Pbx Proteins Research Articles

Spindle function and Wnt pathway inhibition by PBX1 to suppress tumor progression via downregulating DCDC2 in colorectal cancer

PBX1 is a transcription factor that regulates a variety of genes, involved in intracellular lipid metabolism, cell proliferation, and other pathways. The promoting and inhibiting function of PBX1 in various cancer types was extensively discussed, however, there have been no studies on PBX1 proteins in colorectal cancer (CRC). This study aimed to reveal the anti-tumor function of PBX1 in CRC and the underlying molecular mechanism. Bioinformatics analysis revealed that PBX1 is downregulated in CRC, indicating that is a potential antioncogene in CRC. Overexpression of PBX1 suppresses tumor growth and metastasis in vitro and in vivo. Mechanistically, we found that PBX1 acted as a transcription factor that suppressed DCDC2 expression and inhibited spindle function. Moreover, the PBX1-DCDC2 axis controlled the Wnt pathway in CRC cells. Overexpression of DCDC2 restored CRC proliferation, metastasis abilities and Wnt pathway. In conclusion, this study suggests that PBX1 acts as a transcription factor to suppress DCDC2 expression and inhibit cell proliferation and metastasis by disrupting spindle function and the Wnt pathway in CRC.

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.

Analysis of lamprey meis genes reveals that conserved inputs from Hox, Meis and Pbx proteins control their expression in the hindbrain and neural tube

Meis genes are known to play important roles in the hindbrain and neural crest cells of jawed vertebrates. To explore the roles of Meis genes in head development during evolution of vertebrates, we have identified four meis genes in the sea lamprey genome and characterized their patterns of expression and regulation, with a focus on the hindbrain and pharynx. Each of the lamprey meis genes displays temporally and spatially dynamic patterns of expression, some of which are coupled to rhombomeric domains in the developing hindbrain and select pharyngeal arches. Studies of Meis loci in mouse and zebrafish have identified enhancers that are bound by Hox and TALE (Meis and Pbx) proteins, implicating these factors in the direct regulation of Meis expression. We examined the lamprey meis loci and identified a series of cis-elements conserved between lamprey and jawed vertebrate meis genes. In transgenic reporter assays we demonstrated that these elements act as neural enhancers in lamprey embryos, directing reporter expression in appropriate domains when compared to expression of their associated endogenous meis gene. Sequence alignments reveal that these conserved elements are in similar relative positions of the meis loci and contain a series of consensus binding motifs for Hox and TALE proteins. This suggests that ancient Hox and TALE-responsive enhancers regulated expression of ancestral vertebrate meis genes in segmental domains in the hindbrain and have been retained in the meis loci during vertebrate evolution. The presence of conserved Meis, Pbx and Hox binding sites in these lamprey enhancers links Hox and TALE factors to regulation of lamprey meis genes in the developing hindbrain, indicating a deep ancestry for these regulatory interactions prior to the divergence of jawed and jawless vertebrates.

A Computer-Based Methodology to Design Non-Standard Peptides Potentially Able to Prevent HOX-PBX1-Associated Cancer Diseases.

In the last decades, HOX proteins have been extensively studied due to their pivotal role in transcriptional events. HOX proteins execute their activity by exploiting a cooperative binding to PBX proteins and DNA. Therefore, an increase or decrease in HOX activity has been associated with both solid and haematological cancer diseases. Thus, inhibiting HOX-PBX interaction represents a potential strategy to prevent these malignancies, as demonstrated by the patented peptide HTL001 that is being studied in clinical trials. In this work, a computational study is described to identify novel potential peptides designed by employing a database of non-natural amino acids. For this purpose, residue scanning of the HOX minimal active sequence was performed to select the mutations to be further processed. According to these results, the peptides were point-mutated and used for Molecular Dynamics (MD) simulations in complex with PBX1 protein and DNA to evaluate complex binding stability. MM-GBSA calculations of the resulting MD trajectories were exploited to guide the selection of the most promising mutations that were exploited to generate twelve combinatorial peptides. Finally, the latter peptides in complex with PBX1 protein and DNA were exploited to run MD simulations and the ΔGbinding average values of the complexes were calculated. Thus, the analysis of the results highlighted eleven combinatorial peptides that will be considered for further assays.

Bimolecular Fluorescence Complementation (BiFC) and Multiplexed Imaging of Protein-Protein Interactions in Human Living Cells.

Deciphering protein-protein interactions (PPIs) in vivo is crucial to understand protein function. Bimolecular fluorescence complementation (BiFC) makes applicable the analysis of PPIs in many different native contexts, including human live cells. It relies on the property of monomeric fluorescent proteins to be reconstituted from two separate subfragments upon spatial proximity. Candidate partners fused to such complementary subfragments can form a fluorescent protein complex upon interaction, allowing visualization of weak and transient PPIs. It can also be applied for investigation of distinct PPIs at the same time using a multicolor setup. In this chapter, we provide a detailed protocol for analyzing PPIs by doing BiFC in cultured cells. Proof-of-principle experiments rely on the complementation property between the N-terminal fragment of mVenus (designated VN173) and the C-terminal fragment of mCerulean (designated CC155) and the partnership between HOXA7 and PBX1 proteins. This protocol is compatible with any other fluorescent complementation pair fragments and any type of candidate interacting proteins.

Возможная роль гомеодоменсодержащего транскрипционного фактора PREP1 в мезенхимальных клетках сердца

Homeodomain transcription factors play a significant role in mesenchymal stromal cells (MSCs). Previously, the role of Meis1, Pbx1 and Prep1 proteins from the TALE (Three Amino acid Loop Extension) family in adipocytic and osteogenic differentiation of mouse mesenchymal stromal cells was established. In this work, using ChIP-seq and bioinformatic analysis we investigated the binding pattern of PREP1 with the genomic DNA of human heart MSCs, identified nearby genes, and analyzed their ontology. The target genes of the PREP1 factor in cardiac MSCs have been established. Based on the results, the possible involvement of transcription factor PREP1 in the direct reprogramming of fibroblasts into cardiomyocytes is discussed.

A six-amino-acid motif is a major determinant in functional evolution of HOX1 proteins.

Gene duplication and divergence is a major driver in the emergence of evolutionary novelties. How variations in amino acid sequences lead to loss of ancestral activity and functional diversification of proteins is poorly understood. We used cross-species functional analysis of Drosophila Labial and its mouse HOX1 orthologs (HOXA1, HOXB1, and HOXD1) as a paradigm to address this issue. Mouse HOX1 proteins display low (30%) sequence similarity with Drosophila Labial. However, substituting endogenous Labial with the mouse proteins revealed that HOXA1 has retained essential ancestral functions of Labial, while HOXB1 and HOXD1 have diverged. Genome-wide analysis demonstrated similar DNA-binding patterns of HOXA1 and Labial in mouse cells, while HOXB1 binds to distinct targets. Compared with HOXB1, HOXA1 shows an enrichment in co-occupancy with PBX proteins on target sites and exists in the same complex with PBX on chromatin. Functional analysis of HOXA1-HOXB1 chimeric proteins uncovered a novel six-amino-acid C-terminal motif (CTM) flanking the homeodomain that serves as a major determinant of ancestral activity. In vitro DNA-binding experiments and structural prediction show that CTM provides an important domain for interaction of HOXA1 proteins with PBX. Our findings show that small changes outside of highly conserved DNA-binding regions can lead to profound changes in protein function.

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>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>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.

Abstract P3-10-15: Therapeutic targeting of HOX gene dysregulation in triple negative breast cancer

Abstract Introduction: Triple Negative Breast Cancer (TNBC) is a heterogeneous disease, clinically defined for its aggressive tumour behaviour, metastatic ability and poor prognosis. Homeobox (HOX) genes are a family of homeodomain-containing transcription factors, which establish and maintain the identity and fate of cells and tissues during normal embryogenesis and organ development. A total of 39 HOX genes exist, located in four clusters (A-D) on different chromosomes. HOX genes are dysregulated in most cancers and they have an established role in driving key processes in breast carcinogenesis, including the regulation of the cell cycle, apoptosis, angiogenesis and metastasis. In this study, we evaluated the anti-tumour efficacy of a novel inhibitor of HOX protein function, HTL-001, which antagonizes the interactions between HOX proteins and their PBX cofactors. HTL-001, developed by HOX Therapeutics Ltd, is a synthetic peptide of 18 amino acids, comprised of a hexapeptide sequence, which resembles that on HOX proteins of paralogs 1-9, and a short C-terminal polyarginine sequence for cell penetration. We evaluated the efficacy of HTL-001 in vitro as a monotherapy in all cell lines representing all molecular classifications of breast cancer and in combination with chemotherapeutics for TNBC therapy. Most importantly, HTL-001 was examined in MDA-MB-231-BR cells, which have a propensity to metastasise to the brain, reflecting the disease course frequently observed in human patients. Methods: The differential expression of HOX and PBX genes in all subtypes of breast cancer was evaluated using bioinformatics analysis of TCGA and Oncomine data. The sensitivity to HTL-001 of breast cancer-derived cell lines of different molecular classifications, was determined using Cell Proliferation (MTS) Assay and FACS-based Annexin Assay. The anti-tumour effect of HTL-001 in combination with various chemotherapeutics was assessed and analysed for synergy using Bliss Analysis. The expression of genes previously shown to respond to HOX/PBX inhibition was measured from extracted RNA and protein using RT-qPCR and western blotting, respectively. The in vivo efficacy of HTL-001 will be investigated using a mouse flank tumour xenograft model with MDA-MB-231-BR cells, representative of TNBC. Results: TCGA and Oncomine data analysis showed TNBC tumours have a unique signature of HOX gene dysregulation, different to that of all other types of breast tumours, relative to normal breast tissues; most importantly upregulation in HOXB2 and HOXB3 genes. Targeting HOX/PBX dimers with HTL-001, induced apoptotic cell death in all breast cancer cell lines including; MCF-7, ZR-75-1, MDA-MB-231, BT-20 and SK-BR-3, with the highest sensitivity seen in brain seeking MDA-MB-231-BR cells. HTL-001 significantly upregulated the expression of key apoptotic and anti-survival genes including; AIF, cFOS, and DUSP1, resulting in the induction of apoptotic cell death in all breast cancer cell lines tested. Furthermore, we observed high levels of synergy between HTL-001 and the chemotherapeutic agents, Paclitaxel, Palbociclib and 5-Fluorouracil. Conclusion: HTL-001 is a novel inhibitor of HOX and PBX protein interactions capable of marked in vitro cytotoxicity in all types of breast cancer cells, particularly in TNBC. HTL-001 induced rapid apoptosis and triggered a downstream unique signature after target engagement. HTL-001 is currently the only therapy which targets HOX gene dysregulation and in doing so, may help address the current urgent unmet therapeutic need in TNBC through its unique mechanism of action and high levels of synergy with conventional anti-cancer agents. Citation Format: Einthavy Arunachalam, Guy R Simpson, Carla Sofia Möller-Levet, Nicola Annels, Richard Morgan, Timothy Robert Crook, Hardev Singh Pandha. Therapeutic targeting of HOX gene dysregulation in triple negative breast cancer [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P3-10-15.

PBX3 in Cancer.

PBX3 is a homeodomain-containing transcription factor of the pre-B cell leukemia (PBX) family, members of which have extensive roles in early development and some adult processes. A number of features distinguish PBX3 from other PBX proteins, including the ability to form specific and stable interactions with DNA in the absence of cofactors. PBX3 has frequently been reported as having a role in the development and maintenance of a malignant phenotype, and high levels of PBX3 tumor expression have been linked to shorter overall survival in cancer. In this review we consider the similarities and differences in the function of PBX3 in different cancer types and draw together the core signaling pathways involved to help provide a better insight into its potential as a therapeutic target.

Comparative Analysis of Putative Prognostic and Predictive Markers in Neuroblastomas: High Expression of PBX1 Is Associated With a Poor Response to Induction Therapy.

The survival rate for patients with high-risk neuroblastomas remains poor despite new improvements in available therapeutic modalities. A detailed understanding of the mechanisms underlying clinical responses to multimodal treatment is one of the important aspects that may provide precision in the prediction of a patient's clinical outcome. Our study was designed as a detailed comparative analysis of five selected proteins (DDX39A, HMGA1, HOXC9, NF1, and PBX1) in one cohort of patients using the same methodical approaches. These proteins were already reported separately as related to the resistance or sensitivity to retinoids and as useful prognostic markers of survival probability. In the cohort of 19 patients suffering from high-risk neuroblastomas, we analyzed initial immunohistochemistry samples obtained by diagnostic biopsy and post-induction samples taken after the end of induction therapy. The expression of DDX39A, HMGA1, HOXC9, and NF1 showed varied patterns with almost no differences between responders and non-responders. Nevertheless, we found very interesting results for PBX1: non-responders had significantly higher expression levels of this protein in the initial tumor samples when compared with responders; this expression pattern changed inversely in the post-induction samples, and this change was also statistically significant. Moreover, our results from survival analyses reveal the prognostic value of PBX1, NF1, and HOXC9 expression in neuroblastoma tissue. In addition to the prognostic importance of PBX1, NF1, and HOXC9 proteins, our results demonstrated that PBX1 could be used for the prediction of the clinical response to induction chemotherapy in patients suffering from high-risk neuroblastoma.

A Hox-TALE regulatory circuit for neural crest patterning is conserved across vertebrates

In jawed vertebrates (gnathostomes), Hox genes play an important role in patterning head and jaw formation, but mechanisms coupling Hox genes to neural crest (NC) are unknown. Here we use cross-species regulatory comparisons between gnathostomes and lamprey, a jawless extant vertebrate, to investigate conserved ancestral mechanisms regulating Hox2 genes in NC. Gnathostome Hoxa2 and Hoxb2 NC enhancers mediate equivalent NC expression in lamprey and gnathostomes, revealing ancient conservation of Hox upstream regulatory components in NC. In characterizing a lamprey hoxα2 NC/hindbrain enhancer, we identify essential Meis, Pbx, and Hox binding sites that are functionally conserved within Hoxa2/Hoxb2 NC enhancers. This suggests that the lamprey hoxα2 enhancer retains ancestral activity and that Hoxa2/Hoxb2 NC enhancers are ancient paralogues, which diverged in hindbrain and NC activities. This identifies an ancestral mechanism for Hox2 NC regulation involving a Hox-TALE regulatory circuit, potentiated by inputs from Meis and Pbx proteins and Hox auto-/cross-regulatory interactions.

'Building a perfect body': control of vertebrate organogenesis by PBX-dependent regulatory networks.

Pbx genes encode transcription factors that belong to the TALE (three-amino-acid loop extension) superclass of homeodomain proteins. We have witnessed a surge in information about the roles of this gene family as leading actors in the transcriptional control of development. PBX proteins represent a clear example of how transcription factors can regulate developmental processes by combinatorial properties, acting within multimeric complexes to implement activation or repression of transcription depending on their interaction partners. Here, we revisit long-emphasized functions of PBX transcription factors as cofactors for HOX proteins, major architects of the body plan. We further discuss new knowledge on roles of PBX proteins in different developmental contexts as upstream regulators of Hox genes-as factors that interact with non-HOX proteins and can work independently of HOX-as well as potential pioneer factors. Committed to building a perfect body, PBX proteins govern regulatory networks that direct essential morphogenetic processes and organogenesis in vertebrate development. Perturbations of PBX-dependent networks can cause human congenital disease and cancer.

Determination of the Origin of the t(1;19) TCF3-PBX1 Fusion By Genomic Inverse PCR for Exploration of Ligated Breakpoints (GIPFEL)

Pediatric acute lymphoblastic leukemia (ALL) is characterized by recurrent chromosomal translocations. The translocation t(1;19) that fuses the gene encoding the basic helix-loop-helix transcription factor TCF3 with the gene encoding the homeodomain protein PBX1 is the second most common one occurring in approximately 5-10% of precursor B ALL cases. Backtracking of clonotypic TCF3-PBX1 translocations that were identified in leukemia patients by PCR amplification of Guthrie cards from these individuals provided weak evidence for a prenatal origin of a minority of TCF3-PBX1 translocations (2 of 15 cases). The presence of N-nucleotides at the recombination junction, IGH rearrangements and the specific JH and DH segment usage indirectly supported a postnatal origin of the majority of translocations, but could not definitely date the fusion event during development (Wiemels et al. PNAS 2002).We recently developed a novel, DNA-based screening technique (genomic inverse PCR for exploration of ligated breakpoints, GIPFEL) for the detection of translocations without prior knowledge of the exact breakpoint (Fueller et al. PLOS ONE 2015). By GIPFEL screening of 1,000 umbilical cord blood samples, we confirmed a high prevalence (≥5%) of the most frequent ALL associated translocation, t(12;21), in healthy newborns (Schaefer et al. BLOOD 2018). This translocation was 500-fold more frequent than the corresponding leukemia incidence (1/10,000) indicating a low penetrance of the leukemic fusion and a greater importance of secondary oncogenic events. In order to trace the origin of the TCF3-PBX1 fusion and to assess the risk of children bearing the translocation to develop leukemia, we collected 340 cord blood samples of healthy newborns and subjected them to GIPFEL screening.The GIPFEL technique uses stable DNA as a sample and detects a translocation by inverse PCR after restriction enzyme digest of the DNA and circularization of fragments by ligation. For t(1;19) screening, DNA was isolated from CD19+ enriched mononuclear cells, digested with the enzyme MfeI and ligated. Remaining linear DNA fragments were removed by exonuclease digest. After ethanol precipitation of the DNA circles a partially multiplexed, semi-nested PCR was carried out to quantify all possible ligation/junction products specific for the translocation. Samples that screened positive underwent one further demultiplexed PCR, agarose gel electrophoresis and Sanger sequencing to validate the result. An internal PBX1 genomic ligation product served as a positive control. TCF3-PBX1 positive cells at a frequency ≥10-4 to 10-5 would be detected by the GIPFEL method.Of the 340 screened cord bloods, 292 are currently undergoing evaluation and 48 are validated. So far, none of the 48 samples was positive for the TCF3-PBX1 translocation. In case all 340 cord bloods are negative, the result could suggest that TCF3-PBX1 translocations occur very rarely prenatally and that they have a high oncogenic penetrance if they arise in utero, although cooperating secondary mutations are clearly necessary. This would be in line with the strong capability of the TCF3-PBX1 oncoprotein to transform many cell types in vitro and with the generation of diverse (although late occurring) tumors observed in TCF3-PBX1 transgenic mice (reviewed in Aspland et al. Oncogene 2001). These results would support the previous finding of clonotypic TCF3-PBX1 transcripts in 2 of 15 Guthrie cards derived from individuals who later developed leukemia (Wiemels et al. PNAS 2002).Complete results of GIPFEL screening of 340 newborns will be available and presented at the conference. Although the number of healthy newborns investigated is still low, these results will help to determine the origin of the t(1;19) TCF3-PBX1 fusion. DisclosuresNo relevant conflicts of interest to declare.

Targeting HOX/PBX dimers in cancer.

The HOX and PBX gene families encode transcription factors that have key roles in establishing the identity of cells and tissues in early development. Over the last 20 years it has become apparent that they are also dysregulated in a wide range of solid and haematological malignancies and have a predominantly pro-oncogenic function. A key mode of transcriptional regulation by HOX and PBX proteins is through their interaction as a heterodimer or larger complex that enhances their binding affinity and specificity for DNA, and there is growing evidence that this interaction is a potential therapeutic target in malignancies that include prostate, breast, renal, ovarian and lung cancer, melanoma, myeloma, and acute myeloid leukaemia. This review summarizes the roles of HOX and PBX genes in cancer and assesses the therapeutic potential of HOX/PBX dimer inhibition, including the availability of biomarkers for its application in precision medicine.

Identification of proteins associated with transcription factors HOXA9 and E2A-PBX1 by tandem affinity purification

Chimeric transcription factor E2A-PBX1 induces the development of acute lymphoblastic B-cell leukemia in children. Using a transgenic mouse model, we previously demonstrated that homeobox (HOX) gene HOXA9 genetically interact with E2A-PBX1 gene in the development of B-cell leukemia in mice. HOXA9 itself is a potent oncogene resulting in myeloid leukemia when overexpressed, which is strongly accelerated by its collaborator Meis1. HOX, PBX1 and MEIS1 proteins have been shown to form hetero dimeric or trimeric complexes in different combinations. Cooperative interaction between PBX1 and HOX proteins enhances their DNA binding specificity, essential for HOX dependent developmental programs. PBX1 is retained in E2A-PBX1, and thus the strong transcriptional activator properties of E2A-PBX1 may lead to aberrant activation of normally repressed targets of HOX-PBX complexes. However, although there is evidence that E2A-PBX1 could bind to HOX and MEIS1 proteins it is still unclear whether such complexes are actually required for leukemic transformation or whether E2A-PBX1 and HOXA9 are each part of larger protein complexes acting in independent complementing oncogenic pathways. In this study we aim to search for other HOXA9 and E2A-PBX1 interacting proteins. To identify novel proteins interacting with human E2A-PBX1 or HOXA9 we used tandem affinity purification (TAP) of protein complexes from 697 pre-B leukemic and HeLa cell lines transduced to express E2A-PBX1 or HOXA9, respectively, with covalently attached FLAG/HA peptides. The protein composition of each complex was determined using tandem mass-spectrometry. In the E2A-PBX1 containing complex we identified lymphoid transcription factor IKAROS, chromatin remodeling factors of SWI/SNF family while multiple subunits of translation initiation factor eIF3, E3 ubiquitin ligase UBR5 emerged from the HOXA9 complex as potential critical protein partners. This is the first time the protein partners of either E2A-PBX1 or HOXA9 oncoproteins were identified using an unbiased biochemical approach. The identification of translation initiation factors associated with HOXA9 might indicate a novel function for HOX proteins independent of their transcriptional activity.

EXTH-48. SMALL MOLECULAR TARGETING OF HOX-PBX BINDING IN GLIOBLASTOMA AS A NOVEL THERAPEUTIC TREATMENT

The HOX genes encode a family of transcription factors that play an essential role in embryonic patterning, but are dysregulated in numerous cancers, including glioblastoma. Previous research indicates that HOX genes are overexpressed expressed in glioblastoma tumours compared to healthy astrocytes. HXR9, a small hexapeptide, was shown to have strong cytotoxic effects on glioblastoma ‘parental’ and cancer stem cells. In this study we assessed whether a new small molecular mimic (SMM) of HXR9 had similar or increased therapeutic potential. Glioblastoma cell lines were treated with varying doses of HXR9 and SMM, with cell viability determined by MTS assay. Mode of death was determined via annexin-V/7-AAD counterstain assay. RT-PCR and western blot analysis was used to determining the molecular pathway utilised by HXR9 and SMM. SMM was shown to be cytotoxic in, a dose dependent manner, in all cell lines used. Some cell lines exhibited increased sensitivity to SMM when compared to HXR9 treatment. Mode of death was determined to be apoptosis. C-Fos, a sub unit of the pro-apoptotic complex AP-1, was shown to be elevated following treatment with both HOX-PBX inhibitors. C-Fos up regulation was accompanied in all cell lines with a sharp down regulation of Bcl-2. C-Jun, an important subunit in AP-1, was shown to be unaffected by treatment, but was shown to be expressed in all cell lines prior to treatment. Results show that aberrantly expressed HOX and PBX proteins in glioblastoma can be targeted with cytotoxic effect. HXR9 and SMM treatment causes apoptosis through up regulation of C-Fos, and subsequent down regulation of Bcl-2. This mechanism may be caused by an increase of AP-1 activity, which in turn causes suppression in Bcl-2 expression.

Pbx3 and Meis1 cooperate through multiple mechanisms to support Hox-induced murine leukemia.

Hox homeobox transcription factors drive leukemogenesis efficiently only in the presence of Meis or Pbx proteins. Here we show that Pbx3 and Meis1 need to dimerize to support Hox-induced leukemia and we analyze the molecular details of this cooperation. In the absence of Pbx3, Meis1 was highly unstable. As shown by a deletion analysis Meis1 degradation was contingent on a motif coinciding with the Pbx-binding domain. Either deletion of this sequence or binding to Pbx3 prolonged the half-life of Meis1 by preventing its ubiquitination. Meis1 break-down could also be blocked by inhibition of the ubiquitin proteasome system, indicating tight post-transcriptional control. In addition, Meis1 and Pbx3 cooperated genetically as overexpression of Pbx3 induced endogenous Meis1 transcription. These functional interactions translated into in vivo activity. Blocking Meis1/Pbx3 dimerization abrogated the ability to enhance proliferation and colony-forming cell numbers in primary cells transformed by Hoxa9. Furthermore, expression of Meis1 target genes Flt3 and Trib2 was dependent on Pbx3/Meis1 dimerization. This correlated with the requirement of Meis1 to bind Pbx3 in order to form high affinity DNA/Hoxa9/Meis1/Pbx3 complexes in vitro. Finally, kinetics and severity of disease in transplantation assays indicated that Pbx3/Meis1 dimers are rate-limiting factors for Hoxa9-induced leukemia.

Molecular and cellular mechanisms of adipogenesis

The main components of metabolic syndrome include insulin resistance, hypertriglyceridemia and arterial hypertension. Obesity is the cause of metabolic syndrome, mainly as a consequence of the endocrine function of adipose tissue. The volume of adipose tissue depends on the size of individual adipocytes and on their number. The number of adipocytes increases as a result of enhanced adipocyte differentiation. The transcriptional cascade that regulates this differentiation has been well studied. The major adipogenic transcription factor peroxisome proliferator-activated receptor gamma is a ligand-activated nuclear receptor with essential roles in adipogenesis. Its ligands are used to treat metabolic syndrome and type 2 diabetes mellitus. . The present article describes the basic molecular and cellular mechanisms of adipogenesis and discusses the impact of insulin, glucocorticoids, cyclic adenosine monophosphate-activating agents, nuclear receptors and transcription factors on the process of adipogenesis. New regulatory regions of the genome that are capable of binding multiple transcription factors are described, and the most promising drug targets for the treatment of metabolic syndrome and obesity, including the homeodomain proteins Pbx1 and Prep1, are discussed..