Explorando el potencial de la eliminación de las células que expresan PD-L1 en la terapia antitumoral

Tesis Doctoral inédita leída en la Universidad Autónoma de Madrid, Facultad de Medicina, Departamento de Bioquímica. Fecha de Lectura: 02-12-2022Esta tesis tiene embargado el acceso al texto completo hasta el 02-06-2024Inhibition of immune checkpoints such as the PD-1/PD-L1 axis has shown impressive responses in some tumors of otherwise very bad prognosis, such as metastatic melanoma. Unfortunately, and for reasons that remain largely unknown, some patients do not respond to this therapy. A potential limitation is that immunosuppressive cells expressing PD-L1 might likely suppress the immune response through additional inhibitory immune checkpoints or other mechanisms. We have hypothesized that the selective elimination of PD-L1-expressing cells could have more profound effects in cancer therapy than just blocking PD-1/PD-L1 interaction. To address this hypothesis, we have developed an inducible suicidal murine allele of Pd-l1 (PD-L1ATTAC), which allows visualization and selective elimination of PD-L1-expressing cells in adult tissues. Our experiments confirmed the usefulness of the model, which was particularly efficient for the depletion of PDL1+ cells in the peritoneum. In this compartment, the depletion of PD-L1+ cells increases susceptibility to LPS-induced septic shock, proving an intensified immune response in these animals. Interestingly, single-cell RNA sequencing analyses indicate that the depletion of PD-L1-expressing cells in the peritoneum reduces the percentage of B cells and macrophages, mainly immunosuppressive, while increasing the abundance and activity of cytotoxic cells. Once this was confirmed, we evaluated whether the elimination of PD-L1+ cells could similarly increase the antitumor response. To address the impact of our strategy as a potential immunotherapy in the context of cancer, we used a model of peritoneal carcinomatosis based on MC-38 colon cancer cells. Consistent with our original hypothesis, depletion of PD-L1+ cells increases the survival of mice harboring intraperitoneal allografts of colon cancer cells. Together, these results illustrate the usefulness of PD-L1ATTAC mice for studies on immunotherapy and provide initial evidences that support the potential of selectively killing PD-L1-expressing cells in cancer therapyLa inmunoterapia dirigida contra el eje PD-1/PD-L1 ha conseguido respuestas muy satisfactorias en la clínica para el tratamiento de ciertos tumores de muy mal pronóstico, como el melanoma metastásico. Desafortunadamente, y por razones desconocidas, no todos los pacientes responden a este tipo de tratamiento. Un potencial problema es que las células de fenotipo inmunosupresor como las que expresan PD-L1 pueden limitar la intensidad de la respuesta inmune a través de otros puntos de control inmunológicos o múltiples mecanismos adicionales. Es por ello que nuestra hipótesis de trabajo es evaluar si la eliminación de estas células podría ser más eficaz en la terapia contra el cáncer que la simple inhibición de la interacción entre PD-1 y PD-L1. Para demostrar esta hipótesis, hemos desarrollado un modelo de ratón inducible suicida de Pd-l1, denominado PD-L1ATTAC, que permite la visualización y la eliminación selectiva de células que expresan PD-L1 en tejidos adultos. Nuestros resultados confirman la utilidad del modelo, siendo particularmente eficiente en el peritoneo. En este tejido, la eliminación de las células que expresan PD-L1 provoca una mayor sensibilidad al shock séptico inducido por la endotoxina bacteriana LPS, demostrando así que la intensidad de la respuesta inmune está incrementada en estos animales. A nivel de mecanismo, experimentos de secuenciación de ARN de célula única revelaron que la eliminación de células PD-L1+ en el peritoneo reduce el porcentaje de linfocitos B y macrófagos, mayormente inmunosupresores, y simultáneamente aumenta la abundancia y la actividad de las células citotóxicas presentes. Una vez confirmado este hecho, evaluamos si, de una manera similar, la eliminación de células PD-L1+ podría aumentar la respuesta antitumoral. Así, y para abordar el impacto de nuestra estrategia como inmunoterapia antitumoral, utilizamos un modelo de carcinomatosis peritoneal basado en la línea celular de cáncer de colon MC-38. En línea con nuestra hipótesis original, la depleción de las células PD-L1+ aumenta la supervivencia de ratones con aloinjertos intraperitoneales de células tumorales. En conclusión, nuestros resultados ilustran la utilidad del modelo PD-L1ATTAC para estudios preclínicos en inmunoterapia y aportan evidencias iniciales del potencial de eliminar selectivamente las células que expresan PD-L1 como terapia antitumora

PD-L1ATTAC mice reveal the potential of depleting PD-L1 expressing cells in cancer therapy.

Antibodies targeting the PD-1 receptor and its ligand PD-L1 have shown impressive responses in some tumors of bad prognosis. We hypothesized that, since immunosuppressive cells might present several immune checkpoints on their surface, the selective elimination of PD-L1 expressing cells could be efficacious in enabling the activation of antitumoral immune responses. To address this question, we developed an inducible suicidal knock-in mouse allele of Pd-l1 (PD-L1ATTAC) which allows for the tracking and specific elimination of PD-L1-expressing cells in adult tissues. Consistent with our hypothesis, elimination of PD-L1 expressing cells from the mouse peritoneum increased the septic response to lipopolysaccharide (LPS), due to an exacerbated inflammatory response to the endotoxin. In addition, mice depleted of PD-L1+ cells were resistant to colon cancer peritoneal allografts, which was associated with a loss of immunosuppressive B cells and macrophages, concomitant with an increase in activated cytotoxic CD8 T cells. Collectively, these results illustrate the usefulness of PD-L1ATTAC mice for research in immunotherapy and provide genetic support to the concept of targeting PD-L1 expressing cells in cancer.O.F-C. is supported by grants from the Spanish Ministry of Science, Innovation and Universities (PID2021- 128722OB-I00, co-financed with European FEDER funds) and the Spanish Association Against Cancer (AECC; PROYE20101FERN) to O.F-C. and by a Ph.D. fellowship from Mar?a Oliva-Amigos/as del CNIO to E.F-M. The CNIO Bioinformatics Unit (BU) is a member of the Spanish National Bioinformatics Institute (INB) , ISCIII- Bioinformatics platform and is supported by grant PT17/0009/0011, of the Accion Estrategica en Salud 2013-2016 of the Programa Estatal de Investigacion Orientada a los Retos de la Sociedad, funded by the ISCIII and European Regional Development Fund (ERDF-EU) and project RETOS RTI2018-097596-B-I00 funded by AEI-MCIU and cofounded by the ERDF-EU. The authors declare no competing financial interests.S

Activation of the integrated stress response is a vulnerability for multidrug‐resistant FBXW7‐deficient cells

Abstract FBXW7 is one of the most frequently mutated tumor suppressors, deficiency of which has been associated with resistance to some anticancer therapies. Through bioinformatics and genome‐wide CRISPR screens, we here reveal that FBXW7 deficiency leads to multidrug resistance (MDR). Proteomic analyses found an upregulation of mitochondrial factors as a hallmark of FBXW7 deficiency, which has been previously linked to chemotherapy resistance. Despite this increased expression of mitochondrial factors, functional analyses revealed that mitochondria are under stress, and genetic or chemical targeting of mitochondria is preferentially toxic for FBXW7‐deficient cells. Mechanistically, the toxicity of therapies targeting mitochondrial translation such as the antibiotic tigecycline relates to the activation of the integrated stress response (ISR) in a GCN2 kinase‐dependent manner. Furthermore, the discovery of additional drugs that are toxic for FBXW7‐deficient cells showed that all of them unexpectedly activate a GCN2‐dependent ISR regardless of their accepted mechanism of action. Our study reveals that while one of the most frequent mutations in cancer reduces the sensitivity to the vast majority of available therapies, it renders cells vulnerable to ISR‐activating drugs

53BP1 cooperation with the REV7-shieldin complex underpins DNA structure-specific NHEJ.

53BP1 governs a specialized, context-specific branch of the classical non-homologous end joining DNA double-strand break repair pathway. Mice lacking 53bp1 (also known as Trp53bp1) are immunodeficient owing to a complete loss of immunoglobulin class-switch recombination, and reduced fidelity of long-range V(D)J recombination. The 53BP1-dependent pathway is also responsible for pathological joining events at dysfunctional telomeres, and its unrestricted activity in Brca1-deficient cellular and tumour models causes genomic instability and oncogenesis. Cells that lack core non-homologous end joining proteins are profoundly radiosensitive, unlike 53BP1-deficient cells, which suggests that 53BP1 and its co-factors act on specific DNA substrates. Here we show that 53BP1 cooperates with its downstream effector protein REV7 to promote non-homologous end joining during class-switch recombination, but REV7 is not required for 53BP1-dependent V(D)J recombination. We identify shieldin-a four-subunit putative single-stranded DNA-binding complex comprising REV7, c20orf196 (SHLD1), FAM35A (SHLD2) and FLJ26957 (SHLD3)-as the factor that explains this specificity. Shieldin is essential for REV7-dependent DNA end-protection and non-homologous end joining during class-switch recombination, and supports toxic non-homologous end joining in Brca1-deficient cells, yet is dispensable for REV7-dependent interstrand cross-link repair. The 53BP1 pathway therefore comprises distinct double-strand break repair activities within chromatin and single-stranded DNA compartments, which explains both the immunological differences between 53bp1- and Rev7- deficient mice and the context specificity of the pathway

Guidelines for the use and interpretation of assays for monitoring autophagy

In 2008 we published the first set of guidelines for standardizing research in autophagy. Since then, research on this topic has continued to accelerate, and many new scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Accordingly, it is important to update these guidelines for monitoring autophagy in different organisms. Various reviews have described the range of assays that have been used for this purpose. Nevertheless, there continues to be confusion regarding acceptable methods to measure autophagy, especially in multicellular eukaryotes. A key point that needs to be emphasized is that there is a difference between measurements that monitor the numbers or volume of autophagic elements (e.g., autophagosomes or autolysosomes) at any stage of the autophagic process vs. those that measure flux through the autophagy pathway (i.e., the complete process); thus, a block in macroautophagy that results in autophagosome accumulation needs to be differentiated from stimuli that result in increased autophagic activity, defined as increased autophagy induction coupled with increased delivery to, and degradation within, lysosomes (in most higher eukaryotes and some protists such as Dictyostelium) or the vacuole (in plants and fungi). In other words, it is especially important that investigators new to the field understand that the appearance of more autophagosomes does not necessarily equate with more autophagy. In fact, in many cases, autophagosomes accumulate because of a block in trafficking to lysosomes without a concomitant change in autophagosome biogenesis, whereas an increase in autolysosomes may reflect a reduction in degradative activity. Here, we present a set of guidelines for the selection and interpretation of methods for use by investigators who aim to examine macroautophagy and related processes, as well as for reviewers who need to provide realistic and reasonable critiques of papers that are focused on these processes. These guidelines are not meant to be a formulaic set of rules, because the appropriate assays depend in part on the question being asked and the system being used. In addition, we emphasize that no individual assay is guaranteed to be the most appropriate one in every situation, and we strongly recommend the use of multiple assays to monitor autophagy. In these guidelines, we consider these various methods of assessing autophagy and what information can, or cannot, be obtained from them. Finally, by discussing the merits and limits of particular autophagy assays, we hope to encourage technical innovation in the field

Guidelines for the use and interpretation of assays for monitoring autophagy

In 2008 we published the first set of guidelines for standardizing research in autophagy. Since then, research on this topic has continued to accelerate, and many new scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Accordingly, it is important to update these guidelines for monitoring autophagy in different organisms. Various reviews have described the range of assays that have been used for this purpose. Nevertheless, there continues to be confusion regarding acceptable methods to measure autophagy, especially in multicellular eukaryotes. A key point that needs to be emphasized is that there is a difference between measurements that monitor the numbers or volume of autophagic elements (e.g., autophagosomes or autolysosomes) at any stage of the autophagic process vs. those that measure flux through the autophagy pathway (i.e., the complete process); thus, a block in macroautophagy that results in autophagosome accumulation needs to be differentiated from stimuli that result in increased autophagic activity, defined as increased autophagy induction coupled with increased delivery to, and degradation within, lysosomes (in most higher eukaryotes and some protists such as Dictyostelium) or the vacuole (in plants and fungi). In other words, it is especially important that investigators new to the field understand that the appearance of more autophagosomes does not necessarily equate with more autophagy. In fact, in many cases, autophagosomes accumulate because of a block in trafficking to lysosomes without a concomitant change in autophagosome biogenesis, whereas an increase in autolysosomes may reflect a reduction in degradative activity. Here, we present a set of guidelines for the selection and interpretation of methods for use by investigators who aim to examine macroautophagy and related processes, as well as for reviewers who need to provide realistic and reasonable critiques of papers that are focused on these processes. These guidelines are not meant to be a formulaic set of rules, because the appropriate assays depend in part on the question being asked and the system being used. In addition, we emphasize that no individual assay is guaranteed to be the most appropriate one in every situation, and we strongly recommend the use of multiple assays to monitor autophagy. In these guidelines, we consider these various methods of assessing autophagy and what information can, or cannot, be obtained from them. Finally, by discussing the merits and limits of particular autophagy assays, we hope to encourage technical innovation in the field

Erratum to: Guidelines for the use and interpretation of assays for monitoring autophagy (3rd edition) (Autophagy, 12, 1, 1-222, 10.1080/15548627.2015.1100356

non present