bluuebunny/crossref_metadata_2025 huggingface dataset:
Engineering a Controllable and Reversible Switch for CAR-T Cell Immunotherapy Via a Genetic Code Expansion System
Authors: Liu Yue , An Lingna , Zhang Xi
Published: December 2024

Background

As one of the most promising adoptive cell therapies, CAR-T therapy has achieved notable clinical effects in patients with hematological tumors. However, several obstacles remain in CAR-T therapy, such as low killing effects for some tumor targets, high-frequency recurrence, and cytokine release syndrome. These obstacles severely limit the long-term effects and the further application of CAR-T therapy. Therefore, the optimization of CAR-T cells for increased controllability and safety is urgently needed.

Methods

In this study, we engineered a genetic code expansion-based therapeutic system to achieve rapid CAR protein regulation in response to cognate unnatural amino acid at the translational level, in which the termination codon TAG was inserted into the CAR coding region. When the unnatural amino acid, N-ε-((tert-butoxy) carbonyl)-l-lysine (BOCK) is absent, the CAR protein cannot be completely translated, and CAR-T is “closed”. When BOCK is present, it recognizes and combines with the TAG, the whole CAR protein is translated, and CAR-T is “open”. Based on these findings, we subsequently investigated whether BOCK can control CAR protein expression and regulate CAR-T cell function via a series of in vitro experiments and a xenograft mouse tumor model.

Results

First, we verified that the BOCK-induced genetic code expansion system enables regulate protein expression as a controllable switch. Subsequently, we demonstrated that BOCK effectively and precisely controlled CAR protein expression and induced the activation of CAR signaling, the expression of activation markers, CD69 and CD25, and cytokine secretion. When incubated with tumor cells, BOCK regulated CAR-T cell cytotoxicity in a dose-dependent manner. Finally, a NOG mouse model analysis showed that BOCK can control CAR-T cell function and anti-tumor effect in vivo.

Conclusions

We systematically demonstrated that the BOCK-induced genetic code expansion system can effectively and precisely regulate CAR protein expression and control CAR-T cell anti-tumor effects in vitro and in vivo. We conclude that this controllable and reversible switch has a potential for more effective, secure, and clinically available CAR-based cellular immunotherapies.
C-Type Natriuretic Peptide Signaling Promotes Bone Marrow Hematopoietic Regeneration and Alleviates Systemic Lymphopenia
Authors: Gong Pengju , Chen Yamei , Han Leng , Yang Liuqing , Lin Chunru
Published: December 2024

Lymphopenia, a condition of lower-than-normal levels of lymphocytes, is associated with compromised immune function, increased susceptibility to infections, and unfavorable outcomes, particularly for cancer patients administered with chemotherapy. Chemotherapy-induced damage to hematopoietic stem cells (HSC) and bone marrow (BM) microenvironment by genotoxic stress leads to impaired HSC regeneration and lymphoid differentiation, which are fundamental mechanisms underlying treatment-related lymphopenia. The intricate interactions between HSC and BM stromal cells regulate stem cell maintenance and lymphocyte production. However, their dynamic underlying molecular mechanisms remain elusive, and treatment strategies for facilitating HSC regeneration and lymphoid differentiation in patients upon chemotherapy remain underdeveloped. To depict the potential hematopoiesis regulators, by analyzing Single-cell RNA sequencing (scRNA-seq) data from eight healthy donors in the human cell atlas, we found that natriuretic peptide receptor C (NPR-C) is predominantly expressed in HSC and megakaryocyte progenitors but negatively in lymphoid progenitors. We generated single-nucleotide knockin mice harboring NPR-C isoleucine 384 to phenylalanine mutant (NprcI384F/I384F mice), leading to NPR loss of function. These mice exhibited enhanced BM HSC pluripotency, increased lymphoid differentiation, and elevated peripheral lymphocyte status revealed by scRNA-seq, CyTOF and Complete Blood Test. Mechanistically, enhanced C-type natriuretic peptide (CNP) signaling in NprcI384F/I384F mice promotes HSC self-renewal and lymphoid differentiation through stroma cell-mediated HSC maintenance in a cyclic GMP (cGMP)-dependent manner. Importantly, NprcI384F/I384F mice maintained higher peripheral lymphocyte levels post-carboplatin challenge, suggesting that activation of CNP signaling resisted chemotherapy-induced lymphopenia. Clinically, the expression of CNP signaling gene signature is significantly associated with increased presence of HSC and lymphoid progenitor, enhanced mesenchyme development, and improved HSC-supporting networks by RNA-seq data analyses of human BM biopsies. Hence, the CNP-triggered signaling cascade in the BM niche promotes HSC regeneration and lymphoid differentiation, antagonizing systemic lymphopenia following genotoxic stress. Our study elucidates the cooperative mechanisms between HSC and stromal cells mediated by CNP-triggered cascades within the BM microenvironment, providing mechanistic insights regarding mitigating stress-induced lymphopenia.
Critical Roles of LILRB4 in Promoting PIM Kinase Mediated Cell Proliferation and Tumorigenesis in Multiple Myeloma
Authors: Wang Yijie , Ma Jingyuan , Sun Xiyue , Gong Lixin , Liu Lanting , An Gang , Qiu Lu-Gui , Hao Mu
Published: December 2024

Introduction

Multiple myeloma (MM) is the second most common hematologic malignancy. Although multiple targets on MM cells are discovered and applied in clinical treatment, relapse is almost inevitable in MM patients. Our previous study found a high-risk cell cluster from MM patients with overall survival less than 2 years by single-cell RNA sequencing (scRNA seq). In this specific cluster, leukocyte immunoglobulin-like receptor B4 (LILRB4) was highly expressed, indicating the critical role of LILRB4 in myelomagenesis and drug resistance. However, the mechanisms of LILRB4 in MM development has not been fully understood. Here, we investigated the role of LILRB4 in tumorigenesis and MM cell proliferation.

Methods and Results

Our clinical data showed that MM patients with LILRB4 overexpression had poor prognosis and decreased overall survival. Compared with newly-diagnosed MM patients, expression of LILRB4 was higher in the MM patients relapsed after treatment, indicating the significance of LILRB4 in MM progression and drug resistance. In vitro experiment showed that MM cells with LILRB4 overexpression enhanced the cell colony-forming ability and promoted cell proliferation. Conversely, knocking-out LILRB4 induced cell apoptosis. We used MM cells with LILRB4 overexpression to establish myeloma xenograft model and results showed that compared with LILRB4neg MM cells, mice injected with LILRB4high MM cells displayed higher tumor formation rate and lower survival rate. Then, we analyzed RNA-seq data to investigate the mechanisms of LILRB4 in MM proliferation. Transcriptome analysis indicated that overexpression of LILRB4 in MM cell lines activated NF-kB, glycolysis, hypoxia and MTORC1 signaling pathways. Among these, NF-kB pathway has been proved in previous studies. More important, RNA-seq data showed that PIM kinase, correlating with cancer cell proliferation and apoptosis, was significantly upregulated in MM cells with LILRB4 overexpression, which has been confirmed in our in vitro experiments. We also found that LILRB4 upregulated the expression of PIM through activating Src-homology 2-containing protein tyrosine phosphatase (SHP) and signal transducer and activator of transcription (STAT) protein, indicating the underlying mechanism of MM cell proliferation. Interestingly, transcription factors promoting B cell program were upregulated, and the immunophenotypic marker related to B cell differentiation was downregulated after LILRB4 overexpression. These findings indicated that LILRB4 may induce myeloma cell dedifferentiation and promote tumorigenesis.

Conclusion

In conclusion, LILRB4 is highly associated with poor prognosis of MM patients and has the ability of tumorigenesis. LILRB4 promotes MM cell proliferation by activating SHP and STAT to upregulate the expression of PIM kinase, which plays critical roles in MM pathogenesis.
Deciphering Inflammasome Activation in Gaucher Macrophages and Its Role in the Development of Multiple Myeloma
Authors: Reinecke Vivien , Hofbauer Daniel , Bittenbring Jörg-Thomas , Lischer Christopher , Berges Johannes , Nusser Gina , Bitterer Katrin , Voelkl Simon , Mougiakakos Dimitrios , Mackensen Andreas , Bruns Heiko
Published: December 2024

Gaucher disease (GD) is the most common lysosomal storage disease and corresponds to the accumulation of glucocerebroside in macrophages due to defective synthesis of the β-glucocerebrosidase. This lysosomal storage leads to the characteristic engorged macrophages, known as Gaucher macrophages (GMs) and contributes to the secretion of inflammatory cytokines, bone disease, splenomegaly and cytopenia typically encountered in patients with GD. Interestingly, patients with GD have an increased incidence of both polyclonal and monoclonal gammopathies with an increased risk of multiple myeloma (MM).

We have recently been shown in MM that the NLRP3 inflammasome in TAMs is triggered by “frustrated phagocytosis” and represents a critical and early step in the initiation and progression of MM (Hofbauer et al., Immunity, 2021). Therefore, we hypothesize that the inflammasome is activated in GMs, and excessive production of interleukin (IL)-1β and IL-18 by macrophages promotes the development of MM.

We identified subsets of pro-inflammatory macrophages in GD patients with increased transcription of inflammasome associated-markers (AIM2, CASP1, IL-1B, IL-18, and NLRP3) by single-cell RNA sequencing. We verified this inflammasome activation in GMs by immunohistochemistry and flow cytometry through detection of active caspase-1 and ASC aggregation. Mechanistically, we demonstrated in macrophages from healthy donors, that excessive addition of glucocerebroside or inhibition of β-glucocerebrosidase leads to lysosomal rupture, caspase-1 activation and secretion of IL-1β and IL-18. Furthermore, the coculture of primary isolated MM cells with GMs resulted in a significantly higher proliferation and metabolic activity of the tumour cells. Notably, blockade of inflammasome activation by MCC950 (a specific inhibitor of the NLRP3 inflammasome) results in reduced IL-1β and IL-18 secretion from ex vivo cultured GMs.

These data suggest that the inflammasome is activated in GMs by disruption of the lysosomal integrity and that therapeutic inhibition of the NLRP3 inflammasome in GD patients may reduce the risk of clonal evolution of plasma cells.
Characterizing and Targeting Follicular Lymphoma's Common Progenitor Cells
Authors: Atkins Oscar , Zhang Lingling , Hung Miu Shing , Reinhardt Christian , Calado Dinis
Published: December 2024

Follicular lymphoma (FL) is the most prevalent low-grade germinal centre (GC) derived non-Hodgkin lymphoma. Despite being considered an indolent lymphoma its clinical course is heterogenous with some patients experiencing frequent relapses and eventual transformation to a high-grade disease associated with an especially poor prognosis.

Through phylogenetic analysis of successive human FL tumors, evidence has emerged regarding the existence of common progenitor cells (CPCs). These precursor cells serve as a reservoir of cells that are able to evade killing with standard treatments for FL and give rise to relapse and transformed FL. They carry the t(14:18) translocation and are enriched for somatic mutations in chromatin-modifying genes including CREBBP and KMT2D. Developing therapies to specifically target and eliminate these common progenitor cells will be crucial in finding a cure for FL.

To understand this population of therapy-resistant precursor cells, we engineered mouse models that faithfully replicate the early and recurrent gene mutations observed in FL, including BCL2 translocation, CREBBP and KMT2D loss-of-function (LoF) mutations (BCK mice). We then subjected mice to a standard human-like treatment regimen of R-CHOP (Rituximab, Cyclophosphamide, Doxorubicin, Vincristine, Prednisone). Administration of the mouse-adapted R-CHOP resulted in the complete depletion of B cells from control mice. However, treatment of BCK mice revealed a resistant B-cell population. Notably, these resistant cells predominantly displayed a memory B-cell phenotype.

Single cell RNA-seq analysis of these therapy-resistant cells identifies unique transcriptional signatures and enrichment of pathways that can be targeted therapeutically. Comparison with human FL scRNAseq datasets shows that these therapy-resistant precursor cells are enriched in FL tumors of patients with early relapse.

Finally, we have used an innovative in vitro platform that harnesses a GC B-cell culture system to amplify CPCs, facilitating subsequent high-throughput drug screening. This approach has allowed us to identify specific vulnerabilities within the CPC population, paving the way for the development of therapies tailored to overcome FL resistance mechanisms.

Through these concerted efforts, we edge closer to the long-awaited goal of discovering a definitive cure for Follicular Lymphoma.
Venous Thromboembolism in Individuals with Sickle Cell Disease- a Retrospective Review of This Understudied Problem
Authors: Ajayi Ayotola , Zhang Li , Agarwal Shreya , Bhasin Neha
Published: December 2024

Individuals with sickle cell disease (SCD) are proposed to be at a higher risk for venous thromboembolism (VTE) compared to the general population. Observational studies show a 25% increased risk of first VTE in SCD with a mean age of incidence of 30 years, which is much lower than the general population of 65 years. The mechanisms for this increased VTE risk have been proposed to be increased hemolysis causing increased venous stasis and vasculopathy, increased endothelial activation with increased activation of the coagulation pathway and chronic inflammation.

In this study, we reviewed the medical records of 783 individuals with SCD who were followed in our lifelong sickle cell center between May 2013 to May 2023 to determine the prevalence of thrombosis and associations of these events with laboratory and clinical risk factors of thrombosis. We collected retrospective data including patient demographics, clinical history, and laboratory data at the time of VTE events and an evaluation of the presence of a central venous catheter (CVC) in our population. VTE was defined as occurrence of deep vein thrombosis (DVT), pulmonary embolism (PE), cerebral sinus venous thrombosis (CSVT), or right atrial thrombosis.

The prevalence of VTE in our population was 8.2% (n = 64 out of 783), with a median age of 33 years. Out of 64 total patients who developed a VTE during our study period, 21 developed a PE; this was 33% of the VTE events noted. A high body mass index (BMI) (>30 kg/m2) was noted among 22% of the patients in the VTE group compared to 7.6% of the non-VTE group (p<0.001). Smoking was noted in 41% of the VTE group patients compared to 10% in the control group (p<0.001). CVCs were present in 55.6% of the patients in the VTE group compared to those in the non-VTE group (10%) with a p <0.001. Twenty-two patients (34.4%) of the patients in the VTE group had a history of a VTE event prior to our study period and were having a recurrent VTE episode during our study period between 2013 and 2023. In addition, out of those patients who developed a VTE during our study period, 20 patients (31%) developed a recurrent VTE event during our study period.

Our results show that there is a higher rate of obesity and smoke exposure among patients with SCD who develop a VTE event. PE's are a substantial number of VTE events in the SCD population. We also noted a significant correlation between CVCs and VTEs in SCD. There is high recurrence rate of VTEs among SCD patients. Based on these findings, future prospective data are needed to determine if these associations are clear risk factors for VTE in SCD so these risk factors can be minimized in patients at risk for VTE events.
The Addition of Lenalidomide to R-CHOP Is Effective for Prevention of CNS Relapse in High-Risk Patients with Diffuse Large B-Cell Lymphoma
Authors: Wei Xiaolei , Guo Xutao , Wei Qi , Xiong Dan , Zhang Peng , Zhang Zewen , Wei Yongqiang , Feng Ru
Published: December 2024

Central nervous system (CNS) relapse is an uncommon but devastating event in patients in diffuse large B-cell lymphoma (DLBCL). Although several prophylaxis attempts including intrathecal methotrexate and intravenous high-dose methotrexate were conducted to reduce the CNS relapse rate, the optimal management remained uncertain. Herein, we performed an open-label, multicenter trial to explore the efficacy and safety of the combination of lenalidomide and R-CHOP (R2-CHOP) for preventing the CNS relapse in the high-risk diffuse large B cell lymphoma.

Eligible patients were aged 18-80 years, newly diagnosed DLBCL and high risk for CNS recurrence: CNS-IPI:4-6 or involvement of testicular, kidneys or adrenal glands. Lenalidomide orally 25 mg per day was administered on days 1 through 10 of each cycle and delivered concomitantly with standard dose R-CHOP-21 regimen. All patients received aspirin 100mg per day prophylaxis throughout. The primary endpoint was 2-year central nervous system relapse rates. The secondary endpoint were 2-year overall survival, progression-free survival, and treatment-emergent adverse events. This trial is registered with ClinicalTrials.gov, number NCT04544059.

A total of 64 DLBCL patients with high-risk for CNS relapse were included in this study and 56 of them was evaluated. The median age of the patients in this study was 57 years old (range 26-77) and 48.2% patients were female. 91.1% patients had a CNS-IPI score ≥4 and the left patients may be involved with testicular, kidneys or adrenal glands. With a median follow up of 20 months, only 2 (3.6%) patient developed CNS relapse. The 2-year progression-free survival and overall survival were 77.8% and 79.7%, respectively.

In summary, lenalidomide with R-CHOP is effective for prevention of CNS relapse in high-risk patients with diffuse large B-cell lymphoma.

Keywords:

Diffuse large B-cell lymphoma, central nervous system relapse, lenalidomide.
Modulating mRNA Translation Fidelity As a Novel Mechanism for AML Chemoresistance
Authors: Mayerhofer Christina , Li Dan , Kristiansen Trine A. , Mayerhofer Ernst , Sharda Azeem , He Lingli , Mazzola Michael , Schiroli Giulia , Gustafsson Karin , Keyes Sam , Kiem Anna , Crompton Eve , Xu Yanxin , Dou Zhixun , Vidoudez Charles , Miller Peter G. , Van Gastel Nick , Scadden David
Published: December 2024

Acute Myeloid Leukemia (AML) is a highly aggressive blood cancer that initially responds to high-dose chemotherapy, but the majority of patients relapse. To understand the mechanisms underlying chemoresistance of AML, we investigated the metabolic adaptations in AML cells that occur following chemotherapy but before overt disease relapse. In AML patient-derived xenografts (PDX), we applied untargeted metabolomics and showed that chemoresistant AML cells have significantly elevated levels of branched-chain amino acids (BCAAs) compared to untreated cells. Elevated BCAA levels and their catabolism have been implicated in AML but their upregulation to drive chemoresistance is not well studied. Transcriptome analysis and stable isotope tracing supported the upregulation of BCAA pathways during chemotherapy in vivo. Our results showed that AML cells, compared to normal granulocyte-monocyte progenitor cells, had significantly higher valine uptake, suggesting a dependency on BCAA metabolism for survival during chemotherapy. When BCAA restriction was combined with chemotherapy, survival was significantly improved with an accompanying clearance of residual AML cells. However, relapse still occurred.

Unexpectedly, the surviving cells had significantly higher levels of protein synthesis during maximum response to chemotherapy and BCAA restriction. We anticipated that BCAA restriction after chemotherapy would lower mTOR activity and thus protein synthesis rates. Unexpectedly, we found that ribosomal gene transcripts were enriched, and OPP incorporation in newly synthesized peptides increased in AML cells restricted for BCAAs combined with chemotherapy in vivo. Using low-input Ribo-seq to analyze paired transcriptome and translatome data, we found activated ribosomal protein translation and elevated production of the dioxygenase, 2-oxoglutarate-and-iron-dependent oxygenase 1 (OGFOD1). OGFOD1 modulates the fidelity of ribosomal codon-anticodon pairing through ribosome hydroxylation in proximity to the decoding center where this pairing takes place. Accordingly, AML cells with increased OGFOD1 in vivo had reduced ribosome stalling by ribosome profiling. Conversely, inhibiting OGFOD1 led to translation arrest, reduced protein production and improved animal survival following chemotherapy. Our data therefore suggest that OGFOD1 may enable cells to bypass BCAA shortage by permitting less stringent translation accuracy and targeting OGFOD1 and restricting amino acids can counteract this chemoresistance mechanism. Modifying translation fidelity is a previously unrecognized strategy in AML and has important potential implications across other oncologic contexts.
Integrating Spatial Transcriptome, Single-Cell Sequencing and Histopathology Reveals Bone Marrow Spatial Heterogenous in AML Patients
Authors: Lin Xiangjie , Wang Huafeng , Jin Jie
Published: December 2024

Acute myeloid leukemia (AML) is a highly heterogeneous hematologic malignancy characterized by a poor prognosis, primarily due to resistance to primary chemotherapy and a high relapse rate among patients in remission. This underscores the limitations of conventional chemotherapy in achieving a cure for the majority of AML patients. Spatial transcriptome (ST) technology has emerged as a powerful tool for studying the spatial gene expression and cell-cell interaction patterns, yet its application to calcium-rich tissues like bone marrow has been challenging. In this study, we firstly introduced a novel approach for the paraffin embedding and decalcification of bone marrow tissue, which is fully compatible with Visium glass slides. To validate our method, we collected bone marrow tissues form AML patients and performed spatial transcriptomic sequencing alongside single-cell RNA sequencing. We totally got 5364 ± 2049 (AML-b8) and 4892 ± 2096 (AML-b9) unique molecular identifiers (UMIs) per spot, which is significantly higher than the published studies of bone marrow, and equal to those of soft tissues. This demonstrates that our approach overcomes the limitations of spatial transcriptome data collection in calcium-rich tissues. Utilizing marker genes, we accurately mapped the spatial distribution of megakaryocytes (MKs) and bone trabecular in bone marrow, confirmed through HE staining. For the first time, we integrated spatial transcriptome data, single-cell sequencing and histological analysis to provide a comprehensive characterization of bone marrow in AML patients. After confirming the cell type of each spot, we present the maps of bone marrow tissues in AML patients that could provide both spatial information and gene expression level. Furthermore, we found that the proportion of blasts in our ST results more accurately reflects the tumor burden in AML patients compared to bone marrow smear and flow cytometry, due to the preservation of tissue architecture. Our analysis distinguished seven subpopulations within the tumor cells of bone marrow, including leukemia stem cells (LSKs), highlighting the potential of our approach in elucidating the distribution and transcriptomic profiles of LSKs in AML patients. In our ST models, we observed a significant correlation between genes' expression level and their distance to endosteal niche, such as S100A8, S100A9, CYBB, CHMP3, PACS2, UNC93B1, CMKLR1 and TFRC. Functional analysis of intra-tumor hubs revealed key pathways, including Toll-like receptor cascades, apoptosis process and so on, which are pivotal in bone marrow samples. In conclusion, we present a pioneering method for bone marrow spatial transcriptome analysis, offering a complete and detailed profile of bone marrow in AML patients. This work paves the way for a deeper understanding of AML pathophysiology and may lead to the development of more effective therapeutic strategies.
CD47 Upregulation Inhibits Phagocytic Clearance of Alloreactive Donor T Cells after Allo-HCT
Authors: Shaikh Haroon , Flamann Cindy , Matos Carina , Ali Hla , Kern Michael AG , Kreutz Marina , Buettner-Herold Maike , Lischer Christopher , Voelkl Simon , Kellner Christian , Bitterer Katrin , Saul Domenica , Holler Ernst , Zernecke Alma , Mougiakakos Dimitrios , Beilhack Andreas , Bruns Heiko
Published: December 2024

Acute graft-versus-Host disease (aGvHD) is a life-threatening complication that frequently occurs after allogeneic hematopoietic cell transplantation (allo-HCT). Resolving inflammation is a key therapeutic goal in aGvHD. Macrophage-mediated phagocytosis contributes substantially to resolve inflammatory processes. However, the role of phagocytosis after allo-HCT still remains unexplored.

Therefore, we analyzed the expression of the “don´t eat me” signal CD47 on allogeneic T cells from aGvHD patients and in two MHC major mismatch aGvHD mouse models (FVB à C57BL/6, 9 Gy TBI and C57BL/6 à Balb/C, 8 Gy TBI). We evaluated the effect of in vitro and in vivo administration of an anti-CD47 antibody on antibody-dependent cellular phagocytosis (ADCP) of alloreactive T cells using flow cytometry and confocal microscopy, bioluminescence imaging, cytokine measurements and histopathology.

Initial analysis of a global dataset revealed increased CD47 expression on intestinal T cells in patients with chronic gastrointestinal inflammation. Subsequently, we examined CD47 levels on T cells in the allo-HCT setting. Notably, intestinal T cells in patients with severe GvHD (Lerner grade III) significantly upregulated CD47 (p = 0.0079). There were no significant changes in CD24 (another “don´t eat-me”-signal) or in the “eat-me”-signals calreticulin and ATP10A. This upregulation of CD47 correlated with reduced macrophage-mediated phagocytosis. Similarly, intestinal alloreactive donor T cells highly upregulated CD47 in mice developing aGvHD after allo-HCT (p = 0.0317). In vitro studies revealed that CD47 expression is regulated by NF- kB in T cells after TCR activation and primed T cells inhibit ADCP in a CD47-dependent fashion. Transfer of CD47 knock-out T cells and bone marrow resulted in swift clearance of donor T cells after allo-HCT resulting in significantly improved clinical scores and survival compared to controls (p = <0.0001). Crucially, application of an anti-CD47 antibody significantly antagonized the impaired ADCP of alloreactive T cells. In anti-CD47 treated mice, we observed increased phagocytosis of T cells in the gastrointestinal tract (GIT) and induction of immunosuppressive responses in vivo. Besides an increase of the myeloid-derived suppressor cell population, systemic pro-inflammatory cytokine levels such as TNF, IL-1β and IFN-β decreased in the serum.

In conclusion, our data suggest that high CD47 expression impairs phagocytic clearance of alloreactive T cells after allo-HCT, potentially contributing to exaggerated inflammatory responses in the GIT of GvHD patients. Thus, CD47 emerges as potential therapeutic target to eliminate alloreactive T cells, with anti-CD47 treatment aiding the resolution of inflammation in GvHD.
From Harmless to Harmful: Meta-Analysis and Systematic Review of Adverse Clinical Outcomes in Sickle Cell Trait
Authors: Pokima Ngowari , El-gharib Khalil , Khamis Zaid , Marseille Melissa , Keesari Praneeth Reddy , Owolabi Mary , Wang Kai , Odaimi Marcel
Published: December 2024

Background: Sickle cell trait (SCT) has traditionally been considered a benign condition; however, emerging evidence suggests it may have serious clinical consequences. This systematic review and meta-analysis aim to synthesize current research on the adverse clinical outcomes associated with SCT.

Methods: A systematic review search was conducted on PubMed, Embase, and Central Cochrane Registry to identify studies reporting on the clinical outcomes of SCT. Studies were included if they evaluated conditions such as diabetes, end-stage renal disease (ESRD), pulmonary embolism (PE), deep vein thrombosis (DVT), and chronic kidney disease (CKD)(EGFR <60) in individuals with SCT against a matched sample of controls without a history of SCT (matched on age, race, sex). Cross-sectional studies were included. Data extraction and quality assessment were performed independently by two reviewers. From 88 records initially identified from bibliographic databases, 12 studies were eligible and therefore included the meta-analysis. For measuring association between SCT and DVT, PE, CKD, ESRD and diabetes a meta-analysis using the random-effects method was performed to pool weighted odds ratios (OR) of risk estimates, and heterogeneity was assessed using the I² statistic. For effect sizes, a confidence interval of 95% was used, and a p-value of less than 0.05 was used for statistical significance

Results: The meta-analysis included 12 studies, including a total of 1128624 individuals with either SCT or no SCT/SCD. The pooled analysis revealed significant associations between SCT and CKD, ESRD, DVT and PE. Individuals with SCT had an increased risk of ESRD (pooled OR = 2.21, 95% CI: 1.87-2.85, p<0.01), PE (pooled OR = 1.75, 95% CI: 1.53-2.01, p<0.01), DVT (pooled OR = 1.54, 95% CI: 0.99-2.30, p=0.05), and CKD (pooled OR = 1.79, 95% CI: 1.54-2.08). There was no significant association seen with SCT and diabetes. Low heterogeneity was observed in the outcomes of ESRD and CKD (I² = 0% and 43%, respectively), warranting further investigation into underlying factors.

Conclusions: This systematic review and meta-analysis demonstrate that SCT is associated with an increased risk of several serious clinical conditions, challenging the traditional view of SCT as a benign trait. These results may have important public policy implications for risk stratification and genetic counseling of SCT carriers as well as the need for heightened clinical awareness and monitoring of individuals with SCT to prevent and manage potential complications

Keywords: Sickle Cell Trait, Diabetes, End-Stage Renal Disease, Pulmonary Embolism, Deep Vein Thrombosis, Chronic Kidney Disease, Meta-Analysis, Systematic Review
Phosphorylation-Dependent ANXA2 Relocation on the Membrane and TLR4 Engagement: A Novel Signaling Axis Promoted By PIM1 Mutation in Diffuse Large B Cell Lymphoma
Authors: Lu Yaxiao , Zhang Ning , Zhang Yidan , Xu Xiyue , Wang Xianhuo , Zhang Huilai
Published: December 2024

Background：Diffuse large B-cell lymphoma (DLBCL) is the most common subtype of non-Hodgkin lymphoma and has an overall cure rate of approximately 60%. Previously, we observed high PIM1 mutation rates in DLBCL patients with poor outcome. However, the mechanism whether they lead to enhanced PIM1 kinase activity and contribute to lymphomagenesis is currently unknown.

Methods：In this study, a multifaceted approach was employed to elucidate the functional consequences of PIM1 gene mutations and their implications in DLBCL. Recurrent PIM1 gene mutations that exhibited high frequencies across various hematological cancer cohorts were screened from public databases and patient outcomes were stratified by PIM1 mutation status. Liquid chromatography-mass spectrometry (LC-MS) proteomics and bioinformatic tools were utilized to identify proteins involved in critical cellular pathways relevant to PIM1 mutation. A high-throughput drug screening platform was leveraged to find potential therapeutic vulnerabilities unique to PIM1 mutant cells. The antiproliferative effects of PIM1 and PI3K inhibitor were evaluated in DLBCL cell lines and further validated in NSG mouse xenograft models.

Results：We have identified PIM1 mutations, specifically P81S, E135K, L184F, and S97N, as frequently occurring variants, with the former three significantly associated with poor outcome. Our cohort data revealed a trend towards elevated PIM1 protein expression following PIM1 mutations. PIM1-mutated DLBCL tumors exhibited accelerated proliferation and reduced apoptosis, while in vivo animal models demonstrated faster tumor growth. Mechanistically, PIM1 mutations were found to interact with ANXA2, activating it through phosphorylation of serine 26 on the ANXA2 gene. Activated ANXA2 then translocated from the cytoplasm to the cell membrane, binding with TLR4 and further activating the PI3K/AKT/mTOR signaling pathway. Additionally, high-throughput drug screening highlighted the sensitivity of PIM1-mutated cells to multiple PI3K inhibitors. Notably, the PIM1 inhibitor SMI-4a demonstrated synergistic antitumor effects with the PI3K inhibitor Linperlisib in vitro and in vivo, offering a potentially more effective therapeutic strategy for patients harboring PIM1 mutations.

Conclusions: Taken together, we identify recurrent somatic PIM1 L184F mutations that may contribute to the pathogenesis of DLBCL and establish the rationale for therapeutic strategies aimed at targeting the oncogenic pathway activated in PIM1-mutated DLBCL.

Keywords: PIM1, Mutation, ANXA2, Diffuse large B-cell lymphoma, Targeted therapy
HPK1 Inhibition and Venetoclax Combination Suppressed AML By Enhancing Cytotoxic T-Cell Response to Leukaemia
Authors: Zeng Xiaoyuan , Tong Kwui-Wa , Chan Koon Chuen , Zheng Li-Chuan , Lam Stephen , Bray Mark Robert , Mak Tak-Wah , Man Cheuk-Him , Leung Anskar Y. H.
Published: December 2024

Background: Immune escape is one of the major causes of treatment failure in cancer therapy and modulation of immune cell function has become an emerging therapeutic strategy. Haematopoietic progenitor kinase 1 (HPK1) is a negative regulator of T cell signaling and its inhibition promotes T cell function. Venetoclax, a BCL2 inhibitor, has become a standard of care for unfit or elderly patients with acute myeloid leukaemia (AML) and may enhance anti-leukaemic effector T-cell function. We hypothesize that HPK1 inhibitor (HPK1i) may synergize with venetoclax and improve treatment outcome in AML by co-activating cell-death pathway and T cell immunity.

Methods: Human T cells were activated by anti-CD3/CD28 antibodies in vitro. Proliferation, cytokine production, T-cell subsets and exhaustion were examined by flow cytometry after 3-day treatment of HPK1i. Recipient mice engrafting with MLL-AF9 AML were treated with HPK1i singly or in combination with venetoclax. Leukaemic burden, apoptosis and T cell functions and exhaustion were examined.

Results: In vitro, HPK1i treatment of activated human T-cells induced cellular proliferation and expression of TNF-α and INF-γ. It increased effector memory T-cell (CD62L-CD45RA-) and decreased naïve T-cell population (CD62L+CD45RA+). Furthermore, it reduced exhaustion markers including PD-1 and TIM3. In vivo, HPK1i and venetoclax combination significantly decreased the burden and induced apoptosis of leukaemic cells on 18th DPT (days post transplantation), and prolonged survival of non-irradiated mice that were transplanted with donor mouse MLL-AF9 AML cells. Cytotoxic T-cells were activated, associated with increase in TNF-α and INF-γ expression and increase in the effector memory T cell subset. There was a decrease in naïve T cell population and T cell exhaustion marker PD-1. Single cell transcriptomes of serial bone marrow samples from mice treated with the combination were being analyzed and will be presented in the meeting.

Conclusion: HPK1i and venetoclax reduced the leukaemic burden and enhanced the anti-leukemic ability of T cells in MLL-AF9 AML mouse model.

Acknowledgements

This research is supported by the Centre for Oncology and Immunology under the Health@InnoHK Initiative funded by the Innovation and Technology Commission, Theme-based Research Scheme (T12-702/20-N), Hong Kong Jockey Club Charities Trust, Li Shu Fan Medical Foundation, Croucher Foundation, S.K.Yee Medical Foundation (260940161, 260940149, 260940187), Tang King Yin Research Fund, Mr. Ying Wan Leung Research Fund, Madam Madeline Tong Lai-Sheung Cancer Research Fund (A.Y.H.L), The Government of Hong Kong SAR, China.
Combination of Olverembatinib and VP Regimen for Newly Diagnosed Adult Philadelphia Chromosome-Positive Acute Lymphoblastic Leukemia
Authors: Tan Xu , Chen Guo , Ma Yingying , Wang Maihong , Wen Qin , Liu Hong , Zhang Xi , Zhang Cheng
Published: December 2024

Background Philadelphia Chromosome -positive acute lymphoblastic leukemia (PH+ALL) is a common type of adult ALL, and TKI combined with chemotherapy is the main treatment option; early achievement of molecular remission has been found to be an important factor affecting long-term survival. The combination of third-generation TKI Ponatinib can achieve a higher percentage of early molecular remission and significantly improve the prognosis, but the safety is unsatisfactory; Olverembatinib is a novel third-generation TKI, which has demonstrated better efficacy and safety in patients with CML; This study is intended to explore the efficacy and safety of Olverembatinib in combination with VP regimen in the treatment of newly diagnosed adult PH+ALL.

Methods This is a single-arm, prospective trial that enrolled adult patients with newly diagnosed Ph+ ALL. Patients were treated with a combination of Olverembatinib (40mg QOD d1-28), Vindesine 4mg d1,8,15, Prednisone1mg/Kg/d d1-14, and then gradually tapered off for 28d in induction therapy, Post-induction treatment to be decided by each centre.

Results From February 2022 to December 2023, a total of 26 pts were enrolled and all pts completed induction therapy. Among all , Male 12 and Female 14, the median Age was 52(18-70) years old. Sixteen pts (61.5%) expressed the p190 transcript and ten pts (38.4%) expressed the p210 transcript. IKZF1 mutation detected in 5 patients by second-generation sequencing. All patients achieved CR at the end of induction therapy, and no tumor lysis syndrome or treatment-related deaths occurred. No patients had adverse reactions leading to dose reduction or discontinuation, and no treatment-related non-hematological adverse reactions were observed. Molecular response at the end of induction therapy was MMR in 16 patients (53.8%), CMR in 10 (30.8%). The demand for blood transfusion and the incidence of infections were significantly lower compared with our historical data treating with 2nd generation TKI plus chemotherapy treating with low-intensive chemotherapy plus TKIs. During a median follow-up of 354 days, one patient died of transplant complications, while all the remaining survived without relapse.

Conclusion The combination of Olverembatinib and reduced-intensity chemotherapy (VP) is a safe and effective regimen in patients with newly diagnosed Ph+ ALL. The regimen results in high rates of CMR in the absence of intensive chemotherapy, and promises to improve long-term survival in patients with PH+ALL.
Anti-CD38 Monoclonal Antibody Is a Salvage Regimen in Relapsed/Refractory Aplastic Anemia
Authors: Li Weiwang , Zhang Lele , Pan Hong , Gao Zhen , Li Jianping , Nie Neng , Zhao Xin , Huang Jinbo , Zhao Jingyu , Yu Xiao , Kuang Zhexiang , Fang Liwei , Ge Meili , Yuan Weiping , Shi Jun
Published: December 2024

Background: About half of patients with aplastic anemia (AA) are in a refractory or relapsed state after first-line immunosuppressive therapy (IST). The persistent pancytopenia in relapsed/refractory AA (r/r AA) patients affects their quality of life and even is life-threatening. Hematopoietic stem cell transplantation, second course of IST and TPO receptor agonist ameliorate the disease in more than half of these people. But patients not eligible for these treatments are still lack of proper choice.

Methods: We conducted a retrospective study to evaluate the efficacy and safety of daratumumab, a monoclonal CD38 antibody, in treating r/r AA. Daratumumab was given 8mg/kg weekly for 4 weeks to 10 r/r AA patients. Ciclosporin or danazol was given as concurrent treatment to 5 patients who had already received the drug for at least 6 months. Patients achieved response during 12 weeks would receive the second cycle treatment.

Results: The overall response rate was 5 of 10 patients (50%) with multilineage responses in 4 of them. The median response time was 6 weeks (range, 5-10 weeks). Patients sustained response with a median 5.5 months duration time (range, 2-13.5 months), including 2 responders still during follow-up. Infection and infusion-related reaction are the most common adverse events with an 80% and 70% incidence respectively, including 1 patient with grade 3/4 infection. Incidence of hematologic adverse events was 50% and 80% of them were grade 3/4, and the median time of recovering to grade 1/2 was 3 days (range, 1-15 days).

Conclusion: Anti-CD38 monoclonal antibody may be a promising option for r/r AA patients with an appreciable efficacy, rapid response, and accepted adverse events.
Efficacy and Safety of a New Regime Containing PD1 Antibody and Pegaspargase for Advanced-Stage or Relapsed/Refractory Natural Killer/T Cell Lymphoma
Authors: Chen Jie , Zhao Linjun , Wu Chongyang , Yu Haifeng , Li Zhenling , Liu Lihong , Gao Da , Sun Ying , Wei Xiaolei , Wu Yuanbin , Wang Yue , Song Yuqin , Zhu Jun , Liu Weiping
Published: December 2024

Background: Patients with advanced-stage or relapsed/refractory (r/r) natural killer/T cell lymphoma (NKTCL) had a poor prognosis. The anti-programmed death 1 (PD-1) antibody has demonstrated satisfactory efficacy and good tolerance for r/r NKTCL. In this report, we present the efficacy and safety analysis of a new regimen containing dexamethasone, azacytidine, pegaspargase, and tislelizumab (named as DAPT) for advanced-stage or r/r NKTCL in a prospective multicenter phase II study.

Methods: Patients with advanced-stage or r/r NKTCL, aged over 18 years, were eligible for participation. The DAPT regimen chemotherapy was administered as the protocol treatment, which included dexamethasone 10 mg on days 1-3, azacytidine 100 mg on days 1-5, pegaspargase 3,750 IU on day 1, and tislelizumab 200 mg on day 6. Cycles were repeated every 21 days. The primary endpoint was the overall response rate (ORR) .

Results: A total of 39 eligible patients were enrolled. The median age of 52 years (range, 32 to 72 years) and with a male-to-female ratio of 2.42:1. Twenty-four (61.5%) patients had newly diagnosed disease with stage IV, while 15 (38.5%) had r/r disease. Among 31 patients with available response, the ORR and complete remission (CR) rates were 77.4% and 33.3% for the entire cohort, 84.2% and 63.6% for newly diagnosed stage IV disease, and 31.6% and 27.3% for r/r disease, respectively. Ten patients underwent autologous hematopoietic stem cell transplantation (ASCT). The median progression-free survival (PFS) was 7.3 months in patients who completed 6 cycles of DAPT therapy. Moreover, the median PFS were 14.0,3.6 and 2.6 months in patients who achieved CR, PR and non-response, respectively. The median PFS was longer in those who achieved ASCT than those who did not experience transplantation (11.5 vs. 2.6 months). The most common treatment-related adverse events were was neutropenia (63.4%), followed by elevated alanine aminotransferase (43.9%), decreased fibrinogen (39.0%) and hypofibrinogenemia (39.0%). The incidence of ≥3 treatment-related adverse events was 26.8%, which included neutropenia (n=8), immune-related rash (n=2), and drug-induced liver injury (n=1) .No treatment-related deaths were observed.

Conclusion: The DAPT regimen chemotherapy is an effective and tolerable treatment for advanced-stage or r/r NKTCL.

Keywords: Lymphoma; Extranodal NK-T-Cell; Immunotherapy; Therapeutics; Prognosis
Brentuximab Vedotin (BV) and Nivolumab (nivo) for Untreated Patients with Hodgkin Lymphoma (HL): Long-Term Results
Authors: Cheson Bruce D. , Bartlett Nancy L. , LaPlant Betsy R. , Lee Hun Ju , Advani Ranjana H. , Christian Beth , Diefenbach Catherine S. , Feldman Tatyana , Ansell Stephen M.
Published: December 2024

Background: Whereas major improvements have been made in the treatment of advanced HL outcomes of patients > 60 yrs are generally suboptimal compared with younger patients, and generally with more toxicity. To address this issue we conducted the first phase II study of BV-nivo in older pts with HL (ACCRU RU0515051, NCT02758717), with initial results reported at a median of 20.2 mo of follow-up. (Cheson et al, Lancet Haem, 11:e808, 2020). Herein we present the 5-year follow-up for this study.

Methods: Eligible pts were either >60 yrs or considered unsuitable for standard chemotherapy because of cardiac, pulmonary or renal dysfunction. Pts received BV at 1∙8 mg/kg (dose cap at 180 mg) and nivo at 3 mg/kg both intravenously every 21 days for 8 cycles.The primary endpoint was overall response rate (ORR).

Results: Follow-up data were available for 39 of the original 46 pts. At a median follow-up of 58.5 months (0.45-65.4 mo), the median duration of response was 17.2 mo (11.6-45.2). 58.7% of the patients had progressed with the median progression-free survival of 18.3 mo (12.8-46.3 mo); thus 41.3% had still not experienced a progression event. Median overall survival was not yet reached with 84.8% of patients still alive.

Conclusions: BV-nivo is an effective regimen for patients >60 yr, curing approximately 40% of pts. For those not cured by initial therapy, outcome with subsequent treatment is excellent, suggesting check point inhibitor sensitization, or other factors. Optimization of dose and schedule, and incorporation of other novel agents will result in a successful chemo-free approach for older patients with HL, especially those unable to receive an anthracycline.