Hox Loci Research Articles

Genetic modification of a Hox locus drives mimetic color pattern variation in a highly polymorphic bumble bee.

Müllerian mimicry provides natural replicates ideal for exploring mechanisms underlying adaptive phenotypic divergence and convergence, yet the genetic mechanisms underlying mimetic variation remains largely unknown. The current study investigates the genetic basis of mimetic color pattern variation in a highly polymorphic bumble bee, Bombus breviceps (Hymenoptera, Apidae). In South Asia, this species and multiple comimetic species converge onto local Müllerian mimicry patterns by shifting the abdominal setal color from orange to black. Genetic crossing between the orange and black phenotypes suggested the color dimorphism being controlled by a single Mendelian locus, with the orange allele being dominant over black. Genome-wide association suggests that a locus at the intergenic region between two abdominal fate-determining Hox genes, abd-A and Abd-B is associated with the color change. This locus is therefore in the same intergenic region but not the same exact locus as found to drive red-black mid-abdominal variation in a distantly related bumble bee species, B. melanopygus. Gene expression analysis and RNA interferences suggest that differential expression of an intergenic long non-coding RNA between abd-A and Abd-B at the onset setal color differentiation may drive the orange black color variation by causing a homeotic shift late in development. Analysis of this same color locus in comimetic species reveals no sequence association with the same color shift, suggesting that mimetic convergence is achieved through distinct genetic routes. Our study establishes Hox regions as genomic hotspots for color pattern evolution in bumble bees, and demonstrates how pleiotropic developmental loci can drive adaptive radiations in nature.

EPCO-23. DYSFUNCTION OF LARGE H3K9ME3 DOMAINS IN ATRX DEFICIENT GLIOMAS INDUCES GENETIC REARRANGEMENTS AND LATENT DEVELOPMENTAL SIGNALING NETWORKS THROUGH SUPER-ENHANCER LANDSCAPES

Abstract Somatic alterations in ATRX (Alpha Thalassemia/Mental Retardation Syndrome X-linked), a member of SWI/SNF family chromatin regulator has been found to be frequently mutated in diffuse gliomas and defines molecular subtypes with aggressive behavior. Mechanistically, ATRX regulates incorporation of histone H3.3 into chromatin sites across the genome, maintains alternative lengthening of telomeres and establishes genomic distribution of polycomb responsive genes. To understand in depth role of ATRX in gliomas, we performed ChIP-seq and/or Cut-and-tag for histone marks that define active transcription, enhancers, repressors, gene bodies and CTCF on Atrx intact and deficient mNPCs (mouse Neural Progenitor Cells). Our integrated analysis reports depletion of H3K9me3 loci’s that coincidently enriched with Atrx binding sites and Lamina-Associated Domains with genes enriched for cell cycle, motility and chromosome organization. This chromatin perturbations at heterochromatin domains was further confirmed in our Hi-C analysis with switching of A-B and B-A compartments, reorganization of TADs with occasional CTCF marks and gain of novel interacting loops that showed gene expression leakage required for gliomagenesis using Capture Hi-C. Notably, we observed aberrant levels of endogenous retroviral elements (ERVs) family members, including upregulation of Line-1 elements in mNPCs and patient derived glioma stem cells (GSCs) and our analysis shows increased copy number variations in ATRX deficient gliomas as a consequence of Line-1 activation in these subsets of tumors. Finally, our integrated omics- analysis demonstrates enrichment of super-enhancers in Atrx deficient background with several putative druggable candidates for clinical benefits. As an example, we show presence of H3K27ac super-enhancer over HOXA locus and targeting pan-HOXA genes using small inhibitor peptides diminished proliferation and migration of mNPCs and GSCs with increased in cell apoptosis with alterations in downstream developmental signaling pathways. To summarize,our data establishes tangible links between Atrx deficiency and multiscale dysregulated cellular phenotype in gliomas with identifying novel targets for therapy.

TOUCHDOWN PCR FOR INCREASED SENSITIVITY AND SPECIFICITY IN AMPLIFICATION OF MOUSE COGNATE HOMEOBOX

Mammalian system have a large genome with a high level of gene sequence identity from other genomic DNA, making assessment difficult and time-consuming.Our findings describe a simple method for rapidly isolating and amplifying HoxA loci in the mouse genome using degenerate primers. For the semi degenerate primers,they were designed based on cognate gene coding regions of consensus sequences. After assembling sequences from different primer matches amplifying the same HoxA loci, the effects of the universal primer-template match on the efficiency of standard PCR amplification were investigated. Touchdown PCR increased specificity and yield in two consecutive amplifications on different gel concentrations by using high and low annealing temperatures. This method was quick, simple, and inexpensive for amplification of consensus sequences in very large gene sequences.

Transcription factor paralogs orchestrate alternative gene regulatory networks by context-dependent cooperation with multiple cofactors

In eukaryotes, members of transcription factor families often exhibit similar DNA binding properties in vitro, yet orchestrate paralog-specific gene regulatory networks in vivo. The serially homologous first (T1) and third (T3) thoracic legs of Drosophila, which are specified by the Hox proteins Scr and Ubx, respectively, offer a unique opportunity to address this paradox in vivo. Genome-wide analyses using epitope-tagged alleles of both Hox loci in the T1 and T3 leg imaginal discs, the precursors to the adult legs and ventral body regions, show that ~8% of Hox binding is paralog-specific. Binding specificity is mediated by interactions with distinct cofactors in different domains: the Hox cofactor Exd acts in the proximal domain and is necessary for Scr to bind many of its paralog-specific targets, while in the distal leg domain, the homeodomain protein Distal-less (Dll) enhances Scr binding to a different subset of loci. These findings reveal how Hox paralogs, and perhaps paralogs of other transcription factor families, orchestrate alternative downstream gene regulatory networks with the help of multiple, context-specific cofactors.

HOX epimutations driven by maternal SMCHD1/LRIF1 haploinsufficiency trigger homeotic transformations in genetically wildtype offspring

The body plan of animals is laid out by an evolutionary-conserved HOX code which is colinearly transcribed after zygotic genome activation (ZGA). Here we report that SMCHD1, a chromatin-modifying enzyme needed for X-inactivation in mammals, is maternally required for timely HOX expression. Using zebrafish and mouse Smchd1 knockout animals, we demonstrate that Smchd1 haplo-insufficiency brings about precocious and ectopic HOX transcription during oogenesis and embryogenesis. Unexpectedly, wild-type offspring born to heterozygous knockout zebrafish smchd1 mothers exhibited patent vertebrate patterning defects. The loss of maternal Smchd1 was accompanied by HOX epi-mutations driven by aberrant DNA methylation. We further show that this regulation is mediated by Lrif1, a direct interacting partner of Smchd1, whose knockout in zebrafish phenocopies that of Smchd1. Rather than being a short-lived maternal effect, HOX mis-regulation is stably inherited through cell divisions and persists in cultured fibroblasts derived from FSHD2 patients haploinsufficient for SMCHD1. We conclude that maternal SMCHD1/LRIF1 sets up an epigenetic state in the HOX loci that can only be reset in the germline. Such an unusual inter-generational inheritance, whereby a phenotype can be one generation removed from its genotype, casts a new light on how unresolved Mendelian diseases may be interpreted.

A prominent gene activation role for C-terminal binding protein in mediating PcG/trxG proteins through Hox gene regulation.

The evolutionarily conserved C-terminal binding protein (CtBP) has been well characterized as a transcriptional co-repressor. Herein, we report a previously unreported function for CtBP, showing that lowering CtBP dosage genetically suppresses Polycomb group (PcG) loss-of-function phenotypes while enhancing that of trithorax group (trxG) in Drosophila, suggesting that the role of CtBP in gene activation is more pronounced in fly development than previously thought. In fly cells, we show that CtBP is required for the derepression of the most direct PcG target genes, which are highly enriched by homeobox transcription factors, including Hox genes. Using ChIP and co-IP assays, we demonstrate that CtBP is directly required for the molecular switch between H3K27me3 and H3K27ac in the derepressed Hox loci. In addition, CtBP physically interacts with many proteins, such as UTX, CBP, Fs(1)h and RNA Pol II, that have activation roles, potentially assisting in their recruitment to promoters and Polycomb response elements that control Hox gene expression. Therefore, we reveal a prominent activation function for CtBP that confers a major role for the epigenetic program of fly segmentation and development.

Nup93 and CTCF modulate spatiotemporal dynamics and function of the HOXA gene locus during differentiation.

Nucleoporins regulate nuclear transport and are also involved in DNA damage, repair, cell cycle, chromatin organization and gene expression. Here, we studied the role of nucleoporin Nup93 and the chromatin organizer CTCF in regulating expression of the HOXA gene locus during differentiation. ChIP sequencing revealed a significant overlap between Nup93 and CTCF peaks. Interestingly, Nup93 and CTCF are associated with the 3' and 5' HOXA genes, respectively. Depletions of Nup93 and CTCF antagonistically modulate expression levels of 3' and 5' HOXA genes in the undifferentiated human NT2/D1 cell line. Nup93 also regulates the localization of the HOXA gene locus, which disengages from the nuclear periphery upon Nup93 but not CTCF depletion, consistent with its upregulation. The dynamic association of Nup93 and CTCF with the HOXA locus during differentiation correlates with its spatial positioning and expression. Whereas Nup93 tethers the HOXA locus to the nuclear periphery, CTCF potentially regulates looping of the HOXA gene cluster in a temporal manner. In summary, Nup93 and CTCF complement one another in modulating the spatiotemporal dynamics and function of the HOXA gene locus during differentiation. This article has an associated First Person interview with the first authors of the paper.

Mutational synergy during leukemia induction remodels chromatin accessibility, histone modifications and three-dimensional DNA topology to alter gene expression.

Altered transcription is a cardinal feature of acute myeloid leukemia (AML), however, exactly how mutations synergize to remodel the epigenetic landscape and rewire 3-Dimensional (3D) DNA topology is unknown. Here we apply an integrated genomic approach to a murine allelic series that models the two most common mutations in AML, Flt3-ITD and Npm1c. We then deconvolute the contribution of each mutation to alterations of the epigenetic landscape and genome organization, and infer how mutations synergize in the induction of AML. Our studies demonstrate that Flt3-ITD signals to chromatin to alter the epigenetic environment and synergizes with Npm1c mutation to alter gene expression and drive leukemia induction. These analyses also allow the identification of long-range cis-regulatory circuits, including a novel super-enhancer of Hoxa locus, as well as larger and more detailed gene-regulatory networks, driven by transcription factors including PU.1 and IRF8, whose importance we demonstrate through perturbation of network members.

Maternal Transcripts of Hox Genes Are Found in Oocytes of Platynereis dumerilii (Annelida, Nereididae).

Hox genes are some of the best studied developmental control genes. In the overwhelming majority of bilateral animals, these genes are sequentially activated along the main body axis during the establishment of the ground plane, i.e., at the moment of gastrulation. Their activation is necessary for the correct differentiation of cell lines, but at the same time it reduces the level of stemness. That is why the chromatin of Hox loci in the pre-gastrulating embryo is in a bivalent state. It carries both repressive and permissive epigenetic markers at H3 histone residues, leading to transcriptional repression. There is a paradox that maternal RNAs, and in some cases the proteins of the Hox genes, are present in oocytes and preimplantation embryos in mammals. Their functions should be different from the zygotic ones and have not been studied to date. Our object is the errant annelid Platynereis dumerilii. This model is convenient for studying new functions and mechanisms of regulation of Hox genes, because it is incomparably simpler than laboratory vertebrates. Using a standard RT-PCR on cDNA template which was obtained by reverse transcription using random primers, we found that maternal transcripts of almost all Hox genes are present in unfertilized oocytes of worm. We assessed the localization of these transcripts using WMISH.

The RNA-binding protein IGF2BP3 is critical for MLL-AF4-mediated leukemogenesis

Despite recent advances in therapeutic approaches, patients with MLL-rearranged leukemia still have poor outcomes. Here, we find that the RNA-binding protein IGF2BP3, which is overexpressed in MLL-translocated leukemia, strongly amplifies MLL-Af4-mediated leukemogenesis. Deletion of Igf2bp3 significantly increases the survival of mice with MLL-Af4-driven leukemia and greatly attenuates disease, with a minimal impact on baseline hematopoiesis. At the cellular level, MLL-Af4 leukemia-initiating cells require Igf2bp3 for their function in leukemogenesis. At the molecular level, IGF2BP3 regulates a complex posttranscriptional operon governing leukemia cell survival and proliferation. IGF2BP3-targeted mRNA transcripts include important MLL-Af4-induced genes, such as those in the Hoxa locus, and the Ras signaling pathway. Targeting of transcripts by IGF2BP3 regulates both steady-state mRNA levels and, unexpectedly, pre-mRNA splicing. Together, our findings show that IGF2BP3 represents an attractive therapeutic target in this disease, providing important insights into mechanisms of posttranscriptional regulation in leukemia.

There and Back Again: Hox Clusters Use Both DNA Strands.

Bilaterian animals operate the clusters of Hox genes through a rich repertoire of diverse mechanisms. In this review, we will summarize and analyze the accumulated data concerning long non-coding RNAs (lncRNAs) that are transcribed from sense (coding) DNA strands of Hox clusters. It was shown that antisense regulatory RNAs control the work of Hox genes in cis and trans, participate in the establishment and maintenance of the epigenetic code of Hox loci, and can even serve as a source of regulatory peptides that switch cellular energetic metabolism. Moreover, these molecules can be considered as a force that consolidates the cluster into a single whole. We will discuss the examples of antisense transcription of Hox genes in well-studied systems (cell cultures, morphogenesis of vertebrates) and bear upon some interesting examples of antisense Hox RNAs in non-model Protostomia.

Disregulation of CTCF Boundary at the HOX Locus Enhances Sensitity to Multiple Drug Treatment in Myeloid Malignancies

Dysregulation of HOX genes is a dominant mechanism of leukemic transformation by altering self-renewal and differentiation properties of HS/PCs leading to leukemic transformation. The overexpression of posterior HOXA9 gene is also a poor prognostic marker in leukemia patients. Recent studies revealed that CTCF insulator constrains temporal HOX gene expression patterns within confined chromatin domains suggesting that CTCF mediated chromatin boundary activity may involve in regulating HOX gene patterns during AML prognosis.To investigate the role of CTCF in aberrant HOX gene expression and AML prognosis, we employed CRISPR-Cas9 genome editing technology to delete 47 base pairs of the core CTCF motif located between HOXA7 and HOXA9 genes (CBS7/9) in different subtype of AML cells with either MLL-rearrangement or NPM1 mutation. RNA-seq analysis revealed that transcription factors, HOXA9 , RUNX1 , and ZEB1 , as well as MAPK signaling pathway, all of them are involved in AML survival, prognosis and chemo-resistance, are significantly dowregulated in response to the CBS7/9 boundary deletion. Given that these genes and pathways are involved in AML prognosis and chemo-resistance, we then examined whether perturbation of CBS7/9 chromatin boundary resulted in alteration of drug resposes of AML cells. Both WT AML cells and the CBS7/9 defected AML cells derived from MLL-AF9 rearranged MOLM-13 and NPM mutated OCI-AML3 cells were treated with 1 mM of Ara-C or 100 nM of bromodomain (BET) inhibitors. Compared to the WT controls, the defected CBS7/9 boundary resulted in a significantly increase in AML sensitivity to both Ara-C and BET inhibitor treatment in both MOLM-13 and OCI-AML3 cells as evidence by significantly reducing percentage of proliferating cells.To further examine if treatment related drug sensitivity in AML cells is depended on the levels of HOXA9 expression, we overexpressed HOXA9 cDNA in the CBS7/9 deleted MOLM-13 and OCI-AML3 cells. Gain-of-function (GOF) expression of HOXA9 in the CBS7/9 defected AML cells rescued and restored the resistance to the Ara-C treatment of the CBS7/9 KO cells to the levels compatible to that of the WT control MOLM-13 and OCI-AML3 cells, but HOXA9 overexpression did not rescued the resistance of the CBS7/9 KO AML cells to the BET inhibitor treatment. The data suggest that HOXA9 is directly associated with cytotoxic drug Ara-C resistance, but not BET inhibitor mediated drug responses. KO of CBS7/9 specifically decreased enrichment of H3K9ac and H4K5ac, as well as recruitment of BRD4 to the posterior HOXA domain. Furthermore, overexpression of HOXA9 gene in the CBS7/9 KO AML cells indeed rescued the mRNA transcripts affected by CBS7/9 deletion to the levels compatible to that of WT control MOLM-13 or OCI-AML3 cells. Among rescued genes, ZEB1, STAT5A, and RUNX1 play essential roles in pathogenesis of myeloid malignancies and chemo-drug sensitivity.Finally, in order to further explore biological role of CTCF boundaries in AML pathogenesis, we analyze the whole genome sequencing data of 183 primary AML patients obtained from public available Cosmic Database for somatic mutations occurred in the CTCF binding sites. Several core CTCF motifs in the HOXA locus, including CBS5/6 and CBS7/9 boundaries, are highly mutated in AML patients. Thus, disruption of CTCF boundary located between HOXA7 and HOXA9 genes perturbs HOXA9 oncogene transcription and regulates chemo- or epigenetic drug sensitivity of acute myeloid leukemia. Alteration of CTCF boundaries in the oncogene loci may provide a novel therapeutic target for treatment of myeloid malignancies. DisclosuresNo relevant conflicts of interest to declare.

Dot1l Reverses the Abnormal Self-Renewal Conferred on HSPCs through Cohesin Haploinsufficiency By Diminishing Expression of Hoxa7 and Hoxa9

Acute myelogenous leukemia (AML) is a high-risk hematopoietic malignancy with a poor prognosis. Developing novel treatments for AML has been difficult due to the heterogeneity of genetic mutations observed across patients. Recent genomic sequencing of AML patients have identified recurrent somatic mutations in cohesin complex genes in a subset of patients (10-20%). The canonical function of the cohesin complex is to maintain sister chromatid cohesion permitting proper segregation of genomic information to each daughter cell in mitosis. Complete loss of cohesin function causes an early embryonic lethal phenotype in knock-out mice, implying that some level of cohesin function is absolutely required for normal development. Interestingly, somatic cohesin mutations in AML are uniformly heterozygous and not associated with dramatic changes in chromosomal number, implying that cohesin mutations promote AML through a function independent of their role during mitosis.Recent investigations have demonstrated that cohesin haploinsufficiency leads to enhanced self-renewal in Hematopoietic Stem and Progenitor Cells (HSPCs). Previous studies from our lab identified significant increases in the HSPC self-renewal factors Hoxa7 and Hoxa9 in Rad21 -depleted HSPCs concomitant with decreased levels of the repressive histone mark trimethylated lysine 27 on histone H3 (H3K27me3) at the Hoxa7 and Hoxa9 promoters, indicating that Rad21 plays a role in proper PRC2-mediated silencing of these genes. Immunoprecipitation and ChIP experiments revealed that cohesin and PRC2 directly interact and are bound in close proximity to Hoxa7 and Hoxa9 . Interestingly genome-wide levels of PRC2's repressive histone mark H3K27me3 were reduced. Importantly, H3K27me3 levels were significantly decreased at the Hoxa7 / Hoxa9 locus. Knockdown of either Hoxa7 or Hoxa9 in Rad21 -depleted HSPCs suppressed their self-renewal, implying both are critical downstream effectors of reduced cohesin levels.In the present study we sought to delineate additional molecular mechanisms by which cohesin mutations promote enhanced HSPC self-renewal and evaluate possible therapeutic interventions to reverse the phenotype conferred on HSPCs by cohesin haploinsufficiency. We find that reduction of the core cohesin subunit Rad21 using RNAi increases levels of dimethylated lysine 79 on histone H3 (H3K79me2) genome-wide in contrast to the genome-wide reduction of H3K27me3 levels. H3K79me2 is deposited by the Dot1l chromatin modifying complex and is correlated with active gene expression. Given the established role of Dot1l in regulating Hoxa9 expression, we sought to evaluate whether inhibiting Dot1l methyltransferase activity would reverse the phenotype caused by cohesin haploinsufficiency. As anticipated, we observed that inhibition of Dot1l function, using either of two commercially available inhibitors or RNAi mediated knockdown, reverses the enhanced self-renewal capacity of cohesin haploinsufficient HSPCs. Consistent with our proposed mechanism, this reduced self-renewal occurs with a concomitant reduction in Hoxa7 and Hoxa9 expression. We observe a similar trend using HSPCs from Smc3 conditional knockout mice, a more elegant genetic model. RNA-sequencing and ChIP-sequencing studies are ongoing to evaluate the transcriptome and epigenomic changes imparted by cohesin haploinsufficiency and how these changes are affected by Dot1l inhibition. Our preliminary studies are intriguing because cohesin haploinsufficiency appears to have contrasting effects on genome-wide levels H3K27me3 and H3K79me2, two opposing epigenetic marks. In conclusion, our data demonstrate that Dot1l inhibition negatively regulates self-renewal of cohesin haploinsufficient HSPCs, but not control HSPCs, by abbrogating the abberant expression of Hoxa7 and Hoxa9 . This study establishes feasibility for the use of Dot1l inhibition as a targeted therapeutic treatment for AML patients harboring cohesin mutations. DisclosuresLevine:Qiagen: Equity Ownership; Roche: Research Funding; Celgene: Research Funding; Roche: Research Funding; Qiagen: Equity Ownership; Celgene: Research Funding.

A Role for the CRM1 Nuclear Export Receptor in HOXA Gene Deregulation and CALM-AF10 Leukemogenesis

Background:CALM-AF10 translocations are seen in 5-10% of T-cell acute lymphoblastic leukemia (T-ALL) and are characterized by HOXA gene overexpression, a feature shared with leukemias associated with translocations of the Mixed-Lineage Leukemia (MLL) gene. The ability of CALM-AF10 to aberrantly activate HOXA gene transcription is mediated by interaction of the AF10 moiety with the H3K79 methyltransferase DOT1L. We previously demonstrated that a nuclear export signal (NES) within CALM is essential for CALM-AF10's ability to upregulate HOXA genes and cause leukemia in mice. The CRM1 (XPO1) nuclear export receptor specifically interacts with NES motifs, including the CALM NES. Fusing an 18 amino acid NES motif to AF10 recapitulates the oncogenic properties of CALM-AF10. Interfering with the CRM1/CALM-AF10 interaction by either genetic (mutation of the CALM NES motif) or pharmacologic (Leptomycin B - LMB) inhibition abolishes CALM-AF10's ability to bind to HOXA genes and activate their expression. We discovered that CRM1 itself is able to bind to HOXA loci, suggesting that CRM1 may recruit CALM-AF10 to its target genes. The NES binding cleft within CRM1 is maintained in a closed conformation by the C-terminal helix (CRM1 aa 1029-1071), and its truncation greatly enhances the NES-binding affinity of CRM1.Hypothesis: Since the NES motif of CALM is necessary and sufficient for the leukemogenic properties of CALM-AF10, we hypothesized that a CRM1-AF10 fusion protein would also be leukemogenic. To determine the importance of the NES binding ability of CRM1 in leukemogenesis, we investigated a deletion mutant of CRM1-AF10 that lacks the inhibitory C-terminal 43 amino acids of CRM1 (CRM1Δ-AF10).Methods: Confocal immunofluorescence microscopy of transfected HEK293 cells was used to study the intracellular localization of CRM1-AF10 and CRM1Δ-AF10 fusion proteins, and their transcriptional activity wasmeasured with a HOXA7 -luciferase reporter. We used LMB to study the impact of blocking the CRM1/NES interaction on HOXA7 transcriptional activation and cellular localization. Retroviral transduction and transplantation of murine bone marrow were performed to determine the ability of CRM1-AF10 and CRM1Δ-AF10 to induce leukemias.Results: CALM-AF10, CRM1-AF10 and CRM1Δ-AF10 fusion proteins localize to both the cytoplasm and nucleus (in a speckled pattern). While all three fusion proteins induced HOXA gene transcription compared with empty vector, CALM-AF10 (7.3 fold increase) and CRM1Δ-AF10 (7.8 fold) were significantly more potent than CRM1-AF10 (5.1 fold). LMB treatment (4 nM, 25 h) repressed HOXA7 transcription induced by CALM-AF10 (68% repressed), CRM1-AF10 (31% repressed), and CRM1Δ-AF10 (60% repressed). LMB exposure (2 nM, 2 h) induced nuclear retention of all three fusion proteins. In vivo, while all three fusions induced murine myeloid leukemias characterized by overexpression of Hoxa genes, CRM1-AF10 leukemias displayed incomplete penetrance (50%) with prolonged latency (250-300 days) compared with both CALM-AF10 and CRM1Δ-AF10, which demonstrated complete penetrance and similar latencies (100-130 days).Conclusions: We have shown that CRM1 can substitute for CALM to induce HOXA gene expression and cause leukemias when fused to AF10, suggesting that the contribution of the CALM moiety in the CALM-AF10 oncoprotein is to recruit CRM1. Deletion of the C-terminal inhibitory helix of CRM1 potentiates the oncogenic properties of CRM1-AF10. Moreover, pharmacologic inhibition of the NES/CRM1 interaction with LMB counteracts the ability of CRM1-AF10 and CRM1Δ-AF10 to activate the transcription of HOXA genes. These results suggest that binding to an as yet unidentified, NES-containing partner protein plays an important role in CRM1-AF10 leukemogenesis. Of note, the CRM1 C-terminal helix is missing in a CRM1-AF10 translocation described in a T-ALL patient (Bond, BLOOD, 2014). These studies shed light on the importance of CRM1 in leukemias associated with HOXA deregulation, and warrant further studies of the mechanisms underlying the therapeutic efficacy of CRM1 inhibitors (selective inhibitors of nuclear export - SINEs) that target the CRM1/NES interaction. DisclosuresNo relevant conflicts of interest to declare.

Disruption of CTCF Boundary Remodels Chromatin Domain and Reprograms HOX Gene Transcription in Acute Myeloid Leukemia

The organization of the mammalian genome into separate domains either facilitates or restricts interactions between regulatory elements, e.g. enhancers and promoters, for normal gene regulation. CTCF insulator constrains the temporal HOX gene expression patterns within the confined chromatin domain for normal development. However, dysregulation of HOX genes is also a common feature of acute myeloid leukemia (AML). The molecular mechanisms of aberrant HOX expression and associated AML pathogenesis remain unclear. Here, we found that CTCF binding site located between HOXA7 and HOXA9 genes (CBS7/9) acts as a chromatin boundary to establish and maintain AML specific aberrant HOX gene expression patterns and is essential for pathogenesis of myeloid malignancies.To unbiasedly and systematically investigate the mechanism underlying HOX gene regulation in AML, we employed a pooled CRISPR-Cas9 genetic knockout library screening to interrogate intergenic regulatory and chromatin boundary elements in all 4 HOX gene loci. Through this screening, we identified the CBS7/9 chromatin boundary located between HOXA7 and HOXA9 genes that is critical for maintaining aberrant expression of posterior HOXA genes including oncogene HOXA9 and HOXA10 in primary AML patients and cells. To further confirm the role of CBS7/9 in HOX gene regulation, two single-guide RNAs (sgRNAs) flanking 47 base pairs (bps) of the core CTCF motif located between HOXA7 and HOXA9 geneswere employed to guild Cas9 for deletion of the CBS7/9 boundary in 3 different subtypes of AML cells containing MLL rearrangements or NPM1C+ mutation. The defected CBS7/9 resulted in a decrease in CTCF binding to the caudal HOXA domain and an inhibition of posterior HOXA gene expression.To further delineate the role of CBS7/9 chromatin boundary in HOX gene organization and regulation, we then carried out genome wide ChIP-seq, ATAC-seq, 4C, and RNA-seq analysis to examine the changes in chromatin domain organization and corresponding gene expression patterns compared wild-type control and the CBS7/9 KO AML cells. Disruption of CBS7/9 boundary resulted in a spreading of repressive H3K27me3 domain into the caudal HOXA domain that subsequently impaired AML associated active TAD domain by altering enhancer/promoter chromatin accessibility and reducing ectopic interactions among the posterior HOXA genes. Consistent with the role of CBS7/9 boundary in HOX locus chromatin structure and organization, impairment of CBS7/9 boundary function reduced posterior HOXA gene expression and altered myeloid specific transcriptome profiles important for pathogenesis of myeloid malignancies suggesting that the CBS7/9 boundary mediated ectopic HOX expression patterns might be essential for pathogenesis of myeloid malignancies. To further test this possibility, the CBS 7/9 boundary was deleted in both MLL rearranged AML cells and primary patient samples by the CRISPR-Cas9 mediated genome editing and the edited cells were transplanted into NSG mice for the development of human AML in mice. Disruption of CBS chromatin boundary in the HOXA locus reduced human leukemic blast development and invasion as well as enhanced survival of both transplanted AML cell xenograft and patient derived xenograft (PDX) mouse models. Thus, our finding demonstrated that CTCF boundary not only constrains normal gene expression program for memory of cell identity, but also plays a role in maintaining oncogenic transcription program for leukemic transformation. Alteration of CTCF mediated topological boundaries in the oncogene loci provides therapeutic approaches for targeting oncogenic expression program and leukemogenesis. DisclosuresNo relevant conflicts of interest to declare.

Tip60 activates Hoxa9 and Meis1 expression through acetylation of H2A.Z, promoting MLL-AF10 and MLL-ENL acute myeloid leukemia.

Chromosome translocations involving the MLL gene are common rearrangements in leukemia. Such translocations fuse the MLL 5'-region to partner genes in frame, producing MLL-fusions that cause MLL-related leukemia. MLL-fusions activate transcription of target genes such as HoxA cluster and Meis1, but the underlying mechanisms remain to be fully elucidated. In this study, we discovered that Tip60, a MYST-type histone acetyltransferase, was required for the expression of HoxA cluster and Meis1 genes and the development of MLL-fusion leukemia. Tip60 was recruited by MLL-AF10 and MLL-ENL fusions to the Hoxa9 locus, where it acetylated H2A.Z, thereby promoting Hoxa9 gene expression. Conditional deletion of Tip60 prevented the development of MLL-AF10 and MLL-ENL leukemia, indicating that Tip60 is indispensable for the leukemogenic activity of the MLL-AF10 and MLL-ENL-fusions. Our findings provide novel insight about epigenetic regulation in the development of MLL-AF10 and MLL-ENL-fusion leukemia.

Pan-Cancer Genome-Wide DNA Methylation Analyses Revealed That Hypermethylation Influences 3D Architecture and Gene Expression Dysregulation in HOXA Locus During Carcinogenesis of Cancers.

DNA methylation dysregulation during carcinogenesis has been widely discussed in recent years. However, the pan-cancer DNA methylation biomarkers and corresponding biological mechanisms were seldom investigated. We identified differentially methylated sites and regions from 5,056 The Cancer Genome Atlas (TCGA) samples across 10 cancer types and then validated the findings using 48 manually annotated datasets consisting of 3,394 samples across nine cancer types from Gene Expression Omnibus (GEO). All samples’ DNA methylation profile was evaluated with Illumina 450K microarray to narrow down the batch effect. Nine regions were identified as commonly differentially methylated regions across cancers in TCGA and GEO cohorts. Among these regions, a DNA fragment consisting of ∼1,400 bp detected inside the HOXA locus instead of the boundary may relate to the co-expression attenuation of genes inside the locus during carcinogenesis. We further analyzed the 3D DNA interaction profile by the publicly accessible Hi-C database. Consistently, the HOXA locus in normal cell lines compromised isolated topological domains while merging to the domain nearby in cancer cell lines. In conclusion, the dysregulation of the HOXA locus provides a novel insight into pan-cancer carcinogenesis.

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.

The Transcriptomic Landscape of NUP98-Rearranged Pediatric Acute Myeloid Leukemia

Childhood acute myeloid leukemia (AML) is an aggressive myeloid malignancy characterized by mutational and cytogenetic abnormalities. Chromosomal rearrangements involving the NUP98 gene have come to light for its' significant impacts on outcome and response to treatment. NUP98-rearranged (NUP98-R) AML includes NUP98-NSD1, NUP98-KDM5A, and various less common NUP98 fusion partners - such as HOX, SET, and Bromodomain genes. NUP98-R account for 6.8% of patients across Children's Oncology Group studies CCG2961, AAML03P1, AAML0531, and AAML1031. The majority are NUP98-NSD1 (4.6%), then NUP98-KDM5A (1.4%), and the various partners, termed NUP98-X (0.83%). However, the biological implications of NUP98-X have yet to be investigated. We define transcriptional clusters and report transcriptional profiling results to reveal similarities and differences between the diverse NUP98-R fusions. RNA sequencing was completed for 1,492 pediatric AML patients and 68 healthy bone marrow controls (NBM). The algorithms STARfusion, TransAbyss, and CICERO detected 156 NUP98-R from RNA seq: NUP98-NSD1 (N=104), NUP98-KDM5A (N=32), and NUP98-X (N=20). NUP98-X encompassed 13 unique fusion partners and 45% (9/20) had a homeobox gene partner - HOXA9 (N=4), HOXD13 (N=3), HOXA13 (N=1), and PRRX1 (N=1). Unsupervised clustering via uniform manifold approximation and projection (UMAP) with input genes selected by jackstraw PCA revealed NUP98-X cluster more closely with NUP98-NSD1, but their trajectory is dispersed within NUP98-NSD1 and NUP98-KDM5A, indicating a potential hybrid transcriptional profile (Fig 1 A). NUP98-NSD1 clustered remotely from the majority of NUP98-KDM5A, highlighting their unique transcriptional profiles despite sharing NUP98-R. Five transcriptional clusters were identified by the Leiden algorithm, followed by selection of genes significantly associated with cluster assignment. Cluster marker genes were filtered to be unique for each cluster prior gene-set enrichment analysis (Fig 1 B). Cluster C3 encompassed the largest proportion of NUP98-X, with 7 homeobox and both PHF23 cases (9/20, 45%), but also comprised of 19 NUP98-NSD1 and 2 NUP98-KDM5A. Importantly, C3 was highly associated with the expression of long non-coding RNAs, Z69666.2, MT1XP1, and RP11-455O6.2 (≥ 37% specificity, FDR < 0.001), and pathways in enriched in oncogenic microRNAs (FDR < 0.001), suggesting a non-coding signature may define this group. NUP98-NSD1 divided into two primary clusters, C1 (52/104) and C2 (31/104), differentiated by the expression of mannose receptor genes MCR1/ MRC1L1, which detect products of the lysosome pathway, significantly activated in C1 (FDR < 0.001). NUP98-KDM5A also segregated into two clusters, C4 (N=22/32) and C5 (N=7/32) based on an FAB M7/AMKL signature, with 78.6% of M7 NUP98-R located in C4 (p=0.003). Whereas, C5 cases were non-M7 and uniquely enriched in the regulation of stem cells pathway (FDR = 0.005); activation was driven by the expression of POU5F1, BMP4, WNT7, and WNT10A/B. Transcriptional profiling of NUP98-R cohorts, independently compared to other AML (N=1,336), found 27 upregulated differentially expressed genes (DEGs) shared between NUP98-X and NUP98-NSD1, including MYCN oncogene, homeobox PBX3, and DNA methyltransferase DNMT3B; on the other hand, shared overexpression of MLLT3, homeobox IRX3, and CD79a characterized similarities between NUP98-X and NUP98-KDM5A (Fig 1C) and may contribute to the hybrid expression profile observed by UMAP clustering. There were 38 DEGs shared between all NUP98-R cohorts. Differential expression analysis comparing NUP98-R cohorts vs NBM (N=68) showed that 22/38 shared DEGs were dysregulated in normal hematopoiesis and 15/38 genes were concordantly overexpressed in NUP98-R. The minimal set of 15 genes strongly implicates dysregulation at the HOX locus; these targets include hsa-mir-10a, whose genomic locus is within the HOXB cluster, CACNG4 located on chr17q, the same chromosome arm as HOXB, and the remaining shared targets were HOXA/B genes (Fig 1D). In summary, we found that NUP98-X are dispersed between NUP98-NSD1 and NUP98-KDM5A by transcriptional clustering. Clustering revealed significant pathways and marker genes that may contribute to segregation of NUP98-R cohorts, and DEGs that contribute in part to their hybrid transcriptional profile, including HOX genes, PBX3, and IRX3. Disclosures No relevant conflicts of interest to declare.

The RNA Binding Protein IGF2BP3 Is Required for MLL-AF4 Mediated Leukemogenesis

Chromosomal rearrangements of the mixed-lineage leukemia (MLL) gene are observed in acute lymphoblastic leukemias (ALL), acute myeloid leukemias (AML), and in rare mixed-lineage leukemia. Despite recent progress in therapeutic approaches, patients with MLL-rearranged (MLLr) leukemias still have very poor outcomes and a high risk of relapse. Of more than 90 fusion partner genes, MLL-AF4 is the most common MLL fusion protein in patients. Previously, we found that the RNA binding protein IGF2BP3 was specifically overexpressed in MLL-rearranged B-ALL, and enforced expression in vivo led to a pathologic expansion of hematopoietic stem and progenitor cells resulting in B and myeloid cell leukocytosis in the periphery. However, the requirement of IGF2BP3 in MLL-AF4 mediated leukemogenesis remains to be determined. Utilizing our previously generated list of differentially regulated targets with IGF2BP3 knockdown and a published dataset of MLL-Af4 targets, we determined that transcripts modulated by IGF2BP3 showed significant enrichment for MLL-Af4-bound genes. Furthermore, we observed that MLL-AF4 directly binds to and transcriptionally induces IGF2BP3. We performed ChIP-PCR assays on RS4;11 and SEM cell lines, human B-ALL cell lines that carry the MLL-AF4 translocation, and determined that the region in the first intron of IGF2BP3 is strongly bound by MLL-AF4. Furthermore, we observed a dose-dependent increase in luciferase reporter activity when we co-transfected a dual-luciferase reporter vector containing the promoter region of IGF2BP3 with increasing levels of MLL-AF4 expressing retroviral vector. To determine the role of Igf2bp3 in MLL-Af4 driven leukemogenesis, we generated the first Igf2bp3 KO murine model. Surprisingly, Igf2bp3 KO mice maintain normal, steady-state hematopoiesis. However, in striking contrast, deletion of Igf2bp3 in the MLL-Af4 leukemia model, significantly increases the survival of MLL-Af4 transplanted mice and greatly attenuates the disease. Furthermore, Igf2bp3 deficiency significantly reduced the tumor burden and disease severity. We observed significant decreases in WBC counts, spleen weights, and infiltrating leukemic cells visualized in histopathological analysis of hematopoietic tissues and quantified by FACS analysis. Moreover, deletion of Igf2bp3 led to a leukemia-initiating cell (LIC) disadvantage in vivo, demonstrated by significantly reduced engraftment in primary transplanted mice and reconstitution of secondary serially transplanted mice. To identify the transcripts directly regulated by Igf2bp3 in the context of MLL-Af4 driven leukemia, we carried out enhanced crosslinking and immunoprecipitation (eCLIP) transcriptome analysis of MLL-Af4 transformed early stem and progenitor cells and primary cells purified from splenic tumors of MLL-Af4 leukemic mice. We discovered an IGF2BP3-regulated post-transcriptional operon governing leukemic cell survival and proliferation, in which mRNA targets include the Hoxa locus and numerous genes within the Ras signaling pathway. In our study, we provide evidence that Igf2bp3 is required for the initiation of MLL-Af4 driven leukemia. We determined that Igf2bp3 is necessary for the development of and function of MLL-Af4 LICs. Mechanistically, we show that Igf2bp3 binds to and modulates the expression of hundreds of critical target transcripts. In summary, we demonstrate that Igf2bp3 is a positive regulator of MLLr leukemogenesis by targeting Hoxa transcripts such as Hoxa9 and numerous Ras signaling pathway transcripts, thereby controlling multiple downstream effector pathways required for disease initiation and aggressiveness. Together, our findings identify IGF2BP3 as an important, potential therapeutic target in this disease. Disclosures No relevant conflicts of interest to declare.