Objective:To investigate whether daporinad(APO866),a nicotinamide phosphoribosyltransferase(NAMPT)inhibitor,can potently kill Philadelphia chromosome-positive(Ph+)acute lymphoblastic leukemia(ALL)cells via an intrinsic apoptosis-independent pathway.Methods:Ph+ALL SUP-B15 cells were treated with varying concentrations of APO866.After treatment,cell viability was measured via Deep Blue assay,while apoptosis and cell death were determined by Annexin V/7-AAD double staining.Cleavage of Caspase-3 and PARP1 was detected by Western blot,and rescue experiments with the pan-caspase inhibitor Z-VAD-FMK were performed to rule out apoptotic involvement.To determine whether APO866-induced cell killing depends on nicotinamide adenine dinucleotide(NAD+)depletion,the NAD+precursor nicotinic acid(NA)was supplemented and NAD+consuming enzyme CD38 was knocked down.Furthermore,TP53-knockout and BAX/BAK double-knockout SUP-B15 cell models were used to verify that the cytotoxic effect of APO866 is independent of the intrinsic apoptotic pathway.Results:Both Deep Blue and Annexin V/7-AAD staining assays showed that APO866 effectively kills SUP-B15 cells.This cytotoxic effect could not be abrogated by the pan-caspase inhibitor Z-VAD-FMK,and no cleavage of Caspase-3 and PARP1 was detected during the process of cell killing.Intracellular NAD+levels were markedly decreased following APO866 treatment,and supplementation with NA or knockout of CD38 partially reversed such cytotoxicity.Neither TP53 knockout nor BAX/BAK double knockout impaired the killing efficiency of APO866 against SUP-B15 cells.Conclusion:APO866 could potently induce SUP-B15 cell death by depleting intracellular NAD+levels,a process that lacks canonical features of the intrinsic apoptotic pathway.Moreover,its cytotoxicity persists even with deficiencies in key regulators of this pathway,indicating a mechanism of action that is independent of intrinsic apoptosis.

OBJECTIVE:To explore the effect of body mass index (BMI) on the efficiency of hematopoietic stem cell (HSC) mobilization. METHODS:A retrospective analysis was conducted on the clinical data of 158 healthy donors who underwent peripheral blood HSC mobilization in the Affiliated Hospital of Xuzhou Medical University from January 2011 to September 2022. According to the BMI Chinese standard, donors were divided into three groups: normal weight group (BMI<24), overweight group (24≤BMI<28) and obesity group (BMI≥28). The differences in peripheral blood white blood cell (WBC) count, neutrophil count, increase ratios, and peak times obtained from dynamical monitoring between different BMI groups were compared, as well as the differences in mononuclear cell (MNC) count and CD34+ cell count in different age, sex and BMI groups. RESULTS:The median age of 158 donors was 36(18-65) years, with 102 males and 56 females. Donors aged 18-39, 40-49, 50-59 and ≥60 years accounted for 68.4%, 24.1%, 6.3% and 1.3%, respectively. The normal weight, overweight and obesity groups had 68, 56 and 34 cases, respectively. Before collection, the peak WBC count in the normal weight, overweight and obesity groups were 51.30(21.60-84.30)×109/L, 50.30(20.30-85.90)×109/L, and 50.30(24.89-67.80)×109/L, respectively. There was no significant difference between the three groups (P>0.05). The peak neutrophil count in the three groups were 42.59(18.77-76.62)×109/L, 43.74(16.85-72.75)×109/L, and 42.84(17.88-54.73)×109/L, respectively. There was no significant difference between the three groups (P>0.05). The number of MNCs collected in the three groups was 8.55(3.50-19.30)×108/kg, 9.15(3.73-26.20)×108/kg, and 9.79(4.14-30.90)×108/kg, respectively. There was no significant difference between the three groups (P>0.05). The number of CD34+ cells collected in the three groups was 4.56(1.44-11.47)×106/kg, 4.66(1.48-13.48)×106/kg, and 5.27(2.27-12.60)×106/kg, respectively. There was no significant difference between the three groups (P>0.05). CONCLUSION:BMI has no significant effect on the mobilization efficiency of peripheral blood HSCs in healthy donors.

ABSTRACT:Serine/threonine kinase 10 (STK10) is a member of the Ste20 family of serine/threonine kinases and regulates lymphocyte adhesion. Quantitative phosphoproteomic assay showed increased STK10 phosphorylation in activated platelets. However, its role in platelet function remains unclear. In our study, we investigated the expression and role of STK10 in platelet function. We first showed STK10 expression in human and mouse platelets. By establishing megakaryocyte/platelet-specific STK10 knockout mice, we found that the deletion of platelet STK10 impaired hemostasis and arterial thrombosis. Consistently, platelet aggregation, α-granule release, αIIbβ3 activation, procoagulant activity, spreading, and clot retraction were all reduced after the deletion of STK10. Quantitative phosphoproteomic assays revealed several dysregulated phosphoproteins, which were enriched in platelet activation and focal adhesion. Using immunoprecipitation coupled to mass spectrometry and protein phosphorylation profiles screening approaches, we identified that STK10 interacts with integrin-linked protein kinase (ILK) and the deletion of STK10 significantly reduced ILK phosphorylation (Ser343). A subsequent in vitro phosphorylation assay demonstrated that STK10 directly phosphorylated ILK at Ser343. In addition, the inhibition of calcium, protein kinase C, or phosphatidylinositol 3-kinase inhibited STK10 phosphorylation in activated platelets. Moreover, the deletion of platelet STK10 reduced platelet-neutrophil interactions, neutrophil accumulation, and neutrophil extracellular trap formation, ameliorated thromboinflammation, and increased the survival of sepsis mice. Furthermore, an increase in the activation of platelet STK10 and ILK was observed in sepsis mice and patients with sepsis. In conclusion, our study identifies a novel regulatory role of STK10 in platelet function, arterial thrombosis, and thromboinflammation, implying that it might be a potential target for the treatment of thrombotic or cardiovascular diseases.

Abstract Before November 2023, CD19 chimeric antigen receptor (CAR) T-cell therapies had not been approved in China for patients with relapsed or refractory B-cell acute lymphoblastic leukemia (R/R B-ALL), leaving a significant unmet need. In response, inaticabtagene autoleucel (Inati-cel), a novel CD19 CAR T-cell therapy with a distinct single-chain variable fragment (HI19α), was developed and showed promising efficacy in preliminary clinical research. We conducted a phase 2, single-arm, multicenter study of Inati-cel in adult CD19+ R/R B-ALL in China. The primary end point was the overall remission rate (ORR) at the end of month 3. Forty-eight patients who underwent Inati-cel infusion were evaluated for both efficacy and safety. Among them, 34 patients achieved and maintained remission beyond 3 months, with a 3-month ORR of 70.8% (95% confidence interval [CI], 55.9-83.1). The best ORR was 85.4%, with all responders reaching minimal residual disease negativity. With a median follow-up of 23.7 months, the median duration of remission was 20.7 months (95% CI, 6.4 to not reached), and the median overall survival was not reached (95% CI, 13.0 months to not reached). Additionally, grade ≥3 cytokine release syndrome and neurologic events occurred in 12.5% and 6.2% of patients, respectively. The 2-year follow-up data suggest that Inati-cel demonstrates encouraging and durable responses with manageable safety profiles in R/R B-ALL. Based on the data from this pivotal trial, Inati-cel was approved as the first CAR T-cell therapy for adult R/R B-ALL in China and underscores its potential therapeutic benefits for this patient population. This trial was registered at www.ClinicalTrials.gov as #NCT04684147.

Statement of translational relevanceEffects of metachronous primary malignant solid tumor (MPMST) on survival risk and prognosis of multiple myeloma (MM) and differences between MPMST occurring before and after MM remains unclear. Use of well-characterized clinical information of individual patient, we found that older patients with MM (≥ 65 years) had a higher risk of developing MPMST. Patients with MM and MPMST including male patients, aged ≥ 65 years and those with ISS stage III had a worse prognosis. The top three solid cancers occurred before and after MM were the lung, thyroid, and breast cancer. These findings provide detailed information for the precise treatment of patients with MM and MPMST.ObjectiveTo analyze the effects of MPMST on MM and the risk difference of MPMSTs occurring before and after MM.MethodsRetrospective data from patients with MM and MPMST, including sex, age, immunoglobulin isotype, ISS stage, and therapy, were collected from 2015 to 2023. Differences in variables, risk, and survival were compared using the χ² test, logistic regression analysis and the Cox model, respectively.ResultsThe 34 (1.57%) patients with MM and MPMST identified from a total of 2167 MM patients had a shorter overall survival. The survival risk was higher in male patients with MM and MPMST (HR: 3.96, 95% CI: 1.05 -14.96), in those aged ≥ 65 years (HR: 3.30, 95% CI: 1.41 -7.71), and with ISS stage III (HR: 4.08, 95% CI: 0.81-20.65). Patients with MM subsequent to CAR-T cell therapy had neither enhanced incidence rates of second solid cancers nor had longer overall survival time. Furthermore, the top three solid cancers occurred before or after MM were lung, thyroid, and breast cancer.ConclusionMale patients, aged ≥ 65 years and MM patients with ISS stage III and MPMST had a worse prognosis.

Background: Diffuse large B-cell lymphoma (DLBCL) is the most common subtype of non-Hodgkin lymphoma. Polatuzumab vedotin (Pola) has shown significant anti-tumor activity and a manageable safety profile in DLBCL patients. Given the strict eligibility criteria in pivotal trials, real-world evidence in Chinese DLBCL patients is needed to guide clinical practice. Methods: The POLAREAL study (NCT05954910) is a prospective, observational, multicenter registry study designed to evaluate the effectiveness and safety of Pola-based regimens in Chinese patients, with a target enrollment of 1,000 patients. Eligible patients with DLBCL (aged ≥ 18 years) were enrolled and classified into three cohorts: Cohort 1-previously untreated patients classified as unfit or frail (defined as aged ≥ 80 years or < 80 years but with comorbidities and intolerance to standard dose chemotherapy); Cohort 2-previously untreated patients not meeting unfit/frail criteria; and Cohort 3-patients with relapsed/refractory disease. The primary objective was progression-free survival (PFS). Secondary objectives included best response (BCRR/BORR), overall survival (OS) and safety, etc. Results: From August 2023 to February 2025, 794 patients were enrolled and included in the full analysis set, comprising 116 patients in Cohort 1 (median age 76.1; range 44–91), 377 patients in Cohort 2 (median age 59.6; range 18–80), and 301 patients in Cohort 3 (median age 63.2; range 19–90). From cohort 1 to 3, 94.9%, 76.7% and 83.6% of patients had an IPI score of 2-5 respectively. The majority of patients (92.2%, 86.7% and 87.4% for each cohort) had the DLBCL-NOS subtype. The study also included patients typically excluded from clinical trials, such as primary mediastinal B-cell lymphoma and primary cutaneous DLBCL-leg type. The non-GCB subtypes predominated over the GCB subtype, accounted for 61.2% (Cohort 1), 54.9% (Cohort 2) and 54.8% (Cohort 3) of the patients. Double-expressor lymphoma was identified in 28.4%, 27.1% and 24.9% in each group. Additionally, transformed DLBCL-primarily arising from follicular lymphoma-was observed in 5.2%, 6.1% and 11.0% of patients across the three cohorts. In Cohort 3, 27.6% of patients had relapsed disease, while 72.4% had refractory disease. In real-world settings, the median time from the initial DLBCL diagnosis to the initiation of Pola treatment was 0.49 months (range 0-26.3) in Cohort 1 and 0.36 months (range 0-5.1) in Cohort 2, respectively. The most commonly used backbone regimens included Pola-R-mini(reduced) CHP and Pola-R in Cohort 1; Pola-R-CHP and Pola-R-DA EPCH in Cohort 2; Pola-BR, Pola-R-GemOx and Pola-R-ICE in Cohort 3. The median treatment cycles of Pola-based regimen in those patients with EOT were 6, 6 and 3 separately. The median follow-up time was 3.9 months (range 0.0-17.6), 3.2 months (range 0.0-14.7), and 2.9 months (range 0.0-11.6) across the three cohorts, respectively. As the PFS data were not mature at the time of cutoff, BORR and BCRR were reported among patients with evaluable responses including PET-CT data. In Cohort 1, the BORR was 90.7% (95% CI 81.7-96.2), and the BCRR was 62.7% (95% CI 50.7-73.6). In Cohort 2, the BORR and BCRR were 93.5% (95% CI 90.0-96.1) and 69.8% (95% CI 64.0-75.1), respectively. In Cohort 3, the BORR was 78.0% (95% CI 69.7-84.8) and the BCRR was 50.4% (95% CI 41.4-59.4). Among the 801 patients included in the safety analysis set, treatment-emergent adverse events (TEAEs) were reported in 85.4% of patients, with grade≥3 TEAEs occurred in 48.4%. Grade≥2 peripheral neuropathy was observed in 0.7% of cases. Serious adverse events occurred in 17.9% of patients. 67.3% of patients reported Pola related TEAEs. Conclusion: This represents the largest real-world study of Pola to date. In real-world settings featuring greater heterogeneity among DLBCL patients, Pola-based regimens demonstrated promising clinical activity and were well tolerated. These findings offer valuable insights into treatment consideration, with additional evidence expected from continued follow-up and analysis of the POLAREAL study.

The aim of this study was to explore the prognostic value of mean corpuscular volume (MCV) in newly diagnosed aplastic anemia (AA) patients treated with cyclosporine A (CsA) plus androgen or CsA alone. The clinical data of 181 newly diagnosed patients with aplastic anemia from April 2008 to September 2020 in the Affiliated Hospital of Xuzhou Medical University were retrospectively analyzed. According to the MCV levels, the patients were divided into a high-MCV group (107/181) and a normal-MCV group (74/181). We investigated the effect of MCV outcomes in patients with AA. Between the high-MCV and normal-MCV groups, neutrophil count, red blood cell count, platelet count, reticulocyte count, disease severity, lymphocytes, and granulocytes were significantly different (P < 0.05). The overall response rates (CR + PR) were 69.16% and 59.46% in the high-MCV and normal-MCV groups, respectively. The duration of response was not significantly different between MCV groups. The high-MCV patients had an improved 5-year overall survival and progression-free survival compared to the normal-MCV patients (94.40% vs. 68.10%; 71.80% vs. 60.30%, P < 0.001). Regarding hemogram restoration, the leukocyte, neutrophil, hemoglobin, and platelet recovery was accelerated in the high-MCV group (P < 0.05). Furthermore, MCV levels were positively correlated with reticulocyte count, reticulocyte percentage, high-fluorescence reticulocyte, medium-fluorescence reticulocyte, and immature reticulocyte fraction; on the other hand, MCV levels were negatively correlated with low fluorescent reticulocyte. In conclusion, aplastic anemia patients with a high MCV were a better prognostic factor, and the patients with high MCV may have better residual bone marrow hematopoietic function than those with normal MCV.

Background:: Although immunotherapies have greatly improved diffuse large B-cell lymphoma (DLBCL) prognosis, a proportion of patients remain to be relapsed or refractory. Therefore, the identification of novel therapeutic targets and drugs is urgently required. Inhibition of the bromodomain and extra-terminal (BET) proteins has been a promising therapeutic strategy for various haematologic cancers. CPI-0610 is a potent and selective BET inhibitor. The effects of CPI-0610 in DLBCL cells have not been reported yet. Aims:: The aim of this study was to assess the effects of CPI-0610 in DLBCL and its underlying mechanisms. Methods:: DLBCL cells were treated with CPI-0610, followed by measuring cell viability, cell cycle, apoptosis, autophagy, and specific cell signaling pathways. Moreover, immunodeficient mice were engrafted with SUDHL2 cells and then treated with CPI-0610 for analysis of tumor burden. We also analyzed the synergistic effect of CPI-0610 with histone deacetylase inhibitor suberoylanilide hydroxamic acid. Results:: The present study demonstrated that CPI-0610 displayed cell cytotoxicity by arresting the G1 cell cycle and inducing endogenous and exogenous apoptotic pathways. Additionally, CPI-0610 decreased BRD4 and c-Myc expressions and affected MAPK, JAK/STAT, and AKT signalling pathways in human DLBCL cells. An in vivo experiment exhibited that CPI-0610 decreased the primary tumour growth of the DLBCL xenograft model. Furthermore, the use of CPI-0610 in combination with suberoylanilide hydroxamic acid exhibited a specific synergistic effect in inducing apoptosis through the regulation of STAT3 and p38. Conclusion:: Targeting BET may be an effective therapeutic strategy and potentiated by a combination with histone deacetylase inhibition in DLBCL.

Chimeric antigen receptor (CAR)-T cell therapy has proven to be a revolutionizing immunotherapeutic strategy for treating relapsed or refractory lymphoma, achieving remarkable clinical responses. However, there remain some challenges including treatment resistance and early relapse in a minor proportion of patients. The lymphoma tumor microenvironment (TME) is a heterogeneous and dynamic milieu composed of lymphoma cells, immune cells, stromal components, cytokines, and extracellular matrix proteins. CAR-T cell infusion alters the composition of TME and thus impact the endogenous immune response. Additionally, various components of the TME affect the persistence, activity and cytotoxicity of CAR-T cells, which is a key endogenous factor that impeding the efficacy of CAR-T cell therapy in lymphoma. Herein, we review the role of lymphoma TME on CAR-T cells, and discuss strategies targeting TME components to overcome resistance and improve the effectiveness of CAR-T cells.

In recent years, more and more studies have shown that HBV is closely related to DLBCL and is an important adverse prognostic factor. The purpose of this study was to explore the mechanism of BCL-6 on Hepatitis B Virus (HBV) positive diffuse large B cell lymphoma (DLBCL). In vitro, electrotransfection transfers HBV DNA into the DLBCL cell line; Plasmid was transfected into DLBCL cells to construct hepatitis B virus X protein (HBX) overexpressed DLBCL cells; Cell proliferation was detected by Cell Counting Kit-8;Protein expression was measured by Western blot analysis;Cell apoptosis and cycle analysis were evaluated via flow cytometry. Tumor-bearing mice were used to evaluate antitumor efficacy in vivo. The results showed that HBX increased the expression of BCL-6 gene in DLBCL cell line, promoted the proliferation of DLBCL cell line, induced transformation of cell cycle from G1 phase to S phase, and simultaneously promoted the activation of NF-κB pathway. The upregulation of BCL-6 expression induced by HBX were reversed by BCL-6 inhibitors. In addition, BCL-6 inhibitors inhibited the proliferation of DLBCL cell lines overexpressing HBX by promoting apoptosis. According to these findings, HBX might promote cell proliferation through BCL-6 mediated NF-κB signaling in DLBCL cell lines.

Background: Relapsed or refractory B-precursor acute lymphoblastic leukemia (R/R B-ALL) in adults is associated with an unfavorable prognosis. Brexucabtagene autoleucel (KTE-X19) is an autologous anti-CD19 CAR T-cell therapy that has been approved by the US FDA for the treatment of adult patients with R/R B-ALL, based on the results of ZUMA-3 trial. However, the efficacy and safety of KTE-X19 for the treatment of R/R B-ALL in the Chinese population have not been explored. We conducted a single-arm, multi-center, phase II trial (ChiCTR2300073872) to evaluate the efficacy and safety of KTE-X19 in Chinese population with R/R B-ALL, with this report presenting updated outcomes after >6 months median follow-up. Methods: Eligible patients were aged 18 years or older, with Eastern Cooperative Oncology Group performance status (ECOG PS) of 0-1, and morphological disease in the bone marrow (>5% blasts). After leukapheresis and conditioning chemotherapy, patients received a single infusion of KTE-X19 (1 × 10⁶ CAR T cells/kg). The primary endpoint was the overall complete remission (OCR) rate (complete remission [CR] + CR with incomplete hematologic recovery [CRi]) by central assessment. Secondary endpoints included duration of remission (DOR), relapse-free survival (RFS), overall survival (OS), minimal residual disease (MRD) negativity rate, and allogeneic stem cell transplantation (allo-SCT) rate. Results: As of the data cutoff (February 13, 2025), 32 patients had been enrolled and 28 patients received KTE-X19 treatment. The median age of treated patients was 36 years (range 19-66). The median bone marrow blast percentage at baseline was 31.25% (range 0-98%). One patient (3.6%) aged 65 years or older. 61% of patients had ECOG PS of 1. Ten patients (35.7%) had Philadelphia chromosome-positive (Ph+) disease. One patient (3.6%) had CNS-2 disease. One patient (3.6%) had extramedullary disease at screening. Patients had a median of 2 (range 1-5) prior lines of therapy; one patient (3.6%) previously received blinatumomab, four patients (14.3%) previously received inotuzumab ozogamicin, and seven patients (25%) previously received allo-SCT. Median follow-up time for this analysis was 10.3 months (90% CI 5.9, 9.1). Among 28 patients received KTE-X19 treatment, the best OCR rate was 78.6% (90% CI 62.0%, 90.2%) and the best CR rate was 67.9%. The median time to first response was 1 month. All responders had MRD negativity. Nine patients (32.1%) underwent allo-SCT after KTE-X19 infusion. Median RFS, median DOR and median OS were not reached. The estimated 1-year RFS rate was 60.4% and 1-year DOR rate was 76.9%. Among the patients with CR/CRi , the median time to peak CAR T-cell levels in blood post-KTE-X19 was 14 days (range 7-14) and the median peak CAR T-cell levels in blood was 47.64 cells/μL(range 5.13-2725.83). The median area under the curve from Days 0-28 of CAR T-cell levels was 492.98 cell/μL×days (range 81.70-13515.45). No new safety signals were observed in the Chinese population. The most common treatment-emergent adverse events of grade 3 or higher were decreased platelet count (21 [75%] patients), decreased white blood cell count (21 [75%] patients), pyrexia (20 [71.4%]), decreased lymphocyte count (19 [67.9%]) and decreased neutrophil count (19 [67.9%]). Cytokine release syndrome (CRS) of any grade occurred in 26 patients (92.9%), with 6 patients (21.4%) experiencing grade≥3 CRS. Neurological events (NEs) of any grade occurred in 10 patients (35.7%), with 4 patients (14.3%) experiencing grade≥3 NEs. No grade 5 CRS or NEs occurred. Conclusions: After >6 months median follow-up, KTE-X19 demonstrated a high rate of OCR in Chinese adult patients with R/R B-ALL, with median RFS, DOR and OS not reached, and a manageable safety profile. These findings support its therapeutic potential and clinical benefit in the Chinese population.

OBJECTIVE:To investigate the prognostic value of peripheral blood absolute monocyte count(AMC) in non-severe aplastic anaemia(NSAA) patients. METHODS:178 patients with NSAA who attended the Affiliated Hospital of Xuzhou Medical University from April 2008 to September 2020 were retrospectively analyzed, and the optimal cut-off value of peripheral blood AMC was determined by the receiver operating characteristic curve of the subjects, and they were divided into low AMC group (48 patients) and normal AMC group (130 patients), and the differences in clinical characteristics between the two groups were compared. Overall survival(OS) and progression-free survival(PFS) were analyzed by Kaplan-Meier. Univariate and multivariate Cox regression analysis were used to determine the independent prognostic value of AMC. RESULTS:Among 178 NSAA patients, 105(59.0%) were male and 73(41.0%) were female, with a median age of 31(18-87) years old, a median follow-up time of 58 months (range: 6 months-175 months), and a median AMC of 0.15×109/L ［range: (0.01-0.59)×109/L)］. The proportion of granulocytes (27.5% vs 36.0%, P < 0.05), and the proportion of mature monocytes (1% vs 2%, P < 0.05) in the low AMC group were lower than that in the normal AMC group; the proportion of mature lymphocytes in the low AMC group was higher than that in the normal AMC group (54% vs 50%, P < 0.05). However, there was no significantly different in the proportion of erythropoietic cells and stages of the erythropoietic cells between the two groups ( P >0.05). CR (27.7% vs 10.4%) and ORR (75.4% vs 56.3%) in the normal AMC group were higher than that in the low AMC group. Compared with patients in the low AMC group, AA patients in the normal AMC had better 5-year OS (98.5% vs 86.9%, P < 0.01), and the 5-year PFS (86.0% vs 58.9%, P < 0.01). Also, the 10-year survival rate of patients in the normal AMC group was higher than that in the low AMC group (98.5% vs 60.5%,P < 0.01). Univariate analysis showed that age, reticulocyte count, AMC<0.1×109/L and the proportion of bone marrow mature monocytes were related with patients survival. Multivariate Cox regression analysis showed that monocyte count reduction was not an independent poor prognostic factor in NSAA patients （HR =4.474，95%CI ：0.508-44.390； P =0.172）. CONCLUSION:Low AMC level at initial diagnosis is not an independent prognostic factor for NSAA patients, but still suggest potential prognostic value of AMC.

INTRODUCTION:Statins, a class of HMG-CoA reductase inhibitors, exhibit prophylactic benefits against immune rejection induced by allogeneic hematopoietic stem cell transplantation (allo-HSCT). Despite the protective function is confirmed, the precise mechanism to induce immune tolerance of statin in the initial stages of transplantation remains incompletely understood. Given that Treg cells play a critical role in preventing graft versus host response and Foxp3 as a transcription factor of Treg can be induced by statins, we hypothesize that the immunosuppressive effects of statins are partially mediated through regulation of Treg cells expansion. METHODS:T cells were stimulated in vitro under anti-CD3/anti-CD28/IL-2/TGF-β condition or allo-reactive system with or without the addition of statins. The induction of Tregs were detected using flow cytometry. Allo-HSCT models were established by transferring donor cells alone or combined with recipient treated by fluvastatin. The proportions of Treg and phenotypes of effector T cells were identified. Cytokine secretion and antigen-presenting cell (APC) function were tested in irradiated mice. RESULTS:Statins induced higher Treg production in classical and allogeneic cell co-culture conditions in vitro. In the early stage of models treated with fluvastatin only in donors or combined treatment of donors and recipients, a similar phenomenon was observed with elevated levels of Foxp3+ Treg along with increased expression of CCR7, CD62L, and S1P1 on allo-reactive T cells. Fluvastatin treatment suppressed the secretion of pro-inflammatory cytokines IFN-γ and TNF-α by CD4+ and CD8+ T cells in irradiated mice. Furthermore, fluvastatin also contributed to restraining the numbers and activation of APCs, including dentritic cells (DCs) and macrophages in vitro and in vivo. CONCLUSION:Our finding demonstrated that statin exposure modulates immune responses during the initial phase of allo-HSCT by promoting Treg expansion and suppressing inflammatory reactions, which supply a promising strategy for aGVHD prevention.

Background The response rate and long-term survival of anti-CD19 chimeric antigen receptor (CAR) T-cell therapy for relapsed/refractory (r/r) B-cell lymphoma need to be further improved in recent years. B-cell receptor (BCR) signaling pathway plays an indispensable role in the pathogenesis of B cell malignancies. Bruton's tyrosine kinase inhibitors (BTKi) was shown in preclinical study and small scale clinical researches to potentially enhance the efficacy of anti-CD19 CAR-T cell therapy for lymphoma. Aims In this single arm, multi-center pilot study (NCT05744037), we aimed to observe the efficacy and safety of zanubrutinib combined with anti-CD19 CAR-T cell therapy in r/r B-cell lymphoma patients. Methods Patients received a lymphodepletion regimen comprising fludarabine and cyclophosphamide, followed by infusion of autologous anti-CD19 CAR-T cells. All patients received zanubrutinib (160 mg bid) simultaneously, which was maintained for a maximum of 2 years after CAR-T cell infusion. The primary endpoint was overall response rate (ORR), while key secondary endpoints were progression-free survival (PFS), overall survival (OS) and safety. Results We enrolled 10 patients in the preliminary study, with 5 achieving CR and 3 achieving PR, resulting in a CR rate of 50% and an ORR of 80% (8/10). The 1-year OS and PFS rates of patients were 87.5% (95% CI, 38.7%–98.1%) and 78.8% (95% CI, 38.1%–94.3%), respectively. In terms of safety, zanubrutinib combined with anti-CD19 CAR-T strategy is well tolerated, the most frequent toxicities were hematologic adverse events (AEs), with a 70% incidence rate of grade 3 hematologic AEs. Three patients (30%) developed cytokine release syndrome, all of which were grade 1. No instances of immune effector cell-associated neurotoxicity syndrome (ICANS) were observed. All patients had no cardiac events . Conclusion In this single arm clinical trial, zanubrutinib combined with anti-CD19 CAR-T cell therapy demonstrates notable efficacy and good tolerability in r/r B-cell lymphoma patients. A confirmation trial based on larger population is needed in the future.

BACKGROUND:For patients with multiple myeloma progression after anti-BCMA chimeric antigen receptor (CAR) T-cell therapy, the optimal salvage treatment strategies remain unclear. GPRC5D-directed CAR T cell might be a potential option. The aim of this trial was to investigate the activity and safety of anti-GPRC5D CAR T cells in patients with progressive multiple myeloma after anti-BCMA CAR T-cell therapy. METHODS:In this phase 2, open-label, single-arm, phase 2 trial, at the Affiliated Hospital of Xuzhou Medical University in China, we enrolled patients (aged 18-70 years old) with relapsed or refractory multiple myeloma who had progressed disease after anti-BCMA CAR T-cell therapy and a life expectancy of more than 12 weeks without active infections, serious liver, heart, or other diseases. Patients were assigned to receive a single dose of intravenous anti-GPRC5D CAR T cell at 2 × 106 cells per kg. The primary endpoint was the overall response rate, including stringent complete response, complete response, very good partial response, and partial response, according to the standard International Myeloma Working Group response assessment criteria. Activity and safety analyses were done in the patients who received a dose of anti-GPRC5D CAR T cell as defined in the protocol. This trial is registered with the Chinese Clinical Trial Registration Center, ChiCTR2100048888, and is ongoing. FINDINGS:Between Dec 1, 2021, and May 1, 2024, 42 patients were screened, 37 were enrolled and received anti-GPRC5D CAR T-cell therapy. Median age was 59 years (IQR 51-65), 17 (46%) of 37 patients were male and 20 (54%) female. All patients were Asian. At a median follow-up of 12·6 months (IQR 8·2-20·8), the overall response rate was 84% (95% CI 68-94, 31 of 37 patients), including 13 (35%) complete responses or better. The most common grade 3-4 adverse events were haematological toxicities, including leukopenia (34 [92%] of 37 patients), lymphopenia (36 [97%]), neutropenia (29 [78%]), anaemia (23 [62%]), and thrombocytopenia (23 [62%]). 26 (70%) of 37 patients had cytokine release syndrome, which was of grade 3 in two (5%) patients. One case of grade 1 immune effector cell-associated neurotoxicity syndrome was observed. There were no treatment-related deaths in the trial. INTERPRETATION:Anti-GPRC5D CAR T-cell salvage therapy induced a high response rate, and could be a potential treatment option in relapsed or refractory multiple myeloma patients who have progressed after anti-BCMA CAR T-cell treatment. Further investigations are warranted to establish the long-term efficacy and safety of this therapeutic approach. FUNDING:National Natural Science Foundation of China and the General Project of Jiangsu Commission of Health.

Background: Inflammation-induced injury of venous endothelium is the first trigger of deep vein thrombosis (DVT). We previously showed NLRP3 regulates platelet function and arterial thrombosis. However, whether platelet NLRP3 involves in venous thrombosis remains unclear. Objectives: In this study, we intended to investigate the role of NLRP3 in venous thrombosis by using NLRP3 knockout mice and platelet-specific NLRP3 knockout mice. Methods: DVT model was established through ligation of the inferior vena cava. After 48 hours of ligation, inferior vena cava sample was excised to measure thrombi length and weight, the recruitment of platelets, neutrophils, monocytes, and neutrophil extracellular trap (NET) formation by immunofluorescence staining. Results: Deficiency of NLRP3 reduced the incidence and severity of venous thrombosis, inhibited the recruitment and accumulation of platelets, neutrophils, and monocytes in venous thrombi, and reduced NET formation as well as interleukin (IL)-1(3 and IL-18 release. Additionally, platelet NLRP3 is the major source of elevated IL-1(3 in the venous thrombi, and adoptive transfer of platelets to NLRP3-/- mice increased IL-1(3 level in thrombi and promoted venous thrombosis and NET formation. Moreover, platelet NLRP3 deficiency inhibited NET formation induced by activated platelets in vitro. Conclusion: Our study demonstrated that deficiency of NLRP3 reduces the incidence and the severity of venous thrombosis with platelet NLRP3 playing a predominant role, indicating that NLRP3 might be a therapeutic target for the treatment of venous thrombosis.

ABSTRACT:Sepsis is characterized by a systemic inflammation and microvascular thrombosis induced by infection. The nucleotide-oligomerization domain-like receptor family pyrin domain containing 6 protein (NLRP6) possesses both proinflammatory and anti-inflammatory abilities with cell type-specific or tissue-specific functions. However, the role of cell type-specific NLRP6 in sepsis remains poorly understood. In this study, we detected NLRP6 expression in platelets. By using platelet-specific NLRP6 knockout mice and the cecal ligation and puncture model of sepsis, we demonstrated that deletion of platelet NLRP6 increased the mortality; enhanced microvascular thrombosis in the lung and liver; and promoted platelet activation, platelet-neutrophil interactions, as well as the neutrophil extracellular trap (NET) formation after sepsis. Platelet function analysis in vitro showed that deletion of NLRP6 enhanced platelet aggregation, activation, and granules release. In addition, NLRP6 deletion promoted platelet NF-κB signaling via sustaining transforming growth factor-β activated kinase 1-binding protein 1 (TAB1) expression independent of the inflammasome. Moreover, inhibition of NF-κB signaling abolished the aggravated effects of the absence of platelet NLRP6 on the intravascular microthrombosis and NET formation in sepsis and increased the overall survival. Mechanistically, NLRP6 facilitated the interaction between tripartite motif-containing protein 21 (TRIM21) and TAB1 in activated platelets, resulting in K48-linked polyubiquitination of TAB1 and subsequent degradation. Finally, sepsis plasma triggered TAB1 degradation mediated by NLRP6/TRIM21 in normal healthy platelets through toll-like receptor 4/myeloid differentiation primary response 88. Our study identifies a novel protective role of platelet NLRP6 in microvascular thrombosis during sepsis, implying it as a novel target for the treatment of sepsis.

Multiple myeloma (MM) is a common yet incurable hematological malignancy characterized by bone marrow infiltration. A major clinical challenge is the resistance to chemotherapy, highlighting the urgent need to better understand the molecular mechanisms underlying chemotherapeutic resistance to available drugs. Recent studies have emphasized the role of micropeptides in solid tumors and leukemia, but their functions in MM remain unclear. In this study, we identified a novel micropeptide, altKLF4, derived from the transcription factor KLF4, which is highly expressed in newly diagnosed myeloma patient samples. We found that ectopic expression of altKLF4 interfered with chemotherapy sensitivity induced by proteasome inhibitors in myeloma cells. Additionally, confocal microscopy and transcriptome sequencing revealed that altKLF4 co-localizes with the mitochondrial inner marker TOMM20 and participates in mitochondria-related biological processes, suggesting that altKLF4 partially localizes to the mitochondria. Mitochondria may also play a role in regulating ferroptosis. Our results further demonstrated that altKLF4 inhibited drug sensitivity and ferroptosis induced by the GPX4 inhibitor RSL3 in multiple myeloma cells through a direct interaction with GPX4. In vivo experiments showed that RSL3 significantly suppressed primary myeloma growth, which could be rescued by the micropeptide altKLF4. Taken together, our study identifies altKLF4 as a novel micropeptide that serves as a potential biomarker for chemotherapeutic resistance in multiple myeloma, offering insights for diagnosis and management of drug-resistant MM.

Background aims:Chimeric antigen receptor (CAR) T-cell therapy shows remarkable efficacy against relapsed/refractory multiple myeloma (R/R MM). Nutritional status, assessed by objective indices like the Controlling Nutritional Status (CONUT) and Prognostic Nutritional Index (PNI), influences MM prognosis. However, their predictive value for outcomes following CAR-T therapy in R/R MM remains unclear. Methods:We conducted a retrospective analysis of 181 R/R MM patients receiving CAR-T therapy. Patients were stratified by optimal CONUT (cutoff: 6.5) and PNI (cutoff: 42.75) scores determined via ROC analysis. Associations between CONUT/PNI and treatment outcomes were investigated. Results:Patients with low CONUT or high PNI exhibited significantly improved progression-free survival (PFS) and overall survival (OS) compared to their counterparts (high CONUT or low PNI). While objective response rates (ORR) were high overall (approximately 90-95%), they did not differ significantly between CONUT or PNI subgroups. Importantly, low CONUT or high PNI was associated with faster hematopoietic recovery (red blood cells, hemoglobin, platelets, neutrophils, CD4+, CD8 + T-cells), lower incidence of prolonged hematologic toxicity (PHT), and higher peak CAR transgene levels. No significant differences were observed in cytokine release syndrome (CRS) or immune effector cell-associated neurotoxicity syndrome (ICANS) severity between subgroups. Multivariate analysis confirmed high CONUT as an independent risk factor for inferior PFS and OS, while low PNI was an independent risk factor for inferior PFS. Conclusion:This study establishes pretreatment CONUT and PNI as significant prognostic predictors for R/R MM patients undergoing CAR-T therapy. Patients with low CONUT or high PNI experience superior long-term survival outcomes, potentially linked to enhanced hematopoietic recovery and CAR-T cell expansion. These findings underscore the importance of nutritional assessment in prognostication and may guide future strategies to optimize CAR-T outcomes in R/R MM.

OBJECTIVE:To investigate the predictive role of the modified Endothelial Activation and Stress Index (mEASIX) in the efficacy of chimeric antigen receptor T-cell (CAR-T) therapy and cytokine release syndrome (CRS). METHODS:The clinical data of 70 relapsed and refractory (R/R) B-cell tumor patients who were treated with CAR-T therapy from September 1, 2018 to February 28, 2023 in the Department of Hematology, Affiliated Hospital of Xuzhou Medical University, were retrospectively analyzed. The value of log-2 mEASIX before conditioning (-7 d) was calculated, and the patients were divided into a low-mEASIX group (42 patients) and a high-mEASIX group (28 patients) based on the cut-off value of 5.443 determined by the receiver operating characteristic (ROC) curve. Eventually, the predictive role of mEASIX before conditioning on the efficacy of CAR-T cell therapy and CRS was analyzed. RESULTS:The high-mEASIX group exhibited significantly worse median overall survival (OS) and median progression-free survival (PFS) in comparison to the low mEASIX group (OS: 3.2 months vs not reached, P < 0.01; PFS: 1.3 months vs 6.0 months, P =0.009). The incidence of grade ≥2 CRS in the high-mEASIX group was substantially higher than that in the low-mEASIX group (57.1% vs 19.0%, P =0.007). The degree of remission after CAR-T therapy (P =0.001), whether CRS occurs or not (P =0.041), the lactate dehydrogenase (LDH) level before conditioning (P =0.046), and the mEASIX score before conditioning (P =0.047) were independent influencing factors for the OS of patients receiving CAR-T cell therapy. CONCLUSION:The mEASIX score before conditioning can predict OS and the incidence of grade ≥2 CRS in patients with relapsed and refractory B-cell tumors who receive CAR-T cell therapy.