PBX1 Research Articles

Whole Exome Sequencing Revealing a Novel PBX1 Gene Variant in a Chinese Family Causing Recurrent Neonatal Death.

Causative mutations of PBX1 are associated with congenital abnormalities of the kidney and urinary tract (CAKUT), often accompanied by hearing loss, abnormal ear morphology, or developmental delay. The aim of the present investigation was to introduce a novel variant in the PBX1 gene identified in a Chinese family, leading to recurrent neonatal mortality. A pregnant woman (gravida 5, para 0), who had experienced recurrent neonatal deaths, sought genetic etiology diagnosis. Whole exome sequencing (WES) was conducted to identify sequence variants and copy number variants in the fetus presenting with posterior nuchal cystic hygroma and fetal hydrops. A novel NM_002585.4:c.694G>C(p.D232H) in PBX1 was identified in the fetus through trio whole exome sequencing (WES), revealing a paternal mosaic PBX1 variant in blood at 11.54% (6/52 variants reads). Subsequent parental Sanger sequencing confirmed the variant detected by WES. Ultimately, the variant was classified as likely pathogenic, leading the family to elect pregnancy termination at 17 weeks gestation. The novel variant in the PBX1 gene appears to be a significant factor contributing to recurrent neonatal deaths in the Chinese family. Such findings expand the spectrum of PBX1 gene variants and provide valuable perinatal guidance for diagnosing fetuses with PBX1 mutations.

CD45 inhibition in myeloid leukaemia cells sensitizes cellular responsiveness to chemotherapy

Myeloid malignancies are a group of blood disorders characterized by the proliferation of one or more haematopoietic myeloid cell lineages, predominantly in the bone marrow, and are often caused by aberrant protein tyrosine kinase activity. The protein tyrosine phosphatase CD45 is a trans-membrane molecule expressed on all haemopoietic blood cells except that of platelets and red cells. CD45 regulates various cellular physiological processes including proliferation, apoptosis, and lymphocyte activation. However, its role in chemotherapy response is still unknown; therefore, the aim of this study was to investigate the role of CD45 in myeloid malignancies in terms of cellular growth, apoptosis, and response to chemotherapy. The expression of CD45 on myeloid leukaemia primary cells and cell lines was heterogeneous with HEL and OCI-AML3 cells showing the highest level. Inhibition of CD45 resulted in increased cellular sensitivity to cytarabine and ruxolitinib, the two main therapies for AML and MPN. Bioinformatics analysis identified genes whose expression was correlated with CD45 expression such as JAK2, ACTR2, THAP3 Serglycin, and PBX-1 genes, as well as licensed drugs (alendronate, allopurinol, and balsalazide), which could be repurposed as CD45 inhibitors which effectively increases sensitivity to cytarabine and ruxolitinib at low doses. Therefore, CD45 inhibition could be explored as a potential therapeutic partner for treatment of myeloid malignancies in combination with chemotherapy such as cytarabine especially for elderly patients and those showing chemotherapy resistance.

Molecular characterization of TCF3::PBX1 chromosomal breakpoints in acute lymphoblastic leukemia and their use for measurable residual disease assessment

The translocation t(1;19)(q23;p13) with the resulting chimeric TCF3::PBX1 gene is the third most prevalent recurrent chromosomal translocation in acute lymphoblastic leukemia and accounts for 3–5% of cases. The molecular background of this translocation has been incompletely studied, especially in adult cases. We characterized the chromosomal breakpoints of 49 patients with TCF3::PBX1 and the corresponding reciprocal PBX1::TCF3 breakpoints in 15 cases at the molecular level, thus providing an extensive molecular overview of this translocation in a well-defined study patient population. Breakpoints were found to be remarkably clustered not only in TCF3 but also in PBX1. No association with DNA repeats or putative cryptic recombination signal sequence sites was observed. A simplified detection method for breakpoint identification was developed and the feasibility of patient-specific chromosomal break sites as molecular markers for detecting measurable residual disease (MRD) was explored. A highly sensitive generic real-time PCR for MRD assessment using these breakpoint sequences was established that could serve as a useful alternative to the classical method utilizing rearranged immune gene loci. This study provides the first extensive molecular data set on the chromosomal breakpoints of the t(1;19)/TCF3::PBX1 aberration in adult ALL. Based on the obtained data a generic MRD method was developed that has several theoretical advantages, including an on average higher sensitivity and a greater stability of the molecular marker in the course of disease.

Gender-Specific Differences in Human Vertebral Bone Marrow Clot.

Recently, our group described the application of vertebral bone marrow (vBMA) clot as a cell therapy strategy for spinal fusion. Its beneficial effects were confirmed in aging-associated processes, but the influence of gender is unknown. In this study, we compared the biological properties of vBMA clots and derived vertebral mesenchymal stem cells (MSCs) from female and male patients undergoing spinal fusion procedures and treated with vBMA clot. We analyzed the expression of growth factors (GFs) in vBMA clots and MSCs as well as morphology, viability, doubling time, markers expression, clonogenicity, differentiation ability, senescence factors, Klotho expression, and HOX and TALE gene profiles from female and male donors. Our findings indicate that vBMA clots and derived MSCs from males had higher expression of GFs and greater osteogenic and chondrogenic potential compared to female patients. Additionally, vBMA-clot-derived MSCs from female and male donors exhibited distinct levels of HOX and TALE gene expression. Specifically, HOXA1, HOXB8, HOXD9, HOXA11, and PBX1 genes were upregulated in MSCs derived from clotted vBMA from male donors. These results demonstrate that vBMA clots can be effectively used for spinal fusion procedures; however, gender-related differences should be taken into consideration when utilizing vBMA-clot-based studies to optimize the design and implementation of this cell therapy strategy in clinical trials.

Abstract 2231: Epigenomic profiling uncovers distinct enrichment patterns of PBX1 super-enhancer among lung adenocarcinoma

Abstract Small cell lung cancers and squamous cell cancers can be epigenetically subtyped (Kong R et al 2022, Sato T et al, 2019) with focus on super-enhancer (SE) signals within or adjacent to transcription factors (TF). We reasoned that specific epigenomic subsets in lung adenocarcinomas (LUAD) operate with similar cellular programming showing TF dependency for lineage commitment and survival. We hypothesize that these dependencies on lineage programming are associated with a specific epigenomic landscape. Therefore, we investigated enrichment of SEs across the spectrum of LUADs to identify potential regulators for lineage commitment and survival. We obtained data for genome-wide H3K27ac enrichment by chromatin immunoprecipitation followed by sequencing (ChIP-seq) on a total of 53 LUAD cell lines. We performed unsupervised hierarchical clustering of signals on SEs at gene loci annotated with transcriptional regulation identified in two or more cell lines. To Uncover 3D contact information, we employed HiChIP, using antibody against SMC1, a cohesin complex, on NCI-H460. Three LUAD cell lines with distinct enhancer profiles were transfected with five enhancer segments of the SE at the PBX1 gene cloned into a Firefly luciferase plasmid. Lastly, four LUAD cell lines were treated with a PBX1 inhibitor, T417 for 72 hours in varying concentrations ranging from 20uM to 10nM and evaluated for sensitivity to the compound. Clustering analysis identified LUADs with differential enhancer enrichment for TFs. Among those, we focused on the PBX1 gene locus in LUADs harboring SEs proximal to its promoter. SMC1 HiChIP analysis on NCI-H460 suggest all enhancer segments are within a local topologically-associated domain with the PBX1 promoter. Luciferase reporter assay demonstrate activity of enhancer segments within the PBX1 SE, unique to each cell line (NCI-H460, PC-9 and NCI-H1437), with enhancers e4 and e5 rendered the highest level of activity while e2 being the most active in PC-9. The effects of these enhancer segments are congruent to H3K27ac enrichment patterns. Treatment with T417 showed reduction in cell proliferation at an IC50 of 2.5 uM and 0.97 uM for NCI-H460 and PC-9, respectively, whereas T417 did not affect cell viability in NCI-H1792 and NCI-H125. Data indicate a distinct subset of LUADs, enriched for the enhancer segments within the SE at the PBX1 gene locus, at which the HiChIP data displayed the enhancer segments lie within the same topologically-associated domain. The enhancer activity of enhancer segments correlated with H3K27ac enrichment pattern for each cell line, suggesting enrichment correlates with impact. Data with PBX1 T417 exposure suggests dependency of PBX1 in maintaining lineage state and survival for unique subsets of LUADs. Further investigation is needed to determine if T417 dependent inhibition is uniform to all PBX1 active LUADs and if other LUAD subsets are dependent on other TFs. Citation Format: Douglas M. Mansell, Maya Fridrich, Feng Jiang, Benjamin Chen, Ayushi Patel, Tian Li-Wang, Charles A. Powell, Hideo Watanabe. Epigenomic profiling uncovers distinct enrichment patterns of PBX1 super-enhancer among lung adenocarcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2231.

The PBX1/miR-141-miR-200a/EGR2/SOCS3 Axis; Integrative Analysis of Interaction Networks to Discover the Possible Mechanism of MiR-141 and MiR-200a-Mediated Th17 Cell Differentiation.

Overexpression of miR-141 and miR-200a is known to be associated with the differentiation of T helper 17 (Th17) cells, which are key players in the pathophysiology of autoimmune disorders. However, the function and governing mechanism of these two microRNAs (miRNAs) in Th17 cell skewing are poorly defined. The aim of the present study was to identify the common upstream transcription factors and downstream target genes of miR-141 and miR-200a to obtain a better insight into the possible dysregulated molecular regulatory networks driving miR-141/miR-200a-mediated Th17 cell development. A consensus-based prediction strategy was applied for in-silico identification of potential transcription factors and putative gene targets of miR-141 and miR-200a. Thereafter, we analyzed the expression patterns of candidate transcription factors and target genes during human Th17 cell differentiation by quantitative real-time PCR and examined the direct interaction between both miRNAs and their potential target sequences using dual-luciferase reporter assays. According to our miRNA-based and gene-based interaction network analyses, pre-B cell leukemia homeobox (PBX1) and early growth response 2 (EGR2) were respectively taken into account as the potential upstream transcription factor and downstream target gene of miR-141 and miR-200a. There was a significant overexpression of the PBX1 gene during the Th17 cell induction period. Furthermore, both miRNAs could directly target EGR2 and inhibit its expression. As a downstream gene of EGR2, the suppressor of cytokine signaling 3 (SOCS3) was also downregulated during the differentiation process. These results indicate that activation of the PBX1/miR-141-miR-200a/EGR2/SOCS3 axis may promote Th17 cell development and, therefore, trigger or exacerbate Th17-mediated autoimmunity.

Prenatal diagnosis and molecular cytogenetic analyses of a paternal inherited deletion of 1q23.3 encompassing PBX1 gene

BackgroundPatients with deletions involving the long arm of chromosome 1 are rare. The PBX1 gene is located on chromosome 1q23.3. PBX1 encodes a transcription factor which promotes protein–protein interaction and plays a crucial role in several developmental processes. PBX1 haploinsufficiency had been reported to lead syndromic congenital anomalies of kidney and urinary tract (CAKUT) in humans.Case presentationIn this research, a 24-year-old woman (gravida 1, para 0) underwent amniocentesis at 22 weeks’ gestation because of a horseshoe kidney of the fetus on prenatal ultrasound.ResultsChromosomal microarray analysis (CMA) from this family revealed a 1.14 Mb paternal inherited deletion on chromosome 1q23.3, spanning from position 163,620,000 to 164,760,000 (hg19). Trio whole-exome sequencing (WES) showed heterozygous deletions in exons 1–2 of the PBX1 in fetal and paternal samples. At the 3-year follow-up, the baby did not have an abnormal phenotype except a horseshoe kidney.ConclusionWe provide a detailed description of the phenotype in a family with paternal inherited deletion of 1q23.3 encompassing exons 1–2 of the PBX1 gene. Combination of karyotype analysis, CMA, WES, prenatal ultrasound and genetic counseling is helpful for the prenatal diagnosis of chromosomal microdeletions/microduplications.

The TALE never ends: A comprehensive overview of the role of PBX1, a TALE transcription factor, in human developmental defects.

PBX1 is a highly conserved atypical homeodomain transcription factor (TF) belonging to the TALE (three amino acid loop extension) family. Dimerized with other TALE proteins, it can interact with numerous partners and reach dozens of regulating sequences, suggesting its role as a pioneer factor. PBX1 is expressed throughout the embryonic stages (as early as the blastula stage) in vertebrates. In human, PBX1 germline variations are linked to syndromic renal anomalies (CAKUTHED). In this review, we summarized available data on PBX1 functions, PBX1-deficient animal models, and PBX1 germline variations in humans. Two types of genetic alterations were identified in PBX1 gene. PBX1 missense variations generate a severe phenotype including lung hypoplasia, cardiac malformations, and sexual development defects (DSDs). Conversely, truncating variants generate milder phenotypes (mainly cryptorchidism and deafness). We suggest that defects in PBX1 interactions with various partners, including proteins from the HOX (HOXA7, HOXA10, etc.), WNT (WNT9B, WNT3), and Polycomb (BMI1, EED) families are responsible for abnormal proliferation and differentiation of the embryonic mesenchyme. These alterations could explain most of the defects observed in humans. However, some phenotype variability (especially DSDs) remains poorly understood. Further studies are needed to explore the TALE family in greater depth.

Homeodomain protein Pbx1 regulates regulatory T cell development, stability and suppressive function

Abstract Pre-B cell leukemia transcription factor 1 (Pbx1) regulates multiple processes in various cell types. The Pbx1 gene is located in the Sle1a1 lupus susceptibility locus in the NZM2410 lupus-prone mouse model. The Pbx1-d dominant negative isoform lacking the DNA binding domain is more frequent in the CD4+ T cells from NZM240 mice as well as lupus patients as compared to healthy controls. In addition, the transgenic expression of Pbx1-d in CD4+ T cells reproduced the phenotypes of Sle1a1 mice, with impaired Foxp3+ Treg cell homeostasis as well as increased inflammatory CD4+ T cells. To study the role of Pbx1 in Treg cells, we generated Foxp3-specific Pbx1 knock-out mice (Foxp3-Pbx1 KO), which were bred to CD4-Pbx1-d-Tg mice to generate Pbx1-d Tg-KO mice that have Treg cells solely expressing Pbx1-d. Foxp3-Pbx1 KO mice showed reduced numbers of Treg cells with impaired suppressive activity. The numbers of natural Treg (nTrg) cells are decreased in thymus but not in spleen of Foxp3-Pbx1 KO mice, while the number of induced Treg (iTreg) cells are increased in the thymus and decreased in the periphery, suggesting that Pbx1 regulates Treg cell development and stability. The results were enhanced in Pbx1-d Tg-KO mice compared with Foxp3-Pbx1 KO mice, leading to autoimmune phenotypes, suggesting that the expression of Pbx1 functional isoform is necessary for the maintenance and function of Treg cells. RNAseq analyses with Pbx1-KO Treg cells indicated that Pbx1 regulates cell cycle, apoptosis and migration. Overall, our results suggest that Pbx1 regulates Treg cell homeostasis, stability and their suppressive functions and that Pbx1-d promotes lupus development by decreasing Treg cell development and functions. Supported by a grant from the NIH (R01 AI045050)

Dynamic Expression of the Homeobox Factor PBX1 during Mouse Testis Development

Members of the pre-B-cell leukemia transcription factor (PBX) family of homeoproteins are mainly known for their involvement in hematopoietic cell differentiation and in the development of leukemia. The four PBX proteins, PBX1, PBX2, PBX3 and PBX4, belong to the three amino acid loop extension (TALE) superfamily of homeoproteins which are important transcriptional cofactors in several developmental processes involving homeobox (HOX) factors. Mutations in the human PBX1 gene are responsible for cases of gonadal dysgenesis with absence of male sex differentiation while Pbx1 inactivation in the mouse causes a failure in Leydig cell differentiation and function. However, no data is available regarding the expression profile of this transcription factor in the testis. To fill this knowledge gap, we have characterized PBX1 expression during mouse testicular development. Real time PCRs and Western blots confirmed the presence Pbx1 mRNA and PBX1 protein in different Leydig and Sertoli cell lines. The cellular localization of the PBX1 protein was determined by immunohistochemistry and immunofluorescence on mouse testis sections at different embryonic and postnatal developmental stages. PBX1 was detected in interstitial cells and in peritubular myoid cells from embryonic life until puberty. Most interstitial cells expressing PBX1 do not express the Leydig cell marker CYP17A1, indicating that they are not differentiated and steroidogenically active Leydig cells. In adults, PBX1 was mainly detected in Sertoli cells. The presence of PBX1 in different somatic cell populations during testicular development further supports a direct role for this transcription factor in testis cell differentiation and in male reproductive function.

Genome-Wide Analysis of Allele-Specific Expression Genes in Pediatric B-Cell Precursor Acute Lymphoblastic Leukemia

Introduction. Pediatric B-cell precursor acute lymphoblastic leukemia (BCP ALL) is the most common pediatric hematological malignancy and it remains an important cause of morbidity and mortality in children. In this study, we performed an allele-specific expression (ASE) analysis of pediatric BCP ALL with the aim to investigate the relationship between cis-regulatory mutations and gene expression patterns.Materials and methods. Twenty-two high hyperdiploid ALL, twenty ETV6/RUNX1-positive ALL, seven TCF3/PBX1-positive ALL and twenty-eight genetically unclassified BCP ALL (“B-other”) were subjected to whole genome sequencing, SNP array analysis and RNA sequencing. The binomial test was applied to estimate the allelic bias of heterozygous exonic single nucleotide variants (SNVs) in the RNA sequencing data against the genomic data. Allelic ratios >2 or <0.5, and P values <0.05 were used to identify allele-specific expression protein-coding genes.Results. We identified 12,693 expressed genes, of which 9,672 (76%) had heterozygous exonic SNVs (informative genes), in multiple BCP ALL samples (n>2) in 77 of the investigated samples. Genes with ASE were distributed evenly across the autosomal chromosomes in the different subtypes with a range of 30 - 165 ASE genes per case (median number, 86). We found that 630 (6.5%) genes displayed ASE in multiple BCP ALL samples (n>2), of which only eight autosomal genes had monoallelic expression in more than two investigated samples. This suggests that ASE and monoallelic expression are relatively rare in BCP ALL. Gene enrichment analyses showed that pathways involving negative regulation of natural killer cell-mediated cytotoxicity and cell proliferation were enriched, indicating that ASE events possibly were associated with the cell proliferation and leukemia progression in BCP ALL. Furthermore, the hematopoiesis pathway was also enriched in ASE genes that showed high allelic expression bias (allelic ratios >2.5), suggesting that ASE genes might be associated with leukemia development. Somatic genomic aberrations that could cause ASE were also investigated in this study. All informative cases with TCF3/PBX1 rearrangement (n=4) showed monoallelic expression of the PBX1 gene, likely associated with the PBX1 truncation caused by the fusion. Additionally, CHP1, located in 15q15.1, displayed ASE in one case with an inversion involving that chromosome band, indicating a potential cis-acting element in the inversion region that regulated the CHP1 gene expression. Notably, PAX5 displayed various patterns of ASE in BCP ALL. One of three cases with PAX5/ZCCHC7 gene rearrangements displayed PAX5 ASE while the other two did not, indicating a potential uncovered cis-regulatory element around the PAX5/ZCCHC7 breakpoints. Furthermore, two cases with no PAX5 gene rearrangement displayed monoallelic expression of the PAX5 gene, suggesting that there are additional epigenetic alterations were also involved in the regulation of PAX5 gene expression in BCP ALL.Conclusions. In this study, we have characterized genes displaying ASE in childhood BCP ALL. Our data provide new insight into pathogenesis of BCP ALL and may be used to identify novel targets for treatment. DisclosuresNo relevant conflicts of interest to declare.

In Silico Analysis of miRNA-mediated ceRNAs as Potential Molecular Biomarkers in Glioblastoma

Objectives: Glioblastoma multiforme (GBM) is defined as the most frequent and lethal form of the primary brain tumors in the central nervous system (CNS) in adults. Recent studies have focused on the identification of the new targets for the diagnosis and treatment of GBM and resulted in great interest for miRNAs due to their regulatory effects in cancer pathogenesis. Thus, we aimed to characterize novel molecular biomarkers for GBM by computational analysis. Methods: 118 miRNAs that are clinically related with glioblastoma and proven by experimentally were exported through miRTarBase database. 1016 genes projected by these 118 miRNAs were determined via ComiR database. Subsequently, the genes with transcribed ultraconserved regions (T-UCRs) in their exonic regions were designated and the genes which have potential competing endogenous RNA (ceRNA) activities were extracted. Genes with remarkable expression profile differences between glioblastoma and normal brain tissues among ceRNAs that are associated with glioblastoma involving T-UCR were identified. Results: The statistical analysis of the correlation between PBX3 and NRXN3 genes and glioblastoma was carried out by Spearman correlation test. PBX3 and NRXN3 expression was significantly higher and lower in glioblastoma than in normal brain tissues, respectively. On the other hand, the other genes did not have any remarkable differential expression pattern. Conclusion: Based on the findings of the current study, it is determined that NRXN3 acts as a tumor suppressor gene and NRXN3 gene is downregulated in GBM. PBX3 gene functions as an oncogene and is upregulated in GBM.

Abstract 1132: Development and characterization of a novel orally bioavailable menin-MLL inhibitor for treatment of acute leukemia patients with MLL-rearrangement or NPM1 mutation

Abstract DS-M1 is a novel, potent, orally bioavailable small molecule inhibitor of the binding of MLL1 fusion proteins and wild-type MLL1 to Menin in development for the treatment of patients with MLL1-rearranged (MLL1-r) or NPM1-mutated (NPM1-mu) acute leukemia. The interaction of MLL1 fusion proteins with menin plays an important role to enhance the proliferation and block the differentiation of hematopoietic cells, ultimately leading to acute leukemia. Furthermore, patients with MLL1-r acute leukemia respond poorly to currently available treatments, emphasizing the urgent need to develop more effective therapies directly disrupting the menin-MLL1 complex. Here we describe the characterization of DS-M1 and this study demonstrates the mechanism of action of DS-M1 as well as supports the oral dosing regimen planned for the initial clinical studies. DS-M1 selectively inhibited the cellular growth of MLL1-r or NPM1-mu human leukemic cell lines and primary cells from patients with GI50 of less than about 100 nM along with the cellular differentiation in vitro. RNA-seq and ChIP-seq analysis revealed that DS-M1 reduced the expression levels of MEIS1, PBX3 and HOXA9 genes in a concentration-dependent manner and dissociated the menin-MLL1 complex from the MEIS1 gene locus in MV-4-11, MOLM-13, and patient derived AML cells with MLL1-rearrangement. Interestingly, DS-M1 reduced the number of CD34+/CD38- leukemic stem cells (LSCs) in patient derived AML cells and induction of differentiation was also confirmed as measured by increased CD11b or CD14 expression. These results suggest that enhanced differentiation and loss of LSCs via the reduction of MEIS1, HOXA9, and PBX3 gene expression by DS-M1 is one of the main mechanisms of action for the antitumor activity of DS-M1. DS-M1 demonstrates significant survival benefit along with inhibition of MEIS1, HOXA9 and PBX3 expression in aggressive disseminated leukemia models intravenously inoculated with MV-4-11 and MOLM-13 cells as well as in patient-derived xenograft (PDX) models of MLL1-r or NPM1-mu acute leukemia. In disseminated MOLM-13 xenografts, treatment with 100 mg/kg qd of DS-M1 for 19 days produced durable response with event free survival &amp;gt;100 days after last dose (5 out of 6 mice, increase in life span (ILS) &amp;gt;515%). Furthermore, MLL1-r pediatric B-ALL-PDX mice treated with 50 mg/kg bid for 28 days and NPM1-mu AML-PDX mice treated with 100 mg/kg bid for 35 days were all survived (ILS &amp;gt;254% and &amp;gt;300%) without any detectable leukemic blast cells in bone marrow at 120 and 60 days after cessation of therapy, respectively. These data indicate that DS-M1 has a high potency as an antitumor drug with the potential to provide survival advantage in acute leukemia patients with MLL-r and NPM1-mu. Currently, a Phase 1/2 clinical study of DS-M1 is planned in AML and ALL patients with MLL-r or NPM1-mu. Citation Format: Masashi Numata, Machiko Shiroishi, Kenji Yoshikawa, Noriyasu Haginoya, Tsuyoshi Hirata, Yoshimi Takata, Reina Nagase, Kohei Takashima, Akiko Kurimoto, Fumie Tanzawa, Yumiko Tomoe, Tomoaki Hamada, Ryutaro Kanada, Jun Watanabe, Yoshiko Kagoshima, Eri Tokumaru, Kenji Murata, Takayuki Baba, Taeko Shinozaki, Kazuyuki Hashimoto, Motohiro Kato, Shinji Tsutsumi, Mayumi Kitagawa, Kosaku Fujiwara, Yuki Abe. Development and characterization of a novel orally bioavailable menin-MLL inhibitor for treatment of acute leukemia patients with MLL-rearrangement or NPM1 mutation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1132.

MiR-144 inhibits colorectal cancer cell migration and invasion by regulating PBX3.

MicroRNAs (miRNA) are aberrantly expressed in various human cancers, including colorectal cancer (CRC). We aim to investigate the functional role and underlying mechanism of miR-144 in CRC. The expressional level of miR-144 and pre-leukemia transcription factor 3 (PBX3) in CRC tissues and cells was confirmed by quantitative Real Time-Polymerase Chain Reaction (qRT-PCR) and Western blot. The migration and invasion of CRC cells were detected by transwell assay. Luciferase reporter assay was performed to determine the specific target of miR-144 in CRC cells. The results displayed that miR-144 expression was significantly decreased in CRC tissues and cells compared to that in normal controls. Additionally, miR-144 mimic suppressed, while miR-144 inhibitor promoted the ability of CRC cell migration and invasion. More importantly, PBX3 was the direct target of miR-144 in regulating CRC development and PBX3 could reverse the inhibitory effect of miR-144 mimic on CRC cells. PBX3 expression was significantly increased in CRC and negatively correlated with miR-144 expression. In conclusion, miR-144 suppressed CRC cell migration and invasion by targeting PBX3, suggesting its potential value in the diagnosis and treatment of CRC.

MG132 inhibits the expression of PBX3 through miRNAs by targeting Argonaute2 in hepatoma cells.

Cancer stem cells play important roles in the development of tumors also are important targets to therapy of cancer. Former researches had confirmed the pre-leukemia transcription factor 3 (PBX3) was involved in maintaining the characteristics of liver cancer stem cell. We found that PBX3 is an extremely unstable protein with a short half-life in hepatocellular carcinoma cells. Unstable proteins are believed to be susceptible to degradation by ubiquitin-proteasome system. However, when we treated hepatoma cells using the proteasome inhibitor MG132, found the levels of PBX3 protein and mRNA were significantly downregulated, suggesting that PBX3 protein is not degraded by the ubiquitin-proteasome system. Our study aims to investigate the mechanism of MG132 regulation of PBX3. We observed that the levels of miR-424, let-7c, miR-222, miR-200b were upregulated when hepatoma cells were treated with MG132, and this increase was negatively correlated with the levels of PBX3. Using the miRWalk algorithm, previous studies have predicted that these miRNAs target the PBX3 gene. Thus, we investigated the mechanism by which the proteasome inhibitor MG132 regulates these miRNAs. It has been reported that the Argonaute2 protein is an important component of the RNA-induced silencing complex (RISC), and it can regulate the levels of certain miRNAs. Consequently, we also investigated whether the proteasome inhibitor regulates related miRNAs by stabilizing Argonaute2. Using co-infection, co-immunoprecipitation (Co-IP), and western blot assays, we found that MG132 stabilizes the expression of the Argonuate2 protein by inhibiting its degradation via the ubiquitin-proteasome pathway. In summary, the PBX3 protein, which is closely linked to the stemness of hepatoma cells, does not undergo degradation by the ubiquitin-proteasome system (UPM).

P0060COMBINED NOVEL PBX1 AND RET VARIANTS IN AN ADULT WITH SYNDROMIC RENAL HYPOPLASIA: A CASE REPORT

Abstract Background and Aims Congenital abnormalities of the kidney and urinary tract (CAKUT) represent the main cause of pediatric end-stage renal disease with vast pheno- and genotypic heterogeneity. However, its contribution to adult chronic kidney disease (CKD) is less well characterized. We here report on an adult who underwent genetic testing for previously unrecognized CAKUT. Method Phenotypic characterization of a family with mild syndromic form of kidney hypoplasia. Panel-based genetic analysis for CAKUT associated genes in DNA from peripheral blood cells, oral mucosa, and urinary cells. Results A 20-year-old male presented with bilateral hypoplastic kidneys, first diagnosed with nocturnal enuresis at the age of ten. For CKD stage 2 (eGFR 72ml/min/1.73m), hypertension, and moderate albuminuria (300mg/d) he was treated with Ramipril. Additionally, facial dysmorphism with low-set microtia, slight micrognathia, and slightly impaired intelligence was apparent. Bilateral audiograms did not reveal any hearing impairment. His father showed slightly reduced kidney size without any laboratory anomalies or extrarenal symptoms. Further family history including his mother and three half siblings presented unremarkable. Genetic analysis revealed the following two variants: First, a novel heterozygous truncating variant, absent from patient and population databases, was found in PBX1 c.992C&amp;gt;A p.Ser331* (NM_002585.3). As this variant was detected in 25% of NGS reads, we tested for potential mosaicism by direct sequencing in DNA obtained from oral mucosa and urinary epithelia. While the latter showed no PBX1 variation, mucosa cells revealed the mutated allele in 50% of reads. Segregation analysis of the father showed PBX1 wild-type. The mother was not available for genetic testing. Additionally, we identified a novel heterozygous splice site variant in RET: c.1760-5C&amp;gt;A (NM_020975.4), absent from both patient and population databases. This substitution is predicted to affect splicing at the acceptor site of exon 10. Sanger sequencing demonstrated paternal inheritance. Conclusion Pathogenic PBX1 variants were recently associated with CAKUTHED (Congenital Abnormalities of the Kidney and Urinary Tract with or without Hearing loss, abnormal Ears, or Developmental delay) syndrome (Heidet 2017), characterized by highly variable phenotypes. PBX1 encodes a homeodomain transcription factor taking part in transcriptional regulation during embryonic development. Functional studies suggest disturbed PBX1-dependent transactivation ability and altered nuclear translocation, resulting in abnormal interactions between PBX1 proteins and cofactors (Slavotinek 2017). Prevailing PBX1 expression in embryonic compared to adult kidneys support an important function in development (Le Tanno 2017). An aberrant splicing effect of the identified RET variant is supported by the fact that the father, who carries the same variant but PBX1 wild-type, has slightly reduced kidneys. Associations between abnormal kidney development and alterations of RET were first described in stillborn fetuses with renal agenesis (Skinner 2008), but also in patients with vesicoureteral reflux (Yang 2008). RET encodes for a receptor tyrosine-protein kinase, which while binding ligands such as GDNF (glial cell-line derived neurotrophic factor) is involved in signaling pathways and plays an important part during organ development. Particularly, the RET/GDNF signaling pathway plays a crucial role during development of the kidneys (Costantini 2010). Functional analyses indicate an altered phosphorylation status of RET as possible causative mechanism. To the best of our knowledge, this case is the first to report on combined PBX1 and RET gene variants associated with mild syndromic renal hypoplasia, likely mitigated by genetic mosaicism.

Targeting CD49a+PBX1+ NK cells in recurrent spontaneous abortion immunotherapy

Abstract Objective In the first trimester, CD49a+ natural killer (NK) cells are the main immune cells at the maternal-fetal interface. We have demonstrated that uterine NK cells have important physiological functions that can secrete various growth factors to promote fetal development. However, the functional subsets and transcriptional regulation mechanisms of NK cells expressing GPFs are unclear. Result Here, we have identified CD49a+PBX1+ NK cells as a unique subset in which the transcription factor, PBX1, upregulates the expression of the GPFs, pleiotrophin and osteoglycin, to promote fetal development at the maternal-fetal interface of both humans and mice. The results of ChIP experiments with anti-PBX1 antibody in decidual NK cells shown that PBX1 regulates the transcription of pleiotrophin and osteoglycin genes via direct binding. Inactivation of Pbx1 in mouse NK cells caused impaired fetal development due to a significantly decrease in the expression of GPFs. The results of exome sequencing of decidual NK cells from patients with unexplained recurrent spontaneous abortion (URSA), revealed decreased expression of PBX1 correlated with fetal growth restriction and pregnancy failure, suggesting that the impaired PBX1 gene in decidual NK cells is a potential biomarker for URSA. Conclusion Our study revealed that the transcriptional regulation mechanism in NK cells at the maternal-fetal interface, and suggested that impaired CD49a+PBX1+ NK cells contribute to the etiology of URSA, and provided the basis for improved approaches to NK cell-based immunotherapy for pregnancy related diseases.

Long noncoding RNA NORAD promotes the progression of retinoblastoma by sponging miR 136-5p/PBX3 axis.

The article "Long noncoding RNA NORAD promotes the progression of retinoblastoma by sponging miR-136-5p/PBX3 axis, by X.-L. Yang, Y.-J. Hao, B. Wang, X.-L. Gu, X.-X. Wang, J.-F. Sun, published in Eur Rev Med Pharmacol Sci 2020; 24(3):1278-1287. DOI: 10.26355/eurrev_202001_20185. PMID: 32096159" has been withdrawn from the authors. The Publisher apologizes for any inconvenience this may cause.

MiR-655-3p inhibits growth and invasiveness of trophoblasts via targeting PBX3 and thus deteriorates preeclampsia.

The purpose of this study was to elucidate the regulatory effects of microRNA-655-3p (miR-655-3p) on growth and invasiveness of trophoblasts to influence the development of preeclampsia (PE). Relative levels of miR-655-3p and PBX3 in 24 PE pregnant women and 24 healthy ones were examined. Then, the regulatory effects of miR-655-3p and PBX3 on viability and invasiveness in HTR-8/SVneo cells were assessed by Cell Counting Kit-8 (CCK-8) and transwell assay, respectively. Next, the expression levels of apoptosis-associated genes were determined. Finally, the interaction between miR-655-3p and PBX3 was tested by Dual-Luciferase reporter assay. MiR-655-3p was upregulated in placental tissues of PE pregnant women than those of healthy ones. The overexpression of miR-655-3p suppressed viability and invasiveness, and induced apoptosis in HTR-8/SVneo cells. Besides, PBX3 was the target gene binding to miR-655-3p, and more importantly, the overexpression of PBX3 partially reversed the regulatory effects of miR-655-3p on viability and invasiveness in HTR-8/SVneo cells. MiR-655-3p is involved in the development of PE by regulating biological features of trophoblasts by targeting PBX3.

Antitumor effect of baicalin from the Scutellaria baicalensis radix extract in B-acute lymphoblastic leukemia with different chromosomal rearrangements

Acute B-lymphoblastic leukemia (B-ALL) is the most common hematologic malignancy in children. Many cases of B-ALL harbor chromosomal translocations which are often critical determinants of prognosis. Most of them represent altered transcription factors that impact gene transcription or enhance signaling. B-ALLs harboring the mixed-lineage leukemia 1 (MLL1) gene rearrangements represent aggressive, high-risk type of early childhood leukemias that are usually associated with a very poor prognosis. Therefore, there is an urgent need for novel therapeutic agents as well as new treatment strategies.The objective was to examine the vitro inhibitory effects of Scutellaria baicalensis root extract (SBE) in B-ALL cell lines with different chromosomal rearrangements and in leukemic blasts derived from patients’ bone marrow (BMCs).In this study we showed that baicalin which is the main component of the SBE possess antitumor activity against all leukemic cell lines especially those with MLL and PBX1 gene rearrangements. Baicalin inhibited cell proliferation, arrested the cell cycle at the G0/G1 phase, and induced cell death through caspase 3/7 activation. Moreover, baicalin treatment inhibited the glycogen synthase kinase-3β (GSK‐3β) by suppressing its phosphorylation at Y216, and upregulated the downstream mediator of the cell cycle arrest - cyclin dependent kinase inhibitor p27Kip1. Bone marrow derived blasts from B-ALL patients also exhibited varied sensitivity towards baicalin with 72% patients sensitive to the SBE and baicalin treatment.Taken together, our findings provide new insights into the anti-cancer properties of baicalin by showing its diverse mode of action which might be related to the different genetic background.