MALT1 inhibitors are promising therapeutic agents for B-cell lymphomas that are dependent on constitutive or aberrant signaling pathways. However, a potential limitation for signal ...transduction–targeted therapies is the occurrence of feedback mechanisms that enable escape from the full impact of such drugs. Here, we used a functional genomics screen in activated B-cell–like (ABC) diffuse large B-cell lymphoma (DLBCL) cells treated with a small molecule irreversible inhibitor of MALT1 to identify genes that might confer resistance or enhance the activity of MALT1 inhibition (MALT1i). We find that loss of B-cell receptor (BCR)- and phosphatidylinositol 3-kinase (PI3K)-activating proteins enhanced sensitivity, whereas loss of negative regulators of these pathways (eg, TRAF2, TNFAIP3) promoted resistance. These findings were validated by knockdown of individual genes and a combinatorial drug screen focused on BCR and PI3K pathway–targeting drugs. Among these, the most potent combinatorial effect was observed with PI3Kδ inhibitors against ABC-DLBCLs in vitro and in vivo, but that led to an adaptive increase in phosphorylated S6 and eventual disease progression. Along these lines, MALT1i promoted increased MTORC1 activity and phosphorylation of S6K1-T389 and S6-S235/6, an effect that was only partially blocked by PI3Kδ inhibition in vitro and in vivo. In contrast, simultaneous inhibition of MALT1 and MTORC1 prevented S6 phosphorylation, yielded potent activity against DLBCL cell lines and primary patient specimens, and resulted in more profound tumor regression and significantly improved survival of ABC-DLBCLs in vivo compared with PI3K inhibitors. These findings provide a basis for maximal therapeutic impact of MALT1 inhibitors in the clinic, by disrupting feedback mechanisms that might otherwise limit their efficacy.
•Functional genomics screening shows that MALT1 sits at crossroads between multiple oncogenic signaling pathways, including PI3K and MTORC1.•Simultaneous MALT1/MTORC1 inhibition abrogates survival feedback activation of MTORC1 and triggers synergistic killing of ABC-DLBCL.
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Diffuse large B cell lymphomas (DLBCLs) arise from proliferating B cells transiting different stages of the germinal center reaction. In activated B cell DLBCLs (ABC-DLBCLs), a class of DLBCLs that ...respond poorly to current therapies, chromosomal translocations and amplification lead to constitutive expression of the B cell lymphoma 6 (BCL6) oncogene. The role of BCL6 in maintaining these lymphomas has not been investigated. Here, we designed small-molecule inhibitors that display higher affinity for BCL6 than its endogenous corepressor ligands to evaluate their therapeutic efficacy for targeting ABC-DLBCL. We used an in silico drug design functional-group mapping approach called SILCS to create a specific BCL6 inhibitor called FX1 that has 10-fold greater potency than endogenous corepressors and binds an essential region of the BCL6 lateral groove. FX1 disrupted formation of the BCL6 repression complex, reactivated BCL6 target genes, and mimicked the phenotype of mice engineered to express BCL6 with corepressor binding site mutations. Low doses of FX1 induced regression of established tumors in mice bearing DLBCL xenografts. Furthermore, FX1 suppressed ABC-DLBCL cells in vitro and in vivo, as well as primary human ABC-DLBCL specimens ex vivo. These findings indicate that ABC-DLBCL is a BCL6-dependent disease that can be targeted by rationally designed inhibitors that exceed the binding affinity of natural BCL6 ligands.
Diffuse large B cell lymphomas (DLBCLs) are genetically heterogeneous and highly proliferative neoplasms derived from germinal center (GC) B cells. Here, we show that DLBCLs are dependent on ...mitochondrial lysine deacetylase SIRT3 for proliferation, survival, self-renewal, and tumor growth in vivo regardless of disease subtype and genetics. SIRT3 knockout attenuated B cell lymphomagenesis in VavP-Bcl2 mice without affecting normal GC formation. Mechanistically, SIRT3 depletion impaired glutamine flux to the TCA cycle via glutamate dehydrogenase and reduction in acetyl-CoA pools, which in turn induce autophagy and cell death. We developed a mitochondrial-targeted class I sirtuin inhibitor, YC8-02, which phenocopied the effects of SIRT3 depletion and killed DLBCL cells. SIRT3 is thus a metabolic non-oncogene addiction and therapeutic target for DLBCLs.
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•SIRT3 is highly expressed and linked to unfavorable outcome in DLBCL•SIRT3 is required for anaplerotic metabolism in DLBCL by enhancing GDH activity•Loss of Sirt3 impairs lymphomagenesis and prolongs survival of VavP-Bcl2 mice•Selective inhibition of SIRT3 by YC8-02 kills DLBCLs in vitro and in vivo
Li et al. show that SIRT3 is required for diffuse large B cell lymphomas (DLBCLs) but not normal germinal center B cells. SIRT3 depletion induces DLBCL cell death by reducing glutamine flux to the TCA cycle and acetyl-CoA pools. They develop a sirtuin inhibitor that mimics SIRT3 depletion and kills DLBCL cells.
Despite regulating overlapping gene enhancers and pathways, CREBBP and KMT2D mutations recurrently co-occur in germinal center (GC) B cell-derived lymphomas, suggesting potential oncogenic ...cooperation. Herein, we report that combined haploinsufficiency of Crebbp and Kmt2d induces a more severe mouse lymphoma phenotype (vs either allele alone) and unexpectedly confers an immune evasive microenvironment manifesting as CD8
T-cell exhaustion and reduced infiltration. This is linked to profound repression of immune synapse genes that mediate crosstalk with T-cells, resulting in aberrant GC B cell fate decisions. From the epigenetic perspective, we observe interaction and mutually dependent binding and function of CREBBP and KMT2D on chromatin. Their combined deficiency preferentially impairs activation of immune synapse-responsive super-enhancers, pointing to a particular dependency for both co-activators at these specialized regulatory elements. Together, our data provide an example where chromatin modifier mutations cooperatively shape and induce an immune-evasive microenvironment to facilitate lymphomagenesis.
Locus control region (LCR) functions define cellular identity and have critical roles in diseases such as cancer, although the hierarchy of structural components and associated factors that drive ...functionality are incompletely understood. Here we show that OCA-B, a B cell-specific coactivator essential for germinal center (GC) formation, forms a ternary complex with the lymphoid-enriched OCT2 and GC-specific MEF2B transcription factors and that this complex occupies and activates an LCR that regulates the BCL6 proto-oncogene and is uniquely required by normal and malignant GC B cells. Mechanistically, through OCA-B-MED1 interactions, this complex is required for Mediator association with the BCL6 promoter. Densely tiled CRISPRi screening indicates that only LCR segments heavily bound by this ternary complex are essential for its function. Our results demonstrate how an intimately linked complex of lineage- and stage-specific factors converges on specific and highly essential enhancer elements to drive the function of a cell-type-defining LCR.
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•OCT2⋅OCA-B binds heavily across the BCL6 LCR•OCT2, OCA-B, and MEF2B form a ternary complex controlling BCL6 LCR activity•OCA-B directly recruits Mediator, bridging the BCL6 LCR to the BCL6 promoter in cis•Four distinct OCT2⋅OCA-B⋅MEF2B-binding enhancers are essential for LCR function
Chu and Hellmuth et al. reveal hierarchical regulation involving a ternary transcriptional complex, OCT2⋅OCA-B⋅MEF2B, composed of lineage- and stage-specific factors, as a critical component utilizing distinct essential enhancer elements in the LCR, essential for timely induction of BCL6, a master regulator for germinal center B cell differentiation.
The apical junctional complex (AJC), composed of tight and adherens junctions, maintains epithelial barrier function. Since cigarette smoking and chronic obstructive pulmonary disease (COPD), the ...major smoking-induced disease, are associated with increased lung epithelial permeability, we hypothesized that smoking alters the transcriptional program regulating airway epithelial AJC integrity. Transcriptome analysis revealed global down-regulation of physiological AJC gene expression in the airway epithelium of healthy smokers (n = 59) compared to nonsmokers (n = 53) in association with changes in canonical epithelial differentiation pathways such as PTEN signaling accompanied by induction of cancer-related AJC components. The overall expression of AJC-related genes was further decreased in COPD smokers (n = 23). Exposure of airway epithelial cells to cigarette smoke extract in vitro resulted in down-regulation of several AJC genes paralleled by decreased transepithelial resistance. Thus, cigarette smoking induces transcriptional reprogramming of airway epithelial AJC architecture from its physiological pattern necessary for barrier function toward a disease-associated molecular phenotype.
Somatic missense mutations in histone 1 genes occur in ~30% of follicular lymphomas and DLBCL and 85% of Hodgkin's lymphomas, with significant mutual co-occurrence among these alleles, most ...frequently involving H1C and H1E. We crossed constitutive H1C+/-H1E+/- mice with VavP-Bcl2 transgenic mice and observed significant acceleration of lymphomagenesis (p=0.0001). Lymphoma H1 mutations affect the H1 globular domain or C-terminus. We found that the globular domain mutants fail to insert into chromatin whereas C-ter mutants fail to compact chromatin as shown by atomic force microscopy, in vitro assembled nucleosome arrays, and FRET assays in live cells. Hence both types of mutation confer loss of function.
Constitutive H1C/E knockout mice are healthy and have no overt phenotype. However, immunization with T-cell dependent antigen caused significant GC hyperplasia (p=0.013) and disruption of polarity due to expansion in the number of centrocytes. Notably, H1C/EDKO GC B-cells readily outcompeted WT GC B-cells in mixed chimera experiments indicating that they have superior fitness (p=0.0086). To understand the mechanism through which this occurs we performed RNA-seq in H1C/EDKO GC B-cells which revealed an aberrant gene expression signature composed almost entirely of transcriptional activation (n=721 upregulated and n=61 downregulated q=0.05, LFC=log(1.5)). Strikingly, these same genes are upregulated during induced pluripotency (iPS cell) reprogramming, and are normally silenced during early development by the PRC2 complex (p <0.05 to 1e-05). Indeed histone mass spectrometry showed reduced H3K27me3 (p=0.0003) and increased H3K36me2 (p =0.001) in H1C/EDKO GC B-cells.
This prompted us to extensively characterize the epigenome of purified H1C/EDKO vs WT GC B-cells using High-throughput chromosome conformation capture (Hi-C), ATAC-seq, and ChIP-seq for multiple histone marks. From the topological standpoint the genome is distributed in two compartments: Compartment A, consisting of unpacked chromatin available for gene regulatory processes, and compartment B, which is highly packed, silenced and inaccessible. Beyond this organization, sets of genes are organized into boundary delimited domains called TADs that share regulatory information. Remarkably, the primary effect of H1 loss of function was the shifting of approximately 256 TADs from compartment B to compartment A (there was no A to B shift). These TADs manifested highly significant gain of chromatin accessibility by ATACs-seq and featured reciprocal loss of H3K27me3 and gain of H3K36me2. These “B to A” TADs yielded increased looping connectivity and contained the primitive stem cell genes that were upregulated. Indeed, B to A shifting enabled critical stem cell enhancers to interact with and activate various stem cell genes as shown by v4C (e.g. KLF5, PRDM5, MEIS1, etc). Most remarkably, the 3D architecture of H1C/H1EDKO GC B-cells was similar to that of intermediate stages of iPS cell reprogramming.
Consistent with this finding, H1C/EDKO cells exhibited 4-fold greater reprogramming to the iPS state than WT cells after OKSM transduction, and these iPS cells exhibited reduced H3K27me3, stronger pluripotency potential, and impaired differentiation. This effect was rescued by transduction of WT H1, but not globular or C terminal domain mutant H1. Strikingly, the H1C/EDKO primitive stem cell gene expression signature was highly significantly enriched (NES 1.24, FDR<0.001) in the RNA-seq profiles of independent cohorts of DLBCL patients with histone 1 mutations, as well as H1C+/-H1E+/-;VavP-Bcl2 murine lymphomas. Consistent with acquisition of stem cell characteristics, H1C+/-H1E+/-;VavP-Bcl2 but not VavP-Bcl2 primary lymphoma cells manifested lymphoma initiating functionality after secondary transplantation into recipient animals, consistent with the notion that they may have gained stem cell functionality.
Collectively, we find that H1 isoforms are bona fide lymphoma tumor suppressors. We speculate that H1 mutations are limited to GC derived lymphomas due to the stoichiometric need for H1 proteins in these rapidly dividing cells. To the best of our knowledge these are the first data to implicate disruption of topological packing order of chromatin as a cancer driver mechanism, as well as the first data to provide a mechanism whereby mature B-cells can acquire cancer stem cell-like characteristics.
Melnick:Janssen: Research Funding; Epizyme: Consultancy; Constellation: Consultancy.
Microarray technology provides a powerful tool for defining gene expression profiles of airway epithelium that lend insight into the pathogenesis of human airway disorders. The focus of this study ...was to establish rigorous quality control parameters to ensure that microarray assessment of the airway epithelium is not confounded by experimental artifact. Samples (total n = 223) of trachea, large and small airway epithelium were collected by fiberoptic bronchoscopy of 144 individuals and hybridized to Affymetrix microarrays. The pre- and post-chip quality control (QC) criteria established, included: (1) RNA quality, assessed by RNA Integrity Number (RIN) > or = 7.0; (2) cRNA transcript integrity, assessed by signal intensity ratio of GAPDH 3' to 5' probe sets < or = 3.0; and (3) the multi-chip normalization scaling factor < or = 10.0.
Of the 223 samples, all three criteria were assessed in 191; of these 184 (96.3%) passed all three criteria. For the remaining 32 samples, the RIN was not available, and only the other two criteria were used; of these 29 (90.6%) passed these two criteria. Correlation coefficients for pairwise comparisons of expression levels for 100 maintenance genes in which at least one array failed the QC criteria (average Pearson r = 0.90 +/- 0.04) were significantly lower (p < 0.0001) than correlation coefficients for pairwise comparisons between arrays that passed the QC criteria (average Pearson r = 0.97 +/- 0.01). Inter-array variability was significantly decreased (p < 0.0001) among samples passing the QC criteria compared with samples failing the QC criteria.
Based on the aberrant maintenance gene data generated from samples failing the established QC criteria, we propose that the QC criteria outlined in this study can accurately distinguish high quality from low quality data, and can be used to delete poor quality microarray samples before proceeding to higher-order biological analyses and interpretation.
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Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Diffuse large B-cell lymphomas (DLBCL) are broadly dependent on anaplerotic metabolism regulated by mitochondrial SIRT3. Herein we find that translational upregulation of ATF4 is coupled with ...anaplerotic metabolism in DLBCLs due to nutrient deprivation caused by SIRT3 driving rapid flux of glutamine into the tricarboxylic acid (TCA) cycle. SIRT3 depletion led to ATF4 downregulation and cell death, which was rescued by ectopic ATF4 expression. Mechanistically, ATF4 translation is inhibited in SIRT3-deficient cells due to the increased pools of amino acids derived from compensatory autophagy and decreased glutamine consumption by the TCA cycle. Absence of ATF4 further aggravates this state through downregulation of its target genes, including genes for amino acid biosynthesis and import. Collectively, we identify a SIRT3-ATF4 axis required to maintain survival of DLBCL cells by enabling them to optimize amino acid uptake and utilization. Targeting ATF4 translation can potentiate the cytotoxic effect of SIRT3 inhibitor to DLBCL cells. SIGNIFICANCE: We discovered the link between SIRT3 and ATF4 in DLBCL cells, which connected lymphoma amino acid metabolism with ATF4 translation via metabolic stress signals. SIRT3-ATF4 axis is required in DLBCL cells regardless of subtype, which indicates a common metabolic vulnerability in DLBCLs and can serve as a therapeutic target.
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Molecular alterations in the histone methyltransferase EZH2 and the antiapoptotic protein Bcl-2 frequently co-occur in diffuse large B-cell lymphoma (DLBCL). Because DLBCL tumors with these ...characteristics are likely dependent on both oncogenes, dual targeting of EZH2 and Bcl-2 is a rational therapeutic approach. We hypothesized that EZH2 and Bcl-2 inhibition would be synergistic in DLBCL. To test this, we evaluated the EZH2 inhibitor tazemetostat and the Bcl-2 inhibitor venetoclax in DLBCL cells, 3-dimensional lymphoma organoids, and patient-derived xenografts (PDXs). We found that tazemetostat and venetoclax are synergistic in DLBCL cells and 3-dimensional lymphoma organoids that harbor an EZH2 mutation and an IGH/BCL2 translocation but not in wild-type cells. Tazemetostat treatment results in upregulation of proapoptotic Bcl-2 family members and priming of mitochondria to BH3-mediated apoptosis, which may sensitize cells to venetoclax. The combination of tazemetostat and venetoclax was also synergistic in vivo. In DLBCL PDXs, short-course combination therapy resulted in complete remissions that were durable over time and associated with superior overall survival compared with either drug alone.
•EZH2 inhibition combined with Bcl-2 inhibition is synergistic in DLBCL subtypes with EZH2 and BCL2 molecular alterations.•Tazemetostat treatment results in upregulation of proapoptotic Bcl-2 family members and priming of mitochondria to apoptotic cell death.
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