Chromosomal translocations of the mixed-lineage leukemia (MLL) gene with various partner genes result in aggressive leukemia with dismal outcomes. Despite similar expression at the mRNA level from ...the wild-type and chimeric MLL alleles, the chimeric protein is more stable. We report that UBE2O functions in regulating the stability of wild-type MLL in response to interleukin-1 signaling. Targeting wild-type MLL degradation impedes MLL leukemia cell proliferation, and it downregulates a specific group of target genes of the MLL chimeras and their oncogenic cofactor, the super elongation complex. Pharmacologically inhibiting this pathway substantially delays progression, and it improves survival of murine leukemia through stabilizing wild-type MLL protein, which displaces the MLL chimera from some of its target genes and, therefore, relieves the cellular oncogenic addiction to MLL chimeras. Stabilization of MLL provides us with a paradigm in the development of therapies for aggressive MLL leukemia and perhaps for other cancers caused by translocations.
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•UBE2O acts downstream of the interleukin-1 pathway to regulate MLL/COMPASS stability•Stabilizing wild-type MLL protein inhibits MLL leukemia cell proliferation•UBE2O and IRAK inhibition alters a common set of MLL chimera target genes•Targeting the IL-1 pathway is a potential therapeutic strategy for MLL leukemia
Stabilizing wild-type MLL proteins is a potential therapeutic approach for leukemia resulting from MLL translocations.
The spatiotemporal regulation of gene expression is central for cell-lineage specification during embryonic development and is achieved through the combinatorial action of transcription ...factors/co-factors and epigenetic states at cis-regulatory elements. Here, we show that in addition to implementing H3K4me3 at promoters of bivalent genes, Mll2 (KMT2B)/COMPASS can also implement H3K4me3 at a subset of non-TSS regulatory elements, a subset of which shares epigenetic signatures of active enhancers. Our mechanistic studies reveal that association of Mll2's CXXC domain with CpG-rich regions plays an instrumental role for chromatin targeting and subsequent implementation of H3K4me3. Although Mll2/COMPASS is required for H3K4me3 implementation on thousands of loci, generation of catalytically mutant MLL2/COMPASS demonstrated that H3K4me3 implemented by this enzyme was essential for expression of a subset of genes, including those functioning in the control of transcriptional programs during embryonic development. Our findings suggest that not all H3K4 trimethylations implemented by MLL2/COMPASS are functionally equivalent.
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•Mll2/COMPASS occupies and catalyzes H3K4me3 at non-TSS elements•The CXXC domain of Mll2/COMPASS mediates its chromatin targeting•Mll2 regulates transcription of some genes including PGC specification genes•PGC specification requires Mll2/COMPASS’s methyltransferase activity
Hu et al. analyzed the contribution of MLL2’s methyltransferase and CXXC domain in the trimethylation of H3K4 in mouse ES cells and find that while it trimethylates H3K4 at both bivalent gene promoters and non-TSS elements, it regulates transcription at a limited number of genes including those required for PGC specification.
T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive disease, affecting children and adults. Chemotherapy treatments show high response rates but have debilitating effects and carry risk of ...relapse. Previous work implicated NOTCH1 and other oncogenes. However, direct inhibition of these pathways affects healthy tissues and cancer alike. Our goal in this work has been to identify enzymes active in T-ALL whose activity could be targeted for therapeutic purposes.
To identify and characterize new NOTCH1 druggable partners in T-ALL, we coupled studies of the NOTCH1 interactome to expression analysis and a series of functional analyses in cell lines, patient samples, and xenograft models.
We demonstrate that ubiquitin-specific protease 7 (USP7) interacts with NOTCH1 and controls leukemia growth by stabilizing the levels of NOTCH1 and JMJD3 histone demethylase.
is highly expressed in T-ALL and is transcriptionally regulated by NOTCH1. In turn, USP7 controls NOTCH1 levels through deubiquitination. USP7 binds oncogenic targets and controls gene expression through stabilization of NOTCH1 and JMJD3 and ultimately H3K27me3 changes. We also show that USP7 and NOTCH1 bind T-ALL superenhancers, and inhibition of USP7 leads to a decrease of the transcriptional levels of NOTCH1 targets and significantly blocks T-ALL cell growth
and
.
These results provide a new model for USP7 deubiquitinase activity through recruitment to oncogenic chromatin loci and regulation of both oncogenic transcription factors and chromatin marks to promote leukemia. Our studies also show that targeting USP7 inhibition could be a therapeutic strategy in aggressive leukemia.
Abstract The deleterious effects of cannabis use in schizophrenia have been linked, in part, to underlying disturbances in endogenous cannabinoid signaling in the prefrontal cortex. However, while ...receptor autoradiography studies of the primary cannabinoid receptor (CB1R) have consistently found higher CB1R binding in the prefrontal cortex in schizophrenia, deficits in CB1R mRNA levels and protein immunoreactivity have also been reported in the illness. To investigate this apparent discrepancy, we quantified CB1R binding using receptor autoradiography with the selective CB1R ligand 3 H-OMAR in the prefrontal cortex of 21 subjects with schizophrenia who were previously found to have lower levels of both CB1R mRNA using in situ hybridization and CB1R protein using radioimmunocytochemistry relative to matched healthy comparison subjects. We observed higher levels of 3 H-OMAR binding in the prefrontal cortex of schizophrenia subjects that did not appear to be attributable to psychotropic medications or substance abuse. The combination of lower levels of CB1R mRNA and immunoreactivity with higher CB1R receptor binding may reflect 1) altered trafficking of the receptor resulting in higher levels of membrane-bound CB1R or 2) higher CB1R affinity. In either case, greater CB1R receptor availability may contribute to the increased susceptibility of schizophrenia subjects to the deleterious effects of cannabis use.
Chromosomal translocations of the Mixed-lineage leukemia 1 (
) gene generate MLL chimeras that drive the pathogenesis of acute myeloid and lymphoid leukemia. The untranslocated MLL1 is a substrate ...for proteolytic cleavage by the endopeptidase threonine aspartase 1 (taspase1); however, the biological significance of MLL1 cleavage by this endopeptidase remains unclear. Here, we demonstrate that taspase1-dependent cleavage of MLL1 results in the destabilization of MLL. Upon loss of taspase1, MLL1 association with chromatin is markedly increased due to the stabilization of its unprocessed version, and this stabilization of the uncleaved MLL1 can result in the displacement of MLL chimeras from chromatin in leukemic cells. Casein kinase II (CKII) phosphorylates MLL1 proximal to the taspase1 cleavage site, facilitating its cleavage, and pharmacological inhibition of CKII blocks taspase1-dependent MLL1 processing, increases MLL1 stability, and results in the displacement of the MLL chimeras from chromatin. Accordingly, inhibition of CKII in a MLL-AF9 mouse model of leukemia delayed leukemic progression in vivo. This study provides insights into the direct regulation of the stability of MLL1 through its cleavage by taspase1, which can be harnessed for targeted therapeutic approaches for the treatment of aggressive leukemia as the result of MLL translocations.
•The chromatin assembly machinery protects leukemic cell self-renewal by directly regulating transcription of the E3 ubiquitin ligase TRIM13.•TRIM13 is nuclear localized and represses self-renewal by ...driving cell cycle entry through the stabilization of cyclin A1.
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Acute myeloid leukemia (AML) is an aggressive blood cancer that stems from the rapid expansion of immature leukemic blasts in the bone marrow. Mutations in epigenetic factors represent the largest category of genetic drivers of AML. The chromatin assembly factor CHAF1B is a master epigenetic regulator of transcription associated with self-renewal and the undifferentiated state of AML blasts. Upregulation of CHAF1B, as observed in almost all AML samples, promotes leukemic progression by repressing the transcription of differentiation factors and tumor suppressors. However, the specific factors regulated by CHAF1B and their contributions to leukemogenesis are unstudied. We analyzed RNA sequencing data from mouse MLL-AF9 leukemic cells and bone marrow aspirates, representing a diverse collection of pediatric AML samples and identified the E3 ubiquitin ligase TRIM13 as a target of CHAF1B-mediated transcriptional repression associated with leukemogenesis. We found that CHAF1B binds the promoter of TRIM13, resulting in its transcriptional repression. In turn, TRIM13 suppresses self-renewal of leukemic cells by promoting pernicious entry into the cell cycle through its nuclear localization and catalytic ubiquitination of cell cycle–promoting protein, CCNA1. Overexpression of TRIM13 initially prompted a proliferative burst in AML cells, which was followed by exhaustion, whereas loss of total TRIM13 or deletion of its catalytic domain enhanced leukemogenesis in AML cell lines and patient-derived xenografts. These data suggest that CHAF1B promotes leukemic development, in part, by repressing TRIM13 expression and that this relationship is necessary for leukemic progression.
Acute lymphoblastic leukemia (ALL) is a highly aggressive blood cancer affecting children and adults. Certain high-risk disease subsets have poor outcomes and often debilitating therapy-related ...toxicities stemming from direct inhibition of the oncogenes. We hypothesize that the process of oncogenic transformation is driven by aberrant activity of oncogene-associated chromatin modifying partners. These changes create a chromatin environment unique to the malignant state and, therefore, disruption of critical oncogenic chromatin signatures would not likely affect healthy tissues. We have generated strong evidence for the intertwined roles between the NOTCH1 oncogenic pathway and deubiquitinase enzymes in T cell leukemia, members of the ubiquitin-specific proteases (USP) family in particular. Members of the USP family physically interact with NOTCH1 and the lysine 27 on histone H3 (H3K27) demethylase JMJD3 and this methylation-ubiquitination biology-related axis coordinates regulation of transcriptional initiation and elongation, vital for the survival of leukemia cells. Interestingly transcription of USP genes is positively controlled by NOTCH1 creating a feedback loop in leukemia.
We have further characterized this oncogenic axis using a combination of small molecule inhibitors and genetic engineering of USPs in ALL cell lines, primary patient samples and primagraft models of disease. We are able to show that a) USP activity is important for certain oncogenic pathways (such as NOTCH1) in leukemia; b) Oncogenes and USP enzymes co-bind certain areas in the leukemia genome; c) Ubiquitination of histone H2B acts in a combinatorial fashion with H3K27me, is a major epigenetic change affected by the USP activity in leukemia and controls d) transcriptional elongation. Finally we demonstrate that f) chemical inhibition or down-regulation of USPs affect leukemia growth in vitro and in vivo. Ongoing and future studies include manipulation of USP levels in mouse models of leukemia as well as combinatorial use of USP inhibitors with chemotherapeutic regiments in vitro using matched diagnosis-relapsed primary samples and in xenograft studies.
Information gained from these studies will lend rationale towards the use of small molecule inhibitors against USP proteins in clinical trials for the treatment of aggressive and relapsed ALL.
Kumar:Progenra Inc.: Employment. Wang:Progenra Inc.: Employment. Wu:Progenra Inc.: Employment.
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Chromosomal translocations of the mixed‐lineage leukemia (
MLL
) gene with various partner genes result in aggressive MLL leukemia with dismal outcomes. Despite relatively equal ...contributions of the rearranged allele and the remaining wild‐type allele at the mRNA level, the MLL chimeric protein is much more stable than the wild‐type MLL protein. Here, we used Multidimensional Protein Identification Technology (MudPIT) and established a pooled genome‐wide shRNA library screen to identify the factors and pathways involved in regulating the stability of the wild‐type MLL protein. Targeting wild‐type MLL degradation, through blocking of these pathways and factors, preferentially impedes MLL leukemia cell proliferation and downregulates a specific group of target genes of the MLL chimeras and their oncogenic cofactor, the Super Elongation Complex (SEC), as revealed by RNA sequencing and Chromatin Immunoprecipitation Sequencing (ChIP‐Seq). Stabilizing wild‐type MLL protein by inhibition of these pathways displaces the MLL chimera from some of its target genes and therefore relieves the cellular oncogenic addiction to MLL chimeras. Furthermore, pharmacologically inhibition of these pathways with small molecules substantially delays progression and improves survival of murine MLL‐AF9 leukemia in vivo. Therefore, disrupting the balance between wild‐type MLL and MLL chimeras by stabilization of MLL provides us with a paradigm in the development of therapies for aggressive MLL leukemia and perhaps for other cancers driven by chromosomal translocations.
Support or Funding Information
This study was supported by the Samuel Waxman Cancer Research Foundation to John D. Crispino and National Institute of Health grants, CA211428 to Edwin R. Smith, CA117907 to Joaquin M. Espinosa, CA101774 to John D. Crispino. and R35CA197569 to Ali Shilatifard
Studies indicate that decision making and informed consent among patients considering left ventricular assist device (LVAD) support for advanced heart failure could be improved. In the VADDA ...(Ventricular Assist Device Decision Aid) trial, we tested a patient-centered decision aid (DA) to enhance the quality of decision making about LVAD therapy.
After an extensive user-centered design process, we conducted a multisite randomized trial of the DA compared with standard education (SE) among inpatients considering LVAD treatment for advanced heart failure The main outcome was LVAD knowledge at 1 week and 1 month after administration of the DA versus the SE, according to a validated scale. Secondary measures included prespecified quality decision making measures recommended by the International Patient Decision Aid Standards collaboration.
Of 105 eligible patients, 98 consented and were randomly assigned to the DA and SE arms. Patients receiving the VADDA exhibited significantly greater LVAD knowledge than the SE group at 1 week of follow-up (P = .01) but not at 1 month (P = .47). No differences were found between DA and SE patients in rates of acceptance versus decline of LVAD treatment (85% vs 78%; P = .74). Recipients in the DA arm reported greater satisfaction with life after implantation compared with nonrecipients (28 vs 23 out of 30; P = .008), although both arms reported high satisfaction. Patients rated the DA high in acceptability and usability.
The VADDA enhances LVAD knowledge, particularly in the short term (1 week) during the peak period of decision making. The DA does not encourage decision direction and reflects patient, caregiver, and physician preferences for content and format.
https://clinicaltrials.gov/ct2/show/NCT02248974. The trial is registered with clinicaltrials.gov (NCT02248974).
Prenatal exposure to air pollutants is associated with increased risk for neurodevelopmental and neurodegenerative disorders. However, few studies have identified transcriptional changes related to ...air pollutant exposure.
RNA sequencing was used to examine transcriptomic changes in blood and cerebral cortex of three male and three female mouse neonates prenatally exposed to traffic-related nano-sized particulate matter (nPM) compared to three male and three female mouse neonates prenatally exposed to control filter air.
We identified 19 nPM-associated differentially expressed genes (nPM-DEGs) in blood and 124 nPM-DEGs in cerebral cortex. The cerebral cortex transcriptional responses to nPM suggested neuroinflammation involvement, including CREB1, BDNF, and IFNγ genes. Both blood and brain tissues showed nPM transcriptional changes related to DNA damage, oxidative stress, and immune responses. Three blood nPM-DEGs showed a canonical correlation of 0.98 with 14 nPM-DEGS in the cerebral cortex, suggesting a convergence of gene expression changes in blood and cerebral cortex. Exploratory sex-stratified analyses suggested a higher number of nPM-DEGs in female cerebral cortex than male cerebral cortex. The sex-stratified analyses identified 2 nPM-DEGs (Rgl2 and Gm37534) shared between blood and cerebral cortex in a sex-dependent manner.
Our findings suggest that prenatal nPM exposure induces transcriptional changes in the cerebral cortex, some of which are also observed in blood. Further research is needed to replicate nPM-induced transcriptional changes with additional biologically relevant time points for brain development.
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