Meis Proteins Research Articles

Myeloid Ecotropic Viral Integration Site (MEIS) Proteins: Illuminating Cancer Diagnosis and Biomarker Significance

Context: The MEIS proteins, originally described as the "helper groups" of HOX proteins, are now becoming recognized as significant and incredibly diverse controllers of cellular behavior. The MEIS agents can incorporate a variety of epigenetic modulators into chromatin to aid in embryonic growth or tissue homeostasis by adult stem cells. The MEIS gene's expression, binding, stability, post-translational protein modification, and controlled intracellular availability are all fully regulated, and they all react to external stimuli. Objectives: We explored the diverse roles of MEIS proteins in cellular behavior and epigenetic regulation and investigated the potential of MEIS proteins as diagnostic and therapeutic biomarkers in cancers. Results: The MEIS proteins can be diagnostic and therapeutic biomarkers for cancer. The MEIS-PBX heterotrimeric is involved in neurogenesis processes, and the formation of the MEIS2 heterodimer with PAX6 and DLX2 can proliferate periplomerular dopaminergic neurons in the olfactory bulb. The estrogen receptor in breast cancer upregulates MEIS1 and FOXP3. The estrogen receptor pathway's cancer-related genes can express themselves more strongly when MEIS1-FOXP3 interacts with a positive feedback mechanism. Because MEIS1 expression is decreased in clear renal cell carcinoma (ccRCC) cell lines, MEIS1 may function as a tumor suppressor in the development of ccRCC. Conclusions: MEIS1 is recognized as one of the main contributors to the development of leukemias. The MEIS proteins influence the c-MYC signaling pathway, cell proliferation, and invasion of PC by contributing to its development.

Inhibition of Menin, BCL-2, and FLT3 Combined with a Hypomethylating Agent Cures NPM1/FLT3-ITD/-TKD Mutant AML in a PDX Model

Menin inhibition has antileukemia activity in both mixed-lineage leukemia-rearranged (MLL-r) and nucleophosmin (NPM1)-mutated (NPM1c) AML by suppressing the menin-dependent HOX/MEIS gene signature pre-clinically and clinically. However, co-mutations are common in patients with MLL-r or NPM1c AML and menin inhibition in combination with other targeted agents has potential to enhance efficacy. FLT3 is frequently mutated in MLL-r and NPM1c AML. We previously reported that menin inhibition by SNDX-50469 (SNDX) synergized with BCL-2 inhibition by venetoclax (VEN) in vitro using primary AML patient samples and in vivo in a patient-derived xenograft (PDX) model of NPM1/FLT3-ITD/FLT3-TKD mutated AML. However, cells surviving the SNDX and VEN combination at the end of treatment had increased p-FLT3 signaling (Carter BZ et al., Blood 2021). We therefore investigated whether FLT3 inhibition with gilteritinib (Gil) could enhance the efficacy of co-targeting menin and BCL-2 using the same PDX model harboring NPM1/FLT3-ITD/-TKD mutations. We treated PDX-bearing NSG mice with vehicle control, SNDX, Gil, SNDX + Gil, VEN + Gil, or SNDX + Gil + VEN. CyTOF analysis of bone marrow (BM) leukemia cells at the end of treatment demonstrated that the SNDX + Gil + VEN combination targeted leukemia stem/progenitor cells more effectively than SNDX, Gil, or SNDX + Gil. Furthermore, SNDX markedly reduced BCL-2, BCL-XL, BCL2-A1, MEIS1, and PBX3 proteins invivo, which were further reduced by the combination of SNDX, Gil, and VEN (Fig. 1). All treatment arms suppressed leukemia burden in peripheral blood, spleen, and BM and significantly extended survival (Fig. 2) compared to vehicle-treated controls (median 62 days). SNDX (128 days) was significantly more effective than Gil (90.5 days; P = 0.0001). SNDX + Gil (119 days) did not further improve survival compared to SNDX alone, possibly because the two agents have overlapping effects on FLT3 signaling: Gil inhibits FLT3 activity, while menin inhibition suppresses FLT3 levels. The survival duration of the Gil + VEN (121 days) and SNDX + Gil groups did not differ. Even with the reduced Gil (25 vs. 35 mg/kg) and VEN (35 vs. 50 mg/kg) doses compared to the single agent or two-drug combination groups, the SNDX + Gil + VEN combination extended survival (>235 days), significantly longer than SNDX, Gil, SNDX + Gil, or VEN + Gil. Survival in the 3-drug combination group was much longer than in the SNDX + VEN group (143 days), that we previously reported (Carter BZ et al., Blood 2021). To determine if hypomethylating agents further improve survival, we also treated mice with the SNDX + Gil + VEN + 5-Azacytidine (5-AZA) combination. The median survival duration had not been reached when the experiment was terminated on day-414. Importantly, 2/6 mice in the triple- and 3/5 mice in the quadruple-drug treatment groups lived close to the life expectancy of normal NSG mice with no detectable leukemia cells in spleens and BMs when the experiment was terminated on day-414, suggesting that these combinations potentially eliminated all leukemia cells resulting in cures. Conclusions: Findings demonstrate that the combined inhibition of menin, BCL-2, and FLT3 has profound activity against AML and AML stem progenitor cells with NPM1/FLT3-ITD/-TKD mutations. The combination reduces the HOX/MEIS signature and the antiapoptotic BCL-2 proteins, resulting in major survival benefit. The quadruple combination of SNDX + Gil + VEN + 5-AZA resulted in cure of >50% of mice carrying triple mutant AML. Results warrant the clinical development of concomitant inhibition of menin, BCL-2, and FLT3 combined with a hypomethylating agent in NPM1/FLT3-mutant AML. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Cancer Friendly MEIS Proteins

Miyeloid kanser türünden ismini alan MEIS; enerji üretimini teşvik eden, ROS seviyelerini minimum düzeye indirmeye çalışan, kalp kası gelişim evresinde görev alan bir proteindir. Kanserli hücreler MEIS proteini metastaz için kullanmaktadır. MEIS proteini kanserli hücrelerde durdurmak amacıyla ‘kanseri kendi silahıyla vurma’ olarak tanımlanan MEIS protein inhibitörleri (MEISi) Türk akademisyen Fatih KOCABAŞ tarafından keşfedilmiş ve bu çalışma Uluslararası Hemotoloji-Onkoloji ödülüne layık görülmüştür. Yapılan çalışmada MEIS protein inhibitörleri meme ve pankreas gibi kanser türlerinden, %75 ve %95 gibi ciddi oranda kanserli hücrelerin yayılımını engellediği belirtilmiştir. Bu derlemede MEIS proteinlerinin; kanser türleri ve hastalıklar üzerindeki etkisi, MEIS inhibitörlerinin (MEISi) keşfiyle ilgili bilgilerden bahsedilmektedir.

Meis1 supports leukemogenesis through stimulation of ribosomal biogenesis and Myc.

The homeobox transcription factors HoxA9 and Meis1 are causally involved in the etiology of acute myeloid leukemia. While HoxA9 alone immortalizes cells, cooperation with Meis1 is necessary to induce a full leukemic phenotype. Here, we applied degron techniques to elucidate the leukemogenic contribution of Meis1. Chromatin immunoprecipitation experiments revealed that Meis1 localized mainly to H3K27 acetylated and H3K4 mono-methylated enhancers preactivated by HoxA9. Chromatin association of Meis1 required physical presence of HoxA9 and all Meis1 DNA interactions were rapidly lost after HoxA9 degradation. Meis1 controlled a gene expression pattern dominated by Myc, ribosome biogenesis and ribosomal RNA synthesis genes. While Myc accounted for the cell cycle stimulating effect of Meis1, overexpression of this oncogene alone did not accelerate leukemogenesis. Besides its effect on Myc, Meis1 induced transcription of ribosomal biogenesis genes. This was accompanied by an elevated resistance against inhibition of ribosomal RNA synthesis and translation, but without affecting steady-state protein synthesis. Finally, we demonstrate that HoxA9 and Meis1 proteins are stabilized by post-translational modification. Mutation of HoxA9/Meis1 phosphorylation sites or inhibition of casein kinase 2 lead to rapid protein degradation suggesting a potential pathway for pharmacological intervention.

PD07-09 MEIS PROTEINS INHIBIT HOXB13-DEPENDENT PROSTATE CANCER METASTASIS AND ANDROGEN RECEPTOR SIGNALING

You have accessJournal of UrologyCME1 May 2022PD07-09 MEIS PROTEINS INHIBIT HOXB13-DEPENDENT PROSTATE CANCER METASTASIS AND ANDROGEN RECEPTOR SIGNALING Srikanth Perike, Calvin VanOpstall, Mathias Morales, Sophia Lamperis, Steve Kregel, Ryan Brown, and Donald J. Vander Griend Srikanth PerikeSrikanth Perike More articles by this author , Calvin VanOpstallCalvin VanOpstall More articles by this author , Mathias MoralesMathias Morales More articles by this author , Sophia LamperisSophia Lamperis More articles by this author , Steve KregelSteve Kregel More articles by this author , Ryan BrownRyan Brown More articles by this author , and Donald J. Vander GriendDonald J. Vander Griend More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000002526.09AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: Prostate cancer (PCa) is one of the most frequently diagnosed malignancies in men, and its incidence and mortality continue to be a significant clinical problem. Recent evidence from our lab identified MEIS1, an important HOX protein cofactor, as a potential tumor suppressor. Patients bearing MEIS1-positive prostate tumors were significantly less likely to have biochemical recurrence and metastasis compared to men bearing MEIS-negative tumors. Androgen receptor (AR), the major oncogene in PCa, has been shown to interact with HOXB13 to promote prostate cancer progression, but the function of MEIS proteins to antagonize AR/HOXB13 interactions are unknown. We hypothesize that MEIS1 proteins interact with HOXB13 to suppress cancer initiation and progression, and loss of MEIS1 expression in a portion of prostate tumors enables oncogenic AR/HOXB13 interactions. METHODS: We determined the impact of MEIS1 expression and dependency of HOXB13 on AR signaling using cell lines ectopically expressing MEIS1 and/or CRISPR-mediated HOXB13 deletion in both androgen-sensitive LAPC4 and castration-resistant CWR22Rv1 cells. Western blots, qPCR, Proximity Ligation Assay and co-IPs were performed to evaluate the relationship between expression of MEIS1, HOXB13 and AR. Hormonally-intact and castrated male nude mice were used to test the in vivo capability of MEIS1-mediated tumor xenograft formation and rate of tumor growth in the presence and absence of AR ligand. RNA sequencing and informatics analyses was performed on tumor xenografts to determine the global impact of MEIS expression on AR gene targeting in PCa. RESULTS: We found that AR expression was significantly increased when MEIS1 was ectopically expressed compared to controls and HOXB13 knock-out lines. Re-expression of MEIS1 enhanced the binding between HOXB13-MEIS and AR-MEIS, and reduced AR-HOXB13 interaction. In castrated mice, re-expression of MEIS1 significantly decreased the tumor formation and tumor growth rate compared to hormonally intact nude mice. Conversely, xenografts of HOXB13-knockout tumors showed an increased rate of tumor growth and tumor formation compared to MEIS1-expressing cells and controls in both hormonally intact and castrated nude mice. Our preliminary RNA-Seq analysis demonstrate that when MEIS is present, AR pathway activation in response to androgen was associated with increased differentiation and growth suppression. CONCLUSIONS: Our collective data supports our hypothesis that increased MEIS1 expression reduces the AR/HOXB13 interaction and increases the in vivo sensitivity to host castration; this suggests that MEIS-positive cells have decreased oncogenic AR signaling. Future RNA-Seq studies combined with AR ChIP-Seq data will enable us to identify the MEIS-AR targeted genes that are involved in AR pathway modulation that inhibit prostate cancer cell proliferation and suppress tumor progression. These data provide us a strong rationale to support the potential utility of MEIS proteins as predictive clinical biomarkers of metastatic progression. Source of Funding: Funding: This work was supported by the Urology Care Foundation Research Scholar Award sponsored by Chesapeake Urology Associates, DOD grant PC130587, and DOD grant PC180414 © 2022 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 207Issue Supplement 5May 2022Page: e102 Advertisement Copyright & Permissions© 2022 by American Urological Association Education and Research, Inc.MetricsAuthor Information Srikanth Perike More articles by this author Calvin VanOpstall More articles by this author Mathias Morales More articles by this author Sophia Lamperis More articles by this author Steve Kregel More articles by this author Ryan Brown More articles by this author Donald J. Vander Griend More articles by this author Expand All Advertisement PDF DownloadLoading ...

PD44-06 MEIS PROTEINS INHIBIT HOXB13-DEPENDENT PROSTATE CANCER METASTASIS AND ANDROGEN RECEPTOR SIGNALING

You have accessJournal of UrologyProstate Cancer: Basic Research & Pathophysiology II (PD44)1 Sep 2021PD44-06 MEIS PROTEINS INHIBIT HOXB13-DEPENDENT PROSTATE CANCER METASTASIS AND ANDROGEN RECEPTOR SIGNALING Srikanth Perike, Calvin VanOpstall, Ryan Brown, Sophia Lamperis, and Donald J. Vander Griend Srikanth PerikeSrikanth Perike More articles by this author , Calvin VanOpstallCalvin VanOpstall More articles by this author , Ryan BrownRyan Brown More articles by this author , Sophia LamperisSophia Lamperis More articles by this author , and Donald J. Vander GriendDonald J. Vander Griend More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000002058.06AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: Prostate cancer (PCa) is one of the most frequently diagnosed malignancies in men, and its incidence and mortality continue to be a significant clinical problem. Recent evidence from our lab identified MEIS1, an important HOX protein cofactor, as a potential tumor suppressor. Patients bearing MEIS1-positive prostate tumors were less likely to have biochemical recurrence and metastasis compared to men bearing MEIS-negative tumors. Androgen receptor (AR), the major oncogene in PCa, has been shown to interact with HOXB13 to promote prostate cancer progression, but the function of MEIS proteins to antagonize AR/HOXB13 interactions are unknown. We hypothesize that MEIS1 proteins interact with HOXB13 to suppress cancer initiation and progression, and loss of MEIS1 expression in a portion of prostate tumors enables oncogenic AR/HOXB13 interactions. METHODS: We determined the impact of MEIS1 expression and dependency of HOXB13 on AR signaling using cell lines ectopically expressing MEIS1 and/or CRISPR-mediated HOXB13 deletion in both androgen-sensitive LAPC4 and castration-resistant CWR22Rv1 cells. Western blots, qPCR, Proximity Ligation Assay and co-IPs were performed to evaluate the relationship among MEIS1, HOXB13 and AR. Hormonally-intact and castrated male nude mice were used to test the in vivo capability of MEIS1-mediated tumor formation and rate of tumor growth in the presence and absence of AR ligand. RESULTS: We found that AR expression was significantly increased when MEIS1 was ectopically expressed compared to controls and HOXB13 knock-out lines. Re-expression of MEIS1 enhanced the binding between HOXB13-MEIS and AR-MEIS, and reduced the AR-HOXB13 interaction. In castrated mice, re-expression of MEIS1 significantly decreased the tumor formation and tumor growth rate compared to hormonally intact nude mice. Conversely, xenografts of HOXB13-knockout tumors showed an increased rate of tumor growth and tumor formation compared to MEIS1-expressing cells and controls in both hormonally intact and castrated nude mice. CONCLUSIONS: Our collective data supports our hypothesis that increased MEIS1 expression reduces the AR/HOXB13 interaction and increases the in vivo sensitivity to host castration; this suggests that MEIS-positive cells have decreased oncogenic AR signaling. Future RNAseq studies will determine the global impact of MEIS expression on AR gene targeting in PCa and provide us a strong rationale to support the potential utility of MEIS proteins as predictive clinical biomarkers of metastatic progression. Source of Funding: This work was supported by the Urology Care Foundation Research Scholar Award sponsored by Chesapeake Urology Associates Sanford J. Siegel, MD Prostate Cancer Research Award, DOD grant PC130587, and DOD grant PC180414 © 2021 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 206Issue Supplement 3September 2021Page: e739-e739 Advertisement Copyright & Permissions© 2021 by American Urological Association Education and Research, Inc.MetricsAuthor Information Srikanth Perike More articles by this author Calvin VanOpstall More articles by this author Ryan Brown More articles by this author Sophia Lamperis More articles by this author Donald J. Vander Griend More articles by this author Expand All Advertisement Loading ...

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.

Oncogenic and tumor suppressor function of MEIS and associated factors.

MEIS proteins are historically associated with tumorigenesis, metastasis, and invasion in cancer. MEIS and associated PBX-HOX proteins may act as tumor suppressors or oncogenes in different cellular settings. Their expressions tend to be misregulated in various cancers. Bioinformatic analyses have suggested their upregulation in leukemia/lymphoma, thymoma, pancreas, glioma, and glioblastoma, and downregulation in cervical, uterine, rectum, and colon cancers. However, every cancer type includes, at least, a subtype with high MEIS expression. In addition, studies have highlighted that MEIS proteins and associated factors may function as diagnostic or therapeutic biomarkers for various diseases. Herein, MEIS proteins and associated factors in tumorigenesis are discussed with recent discoveries in addition to how they could be modulated by noncoding RNAs or newly developed small-molecule MEIS inhibitors.

MEIS-mediated suppression of human prostate cancer growth and metastasis through HOXB13-dependent regulation of proteoglycans.

The molecular roles of HOX transcriptional activity in human prostate epithelial cells remain unclear, impeding the implementation of new treatment strategies for cancer prevention and therapy. MEIS proteins are transcription factors that bind and direct HOX protein activity. MEIS proteins are putative tumor suppressors that are frequently silenced in aggressive forms of prostate cancer. Here we show that MEIS1 expression is sufficient to decrease proliferation and metastasis of prostate cancer cells in vitro and in vivo murine xenograft models. HOXB13 deletion demonstrates that the tumor-suppressive activity of MEIS1 is dependent on HOXB13. Integration of ChIP-seq and RNA-seq data revealed direct and HOXB13-dependent regulation of proteoglycans including decorin (DCN) as a mechanism of MEIS1-driven tumor suppression. These results define and underscore the importance of MEIS1-HOXB13 transcriptional regulation in suppressing prostate cancer progression and provide a mechanistic framework for the investigation of HOXB13 mutants and oncogenic cofactors when MEIS1/2 are silenced.

Development of Small Molecule MEIS Inhibitors that modulate HSC activity

Meis1, which belongs to TALE-type class of homeobox gene family, appeared as one of the key regulators of hematopoietic stem cell (HSC) self-renewal and a potential therapeutical target. However, small molecule inhibitors of MEIS1 remained unknown. This led us to develop inhibitors of MEIS1 that could modulate HSC activity. To this end, we have established a library of relevant homeobox family inhibitors and developed a high-throughput in silico screening strategy against homeodomain of MEIS proteins using the AutoDock Vina and PaDEL-ADV platform. We have screened over a million druggable small molecules in silico and selected putative MEIS inhibitors (MEISi) with no predicted cytotoxicity or cardiotoxicity. This was followed by in vitro validation of putative MEIS inhibitors using MEIS dependent luciferase reporter assays and analysis in the ex vivo HSC assays. We have shown that small molecules named MEISi-1 and MEISi-2 significantly inhibit MEIS-luciferase reporters in vitro and induce murine (LSKCD34l°w cells) and human (CD34+, CD133+, and ALDHhi cells) HSC self-renewal ex vivo. In addition, inhibition of MEIS proteins results in downregulation of Meis1 and MEIS1 target gene expression including Hif-1α, Hif-2α and HSC quiescence modulators. MEIS inhibitors are effective in vivo as evident by induced HSC content in the murine bone marrow and downregulation of expression of MEIS target genes. These studies warrant identification of first-in-class MEIS inhibitors as potential pharmaceuticals to be utilized in modulation of HSC activity and bone marrow transplantation studies.

MEIS1 and MEIS2 Expression and Prostate Cancer Progression: A Role For HOXB13 Binding Partners in Metastatic Disease

Purpose: Germline mutations within the MEIS-interaction domain of HOXB13 have implicated a critical function for MEIS-HOX interactions in prostate cancer etiology and progression. The functional and predictive role of changes in MEIS expression within prostate tumor progression, however, remain largely unexplored.Experimental Design: Here we utilize RNA expression datasets, annotated tissue microarrays, and cell-based functional assays to investigate the role of MEIS1 and MEIS2 in prostate cancer and metastatic progression.Results: These analyses demonstrate a stepwise decrease in the expression of both MEIS1 and MEIS2 from benign epithelia, to primary tumor, to metastatic tissues. Positive expression of MEIS proteins in primary tumors, however, is associated with a lower hazard of clinical metastasis (HR = 0.28) after multivariable analysis. Pathway and gene set enrichment analyses identified MEIS-associated networks involved in cMYC signaling, cellular proliferation, motility, and local tumor environment. Depletion of MEIS1 and MEIS2 resulted in increased tumor growth over time in vivo, and decreased MEIS expression in both patient-derived tumors and MEIS-depleted cell lines was associated with increased expression of the protumorigenic genes cMYC and CD142, and decreased expression of AXIN2, FN1, ROCK1, SERPINE2, SNAI2, and TGFβ2.Conclusions: These data implicate a functional role for MEIS proteins in regulating cancer progression, and support a hypothesis whereby tumor expression of MEIS1 and MEIS2 expression confers a more indolent prostate cancer phenotype, with a decreased propensity for metastatic progression. Clin Cancer Res; 24(15); 3668-80. ©2018 AACR.

Prep1, A Homeodomain Transcription Factor Involved in Glucose and Lipid Metabolism.

The three-amino acid loop extension (TALE) homeodomain proteins are a family of transcription factor including the mammalian Pbx, MEIS and Prep proteins. TALE proteins can bind other transcription factors such as Pdx-1 and play an important role in the regulation of glucose metabolism. Experiments performed in mutant mice have shown that while the single Pbx1 or Pdx-1 knockout mice feature pancreatic islet malformations, impaired glucose tolerance and hypoinsulinemia, the trans-heterozygous Pbx1+/− Pdx1+/− mice develop age-dependent overt diabetes mellitus. In contrast, Prep1 plays a different role with respect to these proteins. Indeed, Prep1 hypomorphic mice, expressing low levels of protein, feature pancreatic islet hypoplasia accompanied by hypoinsulinemia similar to Pbx1 or Pdx1. Nevertheless, these animals show increased insulin sensitivity in skeletal muscle, liver and adipose tissue accompanied by protection from streptozotocin-induced diabetes. In addition, Prep1 hypomorphic mice feature reduced triglyceride synthesis and do not develop steatohepatitis after a methionine and coline deficient diet. In this review we have underlined how important metabolic functions are controlled by TALE proteins, in particular by Prep1, leading to hypothesis that its suppression might represent beneficial effect in the care of metabolic diseases.

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.

Abstract B33: Alterations in the binding of Meis proteins to HoxB13 or to DNA promote prostate cancer progression

Abstract Introduction: One of the first germline mutations shown to confer an increased risk of early-onset prostate cancer (PrCa) was recently found in the Meis-binding domain of Homeobox B13 (HoxB13). Neither the function of this mutation nor the role of Meis proteins in PrCa has been elucidated. There are three Meis genes, each with variously spliced isoforms, some of which lack a DNA binding domain. Meis proteins normally function as Hox gene cofactors, and regulate proliferation and cell fate. In normal and other disease contexts, Meis proteins with an abrogated homeodomain have been shown to act as a dominant negative and squelch normal Hox gene transcription. The androgen receptor (AR), the major oncogene in PrCa, has been shown to interact with both Meis and Hox proteins. We hypothesize that Meis proteins function as AR-regulated tumor suppressors in advanced prostate cancer, and isoforms lacking the homeodomain prevent HoxB13 DNA binding to promote uncontrolled cell proliferation. Methods: Meis1/2 expression stratified outcomes of men with Gleason 6/7 tumors in a retrospective bioinformatics analysis of microarray data obtained from the Swedish Watchful Waiting cohort. Meis1/2/3 mRNA expression in normal and cancerous prostate cell lines as well as adjacent normal stromal and epithelial patient-derive cell lines was compared by qRT-PCR. Cell number over time was determined by SRB assay for lines stably overexpressing full length or homeodomains-less Meis2 as compared to GFP in castration-sensitive LNCaP and castration-resistant CWR-22Rv1 PrCa cells. Meis2 expression was analyzed by qPCR in androgen-dependent LAPC4 cells. LAPC4 cells were incubated in distinct androgen conditions, including incubation with an AR agonist (R1881) or potent antagonist (enzalutamide). Publicly available microarray data from 19 patients were obtained for analysis from the Gene Expression Omnibus. Results: Through retrospective bioinformatics analysis, we found that men diagnosed with intermediate Gleason scores and low Meis expression had a worse prognosis than men with high Meis levels. qPCR data indicated lower Meis expression in PrCa cell lines as compared to normal prostate epithelial cells. Stromal patient-derived cell lines had significantly higher levels of Meis1 and 2 than their patient-matched epithelial counterparts, while Meis3 expression was consistent across all cell types. Proliferation assays indicated CWR-22Rv1 cells that overexpress full length Meis2 showed significantly lower cell number over five days than GFP- or abrogated homeodomain Meis-expressing cells. qPCR data indicates that PrCa cells exposed to R1881 had significantly lower Meis2 expression as compared to cells treated with enzalutamide. Analysis of publicly available microarrays showed that patients with metastatic prostate cancer were more likely to have lower Meis2 expression than patients with localized or benign disease. Conclusions: Our data indicate that full length Meis protein levels are consistently lower in cancer as compared to normal tissues, and are decreased to a greater extent in more aggressive cancers. Full length Meis proteins may act as tumor suppressors while the homeodomain-less isoforms may act as dominant negatives. Further, we demonstrate a regulatory interaction between Meis expression and AR signaling, providing evidence for a downstream role of Meis proteins in the most important pathway for PrCa progression. We are the first to investigate the functional differences of these isoforms in the context of PrCa. An understanding of how the functional domains of Meis and HoxB13 interact with each other, the AR and the genome are crucial steps towards evaluating the utility of Meis expression as a biomarker to stratify aggressive versus indolent PrCa patients, as well as aid our general understanding of prostate tumor initiation and progression. Citation Format: Hannah Brechka, Raj Bhanvadia, Donald Vander Griend. Alterations in the binding of Meis proteins to HoxB13 or to DNA promote prostate cancer progression. [abstract]. In: Proceedings of the AACR Special Conference: Developmental Biology and Cancer; Nov 30-Dec 3, 2015; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(4_Suppl):Abstract nr B33.

Hox Proteins Coordinate Motor Neuron Differentiation and Connectivity Programs through Ret/Gfrα Genes

The accuracy of neural circuit assembly relies on the precise spatial and temporal control of synaptic specificity determinants during development. Hox transcription factors govern key aspects of motor neuron (MN) differentiation; however, the terminal effectors of their actions are largely unknown. We show that Hox/Hox cofactor interactions coordinate MN subtype diversification and connectivity through Ret/Gfrα receptor genes. Hox and Meis proteins determine the levels of Ret in MNs and define the intrasegmental profiles of Gfrα1 and Gfrα3 expression. Loss of Ret or Gfrα3 leads to MN specification and innervation defects similar to those observed in Hox mutants, while expression of Ret and Gfrα1 can bypass the requirement for Hox genes during MN pool differentiation. These studies indicate that Hox proteins contribute to neuronal fate and muscle connectivity through controlling the levels and pattern of cell surface receptor expression, consequently gating the ability of MNs to respond to limb-derived instructive cues.

The TALE face of Hox proteins in animal evolution.

Hox genes are major regulators of embryonic development. One of their most conserved functions is to coordinate the formation of specific body structures along the anterior-posterior (AP) axis in Bilateria. This architectural role was at the basis of several morphological innovations across bilaterian evolution. In this review, we traced the origin of the Hox patterning system by considering the partnership with PBC and Meis proteins. PBC and Meis belong to the TALE-class of homeodomain-containing transcription factors and act as generic cofactors of Hox proteins for AP axis patterning in Bilateria. Recent data indicate that Hox proteins acquired the ability to interact with their TALE partners in the last common ancestor of Bilateria and Cnidaria. These interactions relied initially on a short peptide motif called hexapeptide (HX), which is present in Hox and non-Hox protein families. Remarkably, Hox proteins can also recruit the TALE cofactors by using specific PBC Interaction Motifs (SPIMs). We describe how a functional Hox/TALE patterning system emerged in eumetazoans through the acquisition of SPIMs. We anticipate that interaction flexibility could be found in other patterning systems, being at the heart of the astonishing morphological diversity observed in the animal kingdom.

Abstract 2243: Alterations in the binding of Meis proteins to HoxB13 or to DNA promote prostate cancer progression

Abstract Intro: One of the first germline mutations shown to confer an increased risk of early-onset prostate cancer (PrCa) is found in the Meis-binding domain of Homeobox B13 (HoxB13). Neither the function of this mutation nor the role of Meis proteins in PrCa has been elucidated. Meis proteins normally function as Hox gene cofactors, and regulate proliferation and cell fate. We hypothesize that disruption in normal Meis binding, either to transcriptional cofactors or DNA elements, is sufficient to alter gene transcription and increase progression of PrCa. Methods: Meis1/2 expression was used to stratify outcomes of men with Gleason 6/7 tumors in a retrospective bioinformatics analysis. Meis1/2 mRNA expression in cell lines was determined by qRT-PCR, and isoform distribution determined by TA-TOPO cloning. Cell number over time was determined for lines stably overexpressing Meis2E or A as compared to GFP in LNCaP and CWR-22Rv1. Results: We found that men diagnosed with intermediate Gleason scores and low expression of Meis had a worse prognosis than men with high levels of Meis. We found lower Meis mRNA expression in PrCa cell lines as compared to normal prostate epithelial cells. We determined that a variety of Meis2 isoforms are expressed in both cancer and normal, and the aggressive PrCa cell line CWR22Rv1 had a higher level of Meis2 isoforms with abrogated DNA-binding homeodomains. LNCaP cells overexpressing Meis2A showed significantly lower cell number over five days than GFP or Meis2E expressing cells. Conclusions: Full length Meis proteins may act as tumor suppressors while the homeodomain-less isoforms may act as oncogenes in the context of PrCa. This is innovative because we are the first to look at the role of Meis proteins in PrCa, and significant because it's linked to evidence involving mutations in HoxB13. Citation Format: Hannah J. Brechka, Masis Isikbay, Raj Bhanvadia, Donald Vander Griend. Alterations in the binding of Meis proteins to HoxB13 or to DNA promote prostate cancer progression. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2243. doi:10.1158/1538-7445.AM2015-2243

Biochemistry of the tale transcription factors PREP, MEIS, and PBX in vertebrates

TALE (three amino acids loop extension) homeodomain transcription factors are required in various steps of embryo development, in many adult physiological functions, and are involved in important pathologies. This review focuses on the PREP, MEIS, and PBX sub-families of TALE factors and aims at giving information on their biochemical properties, i.e., structure, interactors, and interaction surfaces. Members of the three sets of protein form dimers in which the common partner is PBX but they can also directly interact with other proteins forming higher-order complexes, in particular HOX. Finally, recent advances in determining the genome-wide DNA-binding sites of PREP1, MEIS1, and PBX1, and their partial correspondence with the binding sites of some HOX proteins, are reviewed. These studies have generated a few general rules that can be applied to all members of the three gene families. PREP and MEIS recognize slightly different consensus sequences: PREP prefers to bind to promoters and to have PBX as a DNA-binding partner; MEIS prefers HOX as partner, and both PREP and MEIS drive PBX to their own binding sites. This outlines the clear individuality of the PREP and MEIS proteins, the former mostly devoted to basic cellular functions, the latter more to developmental functions. Developmental Dynamics 243:59–75, 2014. © 2013 Wiley Periodicals, Inc.

Distinct regulation of c-myb gene expression by HoxA9, Meis1 and Pbx proteins in normal hematopoietic progenitors and transformed myeloid cells

The proto-oncogenic protein c-Myb is an essential regulator of hematopoiesis and is frequently deregulated in hematological diseases such as lymphoma and leukemia. To gain insight into the mechanisms underlying the aberrant expression of c-Myb in myeloid leukemia, we analyzed and compared c-myb gene transcriptional regulation using two cell lines modeling normal hematopoietic progenitor cells (HPCs) and transformed myelomonocytic blasts. We report that the transcription factors HoxA9, Meis1, Pbx1 and Pbx2 bind in vivo to the c-myb locus and maintain its expression through different mechanisms in HPCs and leukemic cells. Our analysis also points to a critical role for Pbx2 in deregulating c-myb expression in murine myeloid cells cotransformed by the cooperative activity of HoxA9 and Meis1. This effect is associated with an intronic positioning of epigenetic marks and RNA polymerase II binding in the orthologous region of a previously described alternative promoter for c-myb. Taken together, our results could provide a first hint to explain the abnormal expression of c-myb in leukemic cells.

A Conserved Tissue-Specific Homeodomain-Less Isoform of MEIS1 Is Downregulated in Colorectal Cancer

Colorectal cancer is one of the most common cancers in developed nations and is the result of both environmental and genetic factors. Many of the genetic lesions observed in colorectal cancer alter expression of homeobox genes, which encode homeodomain transcription factors. The MEIS1 homeobox gene is known to be involved in several hematological malignancies and solid tumors and recent evidence suggests that expression of the MEIS1 transcript is altered in colorectal cancer. Despite this potential connection, little is known about the role of the gene in the intestines. We probed murine gastrointestinal tissue samples with an N-terminal Meis1 antibody, revealing expression of two previously described isoforms, as well as two novel Meis1 products. A 32 kD Meis1 product was expressed in the nuclei of non-epithelial cells in the stomach and colon, while a 27 kD product was expressed in the cytoplasm of epithelial cells in the proximal colon. Our data suggest that the 27 kD and 32 kD Meis1 proteins are both forms of the Meis1d protein, a homeodomain-less isoform whose transcript was previously identified in cDNA screens. Both the MEIS1D transcript and protein were expressed in human colon mucosa. Expression of the MEIS1D protein was downregulated in 83% (10/12) of primary colorectal cancer samples compared to matched normal mucosa, indicating that MEIS1D is a biomarker of colorectal tumorigenesis. The decreased expression of MEIS1D in colon tumors also suggests that this conserved homeodomain-less isoform may act as a tumor suppressor in human colorectal cancer.