Pax5 Function Research Articles

A SIRT7-dependent acetylation switch regulates early B cell differentiation and lineage commitment through Pax5.

B lymphopoiesis is orchestrated by lineage-specific transcription factors. In B cell progenitors, lineage commitment is mediated by Pax5, which is commonly mutated in B cell acute lymphoblastic leukemia. Despite its essential role in immunity, the mechanisms regulating Pax5 function remain largely unknown. Here, we found that the NAD+-dependent enzyme SIRT7 coordinates B cell development through deacetylation of Pax5 at K198, which promotes Pax5 protein stability and transcriptional activity. Neither Pax5K198 deacetylated nor acetylated mimics rescued B cell differentiation in Pax5-/- pro-B cells, suggesting that B cell development requires Pax5 dynamic deacetylation. The Pax5K198 deacetylation mimic restored lineage commitment in Pax5-/- pro-B cells and B cell differentiation in Sirt7-/- pro-B cells, suggesting the uncoupling of differentiation from lineage commitment. The SIRT7-Pax5 interplay was conserved in B cell acute lymphoblastic leukemia, where SIRT7 expression correlated with good prognosis. Our findings reveal a crucial mechanism for B lymphopoiesis and highlight the relevance of sirtuins in immune function.

Transcriptional function of E2A, Ebf1, Pax5, Ikaros and Aiolos analyzed by in vivo acute protein degradation in early B cell development.

Early B cell lymphopoiesis depends on E2A, Ebf1, Pax5 and Ikaros family members. In the present study, we used acute protein degradation in mice to identify direct target genes of these transcription factors in pro-B, small pre-B and immature B cells. E2A, Ebf1 and Pax5 predominantly function as transcriptional activators by inducing open chromatin at their target genes, have largely unique functions and are essential for early B cell maintenance. Ikaros and Aiolos act as dedicated repressors to cooperatively control early B cell development. The surrogate light-chain genes Igll1 and Vpreb1 are directly activated by Ebf1 and Pax5 in pro-B cells and directly repressed by Ikaros and Aiolos in small pre-B cells. Pax5 and E2A contribute to V(D)J recombination by activating Rag1, Rag2, Dntt, Irf4 and Irf8. Similar to Pax5, Ebf1 also represses the cohesin-release factor gene Wapl to mediate prolonged loop extrusion across the Igh locus. In summary, in vivo protein degradation has provided unprecedented insight into the control of early B cell lymphopoiesis by five transcription factors.

Understanding the function of Pax5 in development of docetaxel-resistant neuroendocrine-like prostate cancers

Resistance to the current Androgen Receptor Signaling Inhibitor (ARSI) therapies has led to higher incidences of therapy-induced neuroendocrine-like prostate cancer (t-NEPC). This highly aggressive subtype with predominant small-cell-like characteristics is resistant to taxane chemotherapies and has a dismal overall survival. t-NEPCs are mostly treated with platinum-based drugs with a combination of etoposide or taxane and have less selectivity and high systemic toxicity, which often limit their clinical potential. During t-NEPC transformation, adenocarcinomas lose their luminal features and adopt neuro-basal characteristics. Whether the adaptive neuronal characteristics of t-NEPC are responsible for such taxane resistance remains unknown. Pathway analysis from patient gene-expression databases indicates that t-NEPC upregulates various neuronal pathways associated with enhanced cellular networks. To identify transcription factor(s) (TF) that could be important for promoting the gene expression for neuronal characters in t-NEPC, we performed ATAC-Seq, acetylated-histone ChIP-seq, and RNA-seq in our NE-like cell line models and analyzed the promoters of transcriptionally active and significantly enriched neuroendocrine-like (NE-like) cancer-specific genes. Our results indicate that Pax5 could be an important transcription factor for neuronal gene expression and specific to t-NEPC. Pathway analysis revealed that Pax5 expression is involved in axonal guidance, neurotransmitter regulation, and neuronal adhesion, which are critical for strong cellular communications. Further results suggest that depletion of Pax5 disrupts neurite-mediated cellular communication in NE-like cells and reduces surface growth factor receptor activation, thereby, sensitizing them to docetaxel therapies. Moreover, t-NEPC-specific hydroxymethylation of Pax5 promoter CpG islands favors Pbx1 binding to induce Pax5 expression. Based on our study, we concluded that continuous exposure to ARSI therapies leads to epigenetic modifications and Pax5 activation in t-NEPC, which promotes the expression of genes necessary to adopt taxane-resistant NE-like cancer. Thus, targeting the Pax5 axis can be beneficial for reverting their taxane sensitivity.

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.

First Description of a Frameshift PAX5 Germline Variant in Two Siblings with B-Cell Precursor Acute Lymphoblastic Leukemia

Introduction: Recent studies have highlighted that about 5% of children with Acute Lymphoblastic Leukemia (ALL) carry a germline mutation that predispose to leukemogenesis. PAX5 somatic mutations are frequently identified on sporadic B-ALL, but recently pathogenic PAX5 germline variants have been described in families with recurrence of B-ALL (Shah et al. 2013; Auer et al. 2014; Yazdanparast et al. 2015, Duployez et al. 2021). We describe the case of two siblings both affected by B-ALL with evidence of a new familial PAX5 variant. Case A was a male, 13 years old, diagnosed with B-II ALL. He was enrolled into the AIEOP-BFM ALL 2009 treatment protocol and according to minimal residual disease (MRD) levels he was stratified as medium risk. Eight years later, his sister (14yrs, named as case B) was diagnosed with B-II ALL, and stratified in the medium risk group of AIEOP-BFM ALL 2017 protocol. The two patients were otherwise healthy and borne from healthy unrelated parents; no history of hematological malignancies was reported across the pedigree. Methods: We performed a custom Next Generation Sequencing ALL predisposition panel on DNA extracted from bone marrow samples of both disease onset and remission phase (defined as negativity by PCR-MRD, 10-4 sensitivity). The libraries were performed following Nextera Flex for Enrichment and sequenced on Illumina Nextseq550 platform; the analyses were carried out by Congenica software. The variants of interest were validated and investigated in the family pedigree through PCR and Sanger sequencing. Digital-MLPA testing 55 ALL hot spot genes (including all PAX5 exons) was performed. Results: Through the NGS analysis, we showed that both patients shared a novel germline heterozygous PAX5 variant (c.548delG, p.Gly183AlafsTer84); moreover, the disease samples of both patients carried an additional somatic PAX5 variant (c.239C>G, p.Pro80Arg; variant fraction: 29% in Case A and 38% in Case B). The Digital-MLPA using the ALL panel, excluded the presence of shared copy number alterations (CNV) and, specifically, did not identified CNV alterations involving the PAX5 locus. The familiar segregation showed a paternal origin of the germline PAX5 variant (p.Gly183AlafsTer84); the mother was found to be wild-type. The PAX5 variant was also found in the DNA sample of the patients' grandmother of the paternal branch; a healthy brother of probands (24 years old) was not tested. To investigate a possible impairment of PAX5 function in the development of the B-cell lineage, we evaluated in both parents the B-cell repertoire on peripheral blood fresh mononuclear cells by FACS analysis. Interestingly, the father has a significant reduction on mature IgD+/CD27+ B-cells with a concomitant accumulation of IgD+/CD27- naïve B-cells. Conclusions: We are reporting a novel germline PAX5 p.Gly183AlafsTer84 in a family with recurrence of B-ALL. In contrast to the previous PAX5 described cases that carry an inherited missense variant resulting in a modest attenuation of PAX5 activity, our patients present a germline frameshift variant that brings to premature stop of the protein, suggesting a more destructive impact on PAX5 activity. As in the other reported cases, our patients presented an additional somatic event on PAX5 that further compromise PAX5 function, leading to the establishment of the leukemic clone. Interestingly and different from the literature, the two siblings share the same PAX5 p.Pro80Arg somatic variant, that has been characterized by Gu as a pathogenic variant impairing B lymphoid development and promoting leukemogenesis (Gu et al. 2019). The detailed molecular characterization, including Whole Transcriptome NGS analysis, is ongoing to investigate whether a PAX5-specific signature can be identified. Moreover, a single-cell NGS approach on remission samples of the two siblings could help to identify specific important pathways and altered gene suppression profiles, preceding full leukemia. Consent-dependent characterization of additional family members are ongoing to assess the penetrance of leukemia associated to this new variant and possible additional features. The identification and characterization of germline PAX5 variants is of high interest in the context of leukemia predisposition because of its clinical implications in terms of familial genetic counselling and because of its contribution in the understanding ALL pathogenesis.

PAX5 Haploinsufficiency Induces Low T Cell Infiltration in the Cancer Microenvironment via Reduced Chemokines.

To investigate the effects of PAXT mutations on tumor immunity. Loss of function of PAX5 plays a key role in the PAX5 mutation tumor. PAX5 haploinsufficiency promoting tumorigenesis is related to immune escape, but there was no report about mechanisms of PAX5 mutation inducing tumor immunological escape. We constructed the PAX5 haplodeletion A20 cell lines using gene-editing technology, built allografted A20 tumor models and evaluated the effect of PAX5 haplodeletion on T cells and chemokines in the tumor microenvironment (TME). Our results from different methods indicated percentages of CD3+ CD4+ T cells and CD3+ CD8+ T cells in TME of PAX5 haplodeletion clones decreased significantly compared with that of PAX5 wild type control. Several chemokines, such as Ccl2, Ccl4, Cxcl9 and Cxcl10, in TME of PAX5. Our study showed that PAX5 haploinsufficiency induced low T cell infiltration in TME using decreased chemokines.

PAX5 alterations in B-cell acute lymphoblastic leukemia.

PAX5, a master regulator of B cell development and maintenance, is one of the most common targets of genetic alterations in B-cell acute lymphoblastic leukemia (B-ALL). PAX5 alterations consist of copy number variations (whole gene, partial, or intragenic), translocations, and point mutations, with distinct distribution across B-ALL subtypes. The multifaceted functional impacts such as haploinsufficiency and gain-of-function of PAX5 depending on specific variants have been described, thereby the connection between the blockage of B cell development and the malignant transformation of normal B cells has been established. In this review, we provide the recent advances in understanding the function of PAX5 in orchestrating the development of both normal and malignant B cells over the past decade, with a focus on the PAX5 alterations shown as the initiating or driver events in B-ALL. Recent large-scale genomic analyses of B-ALL have identified multiple novel subtypes driven by PAX5 genetic lesions, such as the one defined by a distinct gene expression profile and PAX5 P80R mutation, which is an exemplar leukemia entity driven by a missense mutation. Although altered PAX5 is shared as a driver in B-ALL, disparate disease phenotypes and clinical outcomes among the patients indicate further heterogeneity of the underlying mechanisms and disturbed gene regulation networks along the disease development. In-depth mechanistic studies in human B-ALL and animal models have demonstrated high penetrance of PAX5 variants alone or concomitant with other genetic lesions in driving B-cell malignancy, indicating the altered PAX5 and deregulated genes may serve as potential therapeutic targets in certain B-ALL cases.

The Pleiotropy of PAX5 Gene Products and Function.

PAX5, a member of the Paired Box (PAX) transcription factor family, is an essential factor for B-lineage identity during lymphoid differentiation. Mechanistically, PAX5 controls gene expression profiles, which are pivotal to cellular processes such as viability, proliferation, and differentiation. Given its crucial function in B-cell development, PAX5 aberrant expression also correlates with hallmark cancer processes leading to hematological and other types of cancer lesions. Despite the well-established association of PAX5 in the development, maintenance, and progression of cancer disease, the use of PAX5 as a cancer biomarker or therapeutic target has yet to be implemented. This may be partly due to the assortment of PAX5 expressed products, which layers the complexity of their function and role in various regulatory networks and biological processes. In this review, we provide an overview of the reported data describing PAX5 products, their regulation, and function in cellular processes, cellular biology, and neoplasm.

Mechanism of Pax5 Down-Regulation and Its Function in Primary Effusion Lymphoma

Introduction: Primary effusion lymphoma (PEL) is a subtype of aggressive non-Hodgkin lymphoma caused by Kaposi sarcoma-associated herpesvirus (KSHV)/human herpesvirus-8 (HHV-8). PEL develops body cavity effusion without tumor masses especially in immunodeficient patients. The prognosis of PEL is very poor ad there is no standard therapy for PEL. Therefore, the molecular pathogenesis of PEL is needed to be clarified to develop novel therapy. The expressions of Pax5 in PEL patient samples were decreased compared with KSHV/HHV-8-negative B-cell lines in the publically available data set. In this study, we analyzed the mechanism of Pax5 down-regulation and performed the restoration of Pax5 in PEL cells.Methods:Pax5 mRNA expressions of eight PEL cell lines were examined by polymerase chain reaction (PCR). To study the function of Pax5 in PEL, Pax5 was conditionally expressed using tet-on system in three PEL cell lines. To examine the mechanism of Pax5 reduction in PEL, the bisulfite sequencing was performed to analyze the methylation of the Pax5 promoter region in PEL cells. The DNA methyltransferase inhibitor, 5-Azacytidine, was used for demethylation of Pax5 promoter. The anti-proliferative activities of restoring Pax5 in PEL cell lines were analyzed by the methylthiotetrazole method. Cell cycle profiles were analyzed using flow cytometry. Microarray analysis was performed to detect Pax5-regulated genes. The master regulator of KSHV/HHV-8 viral replication, ORF50 , was determined by quantitative PCR.Results: The expression of Pax5 mRNA in PEL cell lines was weaker than in the Burkitt lymphoma cell line, Raji, and normal B-cells derived from peripheral blood mononuclear cells, which express Pax5 mRNA. Although Pax5 promoter region of Raji cells was not methylated, that of PEL cells was heavily methylated. The treatment with 5-Azacytidine induced the demethylation of Pax5 promoter region and restored the expression of Pax5 mRNA. Restoration of Pax5 suppressed cell proliferation via cell cycle arrest. Cell cycle-related genes were down-regulated and several B-cell markers were up-regulated in Pax5-induced PEL cells. Pax5 did not induce ORF50 expression.Conclusions:Pax5 is silenced in PEL cells via the methylation of Pax5 promoter region. Pax5 regulates cell cycle and causes growth inhibition. Reduction of Pax5 has a critical role in the development of PEL and Pax5 is a tumor suppressor in PEL. The targeting of Pax5 with DNA methyltransferase inhibitor could be a novel therapeutic approach in patients with PEL. DisclosuresNo relevant conflicts of interest to declare.

Aberrant HCK Survival Signaling By Mutated MYD88 Requires PAX5 in Waldenstrom's Macroglobulinemia and ABC DLBCL Cells

Activating mutations in MYD88 are present in 95% and 40% of patients with WM and ABC DLBCL, respectively. MYD88 mutations are known to trigger growth and survival of WM and ABC DLBCL cells through BTK/IRAK1/IRAK4 directed NF-kB signaling (Ngo et al, Nature 2011; Yang et al, Blood 2013). Recently, we identified that mutated MYD88 can trigger the SRC family member HCK, a kinase that is downregulated in later stages of B-cell ontogeny (Yang et al, Blood 2016). HCK promotes malignant cell survival through activation of BTK, as well as AKT and ERK1/2, whereas knockdown of HCK leads to decreased survival in WM and ABC DLBCL cells. The regulatory mechanisms that promote mutated MYD88 directed HCK signaling are unknown, and may permit therapeutic advances for MYD88 mutated diseases. To clarify whether HCK expression is regulated by TLRs / MYD88 or BCR signaling, we performed activation studies using triggers of TLR9 (ODN-2006) and TLR4 (LPS-EP) signaling, and BCR (anti-IgM/IgG) signaling in MYD88 wild-type or mutated B-cell lymphoma cells. Stimulation of TLRs but not BCR increased HCK transcription, particularly in wild-type MYD88 expressing B-cell lymphoma cells thereby demonstrating the importance of TLR/MYD88 signaling in driving HCK expression. To better understand the mechanism for the aberrant upregulation of HCK transcription in MYD88 mutated B-cell lymphoma cells, we performed promoter-binding TFs profiling, and observed that among the TFs, PAX5 signal was reduced the most by the addition of HCK promoter into BCWM.1 cell nuclear extracts. The signals of many other TFs that can be regulated by NF-kB (AP1, C\\EBP, Myb, NF-kB), MAPK (AP1, C\\EBP, CREB, HNF4, NF-1) and STAT3 signaling were also reduced. These results indicate that PAX5 and mutated MYD88 directed TFs including NF-kB, AP-1 and STAT3 bind to endogenous HCK promoter sequence. HCK promoter binding consensus sequence analysis and ChIP studies confirmed the presence of PAX5, as well as AP-1, NF-kB and STAT3 binding sites on the HCK promoter. Knockdown of PAX5 reduced HCK mRNA level in BCWM.1 and TMD-8 cells indicating an activating function of PAX5 in the regulation of HCK expression. The deletion of AP-1, NF-kB and STAT3 binding sites on HCK promoter reduced HCK promoter activity, thereby supporting a role for AP-1, NF-kB and STAT3 in HCK gene expression in MYD88 mutated cells. Treatment of BCWM.1 and TMD-8 cells with inhibitors of AP-1, NF-kB and STAT3 decreased HCK promoter activities by luciferase reporter assay, as well as endogenous HCK mRNA transcription levels in MYD88 mutated cells. Our data indicate PAX5 along with mutated MYD88 triggered transcription factors NF-kB, AP-1 and STAT3 orchestrate aberrant HCK expression. The findings provide a framework for efforts directed at blocking MYD88 triggered HCK survival signaling in MYD88 mutated B-cell lymphomas. DisclosuresCastillo:Abbvie: Research Funding; Pharmacyclics: Consultancy, Research Funding; Janssen: Consultancy, Research Funding; Millennium: Research Funding. Treon:Pharmacyclics: Consultancy, Research Funding.

High Expression of MEF2D-Fusion Protein Is Caused By Escaping from the Suppression By miRNA and Leads to Suppress Transcriptional Activity of PAX5

We previously discovered new fusion genes, MEF2D-BCL9 and MEF2D-HNRNPUL1, in 9% of adolescent and young adult B-cell acute lymphoblastic leukemia (ALL; Yasuda T, et al. , Nature Genetics, 2016;48:569-574). Vector driving expression of MEF2D-fusion protein was markedly higher than the expression of wild-type MEF2D (MEF2D W) protein driven by the same expression vector system; however, the mechanism and significance of the high-expression have been yet to be clarified.We first confirmed fusion specific MEF2D high expression was occurred in MEF2D fusion-positive ALL cell lines, Kasumi-7 and Kasumi-9. Protein half-life and the effect of proteasome inhibitor on protein expression were not significantly different between MEF2D W and MEF2D-fusion, suggesting that stability of protein was not different between them. We speculated that MEF2D fusion gene lost the miRNA target that existed in C-terminal half of MEF2D mRNA and that the high-expression of MEF2D fusion protein was caused by escaping from the suppression by miRNA (Figure A). In order to investigate this speculation, we made several expression vectors of MEF2D that express same partial MEF2D protein but transcript different length of mRNA. That is, we introduced a mutation substituting stop codon for D336, the breakpoint of MEF2D-HNRNPUL1 fusion protein, into the expression vector of wild-type MEF2D and designated it as MEF2D (full D336Stop). We deleted C-terminal legion from it with various lengths (Figure B). The protein expression level of MEF2D (1-335) from MEF2D (full D336Stop) vector was similar to that of MEF2D W; however, the protein expression level was markedly increased when DNA region coding amino acid (aa) 377-418 was deleted from the expression vector (Figure C), indicating that protein expression of MEF2D (1-335) was down regulated by miRNA targeting mRNA coding aa 377-418. We identified a candidate of responsible miRNA by database search. Transfection of miRNA mimic of the candidate miRNA decreased the protein expression of MEF2D W but not that of MEF2D fusion. Introduction of mutation at the binding site of the candidate miRNA increased the protein expression of MEF2D W. We examined the effect of MEF2D fusion protein on transcriptional activity of various B cell differentiation regulators and found that MEF2D-fusion protein suppressed transcriptional activity of PAX5 in luciferase assays in dose-dependent manner and that MEF2D W protein did not significantly affect it. These results suggested that the high-expression of MEF2D fusion protein impaired B cell differentiation through the inhibition of PAX5 function. This is the first report that a mutation of a coding gene causes high expression of the protein coded by the gene through escape from suppression by miRNA. These results shed a new insight into the mechanism of leukemia development by MEF2D fusion gene and pathological significance of miRNA-related disorder. [Display omitted] DisclosuresNaoe:Otsuka Pharmaceutical Co.,Ltd.: Honoraria, Research Funding; Nippon Boehringer Ingelheim Co.: Honoraria, Research Funding; Fujifilm Corporation: Patents & Royalties, Research Funding; Dainippon Sumitomo Pharma Co.,LTD.: Research Funding; Astellas Pharma Inc.: Research Funding; Toyama Chemikal Co.,Ltd.: Research Funding. Kiyoi:Bristol-Myers Squibb: Honoraria, Research Funding; Nippon Shinyaku Co., Ltd.: Research Funding; Meiji Seika Pharma Co.,Ltd.: Research Funding; Zenyaku Kogyo Co., Ltd.: Research Funding; MSD K.K.: Research Funding; Celgene Corporation: Consultancy, Research Funding; Kyowa Hakko Kirin Co., Ltd.: Research Funding; Chugai Pharmaceutical Co., Ltd.: Research Funding; Eisai Co., Ltd.: Research Funding; Sumitomo Dainippon Pharma Co., Ltd.: Research Funding; Nippon Boehringer Ingelheim Co., Ltd.: Research Funding; Astellas Pharma Inc.: Consultancy, Research Funding; JCR Pharmaceuticals Co.,Ltd.: Research Funding; Novartis Pharma K.K.: Research Funding; FUJIFILM Corporation: Patents & Royalties, Research Funding; ONO Pharmaceutical Co., Ltd.: Research Funding; Phizer Japan Inc.: Honoraria, Research Funding; Takeda Pharmaceutical Co., Ltd.: Research Funding.

Paraoxonase 2 Enables Initiation of B-ALL By Subverting Metabolic Gatekeeper Functions

Background and significance: The B-lymphoid transcription factors IKZF1 and PAX5 are essential for early B-cell development but also function as metabolic gatekeepers by restricting glucose uptake across the cell membrane (Chan et al., Nature 2017; Xiao et al., Cell 2018). Paraoxonase 2 (PON2) is used as a diagnostic marker and included in a 15 gene diagnostic LDA panel (NCT02883049) for the identification of Ph+ and Ph-like ALL, a B-ALL subgroup with poor outcome and frequent deletion of IKZF1. Pon2 is a member of detoxifying enzymes that are located to the mitochondrial membrane, and hydrolyze lactone metabolites. While PON2 is typically not expressed in normal B-cell precursors, we studied the functional role of high expression levels of Pon2 in B-ALL. Results: Indeed, high Pon2 mRNA levels at diagnosis predicted poor clinical outcomes for both children (COG P9906; n=207; P=1.09e-05) and adults (ECOG; n=215; P=0.003). While PON2 mRNA and protein levels are barely detectable in normal B-cell precursors we found >10-fold increased mRNA and protein levels in multiple B-ALL. Pon2-deficient mice have a number of subtle defects, including propensity to neurogenerative disease and atherosclerosis. However, deletion of Pon2 had profound effects in two models for B-ALL (BCR-ABL1- and NRASG12D). Compared to Pon2 wildtype, Pon2-/- B-ALL cells failed to form colonies in semisolid agar, were prone to cell cycle arrest in G0/G1 phase. Consistent with these observations, Pon2-/- B-ALL cells had a substantially reduced proliferation rate and expressed Arf and p21 checkpoint proteins at substantially increased levels compared to Pon2+/+ ALL cells. These in vitro findings suggest an important role of PON2 in B-lymphoid leukemogenesis, which was confirmed in mouse transplant experiments for BCR-ABL1 and NRASG12D B-ALL models. Pon2-deficiency substantially prolonged survival of recipient mice of BCR-ABL1 B-ALL cells (P=0.0001). Strikingly, NSG mice transplanted with Pon2-deficient NRASG12D B-ALL cells did not develop any leukemia and survived for indefinite periods of time. Based on engineered expression of Cas9 and PON2-specific cRNAs, genetic deletion of PON2 replicated these findings in patient-derived B-ALL xenografts (PDX). Mechanism: Reflecting PAX5 and IKZF1 function, B-lymphoid cells exhibit transcriptional repression of glucose transport (e.g. repression of INSR, GLUT1). Interestingly, deletion of PON2 decreased glucose uptake even further. Intracellular ATP levels in Ph+ ALL cells were drastically reduced and activated the AMPK energy-stress sensor in both murine BCR-ABL1 and NRASG12D B-ALL models and human Ph+ B-ALL cells. Reconstitution of PON2 restored ATP production as early as 3 hours after PON2 induction. Mechanistically, PON2 was previously identified as an inhibitor of STOM and GLUT1 interactions. By disrupting STOM: GLUT1 interactions, PON2 can restore glucose transport. STOM-knockdown in PON2-/- ALL cells replenished cellular ATP levels as did PON2 reconstitution. We conclude that PON2 serves a critical role in B-ALL by subverting the metabolic gatekeeper function of IKZF1 and PAX5 through interference with STOM:GLUT1 interactions. Therapeutic targeting of PON2: While PON2 activity typically results in detoxification of lactone-metabolites, lactone-hydrolysis of the N-(3-oxododecanoyl)-homoserine lactone (3OC12) prodrug results in cytotoxic byproducts. Hence, we tested the concept of leveraging PON2 lactonase activity in a prodrug-approach as a synthetic lethal in Pon2+ B-ALL cells. 3OC12 had profound cytotoxic effects in wildtype but not Pon2-/- B-ALL cells while reconstitution or overexpression of PON2 restored cytotoxic responses to 3OC12. Conclusion: We describe the previously unrecognized function of the detoxifying PON2 lactonase as a central factor in glucose and energy supply in B-ALL, hence subverting metabolic gatekeeper functions of PAX5 and IKZF1. In addition to identifying PON2 as the biomarker for for Ph+ and Ph-like ALL and outcome predictor for patients with B-ALL, our results indicate PON2 facilitate glucose uptake and ATP production via its interaction with STOM. PON2 protects B-ALL cells against glucose depletion and enables malignant growth. On the other hand, we demonstrate a targeted approach that leverages PON2-lactonase as a synthetic lethal to the lactone-prodrug 3OC12. Disclosures No relevant conflicts of interest to declare.

PAX5 is part of a functional transcription factor network targeted in lymphoid leukemia.

One of the most frequently mutated proteins in human B-lineage leukemia is the transcription factor PAX5. These mutations often result in partial rather than complete loss of function of the transcription factor. While the functional dose of PAX5 has a clear connection to human malignancy, there is limited evidence for that heterozygote loss of PAX5 have a dramatic effect on the development and function of B-cell progenitors. One possible explanation comes from the finding that PAX5 mutated B-ALL often display complex karyotypes and additional mutations. Thus, PAX5 might be one component of a larger transcription factor network targeted in B-ALL. To investigate the functional network associated with PAX5 we used BioID technology to isolate proteins associated with this transcription factor in the living cell. This identified 239 proteins out of which several could be found mutated in human B-ALL. Most prominently we identified the commonly mutated IKZF1 and RUNX1, involved in the formation of ETV6-AML1 fusion protein, among the interaction partners. ChIP- as well as PLAC-seq analysis supported the idea that these factors share a multitude of target genes in human B-ALL cells. Gene expression analysis of mouse models and primary human leukemia suggested that reduced function of PAX5 increased the ability of an oncogenic form of IKZF1 or ETV6-AML to modulate gene expression. Our data reveals that PAX5 belong to a regulatory network frequently targeted by multiple mutations in B-ALL shedding light on the molecular interplay in leukemia cells.

MiRNAs 484 and 210 regulate Pax-5 expression and function in breast cancer cells.

Recent studies have enabled the identification of important factors regulating cancer progression, such as paired box gene 5 (Pax-5). This transcription factor has consistently been associated to B-cell cancer lesions and more recently solid tumors including breast carcinoma. Although Pax-5 downstream activity is relatively well characterized, aberrant Pax-5 expression in a cancer-specific context is poorly understood. To investigate the regulation of Pax-5 expression, we turned to micro RNAs (miRNAs), small non-coding RNA molecules that regulate key biological processes. Extensive studies show that miRNA deregulation is prevalent in cancer lesions. In this study, we aim to elucidate a causal link between differentially expressed miRNAs in cancer cells and their putative targeting of Pax-5-dependent cancer processes. Bioinformatic prediction tools indicate that miRNAs 484 and 210 are aberrantly expressed in breast cancer and predicted to target Pax-5 messenger RNA (mRNA). Through conditional modulation of these miRNAs in breast cancer cells, we demonstrate that miRNAs 484 and 210 inhibit Pax-5 expression and regulate Pax-5-associated cancer processes. In validation, we show that these effects are probably caused by direct miRNA/mRNA interaction, which are reversible by Pax-5 recombinant expression. Interestingly, miRNAs 484 and 210, which are both overexpressed in clinical tumor samples, are also modulated during epithelial-mesenchymal transitioning and hypoxia that correlate inversely to Pax-5 expression. This is the first study demonstrating the regulation of Pax-5 expression and function by non-coding RNAs. These findings will help us better understand Pax-5 aberrant expression within cancer cells, creating the possibility for more efficient diagnosis and treatments for cancer patients.

Metabolic gatekeeper function of B-lymphoid transcription factors

B-lymphoid transcription factors (e.g. PAX5, IKZF1) are critical for early B-cell development1–2, yet genetic lesions occur in >80% of cases of B-cell acute lymphoblastic leukemia (ALL)3–4. The significance of these lesions in ALL remained unclear. Combining ChIP-seq and RNA-seq studies, we identified a novel B-lymphoid program for transcriptional repression of glucose and energy supply. Our metabolic analyses revealed that PAX5 and IKZF1 enforce a state of chronic energy deprivation, resulting in constitutive activation of the energy-stress sensor AMPK5–7. Dominant-negative mutants of PAX5 and IKZF1 relieved glucose and energy restriction. Studying a transgenic pre-B ALL mouse model, heterozygous deletion of Pax5 increased glucose uptake and ATP-levels by >25-fold. Reconstitution of PAX5 and IKZF1 in pre-B ALL patient samples restored a non-permissive state and induced energy crisis and cell death. A CRISPR/Cas9-based screen of PAX5- and IKZF1- transcriptional targets identified NR3C1 (glucocorticoid receptor)8, TXNIP (glucose feedback sensor)9 and CNR2 (cannabinoid receptor)10 as central effectors of B-lymphoid restriction of glucose and energy supply. Interestingly, transport-independent lipophilic methyl-conjugates of pyruvate and TCA cycle metabolites bypassed the gatekeeper function of PAX5 and IKZF1 and readily enabled leukemic transformation. Conversely, pharmacological TXNIP- and CNR2-agonists and a small molecule AMPK-inhibitor strongly synergized with glucocorticoids, identifying TXNIP, CNR2 and AMPK as potential therapy-targets. Furthermore, our results provide a mechanistic explanation for the empiric finding that glucocorticoids are effective in the treatment of B-lymphoid but not myeloid malignancies. We conclude that B-lymphoid transcription factors function as metabolic gatekeepers by limiting the amount of cellular ATP to levels that are insufficient for malignant transformation.

Transcriptional Control of Glucose and Energy Supply Prevents Oncogenic Signaling and B Cell Transformation

Background and Hypothesis: B-cell identity is determined by a set of B-cell transcription factors including PAX5, IKZF1 and EBF1. However, B-lineage leukemia clones often carry secondary genetic lesions that result in reduced activity or inactivation of these transcription factors. Studying patient samples from clinical trials for B-lineage childhood (P9906; n=187) and adult (MDACC; n=92) leukemia, we found that genetic defects in one or more B-cell transcription factors represent near-obligate lesions in human acute lymphoblastic leukemia (209 of 279 B-lineage ALL cases). While previously of unknown significance, we found that adult ALL cases with known lesions in one or more of these transcription factors had higher activity of lactate dehydrogenase, and phospho-states indicated higher activity of IRS1, PDK1, and AKT, which contribute to glucose uptake. For this reason, we investigated whether B-cell transcription factors set metabolic constraints of oncogenic signaling in leukemia.Results: Reconstitution of wildtype PAX5 and IKZF1 in patient-derived B-lineage ALL cells carrying PAX5 and IKZF1deletions decreased phospho-AKT levels. Furthermore, protein levels of multiple positive regulators involved in glucose uptake and metabolism (including insulin receptor, glucose transporters and hexokinases) were downregulated upon reconstitution of PAX5 and IKZF1. Conversely, protein levels of negative regulators of glucose uptake and metabolism including NR3C1 and TXNIP were upregulated upon reinstatement of PAX5 and IKZF1 function. Analysis of ChIPseq data of human B cells revealed binding of multiple B-cell transcription factors including PAX5, IKZF1 and EBF1 to promoter regions of genes encoding positive regulators of glucose uptake and metabolism. Binding peaks for B-cell transcription factors were also observed at genes that encode negative regulators of glucose uptake and metabolism (NR3C1 and TXNIP). In addition, direct recruitment of PAX5 was confirmed by single-locus quantitative chromatin immunoprecipitation (qChIP).Reconstitution of wildtype PAX5 and IKZF1 in patient-derived B-lineage ALL cells caused depletion from the cell culture in competitive growth assays, in parallel with reduced glucose consumption and depletion of cellular ATP levels. On the contrary, overexpression of dominant-negative PAX5-ETV6 and IK6 in patient-derived B-lineage ALL cells expressing wildtype PAX5 and IKZF1 resulted in a net survival advantage, concomitant with increases in both glycolytic activity and cellular ATP levels. PAX5-mediated impaired survival fitness was significantly rescued by CRISPR-based activation of gene expression of insulin receptor, glucose transporter and hexokinases in patient-derived B-lineage ALL cells. Conversely, CRISPR/Cas9-mediated deletion of NR3C1 and TXNIP largely reversed the effects of PAX5. Finally, reduced survival fitness upon reconstitution of wild-type PAX5 and IKZF1 in patient-derived pre-B ALL cells was mostly rescued by metabolites that can enter the TCA cycle and thus provide ATP.Conclusions: In summary,B-cell-specific restriction of glycolytic energy supply represents a previously unrecognized metabolic barrier against malignant transformation. Our findings suggest a causative link between impaired glucose uptake and metabolism caused by B-cell transcription factors as well as cell death, and the known tumor suppressive function of PAX5 and IKZF1 in B-lineage ALL. DisclosuresNo relevant conflicts of interest to declare.

Abstract 5025: PAX5 methylation as a novel biomarker of esophageal cancer for both potential tumor malignancy and cisplatin sensitivity

Abstract Therapeutic strategy of esophageal cancer largely depends on histopathological assessment just like tumor differentiation and TNM stage. In order to make appropriate choice for individual case, we have to pay attention to background molecular characteristics about tumor malignancy or chemotherapy sensitivity. In order to find extremely tumor-specific marker of esophageal cancer, we picked several methylation markers from what we previously used in the research of head and neck cancer, which has the same background of smoking and alcohol and the same tissue-type. As a result, we identified PAX5 gene methylation as a very tumor-specific marker also in surgically resected 90 esophageal cancer cases. Then, we continued to examine PAX5 expression, function and other functions. Quantitative MSP (QMSP) assay revealed significantly higher methylation score (100x[PAX5 QMSP]/[ACTB QMSP]) in tumor tissues (16.2±2.4) than in normal tissues (0.4±0.2) (P<0.001, Mann-Whitney U test). Also, 77/90 (85.6%) of these cases showed tumor-specific QMSP elevation. Similarly, all 9 esophageal cancer cell lines (TE1, TE2, TE3, NUEC1, NUEC2, NUEC3, T.T, T.Tn and WSSC) showed high QMSP scores over 10. The inverse correlation of PAX5 methylation and expression was confirmed by Quantitative RT-PCR of clinical samples and 5-aza-dC treated cell lines. PAX5 downregulated cases indicated significantly poor overall survival (P = 0.037, log-rank test). For functional analysis, we examined WST1 assay and BrdU assay using ectopically PAX5 knock down NUEC1 cell line, which has secondly high PAX5 expression. The result showed significantly high cell proliferation and cell cycle acceleration. Since PAX5 is reported to bind p53 promoter and regulate its expression, we hypothesized epigenetically downregulated PAX5 might affect p53 expression and therefore cisplatin sensitivity of esophageal cancer. Actually, PAX5 downregulated NUEC1 cell line acquired significantly cisplatin-resistant character after siRNA transfection. Moreover, among postoperatively cisplatin-treated cases (25 cases), PAX5 hyper-methylated clinical cases (n = 6) demonstrated significantly worse survival than PAX5 hypo-methylated cases (n = 19) (P = 0.002, log-rank test). We also surveyed another downstream mechanism of PAX5 dysregulation. Human Cancer Pathway Finder PCR Assay identified three key molecules including SLC2A1 (Fold change: 4.6), CCL2 (12.5) and IGFBP5 (-5.0). Epigenetic inactivation of PAX5 gene may affects malignant potential of esophageal cancers and cisplatin-based chemotherapy through p53 dysregulation or other carcinogenic pathways. It could be utilized for planning individual multidisciplinary therapy. Citation Format: Keisuke Kurimoto, Masamichi Hayashi, Masahiko Koike, Mitsuro Kanda, Yoko Nishikawa, Naoki Iwata, Yukiko Niwa, Hideki Takami, Daisuke Kobayashi, Chie Tanaka, Suguru Yamada, Goro Nakayama, Hiroyuki Sugimoto, Michitaka Fujiwara, Tsutomu Fujii, Guerrero-Preston Rafael, Yasuhiro Kodera. PAX5 methylation as a novel biomarker of esophageal cancer for both potential tumor malignancy and cisplatin sensitivity. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 5025.

Abstract P4-07-10: MiRNAs are important regulators of Pax-5 expression and function during breast cancer progression

Abstract Recent studies have enabled the identification of important factors regulating cancer progression, one of these being the Pax-5 gene. Pax-5, an essential developmental factor of B cells, is aberrantly expressed in various B cell cancer lesions and solid tumors such as breast carcinoma. Although Pax-5 downstream activity is relatively well characterized, the regulation of aberrant Pax-5 expression in a cancer specific context is poorly understood. To investigate the regulation of Pax-5 expression, we turned our attention to micro-RNAs (miRNAs). MiRNAs are highly conserved, small non-coding RNA molecules that regulate key biological processes. Extensive studies also show their deregulation in multiple cancer lesions. In this study, we aim to elucidate a causal link between differentially expressed miRNAs in cancer cells and their putative targeting of Pax-5-dependent cancer processes. With the help of biobank data and bioinformatics analyses, we observe that miRNAs 484 and 210 are aberrantly expressed in breast cancer cells and cross-reference with their predicted capacity to target the Pax-5 mRNA 3’ untranslated region (3’UTR). Using anti- or pre-miRNAs transfected into Pax-5 expressing breast cancer cell lines (MCF-7 and MB231), we demonstrate that miRNAs 484 and 210 are capable of regulating Pax-5 expression. In addition, miRNA-regulated Pax-5 expression resulted in a concomitant alteration in Pax-5-mediated phenotype and cancer processes. This is the first study demonstrating the regulation of Pax-5 expression and function by non-coding RNAs in cancer cells. We believe that the aberrant expression of Pax-5 in cancer cells is in part due to deregulated miRNA expression profiles. This study will bring insight in regards to cancer regulating processes associated with miRNA and Pax-5 deregulations and help us better understand aberrant Pax-5 expression levels within cancerous states. This study can therefore provide the eventual possibility of earlier, more efficient diagnostics as well as more targeted treatments for cancer patients. Citation Format: Jason M Harquail, Gilles A Robichaud. MiRNAs are important regulators of Pax-5 expression and function during breast cancer progression [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P4-07-10.

Abstract 3094: MiRNAs 484 and 210 control Pax-5 expression and function in breast cancer.

Abstract Every hour, approximately 21 people are diagnosed with cancer where 9 will succumb to their disease. Recent studies have enabled the identification of important factors regulating cancer progression, one of these being the Pax-5 gene. Pax-5, an essential developmental factor, is aberrantly expressed in various B cell cancer lesions and solid tumors such as carcinoma. Although Pax-5 downstream activity is well characterized, the regulation of aberrant Pax-5 expression in a cancer specific context is poorly understood. To investigate the regulation of Pax-5 expression, we turned our attention to micro-RNAs (miRNAs). MiRNAs are highly conserved, small non-coding RNA molecules that regulate key biological processes. Extensive studies also show their deregulation in multiple cancer lesions. This study aims to elucidate a correlation between differentially expressed miRNAs in cancer cells and deregulated Pax-5 expression levels. Using bioinformatics platforms we observe that miRNAs 484, and 210 are aberrantly expressed in breast cancer cells and cross-reference with their capacity to target the Pax-5 mRNA 3’ untranslated region (UTR 3’). Using anti-miRNAs transfected into Pax-5 expressing breast cancer cell lines (MCF-7 and MB231), we demonstrate that miRNAs 484 and 210 are capable of regulating Pax-5 expression. In addition, miRNA-regulated Pax-5 expression resulted in a concomitant alteration in Pax-5-dependant phenotype and cancer processes. This is the first study demonstrating the regulation of Pax-5 expression and function by non-coding RNAs in cancer cells. We believe that the aberrant expression of Pax-5 in cancer cells is in part due to deregulated miRNA expression profiles. This study will bring insight in regards to cancer regulating processes associated with miRNA and Pax-5 deregulations, thus helping us to better understand the aberrant Pax-5 expression levels within cancerous states. This study can therefore provide the eventual possibility of earlier, more efficient diagnostics as well as more targeted treatments for cancer patients. Citation Format: Jason M. Harquail, Gilles A. Robichaud. MiRNAs 484 and 210 control Pax-5 expression and function in breast cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3094. doi:10.1158/1538-7445.AM2013-3094

Abstract B9: Myc represses Ebf1 and Pax5 to impair early B-cell differentiation and drive progenitor B lymphomagenesis

Abstract Activation of Myc oncogenic transcription factors is a hallmark of rapidly dividing malignancies. The Eμ-Myc transgenic mouse is a validated model of MYC activation due to MYC/Ig translocations seen in human Burkitt lymphoma (BL). Normally, these transgenics develop pre-B cell (fractions C-E) stage lymphoma with a mean latency of 3–4 months. Here we report that Myc represses the transcription of Pax5 and Ebf1, which are master transcriptional regulators of B cell fate commitment, and that Pax5 and Ebf1 function as tumor suppressors that impair the tumorigenic potential of Myc-driven lymphoma. Expression analyses of wild-type and premalignant Eμ-Myc fractions A0 through F bone marrow and splenic B cells showed that Pax5 and Ebf1 expression is normally induced in fraction A2 progenitors, but that this response is markedly suppressed in Myc-expressing B cells. In addition, activation of c-Myc in P493-6 B lymphoid cells, which harbor a Tet-regulated c-Myc transgene, repressed Pax5 and Ebf1 mRNA levels, and ChIP analyses demonstrated this was associated with direct binding of c-Myc to the promoter-regulatory regions of Pax5 and Ebf1. Notably, FACS analyses demonstrated marked increases in the numbers of Fraction A1 cells (AA4.1 +B220+CD19−CD4+) and corresponding reductions in Fraction A2 (AA4.1+B220+CD19-CD4-) progenitors in Eμ-Myc versus wild-type mice. Thus, Myc impairs the A1-to-A2 developmental transition of B cells and this is associated with Myc-mediated repression of Pax5 and Ebf1. Collectively these findings, and those of others showing that Pax5 loss promotes the development of progenitor B cell tumors and that inactivating mutations in PAX5 and EBF1 are found in human B-ALL, supported the hypothesis that Ebf1 and Pax5 function as tumor suppressors. To test this, we transduced Eμ-Myc lymphomas harboring inactivating mutations in either Arf or p53 with retroviruses expressing Ebf1 or Pax5 together with a GFP marker, or with control GFP-only virus. Mixtures of GFP+ (transduced) and GFP− (nontransduced) lymphomas were then either cultured ex vivo in medium with IL7, or were directly transplanted into either Nude or NOD/SCID mice. Notably, Ebf1-expressing lymphoma cells underwent rapid growth arrest when cultured ex vivo, indicating Ebf1 cancels Myc's proliferative response. More importantly, enforced expression of Pax5 and especially Ebf1 markedly impaired the tumorigenic potential of Myc-driven lymphoma, irrespective of mutations in Arf or p53, where there was a profound selection against Pax5+GFP+ or Ebf1+GFP+ lymphoma cells in tumor-bearing recipients compared to recipients transplanted with GFP-only expressing lymphoma cells. To assess the effects of Ebf1 and Pax5 in Eμ-expressing (stages A1-F) B cells, as well as their ability to impair the development of lymphomas, we have generated Eμ-Ebf1 and Eμ-Pax5 transgenic mice. These are being crossed to both Eμ-Myc and Eμ-Myc/?μ-Bcl2 transgenics to assess whether Pax5 or Ebf1 will also block lymphoma onset. We predict that Pax5 and Ebf1 will impair lymphomagenesis and override the A1-to-A2 developmental block manifest in Eμ-MycB cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the Second AACR International Conference on Frontiers in Basic Cancer Research; 2011 Sep 14-18; San Francisco, CA. Philadelphia (PA): AACR; Cancer Res 2011;71(18 Suppl):Abstract nr B9.