Role In B-cell Development Research Articles

Pim1 is Critical in T-cell-independent B-cell Response and MAPK Activation in B Cells

The Pim family consists of three members that encode a distinct class of highly conserved serine/threonine kinases. In this study, we generated and examined mice with hematopoiesis-specific deletion of Pim1 and bone marrow (BM) chimeric mice with B-cell-specific targeted deletion of Pim1. Pim1 was expressed at all stages of B-cell development and hematopoietic-specific deletion of Pim1 altered B-cell development in BM, spleen and peritoneal. However, Pim1 deficiency did not affect T-cell development. Studies of BM chimeric mice showed that Pim1 is required in a cell-intrinsic manner to maintain normal B-cell development. Pim1 deficiency led to significant changes in B cell antibody responses. Additionally, Pim1 deficiency resulted in reduced B cell receptor (BCR)-induced cell proliferation and cell cycle progression. Examination of the various BCR-activated signaling pathways in Pim1-deficient B cells reveals defective activation of mitogen-activated protein kinases (MAPKs), which are known to regulate genes involved in cell proliferation and survival. qRT-PCR analysis of BCR-engaged B cells from Pim1-deficient B cells revealed reduced expression of cyclin-dependent kinase (CDK) and cyclin genes, including CDK2, CCNB1 and CCNE1. In conclusion, Pim1 plays a crucial role in B-cell development and B cell activation.

Being associated with multiple diseases, CD79a, as a B-cell marker plays an important role in disease treatment and prognosis

CD79a, a membrane glycoprotein critical for B-cell receptor (BCR) signaling, plays a vital role in B-cell development and immune responses. It serves as a marker for normal and tumor B-cells and is implicated in the progression of various diseases, including B-cell lymphomas, leukemia, autoimmune disorders, and other systemic diseases such as neurological, hematological, and respiratory disorders. This review provides a comprehensive analysis of the discovery, molecular structure, and function of CD79a, along with its regulatory mechanisms at the transcriptional and post-translational levels. CD79a’s involvement in signaling pathways and its potential as a therapeutic target for novel treatments, such as chimeric antigen receptor-T (CAR-T) cell therapy and bispecific antibodies, are also explored. The review highlights emerging therapeutic strategies targeting CD79a, emphasizing its significance in the prognosis of CD79a-related diseases and the ongoing need for further research to optimize clinical interventions.

Inhibiting Bruton's Tyrosine Kinase to Counteract Chemoresistance and Stem Cell-Like Properties in Osteosarcoma.

Osteosarcoma, a highly aggressive bone cancer, often develops resistance to conventional chemotherapeutics, leading to poor prognosis and survival rates. The malignancy and chemoresistance of osteosarcoma pose significant challenges in its treatment, highlighting the critical need for novel therapeutic approaches. Bruton's tyrosine kinase (BTK) plays a pivotal role in B-cell development and has been linked to various cancers, including breast, lung, and oral cancers, where it contributes to tumor growth and chemoresistance. Despite its established importance in these malignancies, the impact of BTK on osteosarcoma remains unexplored. Our study delves into the expression levels of BTK in osteosarcoma tissues by data from the GEO and TCGA database, revealing a marked increase in BTK expression compared with primary osteoblasts and a potential correlation with primary site progression. Through our investigations, we identified a subset of osteosarcoma cells, named cis-HOS, which exhibited resistance to cisplatin. These cells displayed characteristics of cancer stem cells (CSCs), demonstrated a higher angiogenesis effect, and had an increased migration ability. Notably, an upregulation of BTK was observed in these cisplatin-resistant cells. The application of ibrutinib, a BTK inhibitor, significantly mitigated these aggressive traits. Our study demonstrates that BTK plays a crucial role in conferring chemoresistance in osteosarcoma. The upregulation of BTK in cisplatin-resistant cells was effectively countered by ibrutinib. These findings underscore the potential of targeting BTK as an effective strategy to overcome chemoresistance in osteosarcoma treatment.

Abstract PO-020: ZBTB24 is a novel tumor suppressor and cooperates with CDKN2A to suppress B-cell lymphomagenesis

Abstract B-cell lymphoma is the most common hematological malignancy that accounts for the sixth highest mortality among all cancers. Despite recent improvements on combined chemotherapy and immunotherapy, treatments remain challenging due to complex genetic heterogeneity and molecular pathogenesis. To explore novel mechanisms in B-cell lymphomagenesis, we performed a genome-wide CRISPR knockout screen in the murine interleukin-3 (IL-3)-dependent Ba/F3 pro-B cell line and identified Zbtb24 as a putative target whose silencing leads to elevated proliferative signaling and reduced apoptosis, conferring IL-3-independent growth in Ba/F3 cells. Silencing of ZBTB24 also accelerated growth in multiple human B-cell lymphoma cell lines. ZBTB24 is a member of the zinc-finger and BTB domain containing transcription factor that is highly expressed in spleen and bone marrow. While it has been shown that ZBTB24 may play an important role in B-cell development and activation, its involvement in B-cell lymphoma has not been described. Analysis of publicly available datasets found that ZBTB24 copy number deletion occurs in various B-cell lymphomas, which frequently co-occurs with deletion of the tumor suppressor CDKN2A. Hence, we generated B-cell specific Zbtb24 single knockout and Zbtb24/Cdkn2a double knockout mice to explore the role of Zbtb24 in vivo. While Zbtb24 and Cdkn2a single knockout mice remained tumor-free or showed low penetrance of tumorigenesis, the double knockout mice developed lymphadenopathy and splenomegaly accompanied with accelerated mortality. Flow cytometry analysis of bone marrow, spleen, and lymph nodes revealed malignant B-cells. Subsequent transplantation of the splenic cells induced identical malignant B-cell lymphomagenesis and immunophenotype in athymic nude mice, further confirming its malignant identity. These results indicate that ZBTB24 is a novel tumor suppressor in B-cell malignancy. Ongoing studies are being conducted to understand the mechanistic basis for ZBTB24 tumor suppressive function that may involve transcriptional regulation of its target gene expression. This project is supported by the NIH R01CA232246, R01CA129536, and the John Colaizzi Endowment Fund. Citation Format: Rongrong Li, Namratha Sheshadri, Jianliang Shen, Junrong Yan, Jaeyong Jung, Muhammad Usama Tariq, Y Lynn Wang, Ping Xie, Wei-Xing Zong. ZBTB24 is a novel tumor suppressor and cooperates with CDKN2A to suppress B-cell lymphomagenesis [abstract]. In: Proceedings of the Fourth AACR International Meeting on Advances in Malignant Lymphoma: Maximizing the Basic-Translational Interface for Clinical Application; 2024 Jun 19-22; Philadelphia, PA. Philadelphia (PA): AACR; Blood Cancer Discov 2024;5(3_Suppl):Abstract nr PO-020.

CXCR4: from B-cell development to B cell-mediated diseases.

Chemokine receptors are members of the G protein-coupled receptor superfamily. The C-X-C chemokine receptor type 4 (CXCR4), one of the most studied chemokine receptors, is widely expressed in hematopoietic and immune cell populations. It is involved in leukocyte trafficking in lymphoid organs and inflammatory sites through its interaction with its natural ligand CXCL12. CXCR4 assumes a pivotal role in B-cell development, ranging from early progenitors to the differentiation of antibody-secreting cells. This review emphasizes the significance of CXCR4 across the various stages of B-cell development, including central tolerance, and delves into the association between CXCR4 and B cell-mediated disorders, from immunodeficiencies such as WHIM (warts, hypogammaglobulinemia, infections, and myelokathexis) syndrome to autoimmune diseases such as systemic lupus erythematosus. The potential of CXCR4 as a therapeutic target is discussed, especially through the identification of novel molecules capable of modulating specific pockets of the CXCR4 molecule. These insights provide a basis for innovative therapeutic approaches in the field.

SLC39A10 promotes malignant phenotypes of gastric cancer cells by activating the CK2-mediated MAPK/ERK and PI3K/AKT pathways

Solute carrier family 39 member 10 (SLC39A10) belongs to a subfamily of zinc transporters and plays a key role in B-cell development. Previous studies have reported that its upregulation promotes breast cancer metastasis by enhancing the influx of zinc ions (Zn2+); however, its role in gastric cancer remains totally unclear. Here, we found that SLC39A10 expression was frequently increased in gastric adenocarcinomas and that SLC39A10 upregulation was strongly associated with poor patient outcomes; in addition, we identified SLC39A10 as a direct target of c-Myc. Functional studies showed that ectopic expression of SLC39A10 in gastric cancer cells dramatically enhanced the proliferation, colony formation, invasiveness abilities of these gastric cancer cells and tumorigenic potential in nude mice. Conversely, SLC39A10 knockdown inhibited gastric cancer cell proliferation and colony formation. Mechanistically, SLC39A10 exerted its carcinogenic effects by increasing Zn2+ availability and subsequently enhancing the enzyme activity of CK2 (casein kinase 2). As a result, the MAPK/ERK and PI3K/AKT pathways, two major downstream effectors of CK2, were activated, while c-Myc, a downstream target of these two pathways, formed a vicious feedback loop with SLC39A10 to drive the malignant progression of gastric cancer. Taken together, our data demonstrate that SLC39A10 is a functional oncogene in gastric cancer and suggest that targeting CK2 is an alternative therapeutic strategy for gastric cancer patients with high SLC39A10 expression.

Abstract CT128: Phase 1 study of HSK29116, a Bruton tyrosine kinase (BTK) proteolysis-targeting chimera (PROTAC) agent, in patients with relapsed or refractory B-cell malignancies

Abstract BACKGROUND: BTK, a non-receptor kinase in the B-cell receptor signaling pathway, plays an essential role in B-cell development and differentiation. BTK is critical for malignant B-cell survival. Oral BTK inhibitors (BTKis), standard treatments for patients (pts) with B-cell malignancies, prevent proliferation and induce apoptosis of B cells. Currently approved BTKis covalently bind to BTK residue C481, irreversibly inhibiting phosphorylation of downstream kinases and blocking B-cell activation. Because these agents are often given continuously, their use may lead to clinical resistance and unacceptable toxicities. Indeed, acquired resistance to covalent BTKis is common and caused by the expansion of clones with a cysteine-to-serine mutation at residue 481. A novel strategy being developed to overcome BTKi resistance, is PROTAC-induced degradation of BTK. PROTACs are small molecules with one domain that binds the target protein linked to another domain that binds to ubiquitin E3 ligase, resulting in ubiquitin-dependent proteasome degradation of the target protein. HSK29116 is a small BTK PROTAC molecule that is currently undergoing clinical investigation for the treatment of B-cell malignancies. In preclinical studies, HSK29116 overcame drug resistance caused by the C481S BTK mutation. Also, HSK29116 has demonstrated inhibition of B-cell proliferation without measurable loss of other BTK-family kinase activities. This kinase selectivity may limit some of the off-target toxicities seen with currently available BTKis. METHODS: This first-in-human, multicenter, open-label, phase 1 study (NCT04861779) began recruiting in 2021 to evaluate the safety, tolerability, pharmacokinetics (PK), and pharmacodynamics of oral HSK29116. Eligible pts are ≥18 years old with diagnosis of a B-cell malignancy that has relapsed or become refractory to standard therapy (≥2 prior systemic therapies, which may have included a BTKi) and for which no other treatments known to provide clinical benefit exist. Pts must have an Eastern Cooperative Oncology Group performance status ≤2 and life expectancy >3 months. Phase 1a includes dose escalation to identify dose-limiting toxicities and establish the maximum tolerated dose and/or recommended dose of HSK29116 for a phase 1b dose expansion, initiating in the US. Approximately 36 pts will be enrolled in phase 1a and 90 pts in phase 1b (≥40 pts must have previously received covalent BTKis). The primary endpoint is safety. Secondary endpoints include PK and investigator-assessed overall response rate, duration of response, time to response, and progression-free survival. Exploratory analyses will evaluate the relationship between HSK29116 anti-tumor activity and BTK gene mutations, as well as BTK protein degradation effects of HSK29116. Descriptive statistics will be used to summarize all data. Citation Format: Jianyong Li, Wei Xu, Pangke Yan, Yong Cao, Mengyue Hu, William Daley. Phase 1 study of HSK29116, a Bruton tyrosine kinase (BTK) proteolysis-targeting chimera (PROTAC) agent, in patients with relapsed or refractory B-cell malignancies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr CT128.

NONO regulates B-cell development and B-cell receptor signaling.

The paraspeckle protein NONO is a multifunctional nuclear protein participating in the regulation of transcriptional regulation, mRNA splicing and DNA repair. However, whether NONO plays a role in lymphopoiesis is not known. In this study, we generated mice with global deletion of NONO and bone marrow (BM) chimeric mice in which NONO is deleted in all of mature B cells. We found that the global deletion of NONO in mice did not affect T-cell development but impaired early B-cell development in BM at pro- to pre-B-cell transition stage and B-cell maturation in the spleen. Studies of BM chimeric mice demonstrated that the impaired B-cell development in NONO-deficient mice is B-cell-intrinsic. NONO-deficient B cells displayed normal BCR-induced cell proliferation but increased BCR-induced cell apoptosis. Moreover, we found that NONO deficiency impaired BCR-induced activation of ERK, AKT, and NF-κB pathways in B cells, and altered BCR-induced gene expression profile. Thus, NONO plays a critical role in B-cell development and BCR-induced B-cell activation.

CK2β-regulated signaling controls B cell differentiation and function.

Serine-Threonine kinase CK2 supports malignant B-lymphocyte growth but its role in B-cell development and activation is largely unknown. Here, we describe the first B-cell specific knockout (KO) mouse model of the β regulatory subunit of CK2. CK2βKO mice present an increase in marginal zone (MZ) and a reduction in follicular B cells, suggesting a role for CK2 in the regulation of the B cell receptor (BCR) and NOTCH2 signaling pathways. Biochemical analyses demonstrate an increased activation of the NOTCH2 pathway in CK2βKO animals, which sustains MZ B-cell development. Transcriptomic analyses indicate alterations in biological processes involved in immune response and B-cell activation. Upon sheep red blood cells (SRBC) immunization CK2βKO mice exhibit enlarged germinal centers (GCs) but display a limited capacity to generate class-switched GC B cells and immunoglobulins. In vitro assays highlight that B cells lacking CK2β have an impaired signaling downstream of BCR, Toll-like receptor, CD40, and IL-4R all crucial for B-cell activation and antigen presenting efficiency. Somatic hypermutations analysis upon 4-Hydroxy-3-nitrophenylacetyl hapten conjugated to Chicken Gamma Globulin (NP-CGG) evidences a reduced NP-specific W33L mutation frequency in CK2βKO mice suggesting the importance of the β subunit in sustaining antibody affinity maturation. Lastly, since diffuse large B cell lymphoma (DLBCL) cells derive from GC or post-GC B cells and rely on CK2 for their survival, we sought to investigate the consequences of CK2 inhibition on B cell signaling in DLBCL cells. In line with the observations in our murine model, CK2 inactivation leads to signaling defects in pathways that are essential for malignant B-lymphocyte activation.

ARID5B influences B-cell development and function in mouse.

There is growing evidence for an inherited basis of susceptibility to childhood acute lymphoblastic leukemia. Genomewide association studies by us and others have identified non-coding acute lymphoblastic leukemia risk variants at the ARID5B gene locus, but the molecular mechanisms linking ARID5B to normal and malignant hematopoiesis remain largely unknown. Using a Vav1-driven transgenic mouse model, we characterized the role of Arid5b in hematopoiesis in vivo. Arid5b overexpression resulted in a dramatic reduction in the proportion of circulating B cells, immature, and mature Bcell fractions in the peripheral blood and the bone marrow, and also a decrease of follicular B cells in the spleen. There were significant defects in B-cell activation upon Arid5b overexpression in vitro with hyperactivation of B-cell receptor signaling at baseline. In addition, increased mitochondrial oxygen consumption rate of naïve or stimulated B cells of Arid5b-overexpressing mice was observed, compared to the rate of wild-type counterparts. Taken together, our results indicate that ARID5B may play an important role in B-cell development and function.

Abstract 5454: Amdizalisib (HMPL-689), a highly selective PI3Kδ inhibitor, exhibits potent anti-tumor activity in pre-clinical B-cell lymphoma models

Abstract Background: The delta isoform of PI3K (PI3Kδ) plays an important role in B-cell development and function by mediating the signaling of key receptors on B cells. Aberrant activation of PI3Kδ signaling pathway promotes the survival and proliferation of malignant B cells, making selective inhibition of this isoform a promising therapeutic approach for the treatment of B cell malignances. Amdizalisib (HMPL-689, Amdiz), discovered and being currently developed in a pivotal phase II clinical trial (NCT04849351) by HUTCHMED, is a highly potent and selective PI3Kδ inhibitor. Based on encouraging clinical data from the phase Ib study (2021 ESMO, abstract# 8330), Amdiz is a designated breakthrough therapy in China for treatment of relapsed and refractory follicular lymphoma. Herein, we report the pre-clinical anti-tumor activity of Amdiz. Methods: Kinase activity was measured by Transcreener™ Fluorescence Polarization assay. The kinome selectivity profile of Amdiz was evaluated in Eurofins KinaseProfiler™ panel containing 323 kinases. Cell based phosphorylation of AKT was determined by using Acumen Explorer system. CD63 expression in basophils was evaluated by FACS assay. Inhibitory effects of Amdiz alone or in combination with other agents on cell viability were investigated in a panel of B cell lymphoma cell lines by CellTiter-Glo luminescent or CCK-8 assay. The ability of Amdiz to inhibit the activation of B cells in animals was evaluated in naïve rats using an ex vivo assay, and B cell activation was determined by evaluating CD86 expression using FACS analysis. Human B cell lymphoma cell line derived xenograft models were used to determine the anti-tumor activity of Amdiz in combination with other agents such as rituximab, BTK inhibitor, and venetoclax. Results: Amdiz is a highly selective inhibitor of PI3Kδ, showing more than 250-fold selectivity over other PI3K isoforms and no significant inhibition over other 319 protein kinases at the concentration of 1 µM. Amdiz potently inhibited PI3Kδ in biochemical, cellular and human whole blood assays with IC50s ranging from 0.8-3 nM. Its inhibitory effects on cell viability were also evaluated in a panel of B -cell lymphoma cell lines. Results showed that Amdiz potently inhibited cell survival with IC50s from 0.005 to 5 μM. Amdiz showed a long-lasting and strong inhibition on B cell activation in a rat pharmacodynamics (PD) study at a dose as low as 0.1 mg/kg. Moreover, Amdiz significantly improved anti-tumor activity of standard-of-care agents as well as targeted agents in multiple B-cell lymphoma models both in vitro and in vivo. Conclusion: Amdiz is a highly potent and selective PI3Kδ inhibitor with strong activity against B-cell lymphoma in pre-clinical studies, supporting clinical evaluation as either a single agent or in combination with other therapeutic agents for the treatment of B-cell malignancies. Citation Format: Jia Hu, Jianhan Wang, Xiaoming Dai, Jianlin He, Junqing Liang, Ying Yu, Juntao Yu, Na Yang, Linfang Wang, Yu Cai, Xiong Li, Weiguo Qing, Yongxin Ren, Weiguo Su. Amdizalisib (HMPL-689), a highly selective PI3Kδ inhibitor, exhibits potent anti-tumor activity in pre-clinical B-cell lymphoma models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5454.

Genome-Wide ChIP-seq and RNA-seq Analyses of STAT3 Target Genes in TLRs Activated Human Peripheral Blood B Cells.

Toll like receptors (TLRs) induced response plays a vital role in B-cell development and activation, in which TLR7-mediated and TLR9-mediated response interact together and play antagonistic or cooperative roles at different situations. Previous studies showed that the transcription factor signal transducer and activator of transcription (STAT) 3 was one of the key transcriptional factors (TFs) needed for both TLR7 and TLR9 signaling in B cell, and patients with autosomal dominant hyper IgE syndromes (AD-HIES) due to STAT3 mutations having defective TLRs response in B cells. However, how STAT3 affects its target genes and the downstream signaling pathways in B cell upon TLRs stimulation remains unclarified on a genome-wide level. ChIP-seq and RNA-seq was used in this study to identify the STAT3 targets in response to TLRs stimulation in human B cell. STAT3 ChIP-seq results showed a total of 611 and 2,289 differential STAT3-binding sites in human B cell after TLR7 and TLR9 agonists stimulation, respectively. RNA-seq results showed 1,186 and 1,775 differentially expressed genes after TLR7 and TLR9 activation, respectively. We identified 47 primary STAT3 target genes after TLR7 activation and 189 target genes after TLR9 activation in B cell by integration of STAT3 ChIP-seq and RNA-seq data. Among these STAT3 primary targets, we identified 7 TFs and 18 TFs for TLR7 and TLR9 response, respectively. Besides, we showed that STAT3 might regulate TLR9, but not TLR7 response in B cells through directly regulating integrin signaling pathway, which might further affect the antagonism between TLR7 and TLR9 signaling in B cell. Our study provides insights into the molecular mechanism of human TLRs response in B cell and how it can be regulated, which helps to better understand and modulate TLR-mediated pathogenic immune responses in B cell.

Frequency and Correlation of Common Genes Copy Number Alterations in Childhood Acute Lymphoblastic Leukemia with Prognosis.

Objective:It was shown by genomic profiling that despite no detectable chromosomal abnormalities a proportion of children with pre-B acute lymphoblastic leukemia harbors copy number alterations (CNA) of genes playing role in B-cell development and function. The aim of the study was to determine the frequency of CNA in pediatric acute lymphoblastic leukemia and correlate these findings with clinical outcome. Methods:DNA extracted from peripheral blood or bone marrow at diagnosis/relapse of fifty newly diagnosed children with precursor B-cell acute lymphoblastic leukemia was analyzed for CNA with multiplex ligation-dependent probe amplification. Results:The analysis revealed 76 CNA in 24 patients most frequently found in PAR1 (17%), CDKN2A/B (15.7%) and PAX5 (14.4%) genes. There were significant CNA co-occurrences between PAX5, CDKN2A/B, BTG1, ETV6, PAR1 or XP22 genes, (p<0.020) and the high-risk group. There was a significant correlation between EBF1, RB1, and IKZF1 alterations and bone marrow relapse. Patients with CNA in screened genes are more likely to succumb to their disease except for those with PAR1 or XP22 genes (p<0.050). Conclusion:The multiplex ligation-dependent probe amplification could be considered as an independent diagnostic tool allowing prompt identification of patients at high risk of treatment failure and, subsequently, a more adequate treatment approach.

SHC014748M, a novel selective inhi-bitor of PI3Kδ, demonstrates promising preclinical antitumor activity in B cell lymphomas and chronic lymphocytic leukemia

PI3Kδ (phosphatidylinositol 3-kinase-δ), one of the class I PI3Ks, is found expressed primarily in leukocytes and plays an essential role in B-cell development and function. This provides a rationale for the development of small molecule inhibitors that selectively target p110δ for patients with indolent non-Hodgkin lymphomas. Here in this paper, we comprehensively evaluated the in vitro and in vivo antitumor activity of SHC014748M, an oral selective inhibitor of PI3Kδ under Phase I clinical evaluation. Biochemical and cell-based assays were used to measure compound potency and selectivity in lymphoma cell lines as well as primary chronic lymphocytic leukemia (CLL) cells. Scid mice were subcutaneously inoculated with the SU-DHL-6 cell line. SHC014748M was more selective for PI3Kδ inhibition relative to other class I PI3K enzymes and showed in vitro activity in most of 23 B lymphoma cell lines and primary CLL cells. SHC014748M also inhibited phosphorylation of AKT, targets downstream of PI3Kδ, in both lymphoma cells and primary CLL cells. In vivo study revealed that SHC014748M significantly reduced lymphoma cell growth in the treatment group compared with control mice. CCL4, CCL17, CCL22 and CXCL13 in patient serum decreased sharply after SHC014748M treatment. According to the results, SHC014748M appeared to be a novel promising compound in the treatment of B cell lymphomas and CLL.

Drug interactions with Bruton's tyrosine kinase inhibitors: clinical implications and management.

Bruton's tyrosine kinase (BTK) plays an essential role in B-cell development, differentiation and B-cell receptor (BCR) signaling. The use of Bruton's tyrosine kinase inhibitors (BTKi) in the treatment of lymphoid malignancies has dramatically increased, owing to both impressive efficacy and ease of administration. However, BTKi have a range of drug-drug and drug-food interactions, which may alter drug efficacy and/or increase toxicity. Healthcare professionals should be aware of the probability of drug interactions with BTKi and make recommendations accordingly. In this article, we discuss the relevant drug-drug and drug-food interactions associated with ibrutinib, acalabrutinib, and zanubrutinib, and provide clinical practice recommendations for managing these interactions based on the available literature.

Effects of AS2819899, a novel selective PI3Kδ inhibitor, in a NZB/W F1 mouse lupus-like nephritis model

Phosphoinositide 3-kinases generate lipid-based second messengers that control an array of intracellular signaling pathways. In particular, phosphoinositide 3-kinases delta (PI3Kδ) is expressed primarily in hematopoietic cells and plays an important role in B-cell development and function. B cells play a critical role in autoimmune diseases by producing autoantibodies. Studies have therefore increasingly focused on PI3Kδ as a therapeutic target for the treatment of inflammatory and autoimmune diseases. One such autoimmune disease is systemic lupus erythematosus (SLE). SLE is a chronic systemic autoimmune disease with repeated recurrence and remission, and autoantibodies play an important role in its pathogenesis. Here, we examined the pharmacological profile of the novel PI3Kδ selective inhibitor AS2819899 and investigated its therapeutic potential against SLE in a NZB/W F1 mouse lupus-like nephritis model, a widely-used SLE mouse model. AS2819899 prevented B and T cell activation in vitro, and inhibited antibody production in a T-cell independent de novo antibody production mouse model. In the spontaneous NZB/W F1 mouse model, AS2819899 treatment significantly reduced anti-dsDNA antibody titers and improved kidney dysfunction. Further, AS2819899 inhibited the memory recall reaction in a T-cell dependent antibody production mouse model, suggesting that AS2819899 can potentially maintain remission of SLE. Moreover, we identified a pharmacodynamics marker for AS2819899 that may be useful in clinical studies. These results indicate that AS2819899 may be an attractive therapeutic candidate for SLE, including the maintenance of remission.

APRIL-dependent lifelong plasmacyte maintenance and immunoglobulin production in humans

Interactions between the tumor necrosis factor (TNF) ligand superfamily and TNF receptor superfamily play critical roles in B-cell development and maturation. Aproliferation-inducing ligand (APRIL), a member of the TNF ligand superfamily, is secreted from myeloid cells and known to induce the differentiation of memory B cells to plasmacytes. We sought to elucidate the role of APRIL in B-cell differentiation and immunoglobulin production through the analysis of complete APRIL deficiency in human. We performed whole exome sequencing in a patient with adult common variable immunodeficiency. His parents were in a consanguineous marriage. TNFSF13 mRNA and protein expression were analyzed in the primary cells and plasma from the patient and in cDNA-transfected cells and supernatants of the cultures invitro. Immunologic analysis was performed by using flow cytometry and next-generation sequencing. Monocyte-derived dendritic cells differentiated from induced pluripotent stem cells (iPSC-moDCs) were cocultured with memory B cells from healthy controls to examine invitro plasmacyte differentiation. We identified a homozygous frameshift mutation in TNFSF13, the gene encoding APRIL, in the patient. APRIL mRNA and protein were completely absent in the monocytes and iPSC-moDCs of the patient. In contrast to the results of previous animal model studies, the patient showed hypogammaglobulinemia with a markedly reduced level of plasmacytes in peripheral blood and a clearly increased level of circulating marginal zone B cells. Although iPSC-moDC-induced invitro plasmacyte differentiation was reduced in the patient, recombinant APRIL supplementation corrected this abnormality. The first APRIL deficiency in an adult patient with common variable immunodeficiency revealed the role of APRIL in lifelong maintenance of plasmacytes and immunoglobulin production in humans.

Generation and Validation of an Antibody to Canine CD19 for Diagnostic and Future Therapeutic Purposes.

The B-cell coreceptor, CD19 is a transmembrane protein expressed throughout B-cell ontogeny from pro-B cell to plasmablast. It plays an important role in B-cell development and function and is an attractive target for antibody-directed immunotherapies against B-cell malignancies, including acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL), and non-Hodgkin lymphoma (B-NHL) in humans. With the rapid development of next-generation immunotherapies aimed at improving therapeutic efficacy, there is a pressing need for a clinically relevant, immune-competent, spontaneous animal model to derisk these new approaches and inform human immunotherapy clinical trials. Pet dogs develop spontaneous B-cell malignancies, including B-NHL and leukemias that share comparable oncogenic pathways and similar immunosuppressive features to human B-cell malignancies. Despite treatment with multiagent chemotherapy, durable remissions in canine B-NHL are rare and most dogs succumb to their disease within 1 year of diagnosis. Here we report the development and validation of an anti-canine CD19-targeting monoclonal antibody and its single-chain derivatives, which enable next-generation CD19-targeted immunotherapies to be developed and evaluated in client-owned dogs with spontaneous B-NHL. These future in vivo studies aim to provide important information regarding the safety and therapeutic efficacy of CD19-targeted mono- and combination therapies and identify correlative biomarkers of response that will help to inform human clinical trial design. In addition, development of canine CD19-targeted immunotherapies aims to provide better therapeutic options for pet dogs diagnosed with B-cell malignancies.

Hematopoiesis in aged female mice devoid of thyroid hormone receptors.

Hypothyroidism is often associated with anemia and immunological disorders. Similar defects are found in patients and in mice with a mutated dominant-negative thyroid hormone receptor α (TRα) and in knockout mice devoid of this receptor, suggesting that this isoform is responsible for the effects of the thyroid hormones in hematopoiesis. However, the hematological phenotype of mice lacking also TRβ has not yet been examined. We show here that TRα1/TRβ-knockout female mice, lacking all known thyroid hormone receptors with capacity to bind thyroid hormones, do not have overt anemia and in contrast with hypothyroid mice do not present reduced Gata1 or Hif1 gene expression. Similar to that found in hypothyroidism or TRα deficiency during the juvenile period, the B-cell population is reduced in the spleen and bone marrow of ageing TRα1/TRβ-knockout mice, suggesting that TRβ does not play a major role in B-cell development. However, splenic hypotrophy is more marked in hypothyroid mice than in TRα1/TRβ-knockout mice and the splenic population of T-lymphocytes is not significantly impaired in these mice in contrast with the reduction found in hypothyroidism. Our results show that the overall hematopoietic phenotype of the TRα1/TRβ-knockout mice is milder than that found in the absence of hormone. Although other mechanism/s cannot be ruled out, our results suggest that the unoccupied TRs could have a negative effect on hematopoiesis, likely secondary to repression of hematopoietic gene expression.

Glucocorticoids Regulate the Splicing Factor MBNL1, a Potential Control Point for B-Cell Specification

Glucocorticoids, such as dexamethasone and prednisone, are mainstays in the treatment of lymphoid malignancies and autoimmune diseases. To understand the mechanism of how glucocorticoids induce cell death, we integrated a comprehensive, genome-wide shRNA screen with differential gene expression analysis in B-cell acute lymphoblastic leukemia (B-ALL). In addition to activating pro-apoptosis genes and repressing anti-apoptosis genes, we found that repression of B-cell development genes contributes to dexamethasone-induced cell death in B-ALL. Similarly, elevated glucocorticoid levels brought on by stress in stroke victims causes lymphocytopenia by inducing of apoptosis in immature B-cells, but not hemopoietic stem cells or mature B-cells. Additionally, chronic stress has been associated with immune suppression and increased risk for autoimmune disease. Together these findings suggest that glucocorticoids play a normal role in B-cell development, and that elevated glucocorticoid levels in response to stress can alter that development. A control point in this mechanism may be MBNL1. MBNL1 is a sequence-specific RNA binding factor that alters mRNA splicing, stability, and localization. In this work, we show that glucocorticoid suppression of MBNL1 contributes to induction of cell death in B-ALL. Deletion of MBNL1 by CRISPR/Cas9 in the B-ALL cell line NALM-6 induces accumulation and depletion of mRNA levels for hundreds of genes. A large number of B-cell specification genes, including CD19, CD79A, SPI1, and LEF1, are repressed upon MBNL1 deletion, whereas the most highly activated gene is CD34, which is a marker for the less mature lymphoid progenitor cells. Depletion of MBNL1 also changed the splicing of B-cell development genes, including quantitative depletion of LEF1 exon 6. These data suggest that depletion of MBNL1 induces de-differentiation of B-cell precursors. Consistent with this model, an analysis of gene expression in differentiating B-cells revealed a rapid elevation of MBNL1 expression during B-cell specification (after the lymphoid precursor stage), and a concomitant repression of CELF2, an RNA-binding splicing factor that has been shown to oppose MBNL1. We therefore propose that glucocorticoids play a physiological role in B-cell development by restraining B-cell specification through repression of key genes, including MBNL1. Figure Disclosures Tasian: Aleta Biotherapeutics: Membership on an entity's Board of Directors or advisory committees; Gilead Sciences: Research Funding; Incyte Corportation: Research Funding.