Novel PIKfyve/Tubulin Dual-target Inhibitor as a Promising Therapeutic Strategy for B-cell Acute Lymphoblastic Leukemia.

OBJECTIVE: In B-cell acute lymphoblastic leukemia (B-ALL), current intensive chemotherapies for adult patients fail to achieve durable responses in more than 50% of cases, underscoring the urgent need for new therapeutic regimens for this patient population. The present study aimed to determine whether HZX-02-059, a novel dual-target inhibitor targeting both phosphatidylinositol-3-phosphate 5-kinase (PIKfyve) and tubulin, is lethal to B-ALL cells and is a potential therapeutic for B-ALL patients. METHODS: Cell proliferation, vacuolization, apoptosis, cell cycle, and in-vivo tumor growth were evaluated. In addition, Genome-wide RNA-sequencing studies were conducted to elucidate the mechanisms of action underlying the anti-leukemia activity of HZX-02-059 in B-ALL. RESULTS: HZX-02-059 was found to inhibit cell proliferation, induce vacuolization, promote apoptosis, block the cell cycle, and reduce in-vivo tumor growth. Downregulation of the p53 pathway and suppression of the phosphoinositide 3-kinase (PI3K)/AKT pathway and the downstream transcription factors c-Myc and NF-κB were responsible for these observations. CONCLUSION: Overall, these findings suggest that HZX-02-059 is a promising agent for the treatment of B-ALL patients resistant to conventional therapies.

Lactate acid promotes PD-1+ Tregs accumulation in the bone marrow with high tumor burden of Acute myeloid leukemia.

BACKGROUND: Acute myeloid leukemia (AML) displayed poor response to programmed death-1 (PD-1) blockade therapy. Regulatory T cells (Tregs) was one of major immunosuppressive components in Tumor microenvironment and plays a vital role in the resistance of immunotherapy. Coinhibitory receptors regulate function of regulatory Tregs and are associated with resistance of PD-1 blockade. However, the coinhibitory receptors expression and differentiated status of Tregs in AML patients remain to be unclear. METHODS: Phenotypic determination of Tregs and CD8+ T cells in bone marrow of healthy donors and AML patients was performed by flow cytometry. Coculture experiments of AML and Tregs in vitro were performed and the concentrations of lactate acid (LA) in the supernatant were examined by ELISA. RESULTS: More Tregs differentiated into effector subsets in AML patients. However, PD-1 and T-cell immunoglobulin and immunoreceptor tyrosine-based inhibitory motif domain (TIGIT) expression on Tregs were comparable in healthy donors and AML patients. Further analysis showed that PD-1+ and PD-1+TIGIT+Tregs are more abundant in the bone marrow of patients with higher leukemic load. Moreover, PD-1+ Tregs accumulation was associated with higher level of senescent CD4+ T cells and increased frequencies of exhausted CD4+ as well as CD8+ T cells. Notably, neither Tregs nor their effector subsets were decreased among patients in complete remission. PD-1 expression was significantly downregulated in Tregs after achieving complete remission. Mechanistically, both AML cell line (KG-1α) and primary AML blasts produced high concentration of LA. Blockade of LA by lactate transporter inhibitor abrogated the upregulation of PD-1 by AML cells. CONCLUSION: PD-1+ Tregs accumulation in bone marrow in higher leukemic burden setting was linked to lactate acid secreted by AML blasts and decreased after disease remission. Our findings provided a novel insight into Tregs in AML and possible mechanism for resistance of PD-1 blockade in AML.

Enhanced STAT3/PIK3R1/mTOR signaling triggers tubular cell inflammation and apoptosis in septic-induced acute kidney injury: implications for therapeutic intervention.

Septic acute kidney injury (AKI) is a severe form of renal dysfunction associated with high morbidity and mortality rates. However, the pathophysiological mechanisms underlying septic AKI remain incompletely understood. Herein, we investigated the signaling pathways involved in septic AKI using the mouse models of lipopolysaccharide (LPS) treatment and cecal ligation and puncture (CLP). In these models, renal inflammation and tubular cell apoptosis were accompanied by the aberrant activation of the mechanistic target of rapamycin (mTOR) and the signal transducer and activator of transcription 3 (STAT3) signaling pathways. Pharmacological inhibition of either mTOR or STAT3 significantly improved renal function and reduced apoptosis and inflammation. Interestingly, inhibition of STAT3 with pharmacological inhibitors or small interfering RNA blocked LPS-induced mTOR activation in renal tubular cells, indicating a role of STAT3 in mTOR activation. Moreover, knockdown of STAT3 reduced the expression of the phosphoinositide-3-kinase regulatory subunit 1 (PIK3R1/p85α), a key subunit of the phosphatidylinositol 3-kinase for AKT and mTOR activation. Chromatin immunoprecipitation assay also proved the binding of STAT3 to PIK3R1 gene promoter in LPS-treated kidney tubular cells. In addition, knockdown of PIK3R1 suppressed mTOR activation during LPS treatment. These findings highlight the dysregulation of mTOR and STAT3 pathways as critical mechanisms underlying the inflammatory and apoptotic phenotypes observed in renal tubular cells during septic AKI, suggesting the STAT3/ PIK3R1/mTOR pathway as a therapeutic target of septic AKI.

Anlotinib suppresses the DNA damage response by disrupting SETD1A and inducing p53-dependent apoptosis in Transformed Follicular Lymphoma.

Purpose: The high tumor mutational burden (TMB) of transformed follicular lymphoma (tFL) leads to tumor heterogeneity and poor prognosis in follicular lymphoma, in which endogenous DNA damage and epigenetic modification are the key factors. This study aims to evaluate the efficacy of anlotinib in tFL and to investigate its potential therapeutic mechanism. Methods: Cell viability and apoptosis were tested with CCK-8 and annexin V/PI staining kits, respectively. The tumorigenicity test in mice was utilized to further confirm the efficacy of anlotinib in vivo. Western blotting was utilized to explore the molecular mechanisms. Results: Anlotinib induced G2/M phase arrest in tFL cells, inhibited the proliferation of tFL cells and promoted the apoptosis of tFL cells in a dose-dependent manner. Administration of anlotinib markedly reduced tumor mass and weight in an FL xenograft mouse model. The western blot and immunohistochemistry staining results confirmed that the mechanism by which anlotinib promoted tumor cell apoptosis was DNA damage. Further results showed that anlotinib significantly downregulated the expression of SETD1A, leading to its destruction. Anlotinib administration resulted in a significant dose-dependent increase in the level of p-p53. Furthermore, anlotinib greatly downregulated the antiapoptotic proteins Mcl-1 and in parallel upregulated the proapoptotic element BAX and Bak, accompanied by caspase-3 activation and PARP degradation. Conclusion: Anlotinib has a good proapoptotic effect on tumor cells in vitro and in vivo, and its possible mechanism is related to the inhibition of the DNA damage response by disrupting SETD1A.

Angiotensin-converting enzyme - human amniotic mesenchymal stem cells improve pulmonary vascular remodeling in rats with pulmonary hypertension by promoting angiogenesis and counteracting inflammation.

OBJECTIVES: Human Amniotic Mesenchymal Stem Cells (hAMSCs) have strong multidirectional differentiation ability. Studies have found that transfection of target genes into target cells by lentivirus can enhance the differentiation potential of the cells. Angiotensin-Converting Enzyme 2 (ACE2) was found to improve vascular remodeling. Research is lacking on ACE2-hAMSCs. Therefore, this study aimed to investigate the ability to improve pulmonary arterial hypertension using ACE2-hAMSCs. METHODS: Lentiviruses overexpressing ACE2 were mixed with hAMSCs. Then, ACE2-hAMSCs and hAMSCs with good growth in logarithmic growth phase were collected. We detected their migration and angiogenesis. RT-qPCR technology was used to detect the expression levels of genes related to angiogenesis, and inflammation in the two cell lines, and western-blotting was used to detect the expression levels of ACE2. As an animal study, 21 rats were randomly divided into four different groups. Right heart hypertrophy, pulmonary angiogenesis, and serum inflammatory factors were measured before dissection. H&E staining was used to observe the inflammatory infiltration of lung tissues. RESULTS: The migration and angiogenesis of ACE2-hAMSCs were strongerthan that of hAMSCs alone. The expressions of genes in ACE2-hAMSCs were higher, and the expression of ACE2 protein in ACE2-hAMSCs was less. H&E staining showed that the inflammatory infilration of lung tissue in ACE2-hAMSCs groups was significantly improved. In addition, the ACE2-hAMSCs group had stronger pro-angiogenesis and anti-inflammatory effects. CONCLUSION: These results suggest that ACE2-hAMSCs can repair pulmonary vascular endothelial cell injury caused by pulmonary hypertension by promoting angiogenesis and anti-inflammatory ability. This shows that ACE2-hAMSCs have stronger ability to improve pulmonary vascular remodeling than hAMSCs alone.

Nr4a1 marks a distinctive ILC2 activation subset in the mouse inflammatory lung.

BACKGROUND: Group 2 innate lymphoid cells (ILC2s) are critical sources of type 2 cytokines and represent one of the major tissue-resident lymphoid cells in the mouse lung. However, the molecular mechanisms underlying ILC2 activation under challenges are not fully understood. RESULTS: Here, using single-cell transcriptomics, genetic reporters, and gene knockouts, we identify four ILC2 subsets, including two non-activation subsets and two activation subsets, in the mouse acute inflammatory lung. Of note, a distinct activation subset, marked by the transcription factor Nr4a1, paradoxically expresses both tissue-resident memory T cell (Trm), and effector/central memory T cell (Tem/Tcm) signature genes, as well as higher scores of proliferation, activation, and wound healing, all driven by its particular regulons. Furthermore, we demonstrate that the Nr4a1+ILC2s are restrained from activating by the programmed cell death protein-1 (PD-1), which negatively modulates their activation-related regulons. PD-1 deficiency places the non-activation ILC2s in a state that is prone to activation, resulting in Nr4a1+ILC2 differentiation through different activation trajectories. Loss of PD-1 also leads to the expansion of Nr4a1+ILC2s by the increase of their proliferation ability. CONCLUSIONS: The findings show that activated ILC2s are a heterogenous population encompassing distinct subsets that have different propensities, and therefore provide an opportunity to explore PD-1's role in modulating the activity of ILC2s for disease prevention and therapy.

IL-27 regulates NLRP3 inflammasome activation of MDSCs in experimental Sjögren's syndrome.

Excessive NOD-like receptor thermal protein domain associated protein 3 (NLRP3) inflammasome activation has an important function in the pathogenesis of Sjögren's syndrome (SS). Increased and dysfunctional myeloid-derived suppressor cells (MDSCs) promoted SS. However, NLRP3 inflammasome activation of MDSCs in SS and its regulated components are unclear. Splenic MDSCs were purified by immunomagnetic beads and cultured. Western blot was used to assess NLRP3 inflammasomes. Interleukin-1ß (IL-1ß) and IL-18 were measured using enzyme-linked immunosorbent assay. Here we showed that the NLRP3 inflammasome was activated in non-obese diabetic (NOD) mice with SS-like manifestations. We found that NLRP3 inflammasome activation was augmented in MDSCs of SS mice and NLRP3 inflammasome activation was suppressed in IL-27-deficient NOD mice. Consistent with findings of SS mice in vivo, we observed that NLRP3 inflammasome activation by adenosine triphosphate and lipopolysaccharide was remarkably intensified in MDSCs with IL-27 treatment in vitro. Collectively, our data highlighted that IL-27 regulates NLRP3 inflammasome activation of MDSCs in experimental SS.

SIRT2 inhibitor SirReal2 enhances anti-tumor effects of PI3K/mTOR inhibitor VS-5584 on acute myeloid leukemia cells.

BACKGROUND: Acute myeloid leukemia (AML) is a highly aggressive form of cancer that is frequently diagnosed in adults and small molecule inhibitors have gained significant attention as a potential treatment option for AML. METHODS: The up-regulated genes in AML were identified through bioinformatics analysis. Potential candidate agents were selected through pharmacogenomics analysis. Proteomic experiments were conducted to determine the molecular mechanism after inhibitor treatment. To evaluate drug synergy, both cellular functional experiments and an AML mouse model were used. RESULTS: Through bioinformatics analysis, we conducted a screening for genes that are highly expressed in AML, which led to the identification of nine small-molecule inhibitors. Among these inhibitors, the PI3K/mTOR inhibitor VS-5584 demonstrated significant effectiveness in inhibiting AML cell proliferation at low concentrations. Further testing revealed that VS-5584 induced apoptosis and cycle arrest of AML cells in a dose- and time-dependent manner. Proteomics analysis showed significant changes in protein expression profiles of AML cells after VS-5584 treatment, with 287 proteins being down-regulated and 71 proteins being up-regulated. The proteins that exhibited differential expression were primarily involved in regulating the cell cycle and apoptosis, as determined by GO analysis. Additionally, KEGG analysis indicated that the administration of VS-5584 predominantly affected the P53 and SIRT2 signaling pathways. The use of SIRT2 inhibitor SirReal2 alongside VS-5584 caused a significant reduction in the half-maximal inhibitory concentration (IC50 ) of VS-5584 on AML cells. In vivo, experiments suggested that VS-5584 combined with SirReal2 suppressed tumor growth in the subcutaneous model and extended the survival rate of mice injected with tumor cells via tail vein. CONCLUSIONS: Taken together, the PI3K/mTOR inhibitor VS-5584 was effective in suppressing AML cell proliferation. PI3K/mTOR inhibitor combined with SIRT2 inhibitor exhibited a synergistic inhibitory effect on AML cells. Our findings offer promising therapeutic strategies and drug candidates for the treatment of AML.

Bcl-2 inhibition combined with PPARα activation synergistically targets leukemic stem cell-like cells in acute myeloid leukemia.

Persistence of leukemic stem cells (LSCs) is one of the determining factors to acute myeloid leukemia (AML) treatment failure and responsible for the poor prognosis of the disease. Hence, novel therapeutic strategies that target LSCs are crucial for treatment success. We investigated if targeting Bcl-2 and peroxisome proliferator activated receptor α (PPARα), two distinct cell survival regulating mechanisms could eliminate LSCs. This study demonstrate that the Bcl-2 inhibitor venetoclax combined with the PPARα agonist chiglitazar resulted in synergistic killing of LSC-like cell lines and CD34+ primary AML cells while sparing their normal counterparts. Furthermore, the combination regimen significantly suppressed AML progression in patient-derived xenograft (PDX) mouse models. Mechanistically, chiglitazar-mediated PPARα activation inhibited the transcriptional activity of the PIK3AP1 gene promoter and down-regulated the PI3K/Akt signaling pathway and anti-apoptotic Bcl-2 proteins, leading to cell proliferation inhibition and apoptosis induction, which was synergized with venetoclax. These findings suggest that combinatorial Bcl-2 inhibition and PPARα activation selectively eliminates AML cells in vivo and vitro, representing an effective therapy for patients with relapsed and refractory AML.

Glucocorticoid regulation of the mTORC1 pathway modulates CD4+ T cell responses during infection.

Objectives: Conventional glucocorticoid (GC) treatment poses significant risks for opportunistic infections due to its suppressive impact on CD4+ T cells. This study aimed to explore the mechanisms by which GCs modulate the functionality of CD4+ T cells during infection. Methods: We consistently measured FOXP3, inflammatory cytokines and phospho-S6 ribosomal protein levels in CD4+ T cells from patients undergoing conventional GC treatment. Using Foxp3EGFP animals, we investigated the dynamic activation of the mechanistic target of rapamycin complex 1 (mTORC1) pathway and its correlation with the immunoregulatory function of CD4+ T cells under the influence of GCs. Results: GCs dynamically altered the expression pattern of FOXP3 in CD4+ T cells, promoting their acquisition of an active T regulatory (Treg) cell phenotype upon stimulation. Mechanistically, GCs undermined the kinetics of the mTORC1 pathway, which was closely correlated with phenotype conversion and functional properties of CD4+ T cells. Dynamic activation of the mTORC1 signaling modified the GC-dampened immunoregulatory capacity of CD4+ T cells by phenotypically and functionally bolstering the FOXP3+ Treg cells. Interventions targeting the mTORC1 pathway effectively modulated the GC-dampened immunoregulatory capacity of CD4+ T cells. Conclusion: These findings highlight a novel mTORC1-mediated mechanism underlying CD4+ T cell immunity in the context of conventional GC treatment.

Ritanserin suppresses acute myeloid leukemia by inhibiting DGKα to downregulate phospholipase D and the Jak-Stat/MAPK pathway.

Refractory or relapsed (R/R) AML is the most challenging form of AML to treat. Due to frequent genetic mutations, therapy alternatives are limited. Here, we identified the role of ritanserin and its target DGKα in AML. Several AML cell lines and primary patient cells were treated with ritanserin and subjected to cell proliferation, apoptosis and gene analyses with CCK-8 assay, Annexin V/PI assay and Western blotting, respectively. We also evaluated the function of the ritanserin target diacylglycerol kinase alpha (DGKα) in AML by bioinformatics. In vitro experiments have revealed that ritanserin inhibits AML progression in a dose- and time-dependent manner, and it shows an anti-AML effect in xenograft mouse models. We further demonstrated that the expression of DGKα was elevated in AML and correlated with poor survival. Mechanistically, ritanserin negatively regulates SphK1 expression through PLD signaling, also inhibiting the Jak-Stat and MAPK signaling pathways via DGKα. These findings suggest that DGKα may be an available therapeutic target and provide effective preclinical evidence of ritanserin as a promising treatment for AML.

The kinetics of mTORC1 activation associates with FOXP3 expression pattern of CD4+ T cells and outcome of steroid-sensitive minimal change disease.

Minimal change disease (MCD) usually responds to glucocorticoids (GCs) but relapses in most cases. Relapse pathogenesis after complete remission (CR) remains unclear. We hypothesized that FOXP3+ T regulatory cell (Treg) dysregulation may drive early relapses (ER). In this study, a cohort of 23 MCD patients were treated with a conventional GC regimen for the initial onset of nephrotic syndrome. Upon GC withdrawal, seven patients suffered from ER, while 16 patients sustained remission (SR) during the 12-month follow-up. Patients with ER had reduced FOXP3+ Treg proportions compared with healthy controls. Treg reduction, accompanied by IL-10 impairment, was ascribed to a proportional decline of FOXP3medium rather than FOXP3high cells. GC-induced CR was marked by a rise in the proportions of FOXP3+ and FOXP3medium cells compared to baseline levels. These increases declined in patients with ER. The expression level of phosphorylated ribosomal protein S6 was used to track the dynamic shifts in mTORC1 activity within CD4+ T cells of MCD patients at various stages of treatment. Baseline mTORC1 activity was inversely correlated with FOXP3+ and FOXP3medium Treg proportion. The mTORC1 activity in CD4+ T cells served as a reliable indicator for ER and demonstrated improved performance when paired with FOXP3 expression. Mechanically, targeting mTORC1 intervention by siRNAs sufficiently altered the conversion pattern of CD4+ T cell to FOXP3+ Treg. Taken together, the activity of mTORC1 in CD4+ T cells can act as a credible predictor for ER in MCD, especially when combined with FOXP3 expression, and may offer a potential therapeutic avenue for the treatment of podocytopathies.

The STAT1/HMGB1/NF-κB pathway in chronic inflammation and kidney injury after cisplatin exposure.

Rationale: Cisplatin, a potent chemotherapeutic drug, induces side effects in normal tissues including the kidney. To reduce the side effects, repeated low-dose cisplatin (RLDC) is commonly used in clinical setting. While RLDC reduces acute nephrotoxicity to certain extents, a significant portion of patients later develop chronic kidney problems, underscoring the need for novel therapeutics to alleviate the long-term sequelae of RLDC therapy. Methods: In vivo, the role of HMGB1 was examined by testing HMGB1 neutralizing antibodies in RLDC mice. In vitro, the effects of HMGB1 knockdown on RLDC-induced nuclear factor-κB (NF-κB) activation and fibrotic phenotype changes were tested in proximal tubular cells. To study signal transducer and activator of transcription 1 (STAT1), siRNA knockdown and its pharmacological inhibitor Fludarabine were used. We also searched the Gene Expression Omnibus (GEO) database for transcriptional expression profiles and evaluated kidney biopsy samples from CKD patients to verify the STAT1/HMGB1/NF-κB signaling axis. Results: We found that RLDC induced kidney tubule damage, interstitial inflammation, and fibrosis in mice, accompanied by up-regulation of HMGB1. Blockage of HMGB1with neutralizing antibodies and Glycyrrhizin suppressed NF-κB activation and associated production of pro-inflammatory cytokines, reduced tubular injury and renal fibrosis, and improved renal function after RLDC treatment. Consistently, knockdown of HMGB1 decreased NF-κB activation and prevented the fibrotic phenotype in RLDC-treated renal tubular cells. At the upstream, knockdown of STAT1 suppressed HMGB1 transcription and cytoplasmic accumulation in renal tubular cells, suggesting a critical role of STAT1 in HMGB1 activation. Upregulation of STAT1/HMGB1/NF-κB along with inflammatory cytokines was also verified in kidney tissues of CKD patients. Conclusion: These results unravel the STAT1/HMGB1/NF-κB pathway that contributes to persistent inflammation and chronic kidney problems after cisplatin nephrotoxicity, suggesting new therapeutic targets for kidney protection in cancer patients receiving cisplatin chemotherapy.

T-cell immunoglobulin and mucin-domain containing-3 (TIM-3): Solving a key puzzle in autoimmune diseases.

Dysfunctional immune cells participate in the pathogenesis of a variety of autoimmune diseases, although the specific mechanisms remain elusive and effective clinical interventions are lacking. Recent research on immune checkpoint molecules has revealed significant expression of T cell immunoglobulin and mucin domain-containing protein 3 (TIM-3) on the surfaces of various immune cells. These include different subsets of T cells, macrophages, dendritic cells, natural killer cells, and mast cells. Further investigation into its protein structure, ligands, and intracellular signaling pathway activation mechanisms has found that TIM-3, by binding with different ligands, is involved in the regulation of crucial biological processes such as proliferation, apoptosis, phenotypic transformation, effector protein synthesis, and cellular interactions of various immune cells. The TIM-3-ligand axis plays a pivotal role in the pathogenesis of numerous conditions, including autoimmune diseases, infections, cancers, transplant rejection, and chronic inflammation. This article primarily focuses on the research findings of TIM-3 in the field of autoimmune diseases, with a special emphasis on the structure and signaling pathways of TIM-3, its types of ligands, and the potential mechanisms implicated in systemic lupus erythematosus, multiple sclerosis, rheumatoid arthritis, as well as other autoimmune diseases and chronic inflammation. The latest research results in the field of immunology suggest that TIM-3 dysfunction affects various immune cells and participates in the pathogenesis of diseases. Monitoring the activity of its receptor-ligand axis can serve as a novel biological marker for disease clinical diagnosis and prognosis evaluation. More importantly, the TIM-3-ligand axis and the downstream signaling pathway molecules may become key targets for targeted intervention treatment of autoimmune-related diseases.

High-Throughput Drug Screen for Potential Combinations With Venetoclax Guides the Treatment of Transformed Follicular Lymphoma.

Transformed follicular lymphoma (t-FL) is an aggressive malignancy that is refractory and rapidly progressing with poor prognosis. There is currently no effective treatment. High-throughput screening (HTS) platforms are used to profile the sensitivity or toxicity of hundreds of drug molecules, and this approach is applied to identify potential effective treatments for t-FL. We randomly selected a compound panel from the School of Pharmaceutical Sciences Xiamen University, tested the effects of the panel on the activity of t-FL cell lines using HTS and the CCK-8 assay, and identified compounds showing synergistic anti-proliferative activity with the Bcl-2 inhibitor venetoclax (ABT-199). Bioinformatics tools were used to analyze the potential synergistic mechanisms. The single-concentration compound library demonstrated varying degrees of activity across the t-FL cell lines evaluated, of which the Karpas422 cells were the most sensitive, but it was the cell line with the least synergy with ABT-199. We computationally identified 30 drugs with synergistic effects in all cell lines. Molecularly, we found that the targets of these 30 drugs didn't directly regulate Bcl-2 and identified 13 medications with high evidence value above .9 of coordination with ABT-199, further confirming TP53 may play the largest role in the synergistic effect. Collectively, these findings identified the combined regimens of ABT-199 and further suggested that the mechanism is far from directly targeting Bcl-2, but rather through the regulation and synergistic action of p53 and Bcl-2. This study intended to reveal the best synergistic scheme of ABT-199 through HTS to more quickly inform the treatment of t-FL.

NPM1 mutation reprograms leukemic transcription network via reshaping TAD topology.

C-terminal mutation of Nucleophosmin 1 (NPM1C+) was thought to be a primary driving event in acute myeloid leukemia (AML) that reprograms leukemic-associated transcription programs to transform hematopoietic stem and progenitor cells (HSPCs). However, molecular mechanisms underlying NPM1C+-driven leukemogenesis remain elusive. Here, we report that NPM1C+ activates signature HOX genes and reprograms cell cycle regulators by altering CTCF-driven topologically associated domains (TADs). Hematopoietic-specific NPM1C+ knock-in alters TAD topology leading to disrupted regulation of the cell cycle as well as aberrant chromatin accessibility and homeotic gene expression, which results in myeloid differentiation block. Restoration of NPM1 within the nucleus re-establishes differentiation programs by reorganizing TADs critical for myeloid TFs and cell cycle regulators that switch the oncogenic MIZ1/MYC regulatory axis in favor of interacting with coactivator NPM1/p300, and prevents NPM1C+-driven leukemogenesis. In sum, our data reveal that NPM1C+ reshapes CTCF-defined TAD topology to reprogram signature leukemic transcription programs required for cell cycle progression and leukemic transformation.

Activation of CD3 + TIM3 + T cells contributes to excessive inflammatory response during glucocorticoid treatment.

Glucocorticoids (GCs) are widely used to treat autoimmune and inflammatory diseases, but recent research has challenged the notion that GCs are universally anti-inflammatory. In this study, we investigated the effects of long-term GC exposure on circulating T cells in a retrospective cohort of 5,476 patients with primary glomerular diseases. Our results revealed that GCs altered the composition pattern of circulating leukocytes and the correlation between circulating lymphocytes and serum cytokines in response to infections, as well as the subsets of CD4 + T cells. Specifically, GCs promoted the loss of CD4 + T cells and increased the proportions of CD3 + TIM3 + T cells in response to infections, which correlated with the expression of serum inflammatory cytokines, such as IFNG and IL-10. Using animal models of cecal ligation and puncture, we demonstrated that long-term GC exposure exacerbated apoptosis of CD4 + T cells and cytokine storm during sepsis, which was mechanistically linked to the increase of CD3 + TIM3 + T cells. Notably, we found that CD3 + TIM3 + T cells expressed high levels of multiple cytokine genes during infections, suggesting a potent role of TIM3 in the regulation of T cell biology. In vitro studies further showed that engagement of anti-TIM3 treatment enhanced the inflammatory activity of CD3 + T cells. Our findings suggest a causal relationship between chronic exposure to GCs and an excessive inflammatory response mediated by T cells during infections, which is, at least partly, driven by dysregulation of CD3 + TIM3 + T cells.

Loss of CD28 expression associates with severe T-cell exhaustion in acute myeloid leukemia.

Introduction: Despite accumulated evidence in T-cell exhaustion in acute myeloid leukemia (AML), the immunotherapeutic targeting exhausted T cells such as programmed cell death protein 1 (PD-1) blockade in AML failed to achieve satisfying efficacy. Characteristics of exhausted T cells in AML remained to be explored. Methods: Phenotypic analysis of T cells in bone marrow (BM) using flow cytometry combining senescent and exhausted markers was performed in de novo AML patients and healthy donors as well as AML patients with complete remission (CR). Functional analysis of T-cell subsets was also performed in de novo AML patients using flow cytometry. Results: T cells experienced a phenotypic shift to terminal differentiation characterized by increased loss of CD28 expression and decrease of naïve T cells. Additionally, lack of CD28 expression could help define a severely exhausted subset from generally exhausted T cells (PD-1+TIGIT+). Moreover, CD28- subsets rather than CD28+ subsets predominantly contributed to the significant accumulation of PD-1+TIGIT+ T cells in AML patients. Further comparison of de novo and CR AML patients showed that T-cell exhaustion status was improved after disease remission, especially in CD28+ subsets. Notably, higher frequency of CD28-TIGIT-CD4+ T cells correlated with the presence of minimal residual disease in AML-CR group. However, the correlation between CD28- exhausted T cells and cytogenetic risk or white blood cell count was not observed, except for that CD28- exhausted CD4+ T cells correlated with lymphocyte counts. Intriguingly, larger amount of CD28-TGITI+CD8+ T cells at diagnosis was associated with poor treatment response and shorter leukemia free survival. Discussion: In summary, lack of CD28 expression defined a severely exhausted status from exhausted T cells. Accumulation of CD28- exhausted T cells was linked to occurrence of AML, and correlated to poor clinical outcome. Our data might facilitate the development of combinatory strategies to improve the efficacy of PD-1 blockade in AML.

Long-term glucocorticoid exposure persistently impairs CD4+ T cell biology by epigenetically modulating the mTORC1 pathway.

Conventional glucocorticoid (GC) treatment has a long-term influence on T-cell immunity, resulting in an increased risk of opportunistic infection after drug withdrawal. The underlying mechanisms remain ambiguous. This study demonstrated that long-term GC treatment induced persistent lymphopenia in patients with primary glomerular disease. GCs continuously suppressed the proportion of CD4+ T cells even after the daily dose was tapered down to the physiologic equivalences, leading to a significant decline of the CD4/CD8 ratio. Meanwhile, GCs impaired CD4+ T cell biology, leading to enhanced apoptotic cell death, reduced proliferative capacity, downregulated pro-inflammatory genes, and upregulated immunoregulatory genes. Specifically, GCs altered FOXP3 expression pattern in CD4+ T cells and favored their acquisition of an active T regulatory (Treg) cell phenotype with enhanced IL-10 production upon stimulation. Mechanistically, GCs tampered with the transcriptional regulation of mechanistic target of rapamycin complex 1 (mTORC1) pathway, resulting in an inhibitory impact on the signaling activity. Targeting mTORC1 signaling by siRNAs could sufficiently modify the viability of GC-exposed CD4+ T cells. By high-throughput sequencing of genome-wide DNA methylation and mRNA, we further uncovered a causal relationship between the altered DNA methylation level and transcription activity in a subset of mTORC1 pathway genes in long-term GC exposure. Taken together, this study reveals a novel regulation of mTORC1 signaling, which might dominate the long-term influence of GC on CD4+ T cell biology in a dose-independent manner.

Clinical characteristics and outcomes of follicular lymphoma patients with extranodal involvement: analysis of a series of 1090 cases in China.

BACKGROUND: Follicular lymphoma (FL) is the most common indolent B cell lymphoma in the world. The clinical features of extranodal involvement in FL were never extensively described. METHODS: We enrolled 1090 patients diagnosed as newly diagnosed FL at ten medical institutions in China from 2000 to 2020 and conducted this analysis and retrospectively explored clinical characteristics and outcomes of FL patients with extranodal involvement. RESULTS: 400 (36.7%) patients with newly diagnosed FL had no extranodal involvement, 388 (35.6%) patients had one site of extranodal involvement, and 302 (27.7%) had two or more sites of involvement. Patients with >1 extranodal site had significantly worse PFS (p<0.001), as well as OS (p=0.010). The most common site of extranodal involvements was bone marrow (33%), followed by spleen (27.7%) and intestine (6.7%). In patients with extranodal involvement, multivariate Cox analysis found that male patients (p=0.016), poor performance status (p=0.035), increased LDH (p<0.001) and pancreas involvement (p<0.001) was associated with poor PFS, while the latter three factors were also associated with poor OS. Compared to patients with one site of extranodal involvement, patients with >1 site involvement (p=0.012) had 2.04-fold risk to develop POD24. In addition, multivariate Cox analysis found that the usage of rituximab was not associated with better PFS (p=0.787) or OS (p=0.191). CONCLUSIONS: Our cohort is large enough to have statistical significance in FL patients with extranodal involvement. Male sex, increased LDH, poor performance status, >1 extranodal site, as well as pancreas involvement indicated useful prognostic factors in the clinical setting.