Deregulation Of HOX Genes Research Articles

HOX gene regulation in glioma stem cells, mediators of resistance in breast cancer, and paradoxical therapeutic interventions

Therapy resistance remains a major challenge during the treatment of patients with advanced cancer. Due to the complex nature of the cellular and molecular mechanisms underlying therapy resistance, research efforts to tackle this challenge have focused on diverse aspects: from cancer stem cell (CSC) biology, therapy resistance-specific signatures to the development of new combinatorial therapeutic approaches. Among other articles, the August issue of Molecular Oncology includes several studies that directly or indirectly explore therapy resistance in patients with advanced cancer. In some cancers, therapeutic resistance has been linked to the presence of CSCs. In glioma, for example, glioma stem cells (GSCs) represent a tumor cell subpopulation underlying tumor relapse. Understanding the molecular mechanisms behind GSC gene expression may give new insights on tumorigenesis and tumor relapse. In their recent study, Le Boiteux et al. [[1]] analyzed six patient-derived GSC lines, as well as 70 primary adult diffuse glioma samples to map the mechanisms leading to deregulation of the HOX gene clusters, which have been previously implicated in the oncogenic potential of GSCs. The authors identified widespread reactivation of HOX genes in adult IDH wild-type (IDHwt) diffuse glioma samples and GSCs. HOX gene deregulation was associated with self- and cross-regulatory interactions between HOX transcription factors, but not with genomic rearrangements. Integrative analysis of high-throughput DNA methylation analysis, ChIPseq, and RNAseq data demonstrated that the absence of hypermethylation on canonical and alternative bivalent CpG islands in promoters, as well as loss of the H3K27me3 epigenetic mark, underlies aberrant gene expression of HOX clusters in IDHwt gliomas. Besides providing a comprehensive epigenetic analysis of HOX gene regulation in glioma, the study by Le Boiteux et al. highlights the complexity of HOX gene expression via alternative promoters in cancer. In a different setting, Mohammad Sultan and colleagues employed an in vivo genome-wide shRNA screen to identify potential mediators or biomarkers of paclitaxel resistance in patients with advanced breast cancer [[2]]. The transcriptional repressor BCL6 was identified among the top paclitaxel resistance hits, and BCL6 overexpression was detected in tumors of chemotherapy-resistant patients. To further test the role of BCL6 in paclitaxel resistance, the authors performed BCL6 knockdown and BCL6 inhibitor experiments both in vitro and in vivo, in a mouse model of triple-negative breast cancer. BCL6 targeting in preclinical models enhanced the efficacy of paclitaxel treatment. Thus, BCL6 may serve as a biomarker for drug resistance and may potentially be targeted for the treatment of patients with breast cancer that have developed paclitaxel resistance. In addition to chemotherapy, targeted therapies designed to block mitogenic signaling pathways are often used for the treatment of patients with cancer. However, drug resistance to these therapeutic agents represents a major challenge for the treatment of advanced cancer. In their perspective, Matheus Henrique Dias and Rene Bernards explore the prospects of a ‘paradoxical intervention’ strategy for the treatment of therapy-resistant cancers [[3]]. Such a strategy would involve overactivation, rather than inhibition, of mitotic signaling in cancer cells. In their view, such an approach would result in an overload of the stress response pathways and disruption of cancer cell homeostasis and viability. The authors review related data from several independent experimental models, discuss future perspectives, and explore the potential drawbacks of such a paradoxical intervention strategy for the treatment of patients with cancer.

Widespread overexpression from the four DNA hypermethylated HOX clusters in aggressive (IDHwt) glioma is associated with H3K27me3 depletion and alternative promoter usage.

In human, the 39 coding HOX genes and 18 referenced noncoding antisense transcripts are arranged in four genomic clusters named HOXA, B, C, and D. This highly conserved family belongs to the homeobox class of genes that encode transcription factors required for normal development. Therefore, HOX gene deregulation might contribute to the development of many cancer types. Here, we study HOX gene deregulation in adult glioma, a common type of primary brain tumor. We performed extensive molecular analysis of tumor samples, classified according to their isocitrate dehydrogenase (IDH1) gene mutation status, and of glioma stem cells. We found widespread expression of sense and antisense HOX transcripts only in aggressive (IDHwt) glioma samples, although the four HOX clusters displayed DNA hypermethylation. Integrative analysis of expression, DNA methylation, and histone modification signatures along the clusters revealed that HOX gene upregulation relies on canonical and alternative bivalent CpG island promoters that escape hypermethylation. H3K27me3 loss at these promoters emerges as the main cause of widespread HOX gene upregulation in IDHwt glioma cell lines and tumors. Our study provides the first comprehensive description of the epigenetic changes at HOX clusters and their contribution to the transcriptional changes observed in adult glioma. It also identified putative ‘master’ HOX proteins that might contribute to the tumorigenic potential of glioma stem cells.

Roles of the HOX Proteins in Cancer Invasion and Metastasis.

Simple SummaryCancer is the second leading cause of death worldwide, right after cardiovascular diseases, and the invasion and metastatization correspond to the foremost cause of cancer-related deaths. Here, we reviewed the state of the art regarding the importance of HOX transcription factors in these last steps of cancer progression and described five of their complex mechanisms of regulation, including the miRs and lncRNAs interference. This information highlights the importance of HOX in the suppression and induction of disease advancement and point out the potential of HOX products as therapeutic targets for diverse cancer types.Invasion and metastasis correspond to the foremost cause of cancer-related death, and the molecular networks behind these two processes are extremely complex and dependent on the intra- and extracellular conditions along with the prime of the premetastatic niche. Currently, several studies suggest an association between the levels of HOX genes expression and cancer cell invasion and metastasis, which favour the formation of novel tumour masses. The deregulation of HOX genes by HMGA2/TET1 signalling and the regulatory effect of noncoding RNAs generated by the HOX loci can also promote invasion and metastasis, interfering with the expression of HOX genes or other genes relevant to these processes. In this review, we present five molecular mechanisms of HOX deregulation by which the HOX clusters products may affect invasion and metastatic processes in solid tumours.

HOX gene cluster (de)regulation in brain: from neurodevelopment to malignant glial tumours.

HOX genes encode a family of evolutionarily conserved homeodomain transcription factors that are crucial both during development and adult life. In humans, 39 HOX genes are arranged in four clusters (HOXA, B, C, and D) in chromosomes 7, 17, 12, and 2, respectively. During embryonic development, particular epigenetic states accompany their expression along the anterior-posterior body axis. This tightly regulated temporal-spatial expression pattern reflects their relative chromosomal localization, and is critical for normal embryonic brain development when HOX genes are mainly expressed in the hindbrain and mostly absent in the forebrain region. Epigenetic marks, mostly polycomb-associated, are dynamically regulated at HOX loci and regulatory regions to ensure the finely tuned HOX activation and repression, highlighting a crucial epigenetic plasticity necessary for homeostatic development. HOX genes are essentially absent in healthy adult brain, whereas they are detected in malignant brain tumours, namely gliomas, where HOX genes display critical roles by regulating several hallmarks of cancer. Here, we review the major mechanisms involved in HOX genes (de)regulation in the brain, from embryonic to adult stages, in physiological and oncologic conditions. We focus particularly on the emerging causes of HOX gene deregulation in glioma, as well as on their functional and clinical implications.

The stem cell–specific long noncoding RNA HOXA10-AS in the pathogenesis of KMT2A-rearranged leukemia

AbstractHOX genes are highly conserved, and their precisely controlled expression is crucial for normal hematopoiesis. Accordingly, deregulation of HOX genes can cause leukemia. However, despite of intensive research on the coding HOX genes, the role of the numerous long noncoding RNAs (lncRNAs) within the HOX clusters during hematopoiesis and their contribution to leukemogenesis are incompletely understood. Here, we show that the lncRNA HOXA10-AS, located antisense to HOXA10 and mir-196b in the HOXA cluster, is highly expressed in hematopoietic stem cells (HSCs) as well as in KMT2A-rearranged and NPM1 mutated acute myeloid leukemias (AMLs). Using short hairpin RNA– and locked nucleic acid-conjugated chimeric antisense oligonucleotide (LNA-GapmeR)–mediated HOXA10-AS-knockdown and CRISPR/Cas9-mediated excision in vitro, we demonstrate that HOXA10-AS acts as an oncogene in KMT2A-rearranged AML. Moreover, HOXA10-AS knockdown severely impairs the leukemic growth of KMT2A-rearranged patient-derived xenografts in vivo, while high HOXA10-AS expression can serve as a marker of poor prognosis in AML patients. Lentiviral expression of HOXA10-AS blocks normal monocytic differentiation of human CD34+ hematopoietic stem and progenitor cells. Mechanistically, we show that HOXA10-AS localizes in the cytoplasm and acts in trans to induce NF-κB target genes. In total, our data imply that the normally HSC-specific HOXA10-AS is an oncogenic lncRNA in KMT2A-r AML. Thus, it may also represent a potential therapeutic target in KMT2A-rearranged AML.

Paralogous HOX13 Genes in Human Cancers.

Hox genes (HOX in humans), an evolutionary preserved gene family, are key determinants of embryonic development and cell memory gene program. Hox genes are organized in four clusters on four chromosomal loci aligned in 13 paralogous groups based on sequence homology (Hox gene network). During development Hox genes are transcribed, according to the rule of “spatio-temporal collinearity”, with early regulators of anterior body regions located at the 3’ end of each Hox cluster and the later regulators of posterior body regions placed at the distal 5’ end. The onset of 3’ Hox gene activation is determined by Wingless-type MMTV integration site family (Wnt) signaling, whereas 5’ Hox activation is due to paralogous group 13 genes, which act as posterior-inhibitors of more anterior Hox proteins (posterior prevalence). Deregulation of HOX genes is associated with developmental abnormalities and different human diseases. Paralogous HOX13 genes (HOX A13, HOX B13, HOX C13 and HOX D13) also play a relevant role in tumor development and progression. In this review, we will discuss the role of paralogous HOX13 genes regarding their regulatory mechanisms during carcinogenesis and tumor progression and their use as biomarkers for cancer diagnosis and treatment.

Abstract 3873: Novel non-genetic mechanisms drive rhabdomyosarcoma tumorigenesis

Abstract Most pediatric solid tumors originate from perturbations during development, however the mechanisms that drive these disruptions and promote tumor progression are still poorly understood. We believe that epigenetic deregulation of cancer genes and developmental pathways is a common driver in pediatric cancers, especially those with a low mutation rate. To test this novel idea, we are focusing on rhabdomyosarcoma (RMS), a developing muscle tumor with striking genetic differences between two of the major subtypes. We hypothesize that differential epigenetic deregulation of differentiation programs and cancer genes between the major subtypes are essential for tumorigenesis. Indeed, our preliminary integrative analysis reveal distinct epigenetic landscapes between the two subtypes particularly at the loci of HOX cluster genes A-C, including an oncogenic HOXC long non-coding RNA (lncRNA) called HOTAIR. Knockdown of HOTAIR led to derepression of HOXD10, a candidate tumor suppressor, and a decrease in RMS proliferation, migration, and invasion. Thus, we believe HOX gene deregulation is required for tumorigenesis, and their implication in several adult cancers suggests this currently unappreciated mechanism is likely to be more broadly relevant. In addition, we identified a group of RMS specific novel lncRNAs, of which one demonstrates an essential role in cell survival. Our innovative approach to integrate epigenetic data sets from tumor types with varying genetic differences shed light on the mechanisms that drive tumorigenesis, which may lead to identifying previously unknown potential therapeutic targets. Since current therapeutic options have not changed over the last twenty-five years, these data will especially provide a strong translational impact for children with RMS, whose survival outcomes are less than 20% in the case of metastatic disease. Note: This abstract was not presented at the meeting. Citation Format: Justina McEvoy, Michael Dyer, Xiang Chen. Novel non-genetic mechanisms drive rhabdomyosarcoma tumorigenesis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3873. doi:10.1158/1538-7445.AM2017-3873

Abstract A20: Tumorigenicity of Ewing sarcoma is critically dependent on the trithorax proteins MLL and menin

Abstract Ewing sarcoma is driven by the oncogenic fusion protein, EWS-FLI1, and arises via de-regulation of developmental transcriptional programs. Control of normal developmental transcription programs is governed by epigenetic regulation which, in the case of HOX programs, is dependent on coordinated and reciprocal actions of polycomb (PcG) and trithorax (TrxG) proteins. We recently reported that in Ewing sarcoma posterior HOX genes, in particular HOXD13, are abnormally over-expressed and that the promoters of these genes are marked with the TrxG-dependent activating histone modification, H3K4me3. H3K4me3 is deposited by the MLL methyltransferase, a key enzyme that is frequently mutated in leukemia, largely as a result of chromosomal translocations that induce the creation of oncogenic MLL fusion-proteins. In leukemia, MLL fusion-proteins cooperate with wild-type MLL and the scaffolding protein menin to induce malignant transformation via deregulation of HOXA genes. The purpose of this study was to test the hypothesis that MLL and menin contribute to tumorigenesis and to posterior HOX gene deregulation in Ewing sarcoma. Gene and protein expression were determined by microarray, qRT-PCR, western blot and immunohistochemistry. Loss of function was achieved by lentiviral shRNAs and by exposing cells to MI-503, a small molecule inhibitor of menin-MLL protein-protein interactions. Changes in gene and protein expression after MI-503 treatment were assessed by qRT-PCR and western blot. Chromatin immunoprecipitation (ChIP) was used to assess binding of MLL and menin at gene promoters. Our results confirm that MLL, menin and HOXD13 are all highly expressed by Ewing sarcoma tumors and cell lines relative to non-malignant tissues and stem cells, and loss of function studies implicate each as a tumor promoting oncogene. Specifically, knockdown of MLL, menin or HOXD13 resulted in reduced cell proliferation, increased death and/or reduced tumorigenic capacity, as determined by anchorage-independent growth in soft agar and subcutaneous tumor formation in vivo. At a molecular level, knockdown of MLL and menin led to reduced expression of HOXD13, implicating these proteins as key mediators of HOXD13 over-expression. Exposure of Ewing sarcoma cells to MI-503 inhibited proliferation and colony formation in soft agar, and resulted in reduced expression of HOXD13 as well as other posterior HOX genes. In contrast, MI-NC, a control compound with similar structure that lacks affinity for the menin-MLL interaction had no effect on cell growth, viability or HOX gene expression. Significantly, MI-503-treated cells also showed a marked reduction in EWS-FLI1 and wild-type EWSR1 expression, both of which are regulated by the EWSR1 promoter, and of EZH2, a well-established oncogene in Ewing sarcoma. ChIP studies of the EWSR1 and EZH2 promoters demonstrated reduced binding of MLL and menin in MI-503-treated cells. These data demonstrate a key role for MLL and menin in Ewing sarcoma pathogenesis and directly implicate the TrxG complex in regulation of EWS-FLI1, thus highlighting a novel opportunity for therapeutic intervention. Citation Format: Laurie K. Svoboda, Natashay Bailey, Melanie Krook, Raelene Van Noord, Ashley Harris, Rajiv M. Patel, Dafydd Thomas, Tomek Cierpicki, Jolanta Grembecka, Elizabeth R. Lawlor. Tumorigenicity of Ewing sarcoma is critically dependent on the trithorax proteins MLL and menin. [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 A20.

Transcriptional profiling of HOXA9-regulated genes in human glioblastoma cell models.

The data here described pertain to the article by Pojo et al. (2015) [10] titled “A transcriptomic signature mediated by HOXA9 promotes human glioblastoma initiation, aggressiveness and resistance to temozolomide” (Pojoet al., 2015 [10]). HOX genes are part of the homeobox gene family, which encodes transcription factors crucial during embryonic development (Grier et al., 2005; Pearson et al., 2005 [6,9]) and also in post developmental regulation(Neville et al., 2002; Yamamoto et al., 2003; Takahashi et al., 2004;Morgan 2006 [8,14,13,7]). Alterations interfering with the regulation of these genes may lead to tumorigenesis in adults. Due to their contributions in the control of important cellular processes, the deregulation of HOX genes is ultimately correlated with cancer treatment failure and patients' poor prognosis (Golub et al., 1999; Abdel-Fattah et al., 2006 [5,1]; Costa et al.,2010 [4]; Pojo et al., 2015 [10]). Recently, our studies showed that HOXA9 overexpression is associated with poor prognosis in patients with glioblastoma (GBM), the most common and most malignant primary brain tumor. Mechanistically, HOXA9 is associated with resistance to chemotherapy and with pro-proliferative, pro invasive and anti-apoptotic features (Costa et al., 2010 [4]; Pojo et al., 2015 [10]) in GBM in vitro models. Since HOXA9 is a transcription factor, its target genes can be the true biological effectors of its aggressiveness. In this context, whole genome Agilent's microarrays were used to obtain the full transcriptome of HOXA9 in a variety of GBM cell models, including human immortalized astrocytes, established GBM cell lines, and GBM patient derived cell cultures. Here, we provide detailed methods, including experimental design and microarray data analyses,which can be accessed in Gene Expression Omnibus (GEO) under the accession number GSE56517. Additional interpretation of the data is included and supplemented in (Pojo et al., 2015 [10]).

PRMT1 Activates Leukemic Stem Cell Program in MLL-Rearranged Leukemia

In MLL-rearranged leukemia, the Mixed Lineage Leukemia (MLL) gene undergoes chromosomal translocation that results in the loss of C-terminal histone methyltransferase SET domain, whereas the N-terminal of MLL gene fuses in-frame with one of the 60 identified partner genes. The resultant MLL fusion proteins lead to a characteristic aberrant gene expression pattern in human acute myeloid and lymphoblastic leukemia. Epigenetic dysregulation mediated by MLL fusion proteins has been suggested to be a key event in MLL-rearranged leukemia. It has been demonstrated that MLL-EEN/PRMT1 oncogenic complex induces transformation of primary myeloid progenitors via introduction of aberrant H4R3me2 at target Hoxloci. PRMT1 is the predominant protein arginine methyltransferase in mammals and is responsible for over 85% of arginine methylation activity in mammalian cells. Dysregulation of PRMT1 has been implicated in different cancers such as leukemia, suggesting the expression of PRMT1 is positively correlated with cancer progression and clinical parameters. Nevertheless, the leukemogenic role of PRMT1 in the establishment of leukemic stem cell (LSC) remains unclear.Previously we have demonstrated that a MLL fusion protein, MLL-EEN, can strongly enhance the self-renewal ability of murine primary hematopoietic cells through multiple rounds of replating assays. We have created a conditional Mll-Een invertor mouse model (MllEen/+) in which the expression of fusion protein is restricted to hematopoietic progenitors. Immunophenotypic analysis demonstrated a significant increase in the immature myeloid cell population (c-kit+Mac-1+) in bone marrow of MllEen/+ mice, suggesting that the expression of Mll-Een induces the development of acute myeloid leukemia. We have also established an Mll-Een expressing cell line from the bone marrow of MllEen/+ mouse. These leukemic cells can persistently form colonies and they also demonstrated deregulation of Hox genes, which is frequently observed in human leukemia cases. The leukemogenicity of Mll-Een is closely associated with Prmt1, which was demonstrated through knockdown of Prmt1. Strikingly, we discovered a subpopulation of CD41+Mll-Een expressing cells, which showed enhanced self-renewal ability in the serial colony forming assays. The percentage of CD41+ leukemic cells is reduced once Prmt1 was knocked down, suggesting that Prmt1 is crucial in the maintenance of this subpopulation of cells. In addition, the CD41+ cells showed enhanced expression of genes associated with hematopoietic stem cell (HSC) activities (Bmi-1, Runx1, Tal-1 and Lmo2), implying that part of the HSC transcriptional program has been re-activated in these cells. We therefore speculate that the CD41+ cells may represent a group of MLL leukemic cells that harbors strong stem cell features, and presumably functions as LSCs. The CD41+ leukemic cells will be further characterized by their LSC functions and CD41 can potentially serve as a novel LSC marker in MLL-rearranged leukemia. Taken together, studies on the role of PRMT1 can provide novel insights on the establishment of LSC and the development of effective clinical treatment for MLL-rearranged leukemia. DisclosuresNo relevant conflicts of interest to declare.

HOX Genes and Oncogenesis

Class I homeobox genes are a family of transcription factors involved in human development, in the regulation of the cell memory program. Several HOX genes control the normal hematopoietic processes; recent evidence has demonstrated that deregulation of HOX genes plays a crucial role in the leukemogenesis and in many solid tumors mainly by means, the regulation of cell proliferation, cell death, metastasis and DNA repair.

Evidence for a functional role of epigenetically regulated midclusterHOXBgenes in the development of Barrett esophagus

Barrett esophagus (BE) is a human metaplastic condition that is the only known precursor to esophageal adenocarcinoma. BE is characterized by a posterior intestinal-like phenotype in an anterior organ and therefore it is reminiscent of homeotic transformations, which can occur in transgenic animal models during embryonic development as a consequence of mutations in HOX genes. In humans, acquired deregulation of HOX genes during adulthood has been linked to carcinogenesis; however, little is known about their role in the pathogenesis of premalignant conditions. We hypothesized that HOX genes may be implicated in the development of BE. We demonstrated that three midcluster HOXB genes (HOXB5, HOXB6, and HOXB7) are overexpressed in BE, compared with the anatomically adjacent normal esophagus and gastric cardia. The midcluster HOXB gene signature in BE is identical to that seen in normal colonic epithelium. Ectopic expression of these three genes in normal squamous esophageal cells in vitro induces markers of intestinal differentiation, such as KRT20, MUC2, and VILLIN. In BE-associated adenocarcinoma, the activation midcluster HOXB gene is associated with loss of H3K27me3 and gain of AcH3, compared with normal esophagus. These changes in histone posttranslational modifications correlate with specific chromatin decompaction at the HOXB locus. We suggest that epigenetically regulated alterations of HOX gene expression can trigger changes in the transcriptional program of adult esophageal cells, with implications for the early stages of carcinogenesis.

Homeobox A7 increases cell proliferation by up-regulation of epidermal growth factor receptor expression in human granulosa cells

BackgroundHomeobox (HOX) genes encode transcription factors, which regulate cell proliferation, differentiation, adhesion, and migration. The deregulation of HOX genes is frequently associated with human reproductive system disorders. However, knowledge regarding the role of HOX genes in human granulosa cells is limited.MethodsTo determine the role of HOXA7 in the regulation and associated mechanisms of cell proliferation in human granulosa cells, HOXA7 and epidermal growth factor receptor (EGFR) expressions were examined in primary granulosa cells (hGCs), an immortalized human granulosa cell line, SVOG, and a granulosa tumor cell line, KGN, by real-time PCR and Western blotting. To manipulate the expression of HOXA7, the HOXA7 specific siRNA was used to knockdown HOXA7 in KGN. Conversely, HOXA7 was overexpressed in SVOG by transfection with the pcDNA3.1-HOAX7 vector. Cell proliferation was measured by the MTT assay.ResultsOur results show that HOXA7 and EGFR were overexpressed in KGN cells compared to hGCs and SVOG cells. Knockdown of HOXA7 in KGN cells significantly decreased cell proliferation and EGFR expression. Overexpression of HOXA7 in SVOG cells significantly promoted cell growth and EGFR expression. Moreover, the EGF-induced KGN proliferation was abrogated, and the activation of downstream signaling was diminished when HOXA7 was knocked down. Overexpression of HOXA7 in SVOG cells had an opposite effect.ConclusionsOur present study reveals a novel mechanistic role for HOXA7 in modulating granulosa cell proliferation via the regulation of EGFR. This finding contributes to the knowledge of the pro-proliferation effect of HOXA7 in granulosa cell growth and differentiation.

Haploinsufficiency of the MLL and TOB2 genes in lymphoid malignancy

There are many different mechanisms by which the promiscuous MLL gene, with a diversity of partners, can be converted into an oncogene. These include translocations, duplications, insertions and deletions all of which have been linked to HOX gene deregulation as the mediator of haematological malignancy.

A Novel Function of MLL in the S Phase Checkpoint: MLL Is Phosphorylated by ATR Upon DNA Damage, Which Blocks Its Degradation by the SCFskp2 Proteasome, Leading to Its Accumulation That Directly Inhibits DNA Replication.

Abstract 450Cell cycle checkpoints are implemented to safeguard our genome. Accordingly, checkpoint deregulation can result in human cancers. Although the S phase checkpoint plays an essential role in preventing genetic aberrations, the detailed molecular makeup of this signaling cascade, especially how it is executed in higher eukaryotes remains largely unknown. Human chromosome band 11q23 translocation disrupting the MLL gene leads to poor prognostic leukemias. MLL is a histone H3 lysine 4 methyl transferase that maintains HOX gene expression. The importance of HOX gene deregulation in MLL leukemogenesis has been intensively investigated. However, physiological murine MLL leukemia knockin models have indicated that incurred HOX gene aberration alone is insufficient to initiate MLL leukemia. Thus, additional signaling pathways must be involved, which remains to be discovered. Here, we demonstrate a novel function of MLL in executing the S phase DNA damage checkpoint response.We found that MLL was accumulated in the S phase upon DNA damage triggered by various agents including UV, ionizing radiation, etoposide, hydroxyurea and aphidocholin, which was observed in all of the cell lines examined including HeLa, 293T, NIH 3T3, and BJ-1 cells. During a normal cell cycle progression, MLL was recognized and degraded by the SCFskp2 proteasome in the S phase. Upon DNA damage, MLL was phosphorylated and thereby no longer recognized by SCFskp2, leading to its ultimate accumulation in the S phase. To determine the importance of DNA-damage induced MLL accumulation, we investigated whether MLL deficiency compromises S phase checkpoint in response to DNA damage. MLL knockout or knockdown cells displayed radioresistant DNA synthesis (RDS) and chromatid type genomic abnormalities (two hallmarks of S phase checkpoint defect). Using genetically well-defined mouse embryonic fibroblasts (MEFs), we identified ATR, but not ATM or DNA-PK, as the kinase required for the MLL accumulation. Furthermore, MLL with mutation of the ATR phosphorylation site failed to accumulate upon DNA damage and thus was unable to rescue the RDS and genomic instability phenotypes of MLL deficient cells. In summary, MLL is phosphorylated by ATR upon DNA damage, which disrupts its interaction with SCFskp2, leading to its accumulation in the S phase that is essential for the proper DNA damage checkpoint execution.We further dissected the mechanism by which MLL participates in the S phase checkpoint execution. We demonstrated that ATR -mediated phosphorylation of Chk1 remained intact in the absence of MLL, which positions MLL downstream to the DNA damage signaling cascade. CDC45 loading onto the replication origin constitutes the critical step of origin firing and thus ushers DNA replication - a step that is normally inhibited upon DNA damage signaling. Using co-immunoprecipitation and chromatin-immunoprecipitation assays, we demonstrated that S phase-accumulated MLL interacts with the MCM complex at the late replication origin, prevents the loading of CDC45, and thereby inhibits DNA replication. In other words, CDC45 was aberrantly loaded in the absence of MLL, which explains the observed RDS defects associated with the loss of MLL. To determine whether MLL leukemogenic fusions incur S phase checkpoint defects, we employed a MLL-CBP knockin mouse model. The RDS phenotype was observed in murine myeloid progenitor cells (MPCs) with haploinsufficiency of MLL. More importantly, MPCs expressing one knockin allele of MLL-CBP exhibited even greater S phase checkpoint defects, suggesting that MLL fusion further compromised DNA damage checkpoint. Taken together, our study establishes a previously unrecognized activity of MLL in direct inhibition of late origin firing upon DNA damage signaling, the deregulation of which may contribute to the pathogenesis of MLL leukemias. Disclosures:No relevant conflicts of interest to declare.

The Role of MLL in DNA Damage Checkpoint and Its Implication in Leukemogenesis

Cell cycle checkpoints are implemented to safeguard our genome and the deregulation of which results in human cancers. Hence, it is of great significance to discover and investigate novel key constituents of the mammalian DNA damage response network. Human chromosome band 11q23 translocation disrupting the MLL gene leads to poor prognostic leukemias. MLL is a transcription co-activator that maintains HOX gene expression. The importance of HOX gene deregulation in MLL leukemogenesis has been intensively investigated. However, physiological murine MLL leukemia knockin models have indicated that incurred HOX gene aberration alone is insufficient to initiate MLL leukemia. Thus, additional signaling pathway must be involved, which remains to be discovered. Our recent studies demonstrated an intimate relationship between MLL and the cell cycle(Takeda et al. 2006, Genes & Development , 20, 2397–2409; Liu et al. 2007, Genes & Development , 21, 2385–2398). More importantly, our studies uncovered a critical role of MLL in executing the S phase checkpoint. We showed:Over-expression of MLL induces an S phase block.MLL accumulates in the S phase upon DNA damage.MLL deficiency results in radioresistant DNA synthesis (RDS) and chromatid-type chromosomal abnormalities, two signature characteristics of S phase checkpoint defects.We further determined the underlying mechanisms concerning the DNA damage-induced MLL accumulation. Our data showed that MLL is phosphorylated after DNA damage, which in turn blocks its degradation by SCFSkp2 in the S phase and results in the ultimate accumulation. Our data revealed the link between MLL and the S phase checkpoint, which provides novel insights into the mammalian cell cycle checkpoint network and human leukemia pathogenesis. Future studies utilizing murine leukemia models will be performed to examine whether MLL translocation compromises the S phase checkpoint and if the resulted dysfunction contributes to MLL leukemogenesis.

The MLL PHD Fingers of MLL Block MLL Fusion Protein Mediated Transformation.

Mixed Lineage Leukemia (MLL) is a histone H3K4 methyltransferase that is rearranged in both acute myeloid leukemia (AML) and acute lymphoid leukemia (ALL). MLL is required for the maintenance of Hox gene expression. Deregulation of Hox genes by MLL fusion proteins, which fuse MLL in frame to one of over 50 different translocation partners, is critical for transformation. In these translocations, the DNMT homology (CXXC) domain is always included, but the set of adjacent plant homeodomains (PHD), which includes four PHD fingers and a bromodomain, is invariably excluded. PHD fingers have recently been described to bind tri-methylated histone H3K4 and others report PHD domains binding transcriptional co-repressors, such as Mi-2a of the NuRD complex. However, the role of the PHD fingers in MLL is not well understood. To determine the function of the PHD fingers in MLL, we performed bone marrow transduction and colony assays with the MLL fusion protein MLL-AF9, engineered to contain the PHD domain region (MLL-PHD-AF9). These experiments showed that inclusion of the PHD fingers inhibited immortalization as shown by the absence of compact colonies in methylcellulose replating assays and inhibition of proliferation in liquid cultures. Initial experiments with PHD finger deletions to map the inhibiting activity suggest inclusion of any PHD fingers beyond the first PHD finger, results in inhibition of transformation. To monitor the transcriptional activity of the retrovirally infected bone marrow cells, total RNA was isolated from cells harvested after the second replating, when significant differences were seen in colony morphology and size. Consistent with the transformation inhibition, Hoxa9 gene expression was found to be significantly repressed with respect to expression detected in transformed MLL-AF9 cells as determined by qPCR. To confirm this effect is directly due to the MLL fusion proteins, we performed luciferase assays with an MLL responsive myc E-box luciferase construct in MLL −/− MEFs. We found a specific and robust activation of the reporter in the presence of MLL-AF9, which was severely compromised by the inclusion of the PHD fingers. Together, these results suggest the PHD fingers act as transcriptional repressors that inhibit transformation. Our results provide an explanation for the finding that translocations including the coding region for C terminal PHD fingers do not occur in human leukemias and suggest that this region is also involved in the regulation of wild type MLL. We are currently studying the mechanisms of transcriptional repression mediated by the PHD fingers by isolating interacting proteins and assessing their effect on transcription and transformation.

HOX Expression Patterns Identify Major Cytogenetic Groups and Are of Prognostic Importance in Adult AML.

Naturally occurring and experimentally-induced deregulation of HOX genes has been causally linked to leukemia and myeloproliferative states. We developed and validated SYBR-green based real-time qRT-PCR assays for the clustered HOXA-D genes as well as other selected homeodomain, trithorax and polycomb genes. These were used to examine 120 cases of adult AML (median age 58 y) with the following cytogenetic classifications: favorable (FAV) 17, intermediate (INT) 53, unfavorable (UNFAV) 46, no analyzable metaphases 4, plus normal CD34+ sorted cells from 4 bone marrow donors. HOX expression patterns were intimately linked to the major cytogenetic subsets and provide a unifying feature for some AML subsets, particularly within the favorable prognostic group. Twenty genes were identified, with a 95% confidence of no false positives, that distinguished the major cytogenetic groups. In general, levels of HOXA genes and MEIS1 distinguished FAV vs. INT and FAV vs. UNFAV groups, while the HOXB and PBX3 levels distinguished either all three groups or INT vs. UNFAV. Comparing 68 patients who achieved a complete response with 49 patients who had resistant disease (or failed induction therapy), 11 genes were differentially expressed with parametric p-values < 0.05. Based on permutation distribution with a 95% confidence level, there were no more than 5 false positives among these 11 genes. Using a COX proportional hazards model and patients for whom survival data were available, HOXA9 and HOXA10 were predictive of overall survival, with parametric p-values <0.05. Lastly, compared to normal CD34+ cells, homeodomain gene expression was abnormal in nearly every AML. Based on these results and the role of HOX deregulation in experimental leukemogenesis, we propose that differences in HOX expression may be responsible for some of the biological differences observed among major cytogenetic subsets in human AML.

Hox regulation of normal and leukemic hematopoietic stem cells

Herein we focus on recent studies of knock out mice that demonstrate a function for the clustered homeobox (Hox) genes in normal hematopoiesis, on papers that point to their general involvement in human leukemia, and discuss the advances in the understanding of the mechanisms underlying their role in these processes. Expression analysis and gain- or loss- of function studies have shown that Hox play an important role in the regulation of early stages of hematopoiesis, including the self-renewal of hematopoietic stem cells (HSCs)/early progenitors. In the area of leukemia, numerous models of murine leukemia have demonstrated a role for Hox in the pathobiology of the disease. Moreover, the identification of multiple Hox genes as partners of chromosomal translocations and the observed global deregulation of Hox genes and cofactors demonstrated by gene profiling of cells from leukemic patients, have unequivocally shown a major function for Hox genes and cofactors in a wide spectrum of human leukemia. The identification of Hox genes as HSC regulators has been exploited to develop strategies to efficiently expand HSCs ex vivo, a key step to the success of therapies based on HSC transplantation and the understanding of mechanisms underlying HSC regulation. As leukemia is the result of deregulation of normal HSC development, the elucidation of the role of Hox in the pathobiology of the disease is helping to understand how HSCs self-renew and differentiate, and moreover, should facilitate the development of strategies for the management of leukemia.

The CALM/AF10 Fusion: Molecular Analysis of the Fusion Transcripts in 13 Cases of AML and ALL; Gene Expression Profiling Reveals HOX Gene Deregulation.

The t(10;11)(p13;q14) is a recurring translocation associated with the CALM/AF10 fusion gene which is found in undifferentiated leukemia, acute myeloid leukemia, acute lymphoblastic leukemia and malignant lymphoma with poor prognosis. The CALM/AF10 fusion protein was reported to be the most common fusion protein in T-ALL with TCR gamma delta rearrangement. We have analyzed samples from 9 patients with different types of leukemia: case 1 (AML M2), case 2 (AML M0), case 3 (Pre T-ALL), case 4 (Acute Undifferentiated Leukemia), case 5 (PreT-ALL), case 6 and 7 (ProT-ALL), case 8 (T-ALL), case 9 (AML), with a t(10;11) translocation suggesting a CALM/AF10-rearrangement. The samples were analyzed for the presence of the CALM/AF10 and AF10/CALM mRNA by RT-PCR and sequence analysis. All these patients were found positive for the CALM/AF10 fusion.In addition, we analyzed a series of twenty-nine patients with T-ALL with gamma delta rearrangement. Among these patients, four were positive for CALM/AF10 transcripts, indicating a high incidence of CALM/AF10 fusions in this group of leukemia. We found three different breakpoints in CALM at nucleotide 1926, 2091 and a new exon, with 106 bases inserted after nt 2064 of CALM in patient 4. In AF10 four breakpoints were identified: at nucleotide position 424, 589, 883 and 979. In seven patients it was also possible to amplify the reciprocal AF10/CALM fusion transcript (case 1, 3, 4, 8, 9, 10 and 11). There was no correlation between disease phenotype and breakpoint location. The patients were 5 to 46 years old (median 25). Ten CALM/AF10 positive patients were further analyzed using oligonucleotide microarrays representing 33,000 different genes (U133 set, Affymetrix). Analysis of microarray gene expression signatures of these patients revealed high expression levels of the homeobox gene MEIS1 and the HOXA cluster genes HOXA1, HOXA4, HOXA5, HOXA7, HOXA9, and HOXA10. The overexpression of HOX genes seen in these CALM/AF10 positive leukemias is reminiscent of the pattern seen in leukemias with rearrangements of the MLL gene, and complex aberrant karyotypes suggesting a common effector pathway (i.e. HOX gene deregulation) for these diverse leukemias. It is known that alhambra, the Drosophila homologue of AF10 can act on polycomb group responsive elements, which play a critical role in the regulation of the HOX gene clusters. It is thus conceivable that the CALM/AF10 fusion proteins acts in a dominant negative fashion on wild type AF10 function relieving the repression that is presumably normally exerted by AF10 on the expression of HOX genes.