Following DNA-damage, the tumor suppressor p53 activates G1/S blocking and apoptotic genes, and represses other genes, including those involved in G2/M transition. In this latter system, it acts ...through the CCAAT-binding histone-like NF-Y. Several groups have reported that p53 is associated to promoters in unstressed conditions. We developed an oligo-based array containing 179 human promoters, enriched in genes involved in the DNA-damage and ER-stress response. We performed ChIP on chip experiments with p53 and NF-Y in cells under normal growing conditions. We identified 46 new p53 targets and noted (i) a significant enrichment in genes of the ER-stress response, including crucial regulators such as XBP1 and C/EBPβ; (ii) genes whose products are involved in the regulation of p53 function. Several genes were validated by conventional ChIP. DNA-damage dependent PCAF-mediated acetylation was observed on most, but not all promoters. The effect of p53 activation was checked by RT-PCR and transfections in HCT116 wt, E6 and p53-/- cells: most promoters were actively repressed upon Adriamycin treatment or following p53 transfection in p53-/- cells. In particular, the behaviour of some of the genes (BRAC1, RAD23 and RAD17) is consistent with a feed-back loop regulation on p53 levels. Finally, there is a large overlap (66%) between p53 and NF-Y targets. Our data reinstate the physiological importance of p53 promoter recognition and direct transcriptional repression as a mechanism to cope with DNA-damage.
Oncogenic mechanisms in Burkitt lymphoma Schmitz, Roland; Ceribelli, Michele; Pittaluga, Stefania ...
Cold Spring Harbor perspectives in medicine,
02/2014, Letnik:
4, Številka:
2
Journal Article
Recenzirano
Odprti dostop
Burkitt lymphoma is a germinal center B-cell-derived cancer that was instrumental in the identification of MYC as an important human oncogene more than three decades ago. Recently, new genomics ...technologies have uncovered several additional oncogenic mechanisms that cooperate with MYC to create this highly aggressive cancer. The transcription factor TCF-3 is central to Burkitt lymphoma pathogenesis. TCF-3 is rendered constitutively active in Burkitt lymphoma by two related mechanisms: (1) somatic mutations that inactivate its negative regulator ID3, and (2) somatic mutations in TCF-3 that block the ability of ID3 to bind and interfere with its activity as a transcription factor. TCF-3 is also a master regulator of normal germinal center B-cell differentiation. Within the germinal center, TCF-3 up-regulates genes that are characteristically expressed in the rapidly dividing centroblasts, the putative cell of origin for Burkitt lymphoma, while repressing genes expressed in the less proliferative centrocytes. TCF-3 promotes antigen-independent (tonic) B-cell-receptor signaling in Burkitt lymphoma by transactivating immunoglobulin heavy- and light-chain genes while repressing PTPN6, which encodes the phosphatase SHP-1, a negative regulator of B-cell-receptor signaling. Tonic B-cell-receptor signaling sustains Burkitt lymphoma survival by engaging the PI3 kinase pathway. In addition, TCF-3 promotes cell-cycle progression by transactivating CCND3, encoding a D-type cyclin that regulates the G1-S phase transition. Additionally, CCND3 accumulates oncogenic mutations that stabilize cyclin D3 protein expression and drive proliferation. These new insights into Burkitt lymphoma pathogenesis suggest new therapeutic strategies, which are sorely needed in developing regions of the world where this cancer is endemic.
Yondelis is a potent DNA-binding anticancer drug isolated from the tunicate Ecteinascidia turbinata currently undergoing phase III clinical trials. We and others have shown selective inhibition to ...the transcriptional induction
of several genes. We tested the hypothesis that Yondelis specifically targets cell-cycle genes. Our analysis on endogenous
and transfected reporter systems revealed complex patterns of transcriptional inhibition and, surprisingly, activation. Other
inducible systemsâthe metallothionein and the CYP3A4 promotersâwere little affected. We assayed whether interference of DNA
binding of the common nuclear factor Y (NF-Y) activator was responsible for the observed inhibition: in vivo chromatin immunoprecipitation
analysis in NIH3T3 and HCT116 cells indicates that NF-Y binding is little affected by Yondelis addition. Finally, histone
acetylation was modestly affected only on Cdc2 and cyclin B2 but not on other repressed promoters. These data prove that Yondelis
is not a general inhibitor of inducible genes, and its selective effects are exerted downstream from transcription factors
binding and histone acetyl transferases recruitment.
Malignant pirates of the immune system Rui, Lixin; Schmitz, Roland; Ceribelli, Michele ...
Nature immunology,
10/2011, Letnik:
12, Številka:
10
Journal Article
Recenzirano
At great human cost, cancer is the largest genetic experiment ever conducted. This review highlights how lymphoid malignancies have genetically perverted normal immune signaling and regulatory ...mechanisms for their selfish oncogenic goals of unlimited proliferation, perpetual survival and evasion of the immune response.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
B cell receptor (BCR) signalling has emerged as a therapeutic target in B cell lymphomas, but inhibiting this pathway in diffuse large B cell lymphoma (DLBCL) has benefited only a subset of patients
.... Gene expression profiling identified two major subtypes of DLBCL, known as germinal centre B cell-like and activated B cell-like (ABC)
, that show poor outcomes after immunochemotherapy in ABC. Autoantigens drive BCR-dependent activation of NF-κB in ABC DLBCL through a kinase signalling cascade of SYK, BTK and PKCβ to promote the assembly of the CARD11-BCL10-MALT1 adaptor complex, which recruits and activates IκB kinase
. Genome sequencing revealed gain-of-function mutations that target the CD79A and CD79B BCR subunits and the Toll-like receptor signalling adaptor MYD88
, with MYD88(L265P) being the most prevalent isoform. In a clinical trial, the BTK inhibitor ibrutinib produced responses in 37% of cases of ABC
. The most striking response rate (80%) was observed in tumours with both CD79B and MYD88(L265P) mutations, but how these mutations cooperate to promote dependence on BCR signalling remains unclear. Here we used genome-wide CRISPR-Cas9 screening and functional proteomics to determine the molecular basis of exceptional clinical responses to ibrutinib. We discovered a new mode of oncogenic BCR signalling in ibrutinib-responsive cell lines and biopsies, coordinated by a multiprotein supercomplex formed by MYD88, TLR9 and the BCR (hereafter termed the My-T-BCR supercomplex). The My-T-BCR supercomplex co-localizes with mTOR on endolysosomes, where it drives pro-survival NF-κB and mTOR signalling. Inhibitors of BCR and mTOR signalling cooperatively decreased the formation and function of the My-T-BCR supercomplex, providing mechanistic insight into their synergistic toxicity for My-T-BCR
DLBCL cells. My-T-BCR supercomplexes characterized ibrutinib-responsive malignancies and distinguished ibrutinib responders from non-responders. Our data provide a framework for the rational design of oncogenic signalling inhibitors in molecularly defined subsets of DLBCL.
Small cell neuroendocrine cancers (SCNCs) are recalcitrant cancers arising from diverse primary sites that lack effective treatments. Using chemical genetic screens, we identified inhibition of ...ataxia telangiectasia and rad3 related (ATR), the primary activator of the replication stress response, and topoisomerase I (TOP1), nuclear enzyme that suppresses genomic instability, as synergistically cytotoxic in small cell lung cancer (SCLC). In a proof-of-concept study, we combined M6620 (berzosertib), first-in-class ATR inhibitor, and TOP1 inhibitor topotecan in patients with relapsed SCNCs. Objective response rate among patients with SCLC was 36% (9/25), achieving the primary efficacy endpoint. Durable tumor regressions were observed in patients with platinum-resistant SCNCs, typically fatal within weeks of recurrence. SCNCs with high neuroendocrine differentiation, characterized by enhanced replication stress, were more likely to respond. These findings highlight replication stress as a potentially transformative vulnerability of SCNCs, paving the way for rational patient selection in these cancers, now treated as a single disease.
Genomic binding of NF-Y in mouse and human cells Ronzio, Mirko; Bernardini, Andrea; Taglietti, Valentina ...
Genomics (San Diego, Calif.),
September 2024, Letnik:
116, Številka:
5
Journal Article
Recenzirano
Odprti dostop
NF-Y is a Transcription Factor that regulates transcription through binding to the CCAAT-box. To understand its strategy, we analyzed 16 ChIP-seq datasets from human and mouse cells. Shared loci, ...mostly located in promoters of expressed genes of cell cycle, metabolism and gene expression pathways, are associated with histone marks of active chromatin and specific modules of TFs. Other peaks are in enhancers and Transposable Elements -TE- of retroviral origin in human and mouse. We evaluated the relationship with USF1, a common synergistic partner in promoters and MLT1 TEs, upon NF-YB inactivation: USF1 binding decreases in promoters, modestly in MLT1, suggesting a pioneering role of NF-Y in formers, not in the latters. These data define a common set of NF-Y functional targets across different mammalian cell types, suggesting a pioneering role in promoters with respect to TEs.
•NF-Y has a general preference for promoters, in mouse and human cell lines.•NF-Y target genes are of cell cycle, gene expression and metabolism pathways.•NF-Y removal entails a decrease of USF1 binding in promoters, less in repeats.•NF-YB inactivation leads to a drop in expression of USF1+/NF-Y+ targeted genes.
Abstract
We performed a large-scale profiling of currently available kinase inhibitors (KI) to identify “off-targets” that could be repurposed/repositioned as the starting point for the rational ...development of novel and selective kinase inhibitors against underdrugged targets. We profiled 257 KI at a single dose against a panel of 365 wild-type kinases tested individually, for a total of about 100,000 in vitro drug/kinase data points. After unsupervised hierarchical clustering of this dataset based on percent inhibition values, we identified a well-resolved subcluster driven by 5 KI with limited polypharmacology and a clean kinase-inhibition profile, mostly targeting AGC group kinases. These inhibitors were the AKT inhibitors GSK-690693, AZD-5363, and AT-7867; the MSK inhibitor SB-747651-A; and the Rho kinases (ROCK) inhibitor GSK-269962A. Notably, the AGC and hippo kinases LATS1 and LATS2 were part of this subcluster and were both inhibited to significant extents by these 5 compounds. With the regenerative potential of a candidate LATS1/2 inhibitor in mind, we next developed several orthogonal biochemical and cellular assays to prioritize compounds that possessed the desired activity against LATS1/2 without the burden of cytotoxic “off-targets,” such as those resulting from strong AKT inhibition. Based on the results of these assays, we prioritized one of the initially identified kinase inhibitors as the starting point for an ongoing, rational structure-activity relationship (SAR) effort to develop novel, 1st-in-class, highly selective LATS1 and LATS2 inhibitors. We will present data showing how such novel LATS1/2 inhibitors efficiently block LATS1/2-dependent YAP phosphorylation, induce YAP nuclear translocation in a high cell-density setting, and promote the transcription of bona fide YAP target genes such as AMOTL2, CTGF, and CYR61. Finally, consistently with the activation of a YAP-dependent transcriptional program, small-molecule inhibitors of LATS1/2 kinases promoted proliferation/regeneration in an in vitro wound-healing assay. Altogether, we identified novel small molecules that can be used as chemical probes to acutely inactivate the Hippo signaling pathway and validated LATS1 and LATS2 kinases as novel druggable kinases whose inhibition could be exploited for several indications, spanning from regenerative medicine to cancer immunotherapy.
Citation Format: Michele Ceribelli, Patrick Morris, Damien Duveau, Frances A. Tosto, Scott Hoyt, Craig J. Thomas. Development of selective LATS1/LATS2 inhibitors for the pharmacologic modulation of the Hippo signaling pathway abstract. In: Proceedings of the AACR Special Conference on the Hippo Pathway: Signaling, Cancer, and Beyond; 2019 May 8-11; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2020;18(8_Suppl):Abstract nr B06.
Abstract
The Hippo pathway is evolutionarily conserved and plays a critical role in determining organ size and in tumorigenesis. Aberrant genetic alterations in the Hippo pathway genes including ...LATS1/2, NF2, and STK11, as well as increased expression and activity of YAP have been found in some human cancers. Loss of function of Hippo genes results in increased nuclear localization of YAP, which promotes cell proliferation, migration, and invasion. Despite our increasing understanding of molecular phenotypes associated with Hippo aberration and excess YAP activity, there are as yet no FDA-approved therapies targeting this pathway, representing a large, unmet patient need. Previous work from our lab has established an additional role for the pathway in altering cellular response to gemcitabine in pancreatic cancer (PNAS 2017). To uncover therapeutic vulnerabilities caused by increased YAP activity, we performed a quantitative, high-throughput screen using the Mechanism Interrogation PlatE (MIPE) library of compounds. All the drugs in this library are relevant for clinical use in cancer and have known mechanisms of action. Annotated gene families represented in the MIPE library include kinases, GPCRs, and ion channels as well as broad cellular mechanisms including modulators of nuclear receptor transcription factors, lipid metabolism, histone epigenetic machinery, and apoptosis. The screen was carried out in twenty 1,536-well plates consisting of 2,154 compounds at nine different doses on both Panc02.13 cells expressing GFP or constitutively active YAP (YAPS6A). Preliminary data have confirmed our previous work in YAP’s role in increasing sensitivity to gemcitabine and several other chemotherapeutics. Pathway enrichment analysis of drugs based on similar mechanism of action allowed us to identify specific signaling nodes and cellular processes that present points of vulnerability or resistance caused by excess YAP activity. We discovered a profound increase in cellular resistance to MEK inhibitors. AC50 values for seventeen out of eighteen MEK inhibitors tested were on average more than four-fold higher in YAPS6A-expressing cells. We have validated these results in several other pancreatic cell lines in both 2D and 3D spheroid assays. Molecular analysis revealed that nuclear YAP causes sustained PI3K/AKT activation, providing a plausible compensatory pathway for cell survival. Moreover, treatment with PI3K/AKT inhibitors can abrogate this YAP-induced resistance to MEK inhibitors. Overall our data are consistent with previous findings that increases in PI3K and AKT signaling function as an escape mechanism to MEK inhibition. Our data suggest that increased YAP activity may represent a distinct molecular mechanism for how this escape is accomplished in refractory cancers. Cotargeting PI3K/AKT may thus provide therapeutic options for patients carrying mutations in Hippo pathway genes.
Citation Format: Andrew J. Bondesson, Aya Miyaki, Stella Shin, Michele Ceribelli, Craig J. Thomas, Taran S. Gujral. High-throughput chemical screening reveals YAP-mediated alterations in drug sensitivities abstract. In: Proceedings of the AACR Special Conference on the Hippo Pathway: Signaling, Cancer, and Beyond; 2019 May 8-11; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2020;18(8_Suppl):Abstract nr B32.
Adult T cell leukemia/lymphoma (ATLL) is a frequently incurable disease associated with the human lymphotropic virus type I (HTLV-I). RNAi screening of ATLL lines revealed that their proliferation ...depends on BATF3 and IRF4, which cooperatively drive ATLL-specific gene expression. HBZ, the only HTLV-I encoded transcription factor that is expressed in all ATLL cases, binds to an ATLL-specific BATF3 super-enhancer and thereby regulates the expression of BATF3 and its downstream targets, including MYC. Inhibitors of bromodomain-and-extra-terminal-domain (BET) chromatin proteins collapsed the transcriptional network directed by HBZ and BATF3, and were consequently toxic for ATLL cell lines, patient samples, and xenografts. Our study demonstrates that the HTLV-I oncogenic retrovirus exploits a regulatory module that can be attacked therapeutically with BET inhibitors.
Display omitted
•BATF3 and IRF4 are master regulators of ATLL gene expression and proliferation•HBZ drives BATF3 expression by binding to a super-enhancer in the BATF3 locus•BET inhibitors collapse the transcriptional network directed by HBZ and BATF3/IRF4•BET inhibitors are toxic for ATLL cells ex vivo and block ATLL xenograft growth
Nakagawa et al. show that BATF3 and IRF4 cooperatively drive adult T cell leukemia/lymphoma (ATLL)-specific gene expression and that human lymphotropic virus type I encoded HBZ regulates the expression of BATF3 and its downstream targets. BET inhibitors collapse the transcriptional network and kill ATLL cells.
PDF
