DNA methylation regulates B cell activation via repressing Pax5 expression in teleost.

In mammals, the transcription factor Pax5 is a key regulator of B cell development and maturation and specifically expressed in naive/mature B cells but repressed upon B cell activation. Despite the long-standing proposal that Pax5 repression is essential for proper B cell activation, the underlying mechanisms remain largely elusive. In this study, we used a teleost model to elucidate the mechanisms governing Pax5 repression during B cell activation. Treatment with lipopolysaccharide (LPS) and chitosan oligosaccharide (COS) significantly enhanced the antibody secreting ability and phagocytic capacity of IgM+ B cells in large yellow croaker (Larimichthys crocea), coinciding with upregulated expression of activation-related genes, such as Bcl6, Blimp1, and sIgM, and downregulated expression of Pax5. Intriguingly, two CpG islands were identified within the promoter region of Pax5. Both CpG islands exhibited hypomethylation in naive/mature B cells, while CpG island1 was specifically transited into hypermethylation upon B cell activation. Furthermore, treatment with DNA methylation inhibitor 5-aza-2'-deoxycytidine (AZA) prevented the hypermethylation of CpG island1, and concomitantly impaired the downregulation of Pax5 and activation of B cells. Finally, through in vitro methylation experiments, we demonstrated that DNA methylation exerts an inhibitory effect on promoter activities of Pax5. Taken together, our findings unveil a novel mechanism underlying Pax5 repression during B cell activation, thus promoting the understanding of B cell activation process.

EBF1, PAX5, and MYC: regulation on B cell development and association with hematologic neoplasms.

During lymphocyte development, a diverse repertoire of lymphocyte antigen receptors is produced to battle against pathogens, which is the basis of adaptive immunity. The diversity of the lymphocyte antigen receptors arises primarily from recombination-activated gene (RAG) protein-mediated V(D)J rearrangement in early lymphocytes. Furthermore, transcription factors (TFs), such as early B cell factor 1 (EBF1), paired box gene 5 (PAX5), and proto-oncogene myelocytomatosis oncogene (MYC), play critical roles in regulating recombination and maintaining normal B cell development. Therefore, the aberrant expression of these TFs may lead to hematologic neoplasms.

Essential role of the Pax5 C-terminal domain in controlling B cell commitment and development.

The B cell regulator Pax5 consists of multiple domains whose function we analyzed in vivo by deletion in Pax5. While B lymphopoiesis was minimally affected in mice with homozygous deletion of the octapeptide or partial homeodomain, both sequences were required for optimal B cell development. Deletion of the C-terminal regulatory domain 1 (CRD1) interfered with B cell development, while elimination of CRD2 modestly affected B-lymphopoiesis. Deletion of CRD1 and CRD2 arrested B cell development at an uncommitted pro-B cell stage. Most Pax5-regulated genes required CRD1 or both CRD1 and CRD2 for their activation or repression as these domains induced or eliminated open chromatin at Pax5-activated or Pax5-repressed genes, respectively. Co-immunoprecipitation experiments demonstrated that the activating function of CRD1 is mediated through interaction with the chromatin-remodeling BAF, H3K4-methylating Set1A-COMPASS, and H4K16-acetylating NSL complexes, while its repressing function depends on recruitment of the Sin3-HDAC and MiDAC complexes. These data provide novel molecular insight into how different Pax5 domains regulate gene expression to promote B cell commitment and development.

PAX5-miR-142 feedback loop promotes breast cancer proliferation by regulating DNMT1 and ZEB1.

BACKGROUND: Breast cancer is one of the most common malignancies occurred in female around the globe. Recent studies have revealed the crucial characters of miRNA and genes, as well as the essential roles of epigenetic regulation in breast cancer initiation and progression. In our previous study, miR-142-3p was identified as a tumor suppressor and led to G2/M arrest through targeting CDC25C. However, the specific mechanism is still uncertain. METHODS: We identified PAX5 as the upstream regulator of miR-142-5p/3p through ALGGEN website and verified by series of assays in vitro and in vivo. The expression of PAX5 in breast cancer was detected by qRT-PCR and western blot. Besides, bioinformatics analysis and BSP sequencing were performed to analyze the methylation of PAX5 promoter region. Finally, the binding sites of miR-142 on DNMT1 and ZEB1 were predicted by JASPAR, and proved by luciferase reporter assay, ChIP analysis and co-IP. RESULTS: PAX5 functioned as a tumor suppressor by positive regulation of miR-142-5p/3p both in vitro and in vivo. The expression of PAX5 was regulated by the methylation of its promoter region induced by DNMT1 and ZEB1. In addition, miR-142-5p/3p could regulate the expression of DNMT1 and ZEB1 through binding with their 3'UTR region, respectively. CONCLUSION: In summary, PAX5-miR-142-DNMT1/ZEB1 constructed a negative feedback loop to regulate the progression of breast cancer, which provided emerging strategies for breast cancer therapy.

PAX5 fusion genes in acute lymphoblastic leukemia: A literature review.

Acute lymphoblastic leukemia (ALL) is a common cancer affecting children worldwide. The development of ALL is driven by several genes, some of which can be targeted for treatment by inhibiting gene fusions. PAX5 is frequently mutated in ALL and is involved in chromosomal rearrangements and translocations. Mutations in PAX5 interact with other genes, such as ETV6 and FOXP1, which influence B-cell development. PAX5/ETV6 has been observed in both B-ALL patients and a mouse model. The interaction between PAX5 and FOXP1 negatively suppresses the Pax5 gene in B-ALL patients. Additionally, ELN and PML genes have been found to fuse with PAX5, leading to adverse effects on B-cell differentiation. ELN-PAX5 interaction results in the decreased expression of LEF1, MB1, and BLNK, while PML-PAX5 is critical in the early stages of leukemia. PAX5 fusion genes prevent the transcription of the PAX5 gene, making it an essential target gene for the study of leukemia progression and the diagnosis of B-ALL.

EBF1-JAK2 inhibits the PAX5 function through physical interaction with PAX5 and kinase activity.

Gene aberrations of B-cell regulators and growth signal components such as the JAK-STAT pathway are frequently found in B-cell acute lymphoblastic leukemia (B-ALL). EBF1 is a B-cell regulator that regulates the expression of PAX5 and co-operates with PAX5 to regulate B-cell differentiation. Here, we analyzed the function of the fusion protein of EBF1 and JAK2, EBF1-JAK2 (E-J). E-J caused constitutive activation of JAK-STAT and MAPK pathways and induced autonomous cell growth in a cytokine-dependent cell line. E-J did not affect the transcriptional activity of EBF1 but inhibited that of PAX5. Both the physical interaction of E-J with PAX5 and kinase activity of E-J were required for E-J to inhibit PAX5 function, although the detailed mechanism of inhibition remains unclear. Importantly, gene set enrichment analysis using the results of our previous RNA-seq data of 323 primary BCR-ABL1-negative ALL samples demonstrated repression of the transcriptional target genes of PAX5 in E-J-positive ALL cells, which suggests that E-J also inhibited PAX5 function in ALL cells. Our results shed new light on the mechanisms of differentiation block by kinase fusion proteins.

BET inhibition targets ABC-DLBCL constitutive B-cell receptor signaling through PAX5.

B-cell receptor (BCR) signaling is essential for the diffuse large B-cell lymphoma (DLBCL) subtype that originates from activated B-cells (ABCs). ABC-DLBCL cells are sensitive to Bruton tyrosine kinase intervention. However, patients with relapsed or refractory ABC-DLBCL had overall response rates from 33% to 37% for Bruton tyrosine kinase inhibitors, suggesting the evaluation of combination-based treatment for improved efficacy. We investigated the efficacy and mechanism of the bromodomain and extraterminal motif (BET) inhibitor AZD5153 combined with the Bruton tyrosine kinase inhibitor acalabrutinib in ABC-DLBCL preclinical models. AZD5153 is a bivalent BET inhibitor that simultaneously engages the 2 bromodomains of BRD4. Adding AZD5153 to acalabrutinib demonstrated combination benefits in ABC-DLBCL cell line and patient-derived xenograft models. Differential expression analyses revealed PAX5 transcriptional activity as a novel downstream effector of this drug combination. PAX5 is a transcription factor for BCR signaling genes and may be critical for perpetually active BCR signaling in ABC-DLBCL. Our analyses further indicated significant alterations in BCR, RELB/alternative NF-κB, and toll-like receptor/interferon signaling. Validation of these results mapped a positive-feedback signaling loop regulated by PAX5. We demonstrated that AZD5153 decreased PAX5 expression, whereas acalabrutinib disruption of BCR signaling inhibited PAX5 activation. Furthermore, several interferon levels were decreased by AZD5153 and acalabrutinib in tumors. Adding interferon-beta1 (IFNß1) to cells treated with acalabrutinib partially rescued PAX5 activation. Our results demonstrate that AZD5153 enhances the efficacy of acalabrutinib through PAX5 and BCR mechanisms that are critical for ABC-DLBCL.

PAX5 aberrant expression incorporated in MIPI-SP risk scoring system exhibits additive value in mantle cell lymphoma.

Mantle cell lymphoma (MCL) is a subtype of non-Hodgkin lymphoma with highly heterogeneous clinical courses. Paired-box 5 (PAX5), the regulator of B cell differentiation and growth, is abnormally expressed in several types of cancers. Herein, we explored the prognostic value of PAX5 in MCL by comprehensively analyzing the clinical features and laboratory data of 82 MCL cases. PAX5 positivity was associated with shorter overall survival (OS; p = 0.011) and was identified as an independent prognostic factor in MCL patients. The elevated ß2-MG (p = 0.027) and advanced Mantle Cell Lymphoma International Prognostic Index (MIPI) score (p = 0.014) were related to positive PAX5 expression. The MIPI-SP risk scoring system was established and exhibited a superior prognostic value for OS depending on an area under the curve (AUC) of 0.770 (95% CI, 0.658-0.881) than MIPI score. Bioinformatic analysis of PAX5-related genes supported the mechanistic roles of PAX5 in MCL. This study provides insight into the potential role of PAX5 in MCL, and the novel risk scoring system MIPI-SP optimizes the risk stratification and facilitates prognosis evaluation in MCL patients. KEY MESSAGES: • Paired-box 5 positivity indicated adverse prognosis in mantle cell lymphoma patients. • Positive PAX5 expression was related to MIPI score and ß2-MG in MCL patients. • MIPI-SP risk scoring system has superior prognostic value than MIPI score in MCL.

PAX5 and circ1857 affected DLBCL progression and B-cell proliferation through regulating GINS1.

PAX5, a member of the paired box gene family of transcription factors, is a B-cell-specific activator protein that plays important roles during B lymphopoiesis. Two putative PAX5 binding sites in the human GINS1 promoter region were identified. EMSA, ChIP and luciferase assay showed that PAX5 functions as a positive transcription factor for GINS1 expression. Furthermore, coordinated expression of PAX5 and GINS1 was observed in mice B cells under physiological conditions and LPS stimulation situations. A similar pattern was also observed in human DLBCL cell lines under differentiation-inducing conditions. In addition, both PAX5 and GINS1 were highly expressed and significantly correlated in DLBCL specimens and cell lines. These findings suggested that dysregulation of PAX5 played an extremely important role in controlling the universal phenomenon of tumor progression through increased expression of GINS1 in DLBCL. In addition, circ1857 that was generated using back splicing of PAX5 pre-mRNA could further stabilize GINS1 mRNA, modulate GINS1 expression and promote lymphoma progression. To the best of our knowledge, this report is the first to demonstrate the role of GINS1 in DLBCL progression, and the mechanism of GINS1 upregulation using both circ1857 and PAX5 in DLBCL was revealed. Our results suggested that GINS1 may be a possible therapeutic target for DLBCL.

Functional damaging germline variants in ETV6, IKZF1, PAX5 and RUNX1 predisposing to B-cell precursor acute lymphoblastic leukemia.

Recent genome-wide studies have demonstrated that a significant proportion of children with cancer carry predisposing germline variants, with varying incidence according to cancer type. In general, there is a lower incidence of underlying germline predisposing variants among patients with B-cell acute lymphoblastic leukemia (B-ALL) compared to other types of cancer, but higher rates may be found in patients with specific leukemia subtypes. Two categories of ALL-predisposing variants have been described: common polymorphisms, conferring low-penetrance ALL susceptibility, and rare variants, conferring high-penetrance ALL susceptibility. Variants in genes encoding hematopoietic transcription factors are an example of the latter, and include ETV6, IKZF1, PAX5 and RUNX1. Here, we present an overview of the germline variants detected in patients with B-ALL in these four genes and a summary of functional studies analyzing the impacts of these variants upon protein function, and hence their effects with regard to leukemia predisposition. Furthermore, we review specific clinical characteristics of patients with B-ALL, including specific features of the patient or family history and associated somatic genetic characteristics, which are suggestive of underlying germline alterations in one of these genes. This review may be of assistance in the interpretation of patient genetic germline findings, made even more challenging by the absence of a suggestive family history or by an unknown familial cancer history. Despite a low incidence of underlying germline alterations in ETV6, IKZF1, PAX5 and RUNX1 in patients with B-ALL, identification of an underlying ALL predisposition syndrome is relevant to the clinical management of patients and their relatives, as the latter are also at risk of developing cancer.

PAX5 P80R-mutated B-cell acute lymphoblastic leukemia with transformation to histiocytic sarcoma: clonal evolution assessment using NGS-based immunoglobulin clonality and mutation analysis.

Clonality assessment by the detection of immunoglobulin (IG) gene rearrangements is an important method to determine whether two concurrent or subsequent lymphoid malignancies in one patient are clonally related. Here, we report the detailed clonality analysis in a patient with a diagnosis of B-cell acute lymphoblastic leukemia (B-ALL) followed by a histiocytic sarcoma (HS), in which we were able to study clonal evolution by applying next generation sequencing (NGS) to identify IG rearrangements and gene mutations. Using the sequence information of the NGS-based IG clonality analysis, multiple related subclones could be distinguished in the PAX5 P80R-mutated B-ALL. Notably, only one of these subclones evolved into HS after acquiring a RAF1 mutation. This case demonstrates that NGS-based IG clonality assessment and mutation analysis provide clear added value for clonal comparison and thereby improves clinicobiological understanding.

FOXO1 and PAX5 Rearrangement in Alveolar Rhabdomyosarcoma in Saudi Pediatric Patients.

Objective: In this study, we investigate the molecular rearrangement of FOXO1 in alveolar rhabdomyosarcoma (ARHS) in Saudi pediatric patients. Method: We performed a molecular detection of molecular translocation in 30 pediatric cases of ARHS using FOXO1 dual color break-apart FISH probe (ZytoLight®, 13q14.11) and PAX5 dual color break-apart FISH probe (ZytoLight®, 9p13.2). Results: All analyzable cases of ARHS demonstrated FOXO1 translocation whereas PAX5 translocation was not detected in any case. Conclusion: Although the testing for PAX5 rearrangement was based on protein-protein network analysis, our study showed that PAX5 translocation is not conspicuous in ARHS. PAX7/3::FOXO1 fusion genes feature ARMS, rendering crossreactivity between PAX7 and PAX3 a possible explanation. Nevertheless, PAX5 immunoreactivity and molecular translocation could be an adjunctive pathway that is confined to aggressive ARMS.

PAX5 epigenetically orchestrates CD58 transcription and modulates blinatumomab response in acute lymphoblastic leukemia.

Blinatumomab is an efficacious immunotherapeutic agent in B cell acute lymphoblastic leukemia (B-ALL). However, the pharmacogenomic basis of leukemia response to blinatumomab is unclear. Using genome-wide CRISPR, we comprehensively identified leukemia intrinsic factors of blinatumomab sensitivity, i.e., the loss of CD58 as a top driver for resistance, in addition to CD19. Screening 1639 transcription factor genes, we then identified PAX5 as the key activator of CD58. ALL with the PAX5 P80R mutation also expressed the lowest level of CD58 among 20 ALL molecular subtypes in 1988 patients. Genome editing confirmed the effects of this mutation on CD58 expression and blinatumomab sensitivity in B-ALL, with validation in patient leukemic blasts. We described a PAX5-driven enhancer at the CD58 locus, which was disrupted by PAX5 P80R, and the loss of CD58 abolished blinatumomab-induced T cell activation with global changes in transcriptomic/epigenomic program. In conclusion, we identified previously unidentified genetic mechanisms of blinatumomab resistance in B-ALL, suggesting strategies for genomics-guided treatment individualization.

[PAX5-positive plasma cell leukemia presenting as lymphocytosis].

An 82-year-old Japanese male patient was initially diagnosed with lymphocytosis. His complete blood count revealed a white blood cell count of 30.9×109/l with 81% abnormal lymphocytes. The abnormal lymphocytes included monoclonal clones of CD38+ and CD138+cytoplasmic κ+ and IgG-κ M-protein, which led to the final diagnosis of plasma cell leukemia (PCL). Bortezomib and dexamethasone therapy was initiated, but the patient succumbed to the disease on the 8th day of hospitalization. A cytogenetic examination revealed a t (9;14)(p13;q32) translocation and the Western blotting confirmed high PAX5 expression. Similar to our present case, PCL cases with "lymphocytosis" have been widely reported, which some speculating the involvement of PAX5 overexpression in the pathogenesis. Such cases, including ours, may be classified as a unique group of disorders (PCL presenting as "lymphocytosis"), which requires accurate differential diagnosis and subsequent urgent multidisciplinary intensive treatment.

The MBNL1/circNTRK2/PAX5 pathway regulates aerobic glycolysis in glioblastoma cells by encoding a novel protein NTRK2-243aa.

Glioblastoma multiforme (GBM) is the most common tumor of the human central nervous system. Aerobic glycolysis has been strongly related to tumor development and malignant behavior. In this study, we found that MBNL1, circNTRK2, and NTRK2-243aa were markedly downregulated and inhibited glycolysis in GBM, whereas PAX5 was upregulated and promoted glycolysis. Functionally, MBNL1 promoted the expression of circNTRK2 by binding to NTRK2 pre-mRNA, as validated using RNA pull-down and nascent RNA immunoprecipitation assays. Mass spectrometry, western blotting, and immunofluorescence staining methods were used to detect the expression of NTRK2-243aa. NTRK2-243aa-encoded by circNTRK2-phosphorylated PAX5 at Y102, leading to the attenuation of the half-life of PAX5, as validated by in vitro kinase and MG132 rescue assays. Besides, PAX5 transcriptionally facilitated the expression of PKM2 and HK2 by binding to their promoter regions, as verified by luciferase reporter and chromatin immunoprecipitation assays. Finally, overexpression of MBNL1 and circNTRK2 combined with PAX5 knockdown effectively inhibited the formation of GBM xenograft tumors and significantly prolonged the survival of orthotopic nude mice. We have delineated that the MBNL1/circNTRK2/PAX5 pathway plays a crucial role in regulating GBM glycolysis and could provide potential targets and alternative strategies for the treatment of GBM.