B-cell Receptor Signaling Pathway Research Articles

Functional transcriptome analysis revealed upregulation of MAPK-SMAD signalling pathways in chronic heat stress in crossbred cattle.

Animal geneticists and breeders have the impending challenge of enhancing the resilience of Indian livestock to heat stress through better selection strategies. Climate change's impact on livestock is more intense in tropical countries like India where dairy cattle crossbreeds are more sensitive to heat stress. The main reason for this study was to find the missing relative changes in transcript levels in thermo-neutral and heat stress conditions in crossbred cattle through whole-transcriptome analysis of RNA-Seq data. Differentially expressed genes (DEGs) identified based on the minimum log twofold change value and false discovery rate 0.05 revealed 468 up-regulated genes and 2273 down-regulated significant genes. Functional annotation and pathway analysis of these significant DEGs were compared based on Gene Ontology (Biological process), Kyoto Encyclopedia of Genes and Genome (KEGG), and Reactome pathways using g: Profiler, ShinyGO v0.76, and iDEP.951 web tools. On finding network visualization, the most over-represented and correlated pathways were neuronal and sensory organ development, calcium signalling pathway, Mitogen-activated protein kinase (MAPK) and Smad signalling pathway, Ras-proximate-1, or Ras-related protein 1 (Rap 1) signalling pathway, apoptosis, and oxidative stress. Similarly, down-regulated genes were most expressed in mRNA processing, immune system, B-cell receptor signalling pathway, Nucleotide oligomerization domain(NOD)-like receptors (NLRs) signalling pathway and nonsense-mediated decay (NMD) pathway. The heat stress-responsive genes identified in this study will facilitate our understanding of the molecular basis for climate resilience and heat tolerance in Indian dairy crossbreeds.

Single-cell transcriptome revealed dysregulated RNA-binding protein expression patterns and functions in human ankylosing spondylitis

Single-cell transcriptome revealed dysregulated RNA-binding protein expression patterns and functions in human ankylosing spondylitis

BAP31 Plays an Essential Role in Mouse B Cell Development via Regulation of BCR Signaling.

B cell receptor-associated protein 31 (BAP31) is a transmembrane protein that is widely expressed and primarily located in the endoplasmic reticulum (ER). B cells play a crucial role in the immune system, and BAP31 significantly contributes to the functions of various immune cells. However, the specific role of BAP31 in B lymphocytes development remains unknown. In this study, we utilized a mouse model with BAP31 deleted from B cells to investigate its effects. Our findings reveal a block in early B cell development in the bone marrow and a significant decrease in the number of B cells in peripheral lymphoid organs taken from BAP31 B cell conditional knockout (BAP31-BCKO) mice. B cell receptor (BCR) signaling is crucial for the normal development and differentiation of B lymphocytes. BAP31, an endoplasmic reticulum membrane protein, directly regulates the BCR signaling pathway and was shown to be significantly positively correlated with B cell activation and proliferation. These findings establish BAP31 as a crucial regulator of early B cell development.

Analyzing Pooled Microarray Gene Expression Data to Uncover Common Pathways in Periodontitis and Oral Squamous Cell Carcinoma from the Gene Expression Omnibus

ABSTRACT Periodontitis and oral squamous cell carcinoma (OSCC) are prevalent oral diseases with distinct etiologies, yet they share certain molecular and biological characteristics. Gene expression datasets from the gene expression omnibus (GEO) repository (GSE30784 for OSCC and GSE10334 for periodontitis) were analyzed. Data preprocessing and differential gene expression analysis using GEO2R identified common differentially expressed genes (DEGs), and FunRich software facilitated the construction of a protein-protein interaction (PPI) network on the STRING database. Cytoscape, coupled with the CytoHubba plugin, identified Cluster of Differentiation 19 (CD19) and Von Willebrand Factor (VWF) as the top hub genes, with Complement C3 (C3) also highly ranked. Functional enrichment analysis highlighted pathways such as the B-cell receptor signaling pathway, complement and coagulation cascades, and hematopoietic cell lineage. Additionally, miRNet analysis identified key miRNAs, including hsa-mir-26a-5p, hsa-mir-129-2-3p, and hsa-mir-27a-3p, associated with these pathways. These findings suggested an association of molecular mechanisms between periodontitis and OSCC, with identified hub genes and miRNAs potentially serving as therapeutic targets.

Abstract 7586: TOX loss-of-function(TOXLOF)results in BCR hyperactivation andenhanced responses to BCR-targeting in diffuse large B cell lymphomas (DLBCL)

Abstract DLBCL originate from germinal center B cells (GCB). The ABC-DLBCL subtype, characterized by activation of B-cell receptor (BCR) signaling and downstream NF-kB pathway, carries a poor outcome. Up to 30% of ABC-DLBCL present TOXLOF mutations, suggesting that TOX function as a tumor suppressor. Our analysis of TOX expression along the germinal center B-cell differentiation program (naïve → GCB → plasma cells) showed that TOX expression paralleled NF-kB activity with highest levels in plasma cells (P<0.001, vs. either naïve and GCB). We thus propose that TOX acts as a transcriptional gatekeeper in GCB cells to prevent early activation of NF-kB thus enabling differentiation into plasma cells while restraining lymphomagenesis. We generated a conditional GCB TOX KO mouse model, induced the GC reaction with a T-cell antigen and found that TOX KO (vs. TOX WT, 10 mice per condition) had impaired differentiation of plasma cells (p<0.001) and hyperproliferative Ki67+ GCB cells (p<0.05). We then generated (by CRISPR) an isogenic TOXLOF ABC-DLBCL cell line (TMD8) and found upregulation of genes in the NF-kB pathway (q-value <0.05, vs. TMD8). Furthermore, TMD8-TOXLOF cells showed increased secretion of autocrine cytokines IL6 and IL10, and higher STAT3 phosphorylation (p<0.05 for all comparisons, vs. TMD8-TOXWT cells) all compatible with NF-kB activation. Tumor microenvironmental cues can hyperactivate BCR and downstream NF-kB in ABC-DLBCL. We thus implanted isogenic TMD8 cells in mice (n = 10) and determined pathway activation by phospho-flow. We found massive activation of SYK, BTK, PLCG2 (from the BCR) and p65 (from NF-kB) in TMD8-TOXLOF vs. TMD8-TOXWT tumors (all p<0.01), and vs. these cell lines cultured in vitro (all p<0.01). To determine whether these features can be therapeutically exploited, we conducted a viability screening using a panel of 20 clinical-phase targeted therapies relevant to ABC-DLBCL. We found that BTK inhibitor acalabrutinib (BCR pathway) and MALT1 inhibitor JNJ67690246 (NF-kB pathway) were 2-3 fold more active in TMD8-TOXLOF vs. TMD8-TOXWT cells (all p<0.05). We then xenografted isogenic TMD8 cells (n = 10 mice per condition) and, when tumors reached 150 mm3, we randomized mice to vehicle vs. acalabrutinib 30 mg/kg/day by oral gavage (human dose equivalent). Acalabrutinib was significantly more active in TMD8-TOXLOF tumors (vs. TMD8-TOXWT, p=0.0008), which translated into significantly prolonged survival (p<0.05). This acalabrutinib effect was accompanied by significant reduction of BTK, SYK and PLCG2 phosphorylation in TMD8-TOXLOF tumors (p<0.0001, all comparisons). Thus, TOXLOF leads to hyperactivation of the BCR and NF-kB signaling pathways in ABC-DLBCL cells, rendering them hypersensitive to BCR-targeted therapies, delineating a potential mechanism-based therapeutic approach for ABC-DLBCL TOXLOF patients. Citation Format: Eloisi Caldas Lopes, Maria Revuelta, Rossella Marullo, Leandro Cerchietti. TOX loss-of-function(TOXLOF)results in BCR hyperactivation andenhanced responses to BCR-targeting in diffuse large B cell lymphomas (DLBCL) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7586.

Serum Olink Proteomics-Based Identification of Protein Biomarkers Associated with the Immune Response in Ischemic Stroke.

The immune response is considered essential for pathology of ischemic stroke (IS), but it remains unclear which immune response-related proteins exhibit altered expression in IS patients. Here, we used Olink proteomics to examine the expression levels of 92 immune response-related proteins in the sera of IS patients (n = 88) and controls (n = 88), and we found that 59 of these proteins were differentially expressed. Feature variables were screened from the differentially expressed proteins by the least absolute shrinkage and selection operator (LASSO) and the random forest and by determining whether their proteins had an area under the curve (AUC) greater than 0.8. Ultimately, we identified six potential protein biomarkers of IS, namely, MASP1, STC1, HCLS1, CLEC4D, PTH1R, and PIK3AP1, and established a logistic regression model that used these proteins to diagnose IS. The AUCs of the models in the internal validation and the test set were 0.962 (95% confidence interval (CI): 0.895-1.000) and 0.954 (95% CI: 0.884-1.000), respectively, and the same protein detection method was performed in an external independent validation set (AUC: 0.857 (95% CI: 0.801-0.913)). These proteins may play a role in immune regulation via the C-type lectin receptor signaling pathway, the PI3K-AKT signaling pathway, and the B-cell receptor signaling pathway.

Resisting the Resistance: Navigating BTK Mutations in Chronic Lymphocytic Leukemia (CLL).

Bruton's tyrosine kinase (BTK) plays a key role in the B-cell receptor (BCR) signaling pathway and confers anti-apoptotic and proliferative properties to malignant B-cells in chronic lymphocytic leukemia (CLL). Small molecule BTK inhibitors were designed to bind BTK's active site and block downstream signaling. These drugs have now been used in the treatment of thousands of patients with CLL, the most common form of leukemia in the western hemisphere. However, adverse effects of early generations of BTK inhibitors and resistance to treatment have led to the development of newer, more selective and non-covalent BTK inhibitors. As the use of these newer generation BTK inhibitors has increased, novel BTK resistance mutations have come to light. This review aims to discuss previously known and novel BTK mutations, their mechanisms of resistance, and their relationship with patient treatment. Also discussed here are future studies that are needed to investigate the underlying cause allowing these mutations to occur and how they incite resistance. New treatments on the horizon that attempt to maneuver around these resistance mutations can be met with new resistance mutations, creating an unmet need for patients with CLL. Novel therapies and combinations that address all forms of resistance are discussed.

MiR-17-92 cluster-BTG2 axis regulates B-cell receptor signaling in mantle cell lymphoma.

B-cell receptor (BCR) signaling is critically activated and stable for mantle cell lymphoma (MCL), but the underlying mechanism of the activated BCR signaling pathway is not clear. The pathogenic basis of miR-17-92 cluster remains unclear although the oncogenic microRNA (miRNA) miR-17-92 cluster is highly expressed in patients with MCL. We revealed that miR-17-92 cluster overexpression is partly dependent on SOX11 expression and chromatin acetylation of MIR17HG enhancer regions. Moreover, miR-17-92 cluster regulates not only cell proliferation but BCR signaling activation in MCL cell lines. To comprehensively identify miR-17-92 cluster target genes, we performed pulldown-seq, where target RNA of miRNA was captured using the biotinylated miRNA mimics and magnetic bead-coated streptavidin, and quantified using next-generation sequencing. The pulldown-seq identified novel miRNA target genes, including tumor suppressors such as BTG2 (miR-19b), CDKN2A (miR-17), SYNE1 (miR-20a), TET2 (miR-18, miR-19b, and miR-92a), TNFRSF10A (miR-92a), and TRAF3 (miR-17). Notably, the gene expression profile data of patients with MCL revealed that BTG2 expression was negatively associated with that of BCR signature genes, and low BTG2 expression was associated with poor overall survival. Moreover, BTG2 silencing in MCL cell lines significantly induced BCR signaling overactivation and cell proliferation. Our results suggest an oncogenic role of miR-17-92 cluster-activating BCR signaling throughout BTG2 deregulation in MCL. Furthermore, this may contribute to the prediction of the therapeutic efficacy and improved outcomes of MCL.

Establishment and validation of a prognostic immune-related lncRNA risk model for acute myeloid leukemia.

Acute myeloid leukemia (AML) is a cancer arising in the bone marrow and is the most common type of adult leukemia. AML has a poor prognosis, and currently, its prognosis evaluation does not include immune status assessment. This study established an immune-related long non-coding RNA (lncRNA) prognostic risk model for AML based on immune lncRNAs screening. To construct training and validation cohorts, The Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO) public databases were accessed to obtain gene expression profiles and clinical data. The correlation between lncRNAs and immunity genes was analyzed using the "limma" package, and the immune-related lncRNAs were obtained. Through least absolute shrinkage and selection operator regression, a prognostic model was established with immune-related lncRNAs. Using the median risk score, patients were divided into high- and low-risk groups. The Kaplan-Meier method was used for survival analysis, whereas the accuracy of the risk model was evaluated using time-dependent receiver operating characteristic curves, risk score distribution, survival status, and risk heat maps. We utilized univariate and multivariate Cox regression to examine the association between risk score and clinical variables and AML survival and prognosis. In the immune-related lncRNA prognostic risk model, the prognosis was better for low-risk than for high-risk patients, indicating risk score of this model as an independent indicator of prognosis. The area under the curve value for 1-, 3-, and 5-year survival of TCGA patients was 0.817, 0.859, and 0.909, respectively, whereas that of GEO patients (of dataset GPL96-GSE37642) was 0.603, 0.652, and 0.624, respectively. Gene set enrichment analysis revealed the enrichment of multiple pathways, such as antigen processing, B-cell receptor signaling pathway, natural killer cell-mediated cytotoxicity, and chemokines, in high-risk patients. In this study, immune-related lncRNA prognostic risk models effectively predicted AML survival and provided potential treatment targets.

ONO-7018, a First-in-Class MALT1 Inhibitor, Provides Novel Therapeutic Strategies for B Cell Malignancies: Overcoming BTK Inhibitor Acquired Resistance and Enhancing the Antitumor Effect of BTK Inhibitors

Introduction: B cell receptor (BCR) signaling plays a crucial role in promoting survival and growth in B cell malignancies, and several compounds targeting the signaling pathways have been approved. Bruton's tyrosine kinase (BTK) is a key molecule in BCR signaling pathways and BTK inhibitors are one of the essential treatment options for chronic lymphocytic leukemia (CLL) and certain subtypes of B cell Non-Hodgkin Lymphoma. However, there are issues that acquired resistance mutations in the BTK kinase domain and gain of function mutation of its downstream molecules, which lead to BTK inhibitor resistance and limit its efficacy. For example, BTK C481S mutation is well known as a resistant mutation to covalent BTK inhibitors and several mutations of BTK (such as T474I and L528W mutation) have recently been reported in relapsed or refractory CLL patients with acquired resistance to pirtobrutinib, a non-covalent BTK inhibitor. In addition, there are unmet medical needs to improve the duration of response of current standard therapies including BTK inhibitors for B cell malignancies. Therefore, therapeutic strategies to overcome the BTK inhibitor resistance and novel combination therapies to enhance the efficacy of BTK inhibitors are required. Mucosa-associated lymphoid tissue lymphoma translocation protein 1 (MALT1) is known as a key growth and survival regulator in BCR signaling and an emerging therapeutic target. ONO-7018 (formerly known as CTX-177), a potent and selective MALT1 inhibitor, demonstrated preclinical efficacy in several lymphoma models. Aim: Firstly, to explore the potential of ONO-7018 to overcome BTK inhibitor acquired resistance, we tested the antitumor effects of ONO-7018 on cell lines harboring BTK inhibitor-resistant mutations. Secondly, we examined the combination antitumor efficacy of ONO-7018 and tirabrutinib, a second generation BTK inhibitor in vitro and in vivo. Methods: Diffuse large B-cell lymphoma (DLBCL) cell lines harboring BTK inhibitor-resistant mutations (BTK T474I, C481S, or L528: PLCG2 R665W) were generated by continuous passage with BTK inhibitor or CRISPR-Cas9 gene editing followed by single cell cloning. In in vivo study, human DLBCL cell line, TMD8 cells were subcutaneously implanted in immune deficient mice. ONO-7018 and tirabrutinib were orally administered to the mice twice a day. Results: Anti-proliferative activities of ONO-7018 on these BTK inhibitor resistant cell lines were evaluated in vitro. ONO-7018 inhibited the proliferation of these cells with comparable IC 50 values to the parent cell line. Anti-proliferative activities of ONO-7018 alone or in combination with tirabrutinib on DLBCL and mantle cell lymphoma (MCL) cell lines were evaluated in vitro. ONO-7018 exhibited synergistic effects with tirabrutinib in both DLBCL and MCL cell lines. In addition, combination treatment of ONO-7018 with tirabrutinib strongly inhibited the expression of interferon regulatory factor 4, which is a downstream signal molecule in BCR signaling pathways, compared to their single treatment. We then evaluated the antitumor effects of ONO-7018 in combination with tirabrutinib in the xenograft model. Combination treatment of ONO-7018 with tirabrutinib showed dramatic antitumor effect with tumor regression. These results suggest that ONO-7018 could demonstrate antitumor effect on patients with B cell malignancies harboring BTK inhibitor acquired resistant mutations and synergistic antitumor effect with BTK inhibitors in clinic. Conclusion: ONO-7018 would provide novel therapeutic strategies to overcome the BTK inhibitor acquired resistance and enhance the antitumor effect of BTK inhibitors in clinic. Phase 1 study of ONO-7018 (NCT05515406) is currently ongoing.

Sgr-1505 Is a Potent MALT1 Protease Inhibitor with a Potential Best-in-Class Profile

Background: MALT1 (Mucosa-associated lymphoid tissue lymphoma translocation protein 1) is a component of the MALT1-BCL10-CARD11 complex downstream from the Bruton Tyrosine Kinase (BTK) on the B-cell receptor signaling pathway. MALT1 is a key mediator of nuclear factor kappa B (NF-κB) signaling, which is the main driver of a subset of B-cell lymphomas. MALT1 is considered a potential therapeutic target for several subtypes of non-Hodgkin B-cell lymphomas and chronic lymphocytic leukemia (CLL), including tumors with acquired BTK inhibitor (BTKi) resistance. Constitutive activation of the NF-κB is a molecular hallmark of activated B cell-like diffuse large B cell lymphoma (ABC-DLBCL), and MALT1 may have utility as a treatment option for ABC-DLBCL. Previously, we described the discovery of novel MALT1 inhibitors with anti-proliferative effects in non-Hodgkin B-cell lymphoma cells and the strong anti-tumor activity of our MALT1 inhibitors across multiple tumor models as well as combination potential with agents including standard-of-care (ref 1, 2). SGR-1505 is an oral potent small molecule allosteric inhibitor of MALT1 that inhibits MALT1 enzymatic activity and demonstrates anti-proliferative activity in ABC-DLBCL cell lines, both BTKi-sensitive (OCI-LY10) and BTKi-resistant (OCI-LY3). When administered as a single agent and in combination with the approved Bruton's tyrosine kinase (BTK) inhibitor, ibrutinib, SGR-1505 demonstrated tumorostatic and regressive antitumor activity in ABC-DLBCL cell line-derived xenograft and patient-derived xenograft models. These data suggest that SGR-1505-mediated MALT1 inhibition has therapeutic potential for patients with selected B-cell lymphomas. Here we further characterized SGR-1505, in a series of in vitro and ex vivo assays, as well as RNA-seq analysis to examine changes in gene expression from in vivo tumor samples. We also compared SGR-1505 with a competitor Phase I candidate, JNJ-67856633 (JNJ-6633) (ref 3, 4). Results: SGR1505 potency and downstream effects were evaluated in a series of biochemical and cell based assays. SGR-1505 showed excellent potency in the biochemical assay and strong anti-proliferative effects on ABC-DLBCL cells. SGR-1505 was more potent than JNJ-6633 in all assays tested (Table 1). These results are also consistent with the result from a human primary T-cell based assay, where SGR-1505 showed at least ten-fold better potency than JNJ-6633. RNA-seq analysis was conducted to examine changes in gene expression from in vivo tumor samples. Greater modulation of BIOCARTA NF-kB pathway genes was seen with SGR-1505 compared to JNJ-6633, as measured by mean absolute change in gene expression. At 6 hr and later timepoints, we also observed a trend of increases in genes related to cell cycle pathways, such as cell cycle, DNA damage, and apoptosis. SGR-1505 is being evaluated in the SGR-1505-102 phase 1 study, which is an ongoing first-in-human, single center, dose escalation study to evaluate the safety, tolerability, PK and PD of SGR-1505 tablets in healthy participants (ACTRN12623000358640p). Preliminary data showed changes in target engagement markers at concentrations predicted by the in vitro and ex vivo assays, consistent with MALT1 protease inhibition. Conclusions: SGR-1505, a MALT1 protease small molecule inhibitor, consistently demonstrated better potency in in vitro and ex vivo assays when compared to the clinical-stage JNJ-6633 compound and greater effects on NF-kB pathway gene expression in in vivo tumor samples based on RNA-seq analysis. Changes in biological pathways mediated by MALT1 were also observed at relevant doses in the ongoing SGR-1505 healthy volunteer study. Currently, a phase 1 clinical trial in patients with mature B cell neoplasms is also ongoing (NCT05544019). The data presented suggests SGR-1505 has a potential best-in-class profile and supports advancing the ongoing clinical development of SGR-1505.

A Phase 1, Open-Label, Multicenter, Dose-Escalation Study of Sgr-1505 As Monotherapy in Subjects with Mature B-Cell Malignancies

Background and Significance: MALT1 (Mucosa-associated lymphoid tissue lymphoma translocation protein 1) is a component of the MALT1-BCL10-CARD11 complex downstream from the Bruton Tyrosine Kinase (BTK) on the B-cell receptor signaling pathway. MALT1 is a key mediator of nuclear factor kappa B (NF-κB) signaling, which is the main driver of a subset of B-cell lymphomas. MALT1 is considered a potential therapeutic target for several subtypes of non-Hodgkin B-cell lymphomas and chronic lymphocytic leukemia (CLL), including tumors with acquired BTK inhibitor (BTKi) resistance. Constitutive activation of NF-κB is a molecular hallmark of activated B cell-like diffuse large B cell lymphoma (ABC-DLBCL), and MALT1 may have utility as a treatment option for ABC-DLBCL. Furthermore, a MALT1 inhibitor (JNJ-67856633) showed efficacy in mature B cell malignancies from phase 1 studies (ref 1, 2). SGR-1505 is an oral potent small molecule allosteric inhibitor of MALT1 that inhibits MALT1 enzymatic activity and demonstrates anti-proliferative activity in BTKi-sensitive (OCI-LY10) and BTKi-resistant (OCI-LY3) ABC-DLBCL cell lines. SGR-1505 administered as a single agent and in combination with the approved Bruton's tyrosine kinase (BTK) inhibitor, ibrutinib, demonstrates tumorostatic and regressive antitumor activity in ABC-DLBCL cell line-derived and patient-derived xenograft models. These data suggest that SGR-1505-mediated MALT1 inhibition may expand therapeutic options for patients with selected B-cell lymphomas, supporting further evaluation of SGR-1505 in clinical trials. Study Design and Methods: SGR-1505-101 (NCT05544019) is a phase 1, multicenter trial of SGR-1505 as monotherapy in subjects with mature B-cell malignancies (figure 1). At present, the study is open to accrual at multiple investigative sites in the US with plans to expand to Europe. The primary objective is to evaluate safety and tolerability of SGR-1505 as monotherapy and to identify the maximum tolerated dose (MTD) and/or recommended dose (RD). Secondary objectives are to evaluate the pharmacokinetic (PK) profile, food effect, drug-drug interaction, and preliminary anti-tumor activity of SGR-1505. Key inclusion criteria are: history of mature B-cell malignancy (including aggressive and indolent B-cell lymphomas, Waldenström macroglobulinemia, and CLL); measurable or detectable disease according to the applicable disease-specific classification system (Lugano, iwCLL, WWM6); Eastern Cooperative Oncology Group (ECOG) performance status of ≤ 2. Patients with indolent B-cell lymphomas or CLL must have an indication for treatment and not require immediate cytoreductive therapy. Subjects with symptomatic or active CNS involvement, and other conditions or laboratory findings placing them at increased risk to the use of an investigational drug are excluded. SGR-1505 is initially dose-escalated using an accelerated titration design in cohorts of 1-6 subjects, and at higher dose levels using a conventional 3+3 design.

Effectiveness and Safety of Orelabrutinib Combined with Rituximab As First-Line Treatment in Marginal Zone Lymphoma

Background: Marginal zone lymphoma (MZL) collectively is the third most common type of B-cell non-Hodgkin's lymphoma (NHL) worldwide, with an increased trend in incidence during the last two decades (Rossi D E et al., N Engl J Med. 2022). The lack of predominant first-line systemic therapy and toxicities associated with current rituximab-based therapies have inspired the exploration of more optimal treatment strategies (Becnel MR et al., Br J Haematol, 2019). Given the crucial role of bruton tyrosine kinase (BTK) in the B-cell receptor signaling pathway, BTK inhibitor has been proven to yield robust outcomes with improved tolerability in the development of NHL (Dhillon S et al., Drugs, 2021; Xu W et al., Blood, 2019; Song Y et al., Blood, 2019). Orelabrutinib is a novel BTK inhibitor with high selectivity and has been approved in China for relapsed/refractory MZL recently (Dhillon S et al., Drugs, 2021). Meanwhile, several clinical studies reported that orelabrutinib in combination with rituximab in NHL could preserve natural killer cell-mediated antibody-dependent cellular cytotoxicity induced by rituximab and enhance the clinical efficacy (Yu H et al., Mol Ther Oncolytics, 2021; Qu C et al., Blood, 2023). However, orelabrutinib plus rituximab as the first-line treatment for MZL remains largely understudied, thus, we conducted a retrospective study to evaluate its effectiveness and safety. Methods: Between July 27, 2021 and March 18, 2023, 10 patients with MZL who received orelabrutinib (150 mg, qd, po) with rituximab (375 mg/m 2, iv, day 1 of each 28-day cycle) as first-line therapy were included in this retrospective study. After 6 cycles of treatment, patients underwent orelabrutinib monotherapy (150 mg, qd, po) as maintenance for a year. Dynamic dose adjustment was performed according to the patient's clinical status. Effectiveness was assessed by positron emission tomography/computed tomography-based 2014 Lugano criteria. The primary endpoint was the overall response rate (ORR); secondary endpoints were progression-free survival (PFS), overall survival (OS), and safety. Results: Ten patients (6 males; median age 63.5 years, range 45-76) were included in this study, 4 with extranodal MZL of mucosa-associated lymphoid tissue (40.0%), 2 with nodal MZL (20.0%), 1 with splenic MZL (10.0%), and 3 with unknown subtype (30%). Among a total of 10 patients, 3 (30%) achieved complete response (CR) and 6 (60%) attained partial response (PR) as their best response for an ORR of 90% ( Figure 1). After a median follow-up of 13.0 months (range 7.8-24.7), the median PFS was not reached and a 6-month PFS rate was 100% ( Table 1). OS could not be assessed due to there were no deaths occurred. As of the cut-off date (May 6, 2023), 8 patients received orelabrutinib maintenance treatment and the median duration of maintenance treatment was 9.6 months (range 3.0-17.8). The ORR was 75% (6/8) during maintenance treatment, while 1 patient had stable disease (SD) and 1 developed progressive disease (PD). In addition, abnormal hematology due to disease gradually recovered with the therapy within 4 months. No serious adverse events were observed. The off-target related AEs such as atrial fibrillation, diarrhea, and major hemorrhage were not reported. Conclusion: This retrospective data suggests that orelabrutinib with rituximab has an encouraging anti-tumor activity in MZL, with a favorable safety profile. These results provide a potential first-line treatment strategy in MZL. Further verification in prospective clinical trials of clinical outcomes of orelabrutinib in first-line MZL is warranted in the future.

A Phase 2 Study of Acalabrutinib, Umbralisib, and Ublituximab (AU2) in Treatment-Naïve and Relapsed or Refractory Chronic Lymphocytic Leukemia (CLL)

Background Targeting the B-cell receptor (BCR) signaling pathway is highly effective for the treatment of CLL. However, attainment of undetectable minimal residual disease (uMRD) is infrequent with current BCR targeting strategies. Here, we report results of a phase 2 study testing time-limited dual BCR inhibitor therapy with a BTK inhibitor acalabrutinib and a PI3Kδ inhibitor umbralisib. With the goal of enhancing depth of remission, a glycoengineered anti-CD20 antibody ublituximab was initiated during Cycle (C) 7. Methods In this investigator-sponsored trial, eligible patients with treatment-naïve (TN) or relapsed or refractory (RR) CLL received up to 24 cycles of AU2 (NCT04624633). Each cycle was 28 days. Daily oral doses of acalabrutinib (100mg BID) and umbralisib (800mg QD) began on C1 Day (D) 1. Ublituximab IV began on C7 for 6 cycles (D1, 2, 8, and 15 of C7, D1 of C8-12). Treatment was stopped after C12 if patients achieved complete response (CR) by IWCLL criteria, while all others continued acalabrutinib and umbralisib until C24. In March 2023, umbralisib was withdrawn from drug development, and those who were receiving study therapy at the time continued acalabrutinib alone until C24. The primary endpoint was the CR rate after C24. MRD was assessed in peripheral blood (PB) and bone marrow (BM) using multi-color flow cytometry at 10 -4. Results The study enrolled 29 patients from December 2020 to January 2022. The median age at enrollment was 63 years (range 40-77). Most patients were male (62%), had TN CLL (72%), and had unmutated IGHV (79%). RR CLL patients had a median of 1 prior line of therapy, most frequently (88%) with chemoimmunotherapy. Genetic alterations were commonly observed at study enrollment, including deletion 17p or TP53 mutation (21%), deletion 11q or ATM mutation (38%), trisomy 12 (24%), and mutations of NOTCH1(45%), SF3B1 (24%), and RAS/RAF pathway (21%). 7% had complex karyotype defined as ≥3 abnormalities. At a median follow-up of 23 months (range 2-29), with 7 patients still receiving therapy prior to reaching C24, there were no deaths, Richter's transformations, or CLL progression events on the study. The rates of CR, BM uMRD, and PB uMRD at best response were 30%, 44%, and 67%, respectively (Table 1). There were no statistically significant differences in CR and uMRD rates between TN and RR groups at best response. CR and BM uMRD rates improved during the second year of therapy (CR rate: 11% after C12, 28% after C24; BM uMRD rate: 22% after C12, 39% after C24). PB uMRD occurred earlier than BM uMRD, and the rate (44%) remained unchanged between C12 and C24. After completion of study therapy, PB MRD was monitored every 3 months. Five patients had MRD conversion at the end of therapy or shortly after treatment cessation. Median time from the last uMRD to detectable MRD (dMRD) in PB was 3 months (range 3-6). Most (80%, n=4/5) patients with MRD conversion had partial response or dMRD in BM at best response. Two patients stopped treatment early (due to the diagnosis of second cancer and G4 transaminitis, in 1 patient each). In addition, 7 (24%) patients stopped umbralisib between C15-19 due to discontinuation of drug supply; these patients remained on acalabrutinib monotherapy until C24. All others completed up to 24 cycles of treatment including 3 (10%) patients who stopped therapy per protocol after C12 once CR was achieved. Dose reduction of umbralisib (38%) was more common than acalabrutinib (7%) and was most frequently due to diarrhea (21%) and transaminitis (14%). Only 2 (7%) patients required temporary dose reduction of acalabrutinib to avoid drug-drug interaction. Notable adverse events included diarrhea or colitis (76%, 10% Grade 3 or higher [G≥3]), bruising (69%, 0% G≥3), transaminitis (62%, 17% G≥3), COVID-19 infection (55%, 7% G≥3), hypertension (52%, 10% G≥3), rash (24%, 0% G≥3), and pneumonitis (7%, 3% G≥3). One (3%) patient had Pneumocystis jirovecii pneumonia while being off of mandatory prophylaxis due to non-compliance. None had atrial fibrillation. Conclusions Dual inhibition of the BCR signaling pathway with up to 2 years of AU2 provided a time-limited treatment option for TN and RR CLL patients. Immune-related adverse events were frequently observed and required dose reduction of umbralisib in 38% of the patients but most were then able to stay on therapy. Longer follow-up is required to determine durability of remission off therapy.

MS-553 Therapy Inhibits BCR Signaling and Increases Survival Dependence on BCL-2 in BTK-Inhibitor Resistant CLL

B-cell receptor (BCR) signaling pathway inhibitors and B-cell lymphoma-2 (BCL-2) antagonist venetoclax (Ven) have transformed the treatment of chronic lymphocytic leukemia (CLL), including in high-risk patients with 17p deletion and/or TP53 mutations and complex karyotype. Despite the durable remissions observed with inhibitors of Bruton's Tyrosine Kinase (BTKi), a significant proportion of patients develop resistance with acquired mutations in BTK and its downstream target phospholipase Cγ2 ( PLCG2). Patients who progress on BTKi including ibrutinib (ibr) often receive Ven but eventually relapse, underscoring an urgent need for new treatment strategies. Protein kinase C-β (PKC-ß) is a protein downstream of BCR signaling (BTK-PLCG2) and is essential for CLL cell survival and proliferation in vivo. Here we investigate MS-553, an inhibitor of PKC-ß, with a focus of combination strategies in CLL patient samples including relapsed/refractory CLL. We show that CLL cells in contact with stroma upregulate PKC-ßII, its immediate downstream effectors, pGSK and pERK as well as ß-catenin in CLL patients including those with mutant TP53 and 17p (n=8). Correspondingly, MS-553 (5µM, 48h) exposure reduced levels of p-PKC-ßII and its downstream effectors (p-ERK, p-GSK) in a dose dependent manner regardless of TP53 mutation in both CLL cells and those supported by NK.Tert (stromal cell line derived from bone marrow, BM, and mimics BM microenvironment). Pre-treatment (48h, 5µM MS-553) of stromal NK.Tert cells with MS-553 prevented the upregulation of PKC-ßII within adjacent CLL cells implicating the stroma as a major source of PKC-ß activation in CLL cells. We also tested whether MS-553 could inhibit PKC-ß signaling in CLL cells exposed to phorbol myristate acetate (PMA-400nM, 90 min), which simulates BCR activation. MS-553 (5µM, 48h) exposure caused downregulation of BTK effectors (pPKC-ßII, pERK, pGSK), BCL-2 family member proteins (MCL-1 and BCLxl), and ß-catenin (n=10). In parallel experiments, we used BH3 profiling to show that CLL cells' (n=8) exposure to MS-553 in vitro developed increased dependence on BCL-2 and BCLxl for survival (Bim, Bad, XXA1_y4ek, 5-fold± 2-fold increase) in both wild type (WT) and mutant (Mut) CLL cells, suggesting a mechanistic rationale for synergy with Ven. To confirm this, CLL cells were exposed to MS-553, Ven or a combination of MS-553 and Ven. ~3-fold and 9-fold synergistic increase in the mitochondrial dysfunction (measured by cytochrome C release, 12h, 5µM MS-553, 1nM Ven) was observed in stromal supported WT and Mut CLL respectively in MS-553 versus Ven+MS-553 treated group. Similarly, ~3-fold increase was observed in WT and Mut CLL alone group and more than 10-fold increase was found in stromal supported WT and Mut group in cell death determined as percent apoptotic index (measured by annexin V-PI staining, 18h, 5µM MS-553, 1nM Ven) in MS-553 versus Ven+MS-553 group respectively. Finally, we validated our preclinical findings in CLL samples obtained from four patients on MS-553 therapy (NCT03492125, Range = cycles 0-19, MS-553 28 days/cycle). All patients showed an increase in survival dependence towards BCL-2 and BCLxl by Cycle 3-6 of MS-553 therapy. This was accompanied by downregulation of ß-catenin, pPKC-ßII, pGSK, pERK along with BCLxl and MCL-1 after Cycle 10 or later of MS-553 therapy (data summary table1). Overall, our data provides clear evidence that MS-553 inhibits BCR signaling in vitro and in patients on MS-553 therapy. It also provides a rationale for combinations with Ven as a treatment partner based on its ability to increase survival dependence on BCL-2.

Progressive Gut Microbiome Dysbiosis Is Associated with Chronic Lymphocytic Leukemia Pathogenesis and Development in the Eµ-TCL1 Mouse Model

Introduction: The gut microbiome is home to a community of microorganisms such as bacteria, fungi, viruses, archaea, and protozoa. Maintaining a balance between “favorable” and “unfavorable” bacteria within the gut microbiome is essential for homeostatic physiological processes including proper immune system development, regulation of metabolic pathways, protection against pathogens, and gut epithelium integrity. Chronic lymphocytic leukemia (CLL) is a mature B-cell malignancy characterized by the accumulation of CD5 + B-cells in the blood, bone marrow, and secondary lymphoid tissues. The pathogenesis of CLL is related to the activation of critical survival pathways such as B-cell and toll-like receptor signaling pathways, where microbial antigens are known to be key stimulators driving CLL B-cell proliferation. In this study, we investigated how the gut microbiota composition contributes and correlates to CLL disease progression. Methods: Using a well-established murine model representative of CLL development in humans, we profiled the gut microbiota of transgenic Eµ-TCL1 mice and age-matched wild-type B6 (WT B6) mice over the course of 12 months (n = 7-10/group). Fecal pellets were collected aseptically from study mice at four time points (4, 7, 10, and 12 months) for DNA extraction using the QIAamp® PowerFecal® Pro DNA Kit. To characterize the gut microflora, we utilized 16S rRNA sequencing using the Illumina 16S metagenomics protocol. Bacterial taxonomy was assigned and analyzed using the QIIME2™ platform. Linear Discriminant Analysis (LDA) Effect Size (LEfSe) analysis was employed to identify differentially expressed bacterial taxa between cohorts. Beta diversity indices were used to evaluate intersample differences in the fecal microbiota. Mice were monitored for peripheral expansion of leukemia cells (%CD45 +/CD19 +/CD5 + cells) via flow cytometry. Next, we evaluated the microbiome in an aggressive model of CLL. Utilizing the Eµ-TCL1 adoptive transfer murine model, similar methods were adopted to profile the gut microbiome over the course of 7 weeks. Lastly, to evaluate the contribution of the gut microbiota to CLL development, we employed an antibiotic-mediated gut microflora ablation to the adoptive transfer Eµ-TCL1 model, with leukemia expansion monitored post-engraftment on a weekly basis. Results: A unique microbiome signature was identified in both the indolent and aggressive model of CLL. Evaluation of beta diversity, using Bray-Curtis distance metrics, confirmed distinct differences in the gut microbial communities between Eµ-TCL1 and WT B6 cohorts. At the phylum level, the gut microbiota of the Eµ-TCL1 and WT B6 cohorts were similarly dominated by Firmicutes and Bacteroidota. However, Proteobacteria Deferribacterota and Desulfobacterota were more abundant in the Eµ-TCL1 mice over 12 months as compared to age-matched WT B6. Abundance of Actinobacteriota increased specifically at 12 months in the Eµ-TCL1 mice. Additionally, LEfSe analysis showed enrichment of certain phyla in the Eµ-TCL1 mice at 4 months, 10 months, and 12 months. Presence of these bacteria paralleled with expansion of disease. In the aggressive model of CLL, beta diversity confirmed distinct differences in gut microbial communities as CLL disease progressed. Moreover, certain phyla were enriched in leukemic mice at 7 weeks post-engraftment including Proteobacteria and Actinobacteriota. Lastly, in the antibiotic-mediated gut flora ablation model, a delay of disease onset was reflected in mice with ablated gut microbiomes compared to mice with intact gut microbiomes, resulting in prolonged overall survival. Conclusion: We provide novel results associating the gut microbiome with the initiation and progression of CLL disease. In both the indolent and aggressive CLL disease models, we showed that there is a distinct gut microbiome as evident by beta diversity. A dysbiotic signature represented by increasing Proteobacteria abundance was similarly identified in both models. Proteobacteria are known to be evident in diseases characterized by inflammation. These findings support future studies to determine a potential causal mechanism between gut microbiota changes and CLL pathogenesis.

Optogenetic Control of Oncogenic Signaling in B-Cell Malignancies

Background: B-cell receptor (BCR) signals are essential determinants of survival and proliferation throughout normal B-cell development. In B-cell malignancies, these signals are frequently generated by oncogenic mimics of the BCR signaling pathway. For instance, oncogenes in B-ALL, derived from B-cell precursors, typically mimic survival signals from a constitutively active pre-BCR, while tonic and chronic active BCR signaling were identified in mature B-cell lymphomas. Oncogenic BCR-signaling represents an important target of therapeutic intervention: for instance, small molecule inhibitors of SYK (e.g. entospletinib) and BTK (e.g. ibrutinib) have been developed to disrupt oncogenic BCR signaling in mature B-cell lymphomas. Besides SYK and BTK tyrosine kinases, oncogenic BCR-signaling leads to activation of PLCG2, which initiates the release of Ca 2+ from the ER into the cytoplasm. Thereby, calcium flux is decoded by NFATC1 or NF-kB based on fast (NFATC1) or slow (NF-kB) frequencies of Ca 2+-signals. Significance: To interrogate Ca 2+-signals as critical integration point of oncogenic BCR-signaling, we engineered cell lines and PDX with a GCaMP6s-biosensor, which allows tracing of calcium signaling in single cells over time ( Figure A). To cover B-cell malignancies from multiple stages of B-cell development, we engineered B-ALL (pro- and pre-B cells), mantle cell lymphoma (MCL, naïve B-cells), Burkitt's lymphoma (germinal center), DLBCL (post-GC), multiple myeloma (terminally differentiated plasma cells) and Hodgkin's disease (“crippled” BCR-deficient GC-B cells). Strikingly, B-cell malignancies exhibit autonomous Ca 2+-oscillations, which decreased in their frequency from 20 mHz (pro- and pre-B), 11 mHz (naïve), 4 mHz (germinal center) to 0 mHz in post-GC and terminally differentiated plasma cells based on the differentiation stage of their cell of origin ( Figure A). Results: Given the striking differences in oncogenic BCR-signaling as measured by autonomous Ca 2+ oscillations, we developed an optogenetic system to control the frequency and amplitude of Ca 2+ oscillations by blue light pulses ( Figure B). To model frequency-modulated Ca 2+ signaling in B-cells, we engineered murine pre-B cells carrying an optogenetic tool termed OptoCRAC, which enables reversible activation and inactivation of the plasma membrane Ca 2+ channel Orai1 by intermittent blue light irradiation. Time-lapse imaging of a fluorescent Ca 2+ reporter (R-CaMP1.07) confirmed rapid and reversible Ca 2+ influx in response to intermittent blue light pulses. To assess the phenotypic consequence of high-frequency Ca 2+ oscillations, we developed an LED-array-based optogenetic platform for cell culture plates. While low-frequency Ca 2+ oscillations (0.5 mHz) did not impact cell viability, high-frequency Ca 2+ oscillations (20 mHz) rapidly induced NFAT activation (5 min), and eventually cell death within 72 h ( Figure B). Mechanism and conclusions: To elucidate the mechanistic contribution of NFATC1 in B-cell death induced by fast Ca 2+ oscillations, we tested whether genetic deletion of Nfatc1 is sufficient to rescue B-cell death upon fast Ca 2+ oscillation induced by 20 mHz intermittent blue light pulses. As expected, while B-ALL cells retaining intact Nfatc1 rapidly decreased cell viability, the cells with Cre-mediated deletion persistently remained in cell culture upon 20 mHz Ca 2+ oscillation. In addition to genetic deletion of Nfatc1, we also found that inducible expression of mutant activators of NF-kB signaling were able to rescue B-cells from cell death induced by high-frequency oscillations: Concurrent expression of Card11 L232LI, MYD88 L265P and IKK2 S177E/S181E not only reduced the frequency of autonomous Ca 2+ oscillations but also enabled B-cell survival despite delivery of light-pulses and Ca 2+ oscillations at a fast 20 mHz frequency. We conclude that autonomous Ca 2+ oscillations represent a critical integration point of oncogenic BCR-signaling in B-cell malignancies and that optogenetic control of BCR-downstream Ca 2+ oscillations will reveal previously unrecognized vulnerabilities that can be exploited to increase efficacy of BCR-signaling inhibitors (e.g. ibrutinib, entospletinib, idelalisib) that are currently being used for the treatment of B-cell lymphomas.

Molecular complexity of diffuse large B-cell lymphoma: a molecular perspective and therapeutic implications.

Diffuse large B-cell lymphoma (DLBCL) stands as a formidable challenge in the landscape of non-Hodgkin's lymphomas. This review illuminates the remarkable strides made in comprehending DLBCL's molecular intricacies and devising targeted treatments. DLBCL, the most prevalent non-Hodgkin's lymphoma, has seen transformative progress in its characterization. Genetic investigations, led by high-throughput sequencing, have unveiled recurrent mutations in genes such as MYC, BCL2, and BCL6, casting light on the underlying genetic chaos propelling DLBCL's aggressiveness. A pivotal facet of this understanding centers on cell signaling pathways. Dysregulation of B-cell receptor (BCR) signaling, NF-κB, PI3K/Akt/mTOR, JAK/STAT, Wnt/β-Catenin, and Toll-like receptor pathways plays a critical role in DLBCL pathogenesis, offering potential therapeutic targets. DLBCL's complex tumor microenvironment (TME) cannot be overlooked. The dynamic interplay among tumor cells, immune cells, stromal components, and the extracellular matrix profoundly influences DLBCL's course and response to therapies. Epigenetic modifications, including DNA methylation and histone changes, add another layer of intricacy. Aberrant epigenetic regulation plays a significant role in lymphomagenesis, offering prospects for epigenetic-based therapies. Promisingly, these molecular insights have spurred the development of personalized treatments. Targeted therapies and immunotherapies, guided by genomic profiling and molecular classification, are emerging as game-changers in DLBCL management. In conclusion, this review underscores the remarkable strides in understanding DLBCL's molecular underpinnings, spanning genetics, cell signaling, the tumor microenvironment, and epigenetics. These advances pave the way for more effective, personalized treatments, renewing hope for DLBCL patients.

Analysis of Molecular Changes and Features in Rat Knee Osteoarthritis Cartilage: Progress From Cellular Changes to Structural Damage

Objective Although knee osteoarthritis (KOA) is a common disease, there is a lack of specific prevention and early treatment methods. Hence, this study aimed to examine the molecular changes occurring at different stages of KOA to elucidate the dynamic nature of the disease. Design Using a low-force compression model and analyzing RNA sequencing data, we identified molecular changes in the transcriptome of knee joint cartilage, including gene expression and molecular pathways, between the cellular changes and structural damage stages of KOA progression. In addition, we validated hub genes using an external dataset. Results Gene set enrichment analysis (GSEA) identified the following pathways to be associated with KOA: “B-cell receptor signaling pathway,” “cytokine-cytokine receptor interaction,” and “hematopoietic cell lineage.” Expression analysis revealed 585 differentially expressed genes, with 579 downregulated and 6 upregulated genes. Enrichment and clustering analyses revealed that the main molecular clusters were involved in cell cycle regulation and immune responses. Furthermore, the hub genes Csf1r, Cxcr4, Cxcl12, and Ptprc were related to immune responses. Conclusions Our study provides insights into the dynamic nature of early-stage KOA and offers valuable information to support the development of effective intervention strategies to prevent the irreversible damage associated with KOA, thereby addressing a major clinical challenge.

Prognostic value and immune landscapes of cuproptosis-related lncRNAs in esophageal squamous cell carcinoma.

Precisely forecasting the prognosis of esophageal squamous cell carcinoma (ESCC) patients is a formidable challenge. Cuproptosis has been implicated in ESCC pathogenesis; however, the prognostic value of cuproptosis-associated long noncoding RNAs (CuRLs) in ESCC is unclear. Transcriptomic and clinical data related to ESCC were sourced from The Cancer Genome Atlas (TCGA). Using coexpression and Cox regression analysis to identify prognostically significant CuRLs, a prognostic signature was created. Nomogram models were established by incorporating the risk score and clinical characteristics. Tumor Immune Dysfunction and Rejection (TIDE) scores were derived by conducting an immune landscape analysis and evaluating the tumor mutational burden (TMB). Drug sensitivity analysis was performed to explore the underlying molecular mechanisms and guide clinical dosing. Our risk score based on 5 CuRLs accurately predicted poorer prognosis in high-risk ESCC patients across almost all subgroups. The nomogram that included the risk score provided more precise prognostic predictions. Immune pathways, such as the B-cell receptor signaling pathway, were enriched in the datasets from high-risk patients. High TMB in high-risk patients indicated a relatively poor prognosis. High-risk patients with lower TIDE scores were found to benefit more from immunotherapy. High-risk patients exhibited greater responsiveness to Nilotinib, BI-2536, P22077, Zoledronate, and Fulvestrant, as revealed by drug sensitivity analysis. Real-time PCR validation demonstrated significant differential expression of four CuRLs between ESCC and normal cell lines. The above risk score and nomogram can accurately predict prognosis in ESCC patients and provide guidance for chemotherapy and immunotherapy.