the RNA sequence data and clinical characteristics of AML from The Cancer Genome Atlas (TCGA) and Genotype-Tissue Expression database (GTEx) to identify the key factors for prognosis. We found UNC93B1 was highly expressed in AML patients and significantly linked to poor clinical features (p < 0.05). We further validated the high expression of UNC93B1 in another independent AML cohort from GEO datasets (p < 0.001) and performed quantitative PCR of patient samples to confirm the overexpression of UNC93B1 in AML (p < 0.005). Moreover, we discovered high level of UNC93B1 was an independent prognostic factor for poorer outcome both in univariate analysis and multivariate regression (p < 0.001). Then we built a nomogram model based on UNC93B1 expression, age, FAB subtype and cytogenetic risk, the concordance index of which for predicting overall survival was 0.729 (p < 0.001). Time-dependent ROC analysis for predicting survival outcome at different time points by UNC93B1 showed the cumulative 2-year survival rate was 43.7%, and 5-year survival rate was 21.9%. The differentially expressed genes (DEGs) between two groups divided by UNC93B1 expression level were enriched in innate immune signaling and metabolic process pathway. Protein–protein interaction (PPI) network indicated four hub genes (S100A9, CCR1, MRC1 and CD1C) interacted with UNC93B1, three of which were also significantly linked to inferior outcome. Furthermore, we discovered high UNC93B1 tended to be infiltrated by innate immune cells, including Macrophages, Dendritic cells, Neutrophils, Eosinophils, and NK CD56dim cells. We also found UNC93B1 had a significantly positive correlation with CD14, CD68 and almost all Toll-like receptors. Finally, we revealed negatively correlated expression of UNC93B1 and BCL2 in AML and conjectured that high-UNC93B1 monocytic AML is more resistant to venetoclax. And we found high MCL-1 expression compensated for BCL-2 loss, thus, we proposed MCL-1 inhibitor might overcome the resistance of venetoclax in AML. Altogether, our findings demonstrated the utility of UNC93B1 as a powerful poor prognostic predictor and alternative therapeutic target.

The rapid development of computational pathology has brought new opportunities for prognosis prediction using histopathological images. However, the existing deep learning frameworks lack exploration of the relationship between images and other prognostic information, resulting in poor interpretability. Tumor mutation burden (TMB) is a promising biomarker for predicting the survival outcomes of cancer patients, but its measurement is costly. Its heterogeneity may be reflected in histopathological images. Here, we report a two-step framework for prognostic prediction using whole-slide images (WSIs). First, the framework adopts a deep residual network to encode the phenotype of WSIs and classifies patient-level TMB by the deep features after aggregation and dimensionality reduction. Then, the patients' prognosis is stratified by the TMB-related information obtained during the classification model development. Deep learning feature extraction and TMB classification model construction are performed on an in-house dataset of 295 Haematoxylin & Eosin stained WSIs of clear cell renal cell carcinoma (ccRCC). The development and evaluation of prognostic biomarkers are performed on The Cancer Genome Atlas-Kidney ccRCC (TCGA-KIRC) project with 304 WSIs. Our framework achieves good performance for TMB classification with an area under the receiver operating characteristic curve (AUC) of 0.813 on the validation set. Through survival analysis, our proposed prognostic biomarkers can achieve significant stratification of patients' overall survival (P 0.05) and outperform the original TMB signature in risk stratification of patients with advanced disease. The results indicate the feasibility of mining TMB-related information from WSI to achieve stepwise prognosis prediction.

Myeloid cells have been identified as hematopoietic stem cell (HSC)-regulating cells. However, the mechanisms by which myeloid cells regulate the function of HSCs are not fully defined. Our previous study indicated that the HSCs are over-expanded in Vav1-Cre;Rheb1 f l/fl mice. Here, using in vivo and in vitro models, we found that Rheb1 -deficient neutrophils remodeled the bone marrow environment and induced expansion of HSCs in vivo . Further studies showed that loss of Rheb1 impaired neutrophils’ ability to secrete IL-6, led mesenchymal stem cells (MSCs) to produce more SCF, and promote HSC proliferation. We further found that IL-6 suppressed SCF mRNA expression in human MSCs. Interesting, the high level of IL-6 was also related with poor survival of chronic myeloid leukemia (CML) patients, and higher expression of IL-6 in CML cells is associated with the lower expression of SCF in MSCs in patients. Our studies suggested that blocking IL-6 signaling pathway might stimulate MSCs to secrete more SCF, and to support hematopoietic stem/progenitor cells proliferation.

In the process of fetal liver hematopoiesis, the specifically knocking-out the Rictor in hematopoietic system can lead to defect of reconstitution ability, decrease of the functional hematopoietic stem cell numbers and reduction of self-renewal ability of hematopoietic stem cells.

Objective: To explore the role of PDK1 in the transition of endothelial to hematopoietic cells and its effect on the generation and normal function of HSC. Methods: PDK1 was deleted specifically in endothelial cells expressing VEC (Vascular Endothelial Cadherin). CFU-C was performed to detect the effect of PDK1 on the function of hematopoietic progenitor cells using the cells from PDK1(fl/fl), PDK1(fl/+) and Vec-Cre; PDK1(fl/fl) AGM region. Hematopoietic stem cell transplantation assay was conducted to determine the effect of PDK1 on hematopoietic stem cells. Flow cytometry was performed to analyze the influence of PDK1 on percentage, cell cycle and apoptosis of CD31(+)c-Kit(high) cell population. Real-time PCR was conducted to measure the expression of transcription factors involved in process of transition from endothelial to hematopoietic cells. Results: In contrast to the wild type group, the CFU from PDK1-deficient hematopoietic progenitor cells showed smaller in morphology and fewer in quantity. CFU-GM was (24±5)/ee in knockout group, and the control group was (62±1)/ee (P=0.001). PDK1 deletion severely impaired the ability to repopulate hematopoietic cells and differentiate into committed cells. hematopoietic progenitor cells from knockout group was transplanted into 5 recipients without any recipients reconstructed. However, 5 of 7 recipients were reconstructed in control group (P=0.001). The proportion of intra-vascular clusters in the AGM was decreased (the frequency of CD31(+)c-Kit(high) in the knockout group was (0.145±0.017)%, and the control group ratio was (0.385±0.040)% (P=0.001), but not due to the inhibition of cell proliferation and/or increase of apoptosis. Further study found that the absence of endothelial PDK1 causes a decreased expression of RUNX1, P2-RUNX1, GATA2 and other important hematopoietic-related transcription factors in hemogenic cluster. Conclusion: PDK1 deletion impairs the transition of endothelial cells to hematopoietic cells as well as the generation and function of HSC.

OBJECTIVE:To knock out PDK1 by using Vav-Cre and observe the effects of PDK1 knock out on the ratio, number and differentiation of neutrophil.METHODS:The PDK1 expression level of Vav-Cre;PDK1lox/lox mouse in the bone marrow cells was analyzed by RT-PCR. The effect of PDK1 on hematopoietic progenitor was observed by CFU-C assay, and the effect of PDK1 on the ratio and number of neutrophil was detected by flow cytometric analysis.RESULTS:The RNA expression level of bone marrow cells of Vav-Cre;PDK1lox/lox mouse was dramatically lower than that of the control mouse. The number of functional GMP was lower in Vav-Cre;PDK1lox/lox mouse in contrast to controls. The percentage and number of neutrophil were lower, but the percentage of premyelocyte/myelocytes was more than 2 times in Vav-Cre;PDK1lox/lox group compared with control groups.CONCLUSION:PDK1 affects the process of the differention of hematopoietic stem cells to GMP, the neutrophil differention and maturation.

3-Phosphoinositide-dependent protein kinase 1 (PDK1) is a pivotal regulator in the phosphoinositide 3-kinase (PI3K)-Akt signaling pathway that have been shown to play key roles in the functional development of B and T cells via activation of AGC protein kinases during hematopoiesis. However, the role of PDK1 in HSCs has not been fully defined. Here we specifically deleted the PDK1 gene in the hematopoietic system and found that PDK1-deficient HSCs exhibited impaired function and defective lineage commitment abilities. Lack of PDK1 caused HSCs to be less quiescent and to produce a higher number of phenotypic HSCs and fewer progenitors. PDK1-deficient HSCs were also unable to reconstitute the hematopoietic system. Notably, HSC function was more dependent on PDK1 than on mTORC2, which indicates that PDK1 plays a dominant role in the Akt-mediated regulation of HSC function. PDK1-deficient HSCs also exhibited reduced ROS levels, and treatment of PDK1-deficient HSCs with L-butathioninesulfoximine in vitro elevated the low ROS level and promoted colony formation. Therefore, PDK1 appears to contribute to HSC function partially via regulating ROS levels.

mTOR is a serine/threonine (Ser/Thr) protein kinase that responds to multiple signals, including growth factors, amino acids, energy status, stress, and oxygen, regulates cell survival, cell growth, the cell cycle, and cell metabolism, and maintains homeostasis [1]. Increased or decreased mTORC1 activity can alter HSC function and cause hematological disorders [2, 3]. Therefore, a comprehensive knowledge of mTOR is critical to understanding how HSCs function and maintain homeostasis in the hematopoietic system. In this review, we summarize recent advances in the understanding of the mTOR signaling pathway and its roles in hematopoiesis and leukemia. We also discuss pharmacological approaches to manipulate mTOR activity.

Acute myeloid leukemia (AML) with MLL translocation is recognized as a group of aggressive hematopoietic malignancies with poor prognosis. An atypical homeodomain protein family (TALE family, three-amino-acid loop extension) has been reported to be involved in leukemogenesis and leukemia progression. For example, MEIS1 and PBX3 were abnormally expressed in examined AML patient samples when compared with their normal counterparts. While MEIS1 has been studied extensively, the precise role of PBX3 in leukemia development is still largely unknown. In this study, we explored the epigenetic regulatory network of PBX3 and the specific role of PBX3 in leukemia progression. By analyzing the clinical database, we found that the high expression of PBX3 iscorrelated with poor prognosis of AML patients. In MLL-fusion induced AML mouse models that were established by retrovirus-mediated ectopic expression of MLL-AF9 or MLL-NRIP3 genes in murine BM progenitor cells (c-Kit+). PBX3 also showed high expression level in leukemia stem cells (LSC) compared with normal HSPCs. Unlike other homeobox genes such as HOXA9 and MEIS1 that were both highly expressed in LSKs and LSCs, PBX3 only showed extremely high expression in LSCs in comparison with HSPCs or other normal hematopoietic cells. To explore the role of PBX3 in leukemia, we analyzed the epigenetic modification patterns along PBX3 gene. By analyzing the public ChIP-seq database, we found that increased activation mark H3K79me2 and decreased repressive mark H3K27me3 exhibited on PBX3 in LSCs. Additionally, transcription activating modifications H3K79 hypermethylation of PBX3 was only observed in LSCs but not LSKs or GMPs, consistent with increased PBX3 expression level in LSCs compared with HSPCs. To further confirm this, we assessed H3K79me2 level of PBX3 in purified c-Kit+ cells which are enriched with stem cells from leukemic or control mice by ChIP-qPCR. Consistently, ChIP-qPCR results revealed the similar trend with dataset analysis. Moreover, we also detected the transcription repressive mark H3K9me3 on PBX3 in c-Kit+ cells and found a dramatic decrease of H3K9me3 in LSCs in comparison with HSPCs. Thus, our results suggested that high expression level of PBX3 in LSCs might be caused by elevated activation mark H3K79me2 and depressed repressive mark H3K27me3 and H3K9me3. Considering that high expression level of PBX3 in LSCs may contribute to leukemic progression, we used the CRISPR/Cas9 system to delete PBX3 to examine its potential role in MLL-AF9 mediated AML development. We found that PBX3 inactivation significantly prolonged the survival of leukemic mice (median survival was 25 days of control, 31 days of SgPBX3-a, 30 days of SgPBX3-b, 10 mice per group). Morphological analysis revealed more leukemic blast cells with segment nuclei exhibited in PBX3 inactivation group, suggesting that PBX3 deficiency is associated with increased leukemic cell differentiation. Since LSC was considered to be sufficient to drive leukemogenesis and maintain leukemia progression, we investigated the impact of PBX3 inactivation on LSC capability. In vitro colony forming assay showed that PBX3 inactivation group formed significantly fewer colonies of total numbers and type A clones. Flow cytometric analysis showed decreased LSCs frequency in murine leukemia cells of PBX3 inactivation group. Moreover, limiting dilution transplantation assays revealed 4-7 fold decreased of functional LSCs after PBX3 inactivation with the 1/102 of LSCs in control leukemic bone marrow in comparison with 1/442 in SgPBX3-a and 1/741 in SgPBX3-b respectively. Further analysis suggested that depletion of functional LSCs in PBX3 inactivation group mainly through accelerating apoptosis of LSCs, evidenced by both increased Annexin V positive cell frequency of whole BM blasts and LSCs in PBX3 inactivation group. In conclusion, we found PBX3 is epigenetically dis-regulated in LSCs of MLL-r AMLs and is essential for leukemia development. Inactivation of PBX3 could suppress leukemia progression by reducing LSCs frequency and impairing its function. More importantly, the differential expression of PBX3 in normal and malignant hematopoietic cells provides itself potentially as a prognostic marker and therapeutic target for MLL-rearranged leukemia. HD.G and YJ.C contributed equally to this work Disclosures No relevant conflicts of interest to declare.