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SLC9A9 is a sodium hydrogen exchanger present in the recycling endosome and highly expressed in the brain. It is implicated in neuropsychiatric disorders, including autism spectrum disorders (ASDs). Little research concerning its gene expression patterns and biological pathways has been conducted. We sought to investigate its possible biological roles in autism-associated brain regions throughout development. We conducted a weighted gene co-expression network analysis on RNA-seq data downloaded from Brainspan. We compared prenatal and postnatal gene expression networks for three ASD-associated brain regions known to have high SLC9A9 gene expression. We also performed an ASD-associated single nucleotide polymorphism enrichment analysis and a cell signature enrichment analysis. The modules showed differences in gene constituents (membership), gene number, and connectivity throughout time. SLC9A9 was highly associated with immune system functions, metabolism, apoptosis, endocytosis, and signaling cascades. Gene list comparison with co-immunoprecipitation data was significant for multiple modules. We found a disproportionately high autism risk signal among genes constituting the prenatal hippocampal module. The modules were enriched with astrocyte and oligodendrocyte markers. SLC9A9 is potentially involved in the pathophysiology of ASDs. Our investigation confirmed proposed functions for SLC9A9, such as endocytosis and immune regulation, while also revealing potential roles in mTOR signaling and cell survival.. By providing a concise molecular map and interactions, evidence of cell type and implicated brain regions we hope this will guide future research on SLC9A9. Autism Res 2017, 10: 414-429. © 2016 International Society for Autism Research, Wiley Periodicals, Inc., (© 2016 International Society for Autism Research, Wiley Periodicals, Inc.)

Despite major advances in understanding the genetics and epigenetics of acute myelogenous leukemia, there is still a great need to develop more specific and effective therapies. High throughput approaches involving either genetic approaches or small molecule inhibitor screens are beginning to identify promising new therapeutic targets.

The application of microarray technology in schizophrenia research was heralded as paradigm-shifting, as it allowed for high-throughput assessment of cell and tissue function. This technology was widely adopted, initially in studies of postmortem brain tissue, and later in studies of peripheral blood. The collective body of schizophrenia microarray literature contains apparent inconsistencies between studies, with failures to replicate top hits, in part due to small sample sizes, cohort-specific effects, differences in array types, and other confounders. In an attempt to summarize existing studies of schizophrenia cases and non-related comparison subjects, we performed two mega-analyses of a combined set of microarray data from postmortem prefrontal cortices (n=315) and from ex-vivo blood tissues (n=578). We adjusted regression models per gene to remove non-significant covariates, providing best-estimates of transcripts dysregulated in schizophrenia. We also examined dysregulation of functionally related gene sets and gene co-expression modules, and assessed enrichment of cell types and genetic risk factors. The identities of the most significantly dysregulated genes were largely distinct for each tissue, but the findings indicated common emergent biological functions (e.g. immunity) and regulatory factors (e.g., predicted targets of transcription factors and miRNA species across tissues). Our network-based analyses converged upon similar patterns of heightened innate immune gene expression in both brain and blood in schizophrenia. We also constructed generalizable machine-learning classifiers using the blood-based microarray data. Our study provides an informative atlas for future pathophysiologic and biomarker studies of schizophrenia., Competing Interests: The authors have no conflicts of interest to declare., (Published by Elsevier B.V.)

The rs1076560 polymorphism of DRD2 (encoding dopamine receptor D2) is associated with alternative splicing and cognitive functioning; however, a mechanistic relationship to schizophrenia has not been shown. Here, we demonstrate that rs1076560(T) imparts a small but reliable risk for schizophrenia in a sample of 616 affected families and five independent replication samples totaling 4017 affected and 4704 unaffected individuals (odds ratio=1.1; P=0.004). rs1076560(T) was associated with impaired verbal fluency and comprehension in schizophrenia but improved performance among healthy comparison subjects. rs1076560(T) also associated with lower D2 short isoform expression in postmortem brain. rs1076560(T) disrupted a binding site for the splicing factor ZRANB2, diminished binding affinity between DRD2 pre-mRNA and ZRANB2 and abolished the ability of ZRANB2 to modulate short:long isoform-expression ratios of DRD2 minigenes in cell culture. Collectively, this work implicates rs1076560(T) as one possible risk factor for schizophrenia in the Han Chinese population, and suggests molecular mechanisms by which it may exert such influence.

Purpose of Review: HOXA9 is a homeodomain transcription factor that plays an essential role in normal hematopoiesis and acute leukemia, in which its overexpression is strongly correlated with poor prognosis. The present review highlights recent advances in the understanding of genetic alterations leading to deregulation of HOXA9 and the downstream mechanisms of HOXA9-mediated transformation., Recent Findings: A variety of genetic alterations including MLL translocations, NUP98-fusions, NPM1 mutations, CDX deregulation, and MOZ-fusions lead to high-level HOXA9 expression in acute leukemias. The mechanisms resulting in HOXA9 overexpression are beginning to be defined and represent attractive therapeutic targets. Small molecules targeting MLL-fusion protein complex members, such as DOT1L and menin, have shown promising results in animal models, and a DOT1L inhibitor is currently being tested in clinical trials. Essential HOXA9 cofactors and collaborators are also being identified, including transcription factors PU.1 and C/EBPα, which are required for HOXA9-driven leukemia. HOXA9 targets including IGF1, CDX4, INK4A/INK4B/ARF, mir-21, and mir-196b and many others provide another avenue for potential drug development., Summary: HOXA9 deregulation underlies a large subset of aggressive acute leukemias. Understanding the mechanisms regulating the expression and activity of HOXA9, along with its critical downstream targets, shows promise for the development of more selective and effective leukemia therapies.

PRDM16 is a transcription co-factor that plays critical roles in development of brown adipose tissue, as well as maintenance of adult hematopoietic and neural stem cells. Here we report that PRDM16 is a histone H3K4 methyltransferase on chromatin. Mutation in the N-terminal PR domain of PRDM16 abolishes the intrinsic enzymatic activity of PRDM16. We show that the methyltransferase activity of PRDM16 is required for specific suppression of MLL fusion protein-induced leukemogenesis both in vitro and in vivo. Mechanistic studies show that PRDM16 directly activates the SNAG family transcription factor Gfi1b, which in turn downregulates the HOXA gene cluster. Knockdown Gfi1b represses PRDM16-mediated tumor suppression, while Gfi1b overexpression mimics PRDM16 overexpression. In further support of the tumor suppressor function of PRDM16, silencing PRDM16 by DNA methylation is concomitant with MLL-AF9-induced leukemic transformation. Taken together, our study reveals a previously uncharacterized function of PRDM16 that depends on its PR domain activity., (Copyright © 2016 Elsevier Inc. All rights reserved.)

HOXA9 is a homeodomain-containing transcription factor that has an important role in hematopoietic stem cell expansion and is commonly deregulated in acute leukemias. A variety of upstream genetic alterations in acute myeloid leukemia lead to overexpression of HOXA9, which is a strong predictor of poor prognosis. In many cases, HOXA9 has been shown to be necessary for maintaining leukemic transformation; however, the molecular mechanisms through which it promotes leukemogenesis remain elusive. Recent work has established that HOXA9 regulates downstream gene expression through binding at promoter distal enhancers along with a subset of cell-specific cofactor and collaborator proteins. Increasing efforts are being made to identify both the critical cofactors and target genes required for maintaining transformation in HOXA9-overexpressing leukemias. With continued advances in understanding HOXA9-mediated transformation, there is a wealth of opportunity for developing novel therapeutics that would be applicable for greater than 50% of AML with overexpression of HOXA9.

Schizophrenia's (SZ's) heritability and familial transmission have been known for several decades; however, despite the clear evidence for a genetic component, it has been very difficult to pinpoint specific causative genes. Even so genetic studies have taught us a lot, even in the pregenomic era, about the molecular underpinnings and disease-relevant pathways. Recurring themes emerged revealing the involvement of neurodevelopmental processes, glutamate regulation, and immune system differential activation in SZ etiology. The recent emergence of epigenetic studies aimed at shedding light on the biological mechanisms underlying SZ has provided another layer of information in the investigation of gene and environment interactions. However, this epigenetic insight also brings forth another layer of complexity to the (epi)genomic landscape such as interactions between genetic variants, epigenetic marks-including cross-talk between DNA methylation and histone modification processes-, gene expression regulation, and environmental influences. In this review, we seek to synthesize perspectives, including limitations and obstacles yet to overcome, from genetic and epigenetic literature on SZ through a qualitative review of risk factors and prevailing hypotheses. Encouraged by the findings of both genetic and epigenetic studies to date, as well as the continued development of new technologies to collect and interpret large-scale studies, we are left with a positive outlook for the future of elucidating the molecular genetic mechanisms underlying SZ and other complex neuropsychiatric disorders., (Copyright © 2016 Elsevier Inc. All rights reserved.)

In 2009, the U.S. National Institute of Mental Health (NIMH) proposed an approach toward the deconstruction of psychiatric nosology under the research domain criteria (RDoC) framework. The overarching goal of RDoC is to identify robust, objective measures of behavior, emotion, cognition, and other domains that are more closely related to neurobiology than are diagnoses. A preliminary framework has been constructed, which has connected molecules, genes, brain circuits, behaviors, and other elements to dimensional psychiatric constructs. Although the RDoC framework has salience in emerging studies, foundational literature that pre-dated this framework requires synthesis and translation to the evolving objectives and nomenclature of RDoC. Toward this end, we review the candidate-gene association, linkage, and genome-wide studies that have implicated a variety of loci and genetic polymorphisms in selected Positive Valence Systems (PVS) constructs. Our goal is to review supporting evidence to currently listed genes implicated in this domain and novel candidates. We systematically searched and reviewed literature based on keywords listed under the June, 2011, edition of the PVS matrix on the RDoC website (http://www.nimh.nih.gov/research-priorities/rdoc/positive-valence-systems-workshop-proceedings.shtml), which were supplemented with de novo keywords pertinent to the scope of our review. Several candidate genes linked to the PVS framework were identified from candidate-gene association studies. We also identified novel candidates with loose association to PVS traits from genome-wide studies. There is strong evidence suggesting that PVS constructs, as currently conceptualized under the RDoC initiative, index genetically influenced traits; however, future research, including genetic epidemiological, and psychometric analyses, must be performed., (© 2015 Wiley Periodicals, Inc.)

Chromosomal translocations affecting mixed lineage leukemia gene (MLL) result in acute leukemias resistant to therapy. The leukemogenic activity of MLL fusion proteins is dependent on their interaction with menin, providing basis for therapeutic intervention. Here we report the development of highly potent and orally bioavailable small-molecule inhibitors of the menin-MLL interaction, MI-463 and MI-503, and show their profound effects in MLL leukemia cells and substantial survival benefit in mouse models of MLL leukemia. Finally, we demonstrate the efficacy of these compounds in primary samples derived from MLL leukemia patients. Overall, we demonstrate that pharmacologic inhibition of the menin-MLL interaction represents an effective treatment for MLL leukemias in vivo and provide advanced molecular scaffold for clinical lead identification., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Advances in molecular genetics, fueled by the results of large-scale genome-wide association studies, meta-analyses, and mega-analyses, have provided the means of identifying genetic risk factors for human disease, thereby enriching our understanding of the functionality of the genome in the post-genomic era. In the past half-decade, research on neuropsychiatric disorders has reached an important milestone: the identification of susceptibility genes reliably associated with complex psychiatric disorders at genome-wide levels of significance. This age of discovery provides the groundwork for follow-up studies designed to elucidate the mechanism(s) by which genetic variants confer susceptibility to these disorders. The gene encoding zinc-finger protein 804 A (ZNF804A) is among these candidate genes, recently being found to be strongly associated with schizophrenia and bipolar disorder via one of its non-coding mutations, rs1344706. Neurobiological, molecular, and bioinformatic analyses have improved our understanding of ZNF804A in general and this variant in particular; however, more work is needed to establish the mechanism(s) by which ZNF804A variants impinge on the biological substrates of the two disorders. Here, we review literature recently published on ZNF804A, and analyze critical concepts related to the biology of ZNF804A and the role of rs1344706 in schizophrenia and bipolar disorder. We synthesize the results of new bioinformatic analyses of ZNF804A with key elements of the existing literature and knowledge base. Furthermore, we suggest some potentially fruitful short- and long-term research goals in the assessment of ZNF804A., (© 2014 Wiley Periodicals, Inc.)

Lens epithelium-derived growth factor (LEDGF) is a chromatin-associated protein implicated in leukemia and HIV type 1 infection. LEDGF associates with mixed-lineage leukemia (MLL) fusion proteins and menin and is required for leukemic transformation. To better understand the molecular mechanism underlying the LEDGF integrase-binding domain (IBD) interaction with MLL fusion proteins in leukemia, we determined the solution structure of the MLL-IBD complex. We found a novel MLL motif, integrase domain binding motif 2 (IBM2), which binds to a well-defined site on IBD. Point mutations within IBM2 abolished leukemogenic transformation by MLL-AF9, validating that this newly identified motif is essential for the oncogenic activity of MLL fusion proteins. Interestingly, the IBM2 binding site on IBD overlaps with the binding site for the HIV integrase (IN), and IN was capable of efficiently sequestering IBD from the menin-MLL complex. A short IBM2 peptide binds to IBD directly and inhibits both the IBD-MLL/menin and IBD-IN interactions. Our findings show that the same site on IBD is involved in binding to MLL and HIV-IN, revealing an attractive approach to simultaneously target LEDGF in leukemia and HIV., (© 2014 by The American Society of Hematology.)

Homeobox A9 (HOXA9) is a homeodomain-containing transcription factor that plays a key role in hematopoietic stem cell expansion and is commonly deregulated in human acute leukemias. A variety of upstream genetic alterations in acute myeloid leukemia (AML) lead to overexpression of HOXA9, almost always in association with overexpression of its cofactor meis homeobox 1 (MEIS1) . A wide range of data suggests that HOXA9 and MEIS1 play a synergistic causative role in AML, although the molecular mechanisms leading to transformation by HOXA9 and MEIS1 remain elusive. In this study, we identify CCAAT/enhancer binding protein alpha (C/EBPα) as a critical collaborator required for Hoxa9/Meis1-mediated leukemogenesis. We show that C/EBPα is required for the proliferation of Hoxa9/Meis1-transformed cells in culture and that loss of C/EBPα greatly improves survival in both primary and secondary murine models of Hoxa9/Meis1-induced leukemia. Over 50% of Hoxa9 genome-wide binding sites are cobound by C/EBPα, which coregulates a number of downstream target genes involved in the regulation of cell proliferation and differentiation. Finally, we show that Hoxa9 represses the locus of the cyclin-dependent kinase inhibitors Cdkn2a/b in concert with C/EBPα to overcome a block in G1 cell cycle progression. Together, our results suggest a previously unidentified role for C/EBPα in maintaining the proliferation required for Hoxa9/Meis1-mediated leukemogenesis.

Here we report a comprehensive characterization of our recently developed inhibitor MM-401 that targets the MLL1 H3K4 methyltransferase activity. MM-401 is able to specifically inhibit MLL1 activity by blocking MLL1-WDR5 interaction and thus the complex assembly. This targeting strategy does not affect other mixed-lineage leukemia (MLL) family histone methyltransferases (HMTs), revealing a unique regulatory feature for the MLL1 complex. Using MM-401 and its enantiomer control MM-NC-401, we show that inhibiting MLL1 methyltransferase activity specifically blocks proliferation of MLL cells by inducing cell-cycle arrest, apoptosis, and myeloid differentiation without general toxicity to normal bone marrow cells or non-MLL cells. More importantly, transcriptome analyses show that MM-401 induces changes in gene expression similar to those of MLL1 deletion, supporting a predominant role of MLL1 activity in regulating MLL1-dependent leukemia transcription program. We envision broad applications for MM-401 in basic and translational research., (Copyright © 2014 Elsevier Inc. All rights reserved.)

The gene that encodes zinc finger protein 804A (ZNF804A) became a candidate risk gene for schizophrenia (SZ) after surpassing genome-wide significance thresholds in replicated genome-wide association scans and meta-analyses. Much remains unknown about this reported gene expression regulator; however, preliminary work has yielded insights into functional and biological effects of ZNF804A by targeting its regulatory activities in vitro and by characterizing allele-specific interactions with its risk-conferring single nucleotide polymorphisms (SNPs). There is now strong epidemiologic evidence for a role of ZNF804A polymorphisms in both SZ and bipolar disorder (BD); however, functional links between implicated variants and susceptible biological states have not been solidified. Here we briefly review the genetic evidence implicating ZNF804A polymorphisms as genetic risk factors for both SZ and BD, and discuss the potential functional consequences of these variants on the regulation of ZNF804A and its downstream targets. Empirical work and predictive bioinformatic analyses of the alternate alleles of the two most strongly implicated ZNF804A polymorphisms suggest they might alter the affinity of the gene sequence for DNA- and/or RNA-binding proteins, which might in turn alter expression levels of the gene or particular ZNF804A isoforms. Future work should focus on clarifying the critical periods and cofactors regulating these genetic influences on ZNF804A expression, as well as the downstream biological consequences of an imbalance in the expression of ZNF804A and its various mRNA isoforms., (© 2013 Wiley Periodicals, Inc.)

The MLL fusion proteins, AF9 and ENL, activate target genes in part via recruitment of the histone methyltransferase DOT1L (disruptor of telomeric silencing 1-like). Here we report biochemical, biophysical, and functional characterization of the interaction between DOT1L and MLL fusion proteins, AF9/ENL. The AF9/ENL-binding site in human DOT1L was mapped, and the interaction site was identified to a 10-amino acid region (DOT1L865-874). This region is highly conserved in DOT1L from a variety of species. Alanine scanning mutagenesis analysis shows that four conserved hydrophobic residues from the identified binding motif are essential for the interactions with AF9/ENL. Binding studies demonstrate that the entire intact C-terminal domain of AF9/ENL is required for optimal interaction with DOT1L. Functional studies show that the mapped AF9/ENL interacting site is essential for immortalization by MLL-AF9, indicating that DOT1L interaction with MLL-AF9 and its recruitment are required for transformation by MLL-AF9. These results strongly suggest that disruption of interaction between DOT1L and AF9/ENL is a promising therapeutic strategy with potentially fewer adverse effects than enzymatic inhibition of DOT1L for MLL fusion protein-associated leukemia.

MLL rearrangements are common in leukemia and considered an adverse risk factor. Through interactions with the polymerase-associated factor complex (PAFc), mixed lineage leukemia (MLL) fusion proteins activate genes critical for blocking differentiation, such as HOXA9. Here we investigate whether the MLL-PAFc interaction can be exploited therapeutically using both genetic and biochemical approaches. We tested the genetic requirement of the PAFc in acute myeloid leukemia (AML) using a conditional allele of the PAFc subunit, Cdc73. We show that the PAFc is indiscriminately necessary for the proliferation of AML cells through the epigenetic regulation of proleukemogenic target genes, such as MEIS1 and Bcl2. To investigate the therapeutic potential of targeting the MLL-PAFc interaction, we engineered a dominant negative fragment of MLL capable of binding to the PAFc. Disruption of the MLL-PAFc interaction selectively inhibits the proliferation of MLL leukemic cells without affecting cells transformed by an unrelated E2A-HLF fusion protein. Using in vivo hematopoietic reconstitution assays, we demonstrate that disruption of the MLL-PAFc does not alter normal hematopoietic stem cell function. Together, our data show a selective growth inhibition of MLL-associated leukemic cells and tolerance of normal hematopoiesis to disruption of the MLL-PAFc interaction establishing the MLL-PAFc interaction as an attractive therapeutic target.

Stimulation of transcriptional elongation is a key activity of leukemogenic MLL fusion proteins. Here, we provide evidence that MLL-ENL also inhibits Polycomb-mediated silencing as a prerequisite for efficient transformation. Biochemical studies identified ENL as a scaffold that contacted the elongation machinery as well as the Polycomb repressive complex 1 (PRC1) component CBX8. These interactions were mutually exclusive in vitro, corresponding to an antagonistic behavior of MLL-ENL and CBX8 in vivo. CBX8 inhibited elongation in a specific reporter assay, and this effect was neutralized by direct association with ENL. Correspondingly, CBX8-binding-defective MLL-ENL could not fully activate gene loci necessary for transformation. Finally, we demonstrate dimerization of MLL-ENL as a neomorphic activity that may augment Polycomb inhibition and transformation., (Copyright © 2013 The Authors. Published by Elsevier Inc. All rights reserved.)

The mixed lineage leukemia protein MLL1 contains four highly conserved plant homeodomain (PHD) fingers, which are invariably deleted in oncogenic MLL1 fusion proteins in human leukemia. Here we show that the second PHD finger (PHD2) of MLL1 is an E3 ubiquitin ligase in the presence of the E2-conjugating enzyme CDC34. This activity is conserved in the second PHD finger of MLL4, the closest homolog to MLL1 but not in MLL2 or MLL3. Mutation of PHD2 leads to MLL1 stabilization, as well as increased transactivation ability and MLL1 recruitment to the target gene loci, suggesting that PHD2 negatively regulates MLL1 activity.

It is difficult to imagine a field that is changing as rapidly as pathology. A convergence of factors including not only scientific and technological advances but also changes in business models is transforming the field, particularly in the area of cancer diagnostics. The authors examine 8 themes, or "forces of change," in pathology and speculate on how these will affect pathology sign-out and the future role of pathologists in patient care., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Mixed lineage leukemia (MLL) is a key epigenetic regulator of normal hematopoietic development and chromosomal translocations involving MLL are one of the most common genetic alterations in human leukemia. Here we show that ASB2, a component of the ECS(ASB) E3 ubiquitin ligase complex, mediates MLL degradation through interaction with the PHD/Bromodomain region of MLL. Forced expression of ASB2 degrades MLL and reduces MLL transactivation activity. In contrast, the MLL-AF9 fusion protein does not interact with ASB2 and is resistant to ASB2 mediated degradation. Increased expression of ASB2 during hematopoietic differentiation is associated with decreased levels of MLL protein and down-regulation of MLL target genes. Knockdown of ASB2 leads to increased expression of HOXA9 and delayed cell differentiation. Our data support a model whereby ASB2 contributes to hematopoietic differentiation, in part, through MLL degradation and HOX gene down-regulation. Moreover, deletion of the PHD/Bromo region renders MLL fusion proteins resistant to ASB2-mediated degradation and may contribute to leukemogenesis.

Translocations involving the mixed lineage leukemia (MLL) gene result in human acute leukemias with very poor prognosis. The leukemogenic activity of MLL fusion proteins is critically dependent on their direct interaction with menin, a product of the multiple endocrine neoplasia (MEN1) gene. Here we present what are to our knowledge the first small-molecule inhibitors of the menin-MLL fusion protein interaction that specifically bind menin with nanomolar affinities. These compounds effectively reverse MLL fusion protein-mediated leukemic transformation by downregulating the expression of target genes required for MLL fusion protein oncogenic activity. They also selectively block proliferation and induce both apoptosis and differentiation of leukemia cells harboring MLL translocations. Identification of these compounds provides a new tool for better understanding MLL-mediated leukemogenesis and represents a new approach for studying the role of menin as an oncogenic cofactor of MLL fusion proteins. Our findings also highlight a new therapeutic strategy for aggressive leukemias with MLL rearrangements.

The clustered homeobox proteins play crucial roles in development, hematopoiesis, and leukemia, yet the targets they regulate and their mechanisms of action are poorly understood. Here, we identified the binding sites for Hoxa9 and the Hox cofactor Meis1 on a genome-wide level and profiled their associated epigenetic modifications and transcriptional targets. Hoxa9 and the Hox cofactor Meis1 cobind at hundreds of highly evolutionarily conserved sites, most of which are distant from transcription start sites. These sites show high levels of histone H3K4 monomethylation and CBP/P300 binding characteristic of enhancers. Furthermore, a subset of these sites shows enhancer activity in transient transfection assays. Many Hoxa9 and Meis1 binding sites are also bound by PU.1 and other lineage-restricted transcription factors previously implicated in establishment of myeloid enhancers. Conditional Hoxa9 activation is associated with CBP/P300 recruitment, histone acetylation, and transcriptional activation of a network of proto-oncogenes, including Erg, Flt3, Lmo2, Myb, and Sox4. Collectively, this work suggests that Hoxa9 regulates transcription by interacting with enhancers of genes important for hematopoiesis and leukemia.

Aggressive leukemias arise in both children and adults as a result of rearrangements to the mixed-lineage leukemia gene (MLL) located on chromosome 11q23. MLL encodes a large histone methyltransferase that directly binds DNA and positively regulates gene transcription, including homeobox (HOX) genes. MLL is involved in chromosomal translocations, partial tandem duplications, and amplifications, all of which result in hematopoietic malignancies due to sustained HOX expression and stalled differentiation. MLL lesions are associated with both acute myeloid leukemia and acute lymphoid leukemia and are usually associated with a relatively poor prognosis despite improved treatment options such as allogeneic hematopoietic stem cell transplantation, which underscores the need for new treatment regimens. Recent advances have begun to reveal the molecular mechanisms that drive MLL-associated leukemias, which, in turn, have provided opportunities for therapeutic development. Here, we discuss the etiology of MLL leukemias and potential directions for future therapy.

We have developed an enhanced form of reduced representation bisulfite sequencing with extended genomic coverage, which resulted in greater capture of DNA methylation information of regions lying outside of traditional CpG islands. Applying this method to primary human bone marrow specimens from patients with Acute Myelogeneous Leukemia (AML), we demonstrated that genetically distinct AML subtypes display diametrically opposed DNA methylation patterns. As compared to normal controls, we observed widespread hypermethylation in IDH mutant AMLs, preferentially targeting promoter regions and CpG islands neighboring the transcription start sites of genes. In contrast, AMLs harboring translocations affecting the MLL gene displayed extensive loss of methylation of an almost mutually exclusive set of CpGs, which instead affected introns and distal intergenic CpG islands and shores. When analyzed in conjunction with gene expression profiles, it became apparent that these specific patterns of DNA methylation result in differing roles in gene expression regulation. However, despite this subtype-specific DNA methylation patterning, a much smaller set of CpG sites are consistently affected in both AML subtypes. Most CpG sites in this common core of aberrantly methylated CpGs were hypermethylated in both AML subtypes. Therefore, aberrant DNA methylation patterns in AML do not occur in a stereotypical manner but rather are highly specific and associated with specific driving genetic lesions., Competing Interests: GPS, IK, LZ, and JB are currently or have until recently been employed by Illumina.

Imidazolate-containing {Fe(NO)(2)}(9) molecular squares have been synthesized by oxidative CO displacement from the reduced Fe(CO)(2)(NO)(2) precursor. The structures of complex 1 [(imidazole)Fe(NO)(2)](4), (Ford, Li, et al.; Chem. Commun.2005, 477-479), 2 [(2-isopropylimidazole)Fe(NO)(2)](4), and 3 [(benzimidazole)Fe(NO)(2)](4), as determined by X-ray diffraction analysis, find precise square planes of irons with imidazolates bridging the edges and nitrosyl ligands capping the irons at the corners. The orientation of the imidazolate ligands in each of the complexes results in variations of the overall structures, and molecular recognition features in the available cavities of 1 and 3. Computational studies show multiple low energy structural isomers and confirm that the isomers found in the crystallographic structures arise from intermolecular interactions. EPR and IR spectroscopic studies and electrochemical results suggest that the tetramers remain intact in solution in the presence of weakly coordinating (THF) and noncoordinating (CH(2)Cl(2)) solvents. Mössbauer spectroscopic data for a set of reference dinitrosyl iron complexes, reduced {Fe(NO)(2)}(10) compounds A ((NHC-iPr)(2)Fe(NO)(2)), and C ((NHC-iPr)(CO)Fe(NO)(2)), and oxidized {Fe(NO)(2)}(9) compounds B ([(NHC-iPr)(2)Fe(NO)(2)][BF(4)]), and D ((NHC-iPr)(SPh)Fe(NO)(2)) (NHC-iPr = 1,3-diisopropylimidazol-2-ylidene) demonstrate distinct differences of the isomer shifts and quadrupole splittings between the oxidized and reduced forms. The reduced compounds have smaller positive isomer shifts as compared to the oxidized compounds ascribed to the greater π-backbonding to the NO ligands. Mössbauer data for the tetrameric complexes 1-3 demonstrate larger isomer shifts, most comparable to compound D; all four complexes contain cationic {Fe(NO)(2)}(9) units bound to one anionic ligand and one neutral ligand. At room temperature, the paramagnetic, S = (1)/(2) per iron, centers are not coupled., (© 2011 American Chemical Society)

Chromosomal translocations involving the mixed lineage leukemia (MLL) gene lead to the development of acute leukemias. Constitutive HOX gene activation by MLL fusion proteins is required for MLL-mediated leukemogenesis; however, the underlying mechanisms remain elusive. Here, we show that chromobox homolog 8 (CBX8), a Polycomb Group protein that interacts with MLL-AF9 and TIP60, is required for MLL-AF9-induced transcriptional activation and leukemogenesis. Conversely, both CBX8 ablation and specific disruption of the CBX8 interaction by point mutations in MLL-AF9 abrogate HOX gene upregulation and abolish MLL-AF9 leukemic transformation. Surprisingly, Cbx8-deficient mice are viable and display no apparent hematopoietic defects. Together, our findings demonstrate that CBX8 plays an essential role in MLL-AF9 transcriptional regulation and leukemogenesis., (2011 Elsevier Inc. All rights reserved.)

N-heterocyclic carbenes (NHCs) are shown to be reasonable mimics of imidazole ligands in dinitrosyl iron complexes determined through the synthesis and characterization of a series of {Fe(NO)(2)}(10) and {Fe(NO)(2)}(9) (Enemark-Feltham notation) complexes. Monocarbene complexes (NHC-iPr)(CO)Fe(NO)(2) (1) and (NHC-Me)(CO)Fe(NO)(2) (2) (NHC-iPr = 1,3-diisopropylimidazol-2-ylidene and NHC-Me = 1,3-dimethylimidazol-2-ylidene) are formed from CO/L exchange with Fe(CO)(2)(NO)(2). An additional equivalent of NHC results in the bis-carbene complexes (NHC-iPr)(2)Fe(NO)(2) (3) and (NHC-Me)(2)Fe(NO)(2) (4), which can be oxidized to form the {Fe(NO)(2)}(9) bis-carbene complexes 3(+) and 4(+). Treatment of complexes 1 and 2 with [NO]BF(4) results in the formation of uncommon trinitrosyl iron complexes, (NHC-iPr)Fe(NO)(3)(+) (5(+)) and (NHC-Me)Fe(NO)(3)(+) (6(+)), respectively. Cleavage of the Roussin's Red "ester" (μ-SPh)(2)[Fe(NO)(2)](2) with either NHC or imidazole results in the formation of (NHC-iPr)(PhS)Fe(NO)(2) (7) and (Imid-iPr)(PhS)Fe(NO)(2) (10) (Imid-iPr = 2-isopropylimidazole). The solid-state molecular structures of complexes 1, 2, 3, 4, 5(+), and 7 show that they all have pseudotetrahedral geometry. Infrared spectroscopic data suggest that NHCs are slightly better electron donors than imidazoles; electrochemical data are also consistent with what is expected for typical donor/acceptor abilities of the spectator ligands bound to the Fe(NO)(2) unit. Although the monoimidazole complex (Imid-iPr)(CO)Fe(NO)(2) (8) was observed via IR spectroscopy, attempts to isolate this complex resulted in the formation of a tetrameric {Fe(NO)(2)}(9) species, [(Imid-iPr)Fe(NO)(2)](4) (9), a molecular square analogous to the unsubstituted imidazole reported by Li and Wang et al. Preliminary NO-transfer studies demonstrate that the {Fe(NO)(2)}(9) bis-carbene complexes can serve as a source of NO to a target complex, whereas the {Fe(NO)(2)}(10) bis-carbenes are unreactive in the presence of a NO-trapping agent.

Disruptor of telomeric silencing 1-like (Dot1l) is a histone 3 lysine 79 methyltransferase. Studies of constitutive Dot1l knockout mice show that Dot1l is essential for embryonic development and prenatal hematopoiesis. DOT1L also interacts with translocation partners of Mixed Lineage Leukemia (MLL) gene, which is commonly translocated in human leukemia. However, the requirement of Dot1l in postnatal hematopoiesis and leukemogenesis of MLL translocation proteins has not been conclusively shown. With a conditional Dot1l knockout mouse model, we examined the consequences of Dot1l loss in postnatal hematopoiesis and MLL translocation leukemia. Deletion of Dot1l led to pancytopenia and failure of hematopoietic homeostasis, and Dot1l-deficient cells minimally reconstituted recipient bone marrow in competitive transplantation experiments. In addition, MLL-AF9 cells required Dot1l for oncogenic transformation, whereas cells with other leukemic oncogenes, such as Hoxa9/Meis1 and E2A-HLF, did not. These findings illustrate a crucial role of Dot1l in normal hematopoiesis and leukemogenesis of specific oncogenes.

Objective: The aim of this study was to better understand how mixed lineage leukemia (MLL) fusion proteins deregulate the expression of genes critical for leukemia., Materials and Methods: The transforming domain of one of the most common MLL fusion partners, AF9, was immunopurified after expression in myeloblastic M1 cells, and associating proteins were identified by mass spectrometric analysis. Chromatin immunoprecipitation followed by quantitative polymerase chain reaction was used to determine how binding of associating proteins compare across Hoxa9 and Meis1 in cell lines with and without MLL fusion proteins and how binding is altered during gene down-regulation and differentiation., Results: Consistent with earlier purifications of ENL and AF4 from 293 cells, the 90 amino acid C-terminal domain of AF9 associates with many other MLL translocation partners including Enl, Af4, Laf4, Af5q31, Ell, and Af10. This complex, termed elongation assisting proteins (EAPs), also contains the RNA polymerase II C-terminal domain kinase Cdk9/Cyclin T1/T2 (pTEFb) and the histone H3 lysine 79 methyltransferase Dot1L. Myeloid cells transformed by MLL fusions show higher levels and a broader distribution of EAP components at genes critical for leukemia. Inhibition of EAP components pTEFb and Dot1l show that both contribute significantly to activation of Hoxa9 and Meis1 expression. EAP is dynamically associated with the Hoxa9 and Meis1 loci in hematopoietic cells and rapidly dissociates during induction of differentiation. In the presence of MLL fusion proteins, its dissociation is prevented., Conclusions: The findings suggest that MLL fusion proteins deregulate genes critical for leukemia by excessive recruitment and impaired dissociation of EAP from target loci., (Copyright © 2011 ISEH - Society for Hematology and Stem Cells. Published by Elsevier Inc. All rights reserved.)

Recent studies identified an interaction between the Polymerase Associated Factor complex (PAFc) and Mixed Lineage Leukemia (MLL), including MLL-rearranged oncoproteins. This interaction is critical for MLL transcriptional activity and MLL-rearranged leukemogenesis. Here, we discuss the potential molecular role of the PAFc in transcriptional dysregulation of MLL target genes and the interplay between PAFc and MLL-rearranged oncoproteins in leukemogenesis.

Context: Rapid advances in understanding the molecular biology of acute myeloid leukemia are transforming the approach to diagnosis, prognostication, and treatment of these cases., Objective: To briefly review the current state of AML classification with a particular emphasis on the role of molecular studies and their impact on the management of acute myeloid leukemia and other malignancies., Data Sources: Current literature and experience of the authors., Conclusions: While morphology, immunophenotyping, cytogenetics, and clinical history continue to play an important role, an increasing number of molecular tests are now required to properly classify these cases.

MLL is involved in chromosomal rearrangements that generate fusion proteins with deregulated transcriptional activity. The mechanisms of MLL fusion protein-mediated transcriptional activation are poorly understood. Here we show MLL interacts directly with the polymerase associated factor complex (PAFc) through sequences flanking the CxxC domain. PAFc interacts with RNA polymerase II and stimulates posttranslational histone modifications. PAFc augments MLL and MLL-AF9 mediated transcriptional activation of Hoxa9. Conversely, knockdown of PAFc disrupts MLL fusion protein-mediated transcriptional activation and MLL recruitment to target loci. PAFc gene expression is downregulated during hematopoiesis and likely serves to regulate MLL function. Deletions of MLL that abolish interactions with PAFc also eliminate MLL-AF9 mediated immortalization indicating an essential function for this interaction in leukemogenesis.

The Additional sex combs like 1 (Asxl1) gene is 1 of 3 mammalian homologs of the Additional sex combs (Asx) gene of Drosophila. Asx is unusual because it is required to maintain both activation and silencing of Hox genes in flies and mice. Asxl proteins are characterized by an amino terminal homology domain, by interaction domains for nuclear receptors, and by a C-terminal plant homeodomain protein-protein interaction domain. A recent study of patients with myelodysplastic syndrome (MDS) and chronic myelomonocytic leukemia (CMML) revealed a high incidence of truncation mutations that would delete the PHD domain of ASXL1. Here, we show that Asxl1 is expressed in all hematopoietic cell fractions analyzed. Asxl1 knockout mice exhibit defects in frequency of differentiation of lymphoid and myeloid progenitors, but not in multipotent progenitors. We do not detect effects on hematopoietic stem cells, or in peripheral blood. Notably, we do not detect severe myelodysplastic phenotypes or leukemia in this loss-of-function model. We conclude that Asxl1 is needed for normal hematopoiesis. The mild phenotypes observed may be because other Asxl genes have redundant function with Asxl1, or alternatively, MDS or oncogenic phenotypes may result from gain-of-function Asxl mutations caused by genomic amplification, gene fusion, or truncation of Asxl1.

The Additional sex combs (Asx) gene of Drosophila behaves genetically as an enhancer of trithorax and polycomb (ETP) in displaying bidirectional homeotic phenotypes, suggesting that is required for maintenance of both activation and silencing of Hox genes. There are three murine homologs of Asx called Additional sex combs-like1, 2, and 3. Asxl1 is required for normal adult hematopoiesis; however, its embryonic function is unknown. We used a targeted mouse mutant line Asxl1(tm1Bc) to determine if Asxl1 is required to silence and activate Hox genes in mice during axial patterning. The mutant embryos exhibit simultaneous anterior and posterior transformations of the axial skeleton, consistent with a role for Asxl1 in activation and silencing of Hox genes. Transformations of the axial skeleton are enhanced in compound mutant embryos for the polycomb group gene M33/Cbx2. Hoxa4, Hoxa7, and Hoxc8 are derepressed in Asxl1(tm1Bc) mutants in the antero-posterior axis, but Hoxc8 expression is reduced in the brain of mutants, consistent with Asxl1 being required both for activation and repression of Hox genes. We discuss the genetic and molecular definition of ETPs, and suggest that the function of Asxl1 depends on its cellular context.

One of the great paradoxes in cellular differentiation is how cells with identical DNA sequences differentiate into so many different cell types. The mechanisms underlying this process involve epigenetic regulation mediated by alterations in DNA methylation, histone posttranslational modifications, and nucleosome remodeling. It is becoming increasingly clear that disruption of the "epigenome" as a result of alterations in epigenetic regulators is a fundamental mechanism in cancer. This has major implications for the future of both molecular diagnostics as well as cancer chemotherapy.

Mammalian telomeres consist of TTAGGG repeats organized in nucleosomes and associated with a six-protein complex known as shelterin, which preserves telomere structure and protects chromosome ends from the cellular DNA damage response. Recent studies have found that telomeres are transcribed into telomeric UUAGGG repeat-containing RNA (TERRA) starting from subtelomeric regions. TERRA binding at telomeres appears to be involved in cis-based mechanisms of telomeric chromatin organization and maintenance. A number of histone methyltransferases (HMTs) are known to influence telomeric chromatin status; however, the regulatory mechanisms of telomere transcription are poorly understood. Here, we show that the histone 3/lysine 4 (H3/K4) HMT and the transcriptional regulator MLL associate with telomeres and contribute to their H3/K4 methylation and transcription in a telomere length-dependent manner. In human diploid fibroblasts, RNA interference-mediated MLL depletion affects telomere chromatin modification and transcription and induces the telomere damage response. Telomere uncapping through either TRF2 shelterin protein knockdown or exposure to telomere G-strand DNA oligonucleotides significantly increases the transcription of TERRA, an effect mediated by the functional cooperation between MLL and the tumor suppressor p53. In total, our findings identify a previously unrecognized role of MLL in modifying telomeric chromatin and provide evidence for the functional interaction between MLL, p53, and the shelterin complex in the regulation of telomeric transcription and stability.

The immobilization of synthetic analogues of the [FeFe]-hydrogenase, [FeFe]H(2)ase, enzyme active site on polyethyleneglycol-rich polystyrene beads is described. Using the reactivity of the amine termini of the PEG chains with carboxylates incorporated into (mu-SRS)[Fe(CO)(3)](2) or (mu-SR)(2)[Fe(CO)(3)](2) derivative, nu(CO)IR signatures can be used to interrogate the structure and properties of the diiron carbonyl complexes once incorporated into the PEG environment of the polymer beads. Alternatively, the SRS dithiolate was first attached to the resin and the diiron unit assembled via an in situ process on the bead.

In the hematopoietic system, menin was found to interact with MLL, a large protein encoded by the mixed linage leukemia gene that acts as a histone H3 methyltransferase. The MLL gene is a recurrent target for translocations in both acute myeloid and acute lymphoid leukemias. MLL gene rearrangements involve a variety of translocation partners, giving rise to MLL fusion proteins whose transforming ability is mediated through upregulated expression of Homeobox (Hox) genes as well as other targets. Recent work indicates that menin is an essential partner of MLL fusion proteins in leukemic cells and that it regulates normal hematopoiesis. In the absence of menin, steady-state hematopoiesis is largely preserved; however, menin-deficient hematopoietic stem cells are markedly deficient in situations of hematopoietic stress, such as during recovery after bone marrow transplantation. In leukemias driven by MLL fusion proteins, menin is essential for transformation and growth of the malignant cells. Thus, menin-MLL interactions represent a promising therapeutic target in leukemias with MLL rearrangements.

Disruption of epigenetic regulators of transcription is a central mechanism of oncogenesis. Many of the advances in the understanding of these mechanisms are attributable to the successful development of chromatin immunoprecipitation (ChIP) for in vivo detection of histone modifications as well as chromatin binding regulatory proteins. This is a powerful technique for analyzing histone modifications as well as binding sites for proteins that bind either directly or indirectly to DNA. Here we present two ChIP protocols. The first is particularly useful for identifying histone modifications or binding at specific, known genomic sites. The second, employing serial analysis of gene expression, is particularly powerful for the discovery of previously unidentified sites of modification or binding.

Chromosomal translocations involving the mixed lineage leukemia (MLL) gene are associated with aggressive acute lymphoid and myeloid leukemias. These translocations are restricted to an 8.3-kb breakpoint region resulting in fusion of amino terminal MLL sequences in frame to 1 of more than 60 different translocation partners. The translocations consistently delete the plant homeodomain (PHD) fingers and more carboxyl terminal MLL sequences. The function of the PHD fingers is obscure and their specific role in transformation has not been explored. Here we show that inclusion of the PHD fingers in the MLL fusion protein MLL-AF9 blocked immortalization of hematopoietic progenitors. Inclusion of 2 or more PHD fingers reduced association with the Hoxa9 locus and suppressed Hoxa9 up-regulation in hematopoietic progenitors. These data provide an explanation for why MLL translocation breakpoints exclude the PHD fingers and suggest a possible role for these domains in regulating the function of wild-type MLL.

MLL rearrangements in humans lead to both acute myeloid leukemia (AML) and acute lymphoid leukemia (ALL). While AML has been successfully produced in mice, modeling ALL has been more difficult. In this issue of Cancer Cell, Wei et al. (2008) describe generation of AML, ALL, and biphenotypic leukemia by manipulating the cytokine milieu of human progenitor cells expressing MLL-AF9. They demonstrate that both multipotent and lineage-restricted progenitors are targeted by MLL-AF9 fusion proteins and that Rac signaling is crucial for survival. This study demonstrates the heterogeneity of MLL-AF9 leukemic stem cells and the importance of the microenvironment in determining lineage outcome.

HOX and three amino acid loop extension (TALE) proteins cooperate to induce transformation in mouse leukemia models, and are dysregulated in a variety of human leukemias. Despite decades of research, the mechanism of action for Hox proteins in embryogenesis and hematopoiesis remains unclear. Recent studies on the roles of Hoxa9 and Meis1 in leukemia has led to a wealth of new data, but their molecular mechanisms of action and synergy remain obscure. Advances in genome-wide technologies offer new avenues for understanding how homeodomain-containing transcription factors exert their programs in normal and neoplastic development.

Heterobimetallic complexes comprised of W(CO)4 adducts of (N2S2)M(NO) have been isolated and characterized by nu(CO) and nu(NO)IR spectroscopies and X-ray diffraction. The molecular structures of (N2S2)M(NO) compounds (bme-dach)Co(NO), [(bme-dach)Co(NO)]W(CO)4, and [(bme-dach)Fe(NO)]W(CO)4 [bme-dach = N, N'-bis(2-mercaptoethyl)-1,4-diazacycloheptane)] find the square-pyramidal (bme-dach)M(NO) unit to serve as a bidentate ligand via the cis-dithiolato sulfurs, with a hinge angle of the butterfly bimetallic structures of ca. 130 degrees . The W(CO)4 moiety is used as a probe of the electron-donor ability of the nitrosyl complexes through CO stretching frequencies that display a minor increase as compared to analogous [(N2S2)Ni]W(CO)4 complexes. These findings are consistent with the electron-withdrawing influence of the {Co(NO)}(8) and {Fe(NO)}(7) units on the bridging thiolate sulfurs relative to Ni(2+). Also sensitive to derivatization by W(CO)4 is the NO stretch, which blue shifts by ca. 30 and 50 cm(-1) for the Co and Fe complexes, respectively. Cyclic voltammetry studies find similar reduction potentials (-1.08 V vs NHE in N, N-dimethylformamide solvent) of the (bme-dach)Co(NO) and (bme-dach)Fe(NO) free metalloligands, which are positively shifted by ca. 0.61 and 0.48 V, respectively, upon complexation to W(CO)4.

Fusion of the mixed lineage leukemia protein (MLL) to one of over 50 different translocation partners converts it into a potent leukemogenic oncoprotein. The resulting fusion proteins transform primarily through upregulation of A-cluster Hox genes, including Hoxa9 and the Hox cofactor Meis1. Considerable progress has been made in delineating the differences between normal Hox gene regulation by MLL and deregulated transcription in MLL-induced leukemias. Some MLL translocation partners dimerize the truncated MLL molecule. Other translocation partners appear to recruit nuclear coactivator complexes that have diverse enzymatic activities that impinge on transcriptional elongation pathways. These enzymatic activities, including RNA polymerase II phosphorylation as well as histone H3 lysine 79 methylation present attractive targets for the development of future MLL-directed therapy.

Chimeric proteins joining the histone methyltransferase MLL with various fusion partners trigger distinctive lymphoid and myeloid leukemias. Here, we immunopurified proteins associated with ENL, a protein commonly fused to MLL. Identification of these ENL-associated proteins (EAPs) by mass spectrometry revealed enzymes with a known role in transcriptional elongation (RNA polymerase II C-terminal domain kinase [RNAPolII CTD] positive transcription elongation factor b [pTEFb]), and in chromatin modification (histone-H3 methyltransferase DOT1L) as well as other frequent MLL partners (AF4, AF5q31, and LAF4), and polycomb group members (RING1, CBX8, and BCoR). The composition of EAP was further verified by coimmunoprecipitation, 2-hybrid analysis, pull-down, and colocalization experiments. Purified EAP showed a histone H3 lysine 79-specific methylase activity, displayed a robust RNAPolII CTD kinase function, and counteracted the effect of the pTEFb inhibitor 5,6-dichloro-benzimidazole-riboside. In vivo, an ENL knock-down diminished genome-wide as well as gene-specific H3K79 dimethylation, reduced global run-on elongation, and inhibited transient transcriptional reporter activity. According to structure-function data, DOT1L recruitment was important for transformation by the MLL-ENL fusion derivative. These results suggest a function of ENL in histone modification and transcriptional elongation.

The immunoglobulin degrading enzyme of Streptococcus pyogenes, IdeS, is an unusual cysteine protease produced by group A streptococci for which the only known substrate is immunoglobulin G (IgG). To date, IdeS has not been found to cleave any of the known synthetic substrates that other cysteine proteases hydrolyse, thus making the development of an IdeS detection assay difficult. Furthermore, at high doses of substrate, product generation is inhibited potentially due to the need for a dimeric enzyme complex with IgG. In this study we have developed a mass spectral assay for IdeS activity based on the detection of an Mr approximately 25,300 Fc fragment that retains the ability to bind streptococcal protein G. Using this assay procedure, evidence for a multimeric enzyme-substrate complex was obtained as well as identifying isolated heavy chains as a non-substrate inhibitor of IdeS activity. Under appropriate experimental conditions the assay could be used to detect IdeS activity in bacterial culture media or in human plasma without a requirement for purified reactants. The availability of a rapid and sensitive assay for IdeS should facilitate the detailed biochemical characterization of this unusual bacterial cysteine protease.