RESUMEN
Aberrant activity of the SUMOylation pathway has been associated with MYC overexpression and poor prognosis in aggressive B-cell lymphoma (BCL) and other malignancies. Recently developed small-molecule inhibitors of SUMOylation (SUMOi) target the heterodimeric E1 SUMO activation complex (SAE1/UBA2). Here, we report that activated MYC signaling is an actionable molecular vulnerability in vitro and in a preclinical murine in vivo model of MYC-driven BCL. While SUMOi conferred direct effects on MYC-driven lymphoma cells, SUMO inhibition also resulted in substantial remodeling of various subsets of the innate and specific immunity in vivo. Specifically, SUMOi increased the number of memory B cells as well as cytotoxic and memory T cells, subsets that are attributed a key role within a coordinated anti-tumor immune response. In summary, our data constitute pharmacologic SUMOi as a powerful therapy in a subset of BCL causing massive remodeling of the normal B-cell and T-cell compartment.
Asunto(s)
Linfoma de Células B , Linfoma , Humanos , Ratones , Animales , Proteínas Proto-Oncogénicas c-myc/metabolismo , Transducción de Señal , Linfoma/tratamiento farmacológico , Linfoma de Células B/tratamiento farmacológico , Biomarcadores , Enzimas Activadoras de Ubiquitina/metabolismoRESUMEN
Activated SUMOylation is a hallmark of cancer. Starting from a targeted screening for SUMO-regulated immune evasion mechanisms, we identified an evolutionarily conserved function of activated SUMOylation, which attenuated the immunogenicity of tumor cells. Activated SUMOylation allowed cancer cells to evade CD8+ T cell-mediated immunosurveillance by suppressing the MHC class I (MHC-I) antigen-processing and presentation machinery (APM). Loss of the MHC-I APM is a frequent cause of resistance to cancer immunotherapies, and the pharmacological inhibition of SUMOylation (SUMOi) resulted in reduced activity of the transcriptional repressor scaffold attachment factor B (SAFB) and induction of the MHC-I APM. Consequently, SUMOi enhanced the presentation of antigens and the susceptibility of tumor cells to CD8+ T cell-mediated killing. Importantly, SUMOi also triggered the activation of CD8+ T cells and thereby drove a feed-forward loop amplifying the specific antitumor immune response. In summary, we showed that activated SUMOylation allowed tumor cells to evade antitumor immunosurveillance, and we have expanded the understanding of SUMOi as a rational therapeutic strategy for enhancing the efficacy of cancer immunotherapies.
Asunto(s)
Presentación de Antígeno , Neoplasias , Antígenos de Histocompatibilidad Clase I , Humanos , Evasión Inmune , Neoplasias/patología , SumoilaciónRESUMEN
Evasion from drug-induced apoptosis is a crucial mechanism of cancer treatment resistance. The proapoptotic protein NOXA marks an aggressive pancreatic ductal adenocarcinoma (PDAC) subtype. To identify drugs that unleash the death-inducing potential of NOXA, we performed an unbiased drug screening experiment. In NOXA-deficient isogenic cellular models, we identified an inhibitor of the transcription factor heterodimer CBFß/RUNX1. By genetic gain and loss of function experiments, we validated that the mode of action depends on RUNX1 and NOXA. Of note is that RUNX1 expression is significantly higher in PDACs compared to normal pancreas. We show that pharmacological RUNX1 inhibition significantly blocks tumor growth in vivo and in primary patient-derived PDAC organoids. Through genome-wide analysis, we detected that RUNX1-loss reshapes the epigenetic landscape, which gains H3K27ac enrichment at the NOXA promoter. Our study demonstrates a previously unknown mechanism of NOXA-dependent cell death, which can be triggered pharmaceutically. Therefore, our data show a way to target a therapy-resistant PDAC, an unmet clinical need.
Asunto(s)
Apoptosis/genética , Carcinoma Ductal Pancreático/genética , Subunidad alfa 2 del Factor de Unión al Sitio Principal/genética , Expresión Génica , Neoplasias Pancreáticas/genética , Proteínas Proto-Oncogénicas c-bcl-2/genética , Mutaciones Letales Sintéticas , Carcinoma Ductal Pancreático/patología , Subunidad alfa 2 del Factor de Unión al Sitio Principal/antagonistas & inhibidores , Humanos , Neoplasias Pancreáticas/patología , Regiones Promotoras Genéticas , Regulación hacia ArribaRESUMEN
A ring-shaped helicase unwinds DNA during chromosome replication in all organisms. Replicative helicases generally unwind duplex DNA an order of magnitude slower compared to their in vivo replication fork rates. However, the origin of slow DNA unwinding rates by replicative helicases and the mechanism by which other replication components increase helicase speed are unclear. Here, we demonstrate that engagement of the eukaryotic CMG helicase with template DNA at the replication fork impairs its helicase activity, which is alleviated by binding of the single-stranded DNA binding protein, RPA, to the excluded DNA strand. Intriguingly, we found that, when stalled due to interaction with the parental duplex, DNA rezipping-induced helicase backtracking reestablishes productive helicase-fork engagement, underscoring the significance of plasticity in helicase action. Our work provides a mechanistic basis for relatively slow duplex unwinding by replicative helicases and explains how replisome components that interact with the excluded DNA strand stimulate fork rates.
Asunto(s)
ADN Helicasas/metabolismo , Replicación del ADN/fisiología , ADN/química , ADN/metabolismo , Animales , Bacteriófago T4 , Microscopía por Crioelectrón , ADN de Cadena Simple/metabolismo , Proteínas de Unión al ADN/metabolismo , Drosophila melanogaster/genética , Escherichia coli/genéticaRESUMEN
In the eukaryotic replisome, DNA unwinding by the Cdc45-MCM-Go-Ichi-Ni-San (GINS) (CMG) helicase requires a hexameric ring-shaped ATPase named minichromosome maintenance (MCM), which spools single-stranded DNA through its central channel. Not all six ATPase sites are required for unwinding; however, the helicase mechanism is unknown. We imaged ATP-hydrolysis-driven translocation of the CMG using cryo-electron microscopy (cryo-EM) and found that the six MCM subunits engage DNA using four neighboring protomers at a time, with ATP binding promoting DNA engagement. Morphing between different helicase states leads us to suggest a non-symmetric hand-over-hand rotary mechanism, explaining the asymmetric requirements of ATPase function around the MCM ring of the CMG. By imaging of a higher-order replisome assembly, we find that the Mrc1-Csm3-Tof1 fork-stabilization complex strengthens the interaction between parental duplex DNA and the CMG at the fork, which might support the coupling between DNA translocation and fork unwinding.
Asunto(s)
Adenosina Trifosfato/metabolismo , ADN Helicasas/metabolismo , ADN Polimerasa Dirigida por ADN/metabolismo , ADN/metabolismo , Eucariontes/enzimología , Complejos Multienzimáticos/metabolismo , Adenosina Trifosfatasas/metabolismo , Secuencia de Aminoácidos , Animales , Microscopía por Crioelectrón , ADN/ultraestructura , ADN Helicasas/química , ADN Helicasas/ultraestructura , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/enzimología , Hidrólisis , Modelos Moleculares , Dominios Proteicos , Saccharomyces cerevisiae/metabolismoRESUMEN
Accurate DNA replication is tightly regulated in eukaryotes to ensure genome stability during cell division and is performed by the multi-protein replisome. At the core an AAA+ hetero-hexameric complex, Mcm2-7, together with GINS and Cdc45 form the active replicative helicase Cdc45/Mcm2-7/GINS (CMG). It is not clear how this replicative ring helicase translocates on, and unwinds, DNA. We measure real-time dynamics of purified recombinant Drosophila melanogaster CMG unwinding DNA with single-molecule magnetic tweezers. Our data demonstrates that CMG exhibits a biased random walk, not the expected unidirectional motion. Through building a kinetic model we find CMG may enter up to three paused states rather than unwinding, and should these be prevented, in vivo fork rates would be recovered in vitro. We propose a mechanism in which CMG couples ATP hydrolysis to unwinding by acting as a lazy Brownian ratchet, thus providing quantitative understanding of the central process in eukaryotic DNA replication.
Asunto(s)
ADN Helicasas/metabolismo , Replicación del ADN , Proteínas de Drosophila/metabolismo , Modelos Moleculares , ADN Helicasas/aislamiento & purificación , Proteínas de Drosophila/aislamiento & purificación , Fenómenos Magnéticos , Pinzas Ópticas , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Imagen Individual de Molécula/métodosRESUMEN
Progression of DNA replication depends on the ability of the replisome complex to overcome nucleoprotein barriers. During eukaryotic replication, the CMG helicase translocates along the leading-strand template and unwinds the DNA double helix. While proteins bound to the leading-strand template efficiently block the helicase, the impact of lagging-strand protein obstacles on helicase translocation and replisome progression remains controversial. Here, we show that CMG and replisome progressions are impaired when proteins crosslinked to the lagging-strand template enhance the stability of duplex DNA. In contrast, proteins that exclusively interact with the lagging-strand template influence neither the translocation of isolated CMG nor replisome progression in Xenopus egg extracts. Our data imply that CMG completely excludes the lagging-strand template from the helicase central channel while unwinding DNA at the replication fork, which clarifies how two CMG helicases could freely cross one another during replication initiation and termination.