Results of an early access treatment protocol of daratumumab monotherapy in spanish patients with relapsed or refractory multiple myeloma

Daratumumab is a human CD38-targeted monoclonal antibody approved as monotherapy for heavily pretreated relapsed and refractory multiple myeloma. We report findings for the Spanish cohort of an open-label treatment protocol that provided early access to daratumumab monotherapy and collected safety and patient-reported outcomes data for patients with relapsed or refractory multiple myeloma. At 15 centers across Spain, intravenous daratumumab (16mg/kg) was administered to 73 patients who had ≥3 prior lines of therapy, including a proteasome inhibitor and an immunomodulatory drug, or who were double refractory to both. The median duration of daratumumab treatment was 3.3 (range: 0.03–13.17) months, with a median number of 12 (range: 1–25) infusions. Grade 3/4 treatment-emergent adverse events were reported in 74% of patients and included lymphopenia (28.8%), thrombocytopenia (27.4%), neutropenia (21.9%), leukopenia (19.2%), and anemia (15.1%). Common (>5%) serious treatmentemergent adverse events included respiratory tract infection (9.6%), general physical health deterioration (6.8%), and back pain (5.5%). Infusion-related reactions occurred in 45% of patients. The median change from baseline in all domains of the EQ-5D-5L and EORTC QLQ-C30 was mostly 0. A total of 18 (24.7%) patients achieved a partial response or better, with 10 (13.7%) patients achieving a very good partial response or better. Median progression-free survival was 3.98 months. The results of this early access treatment protocol are consistent with previously reported trials of daratumumab monotherapy and confirm its safety and antitumoral efficacy in Spanish patients with heavily treated relapsed or refractory multiple myeloma

Papel de las tetraspaninas en la internalización y el tráfico de moléculas asociadas en modelos tumorales y de infección viral

Tesis doctoral inédita Universidad Autónoma de Madrid, Facultad de Ciencias, Departamento de Biología Molecular. Fecha de lectura: 11-10-2019Esta tesis tiene embargado el acceso al texto completo hasta el 11-04-2021Tetraspanins are a superfamily of ubiquitous transmembrane molecules that mediate the formation of specialized microdomains in the plasma and intracellular membranes. These microdomains play a role in cell adhesion and membrane fusion events by regulating local concentration of adhesion receptors and transmembrane enzymes, as well as their intracellular trafficking. This thesis first focused in the characterization of the molecular association previously observed in our group, between tetraspanin CD81 and the dNTP hydrolase SAMHD1 that acts as a restriction factor for HIV infection. We demonstrated that CD81 could regulate intracellular dNTP content and thus viral retrotranscription by an inhibition of SAMHD1 proteasomal degradation. CD81 gene deletion does not affect extracellular vesicle secretion but only mildly impacts on their composition. Thus, this thesis explored the role of CD9, another tetraspanin highly enriched in extracellular vesicles and commonly employed as exosome marker. We demonstrated that blocking peptides do not affect exosome secretion, while CRISPR/Cas9 deletion of CD9 gene results in an increase in extracellular vesicle secretion, however, in both cases, a similar profile was observed in proteomic analyses. These analyses revealed a decrease in tetraspanins, integrins and exosome markers while complement molecules and extracellular matrix components were increased, suggesting that CD9 blockade inhibits the secretion of a subpopulation among exosomes. Detailed characterization of CD9 KO cells revealed an increment in multivesicular bodies and a diminution of amphisomes, usually very prominent in melanoma cells. Lysosomal function was competent in these cells while they showed an impaired mitophagy. MT1-MMP is sorted into exosomes and this form of secretion played a significant role in tumor invasion. Previous results from Dr Arroyo´s group showed a direct interaction of MT1-MMP cytoplasmic domain with actin linkers from the ERM family. Since both ERM and tetraspanins are among the most abundant components of exosomes, we thought that this system could be optimal to study the involvement of both mechanisms in exosome component sorting. The RRH motif in the cytoplasmic domain of MT1-MMP is the most proximal to the juxtamembrane region, and it was shown to be important for its binding and colocalization with ERMs at protrusive cell structures. Mutation of this motif impaired MT1-MMP autoprocessing. As a result, enhanced gelatinolytic capacity even though expression levels at the plasma membrane were lower than with wild-type MT1-MMP. Both association to ERM, or CD151 expression regulated MT1-MMP internalization. In sum, this thesis reveal a fundamental role for tetraspanins in the regulation of the fate of the different intracellular compartments along the endocytic/lysosomal/autophagy pathway.La superfamilia de las tetraspaninas engloba un ubicuo conjunto de proteínas implicadas en la formación de microdominios especializados en la membrana plasmáticas membranas intracelulares. Estos microdominios son fundamentales en procesos de adhesión celular y fusión de membranas al regular tanto la concentración local de receptores de adhesión y enzimas transmembrana, como su tráfico intracelular. Esta tesis se ha centrado en caracterizar la interacción molecular previamente descrita en nuestro laboratorio entre la tetraspanina CD81 y la hidrolasa de dNTPs SAMHD1, que actúa como un factor de restricción de la infección por VIH. Hemos demostrado que CD81 puede regular la concentración de dNTPs en la célula y la retrotranscripción viral, al inhibir la degradación de SAMHD1 por el proteasoma. La deleción del gen de CD81 no afecta la secreción de vesículas extracelulares pero sí tiene efectos en su composición. Por esta razón, estudiamos el papel de CD9 especialmente enriquecida en exovesículas y comúnmente empleada como marcador de exosomas. Hemos demostrado que con el uso de péptidos bloqueantes del extremo carboxilo terminal la secreción no cambia y sin embargo aumenta drásticamente cuando se elimina el gen mediante el sistema CRISPR/Cas9. No obstante, el análisis proteómico sí mostró cambios similares en ambos casos, alterando la composición de las vesículas de manera que descendía la presencia de ciertas tetraspaninas, integrinas y marcadores exosomales, mientras que aumentaban moléculas del complemento y componentes de matriz extracelular, sugiriendo que el bloqueo de CD9 inhibe la secreción de una subpoblación específica de exosomas. La caracterización detallada de las células CRISPR/Cas9 CD9 reveló un aumento en el número de cuerpos multivesiculares y una disminución en la formación de anfisomas. Sin embargo, la función lisosomal era correcta pero la mitofagia estaba inhibida. MT1-MMP es una metaloproteinasa incluida en exosomas. Esta forma de secreción de la proteasa juega un papel importante en la invasión tumoral. Resultados previos del grupo de la Dra. G. Arroyo revelaron que la región citosólica de MT1-MMP interacciona con proteínas ERM que sirven de unión con el citoesqueleto de actina. Las ERMs y las tetraspaninas son componentes típicos de exosomas por ello pensamos que el estudio de esta proteasa podría ser un modelo óptimo para determinar cuál de los dos mecanismos (conexión con ERMs o inclusión en TEMs) podría ser relevante en la inclusión en exosomas. El motivo RRH en la región citosólica de MT1-MMP, más próximo a la membrana, es crítico para la unión y colocalización con ERMs en estructuras protrusivas. La mutación de esta región altera el autoprocesamiento de MT1-MMP y aumenta su actividad gelatinolítica aunque sus niveles de expresión en la superficie sean menores. Tanto la asociación con ERMs como la expresión de CD151 regulan la internalización de MT1-MMP. Esta tesis revela el papel de las tetraspaninas en la regulación del destino final de diferentes componentes intracelulares a lo largo de las vías endocíticas, lisosomal y de autofagiaThis work was supported by grants BFU2014-55478-R; BIO2017- 86500-R; Fundación Ramón Areces and RYC-2012-11025 to MY-M; and was co-funded by Fondo Europeo de Desarrollo Regional (FEDER). HS was supported by a FPI-UAM Fellowship

CD9 inhibition reveals a functional connection of extracellular vesicle secretion with mitophagy in melanoma cells

Tetraspanins are often used as Extracellular Vesicle (EV) detection markers because of their abundance on these secreted vesicles. However, data on their function on EV biogenesis are controversial and compensatory mechanisms often occur upon gene deletion. To overcome this handicap, we have compared the effects of tetraspanin CD9 gene deletion with those elicited by cytopermeable peptides with blocking properties against tetraspanin CD9. Both CD9 peptide or gene deletion reduced the number of early endosomes. CD9 peptide induced an increase in lysosome numbers, while CD9 deletion augmented the number of MVB and EV secretion, probably because of compensatory CD63 expression upregulation. In vivo, CD9 peptide delayed primary tumour cell growth and reduced metastasis size. These effects on cell proliferation were shown to be concomitant with an impairment in mitochondrial quality control. CD9 KO cells were able to compensate the mitochondrial malfunction by increasing total mitochondrial mass reducing mitophagy. Our data thus provide the first evidence for a functional connection of tetraspanin CD9 with mitophagy in melanoma cells.Ministerio Espanol de Economia y Competitividad (MINECO), grant from the Fundacion Ramon Areces and Leonardo Grant from fBBVA to Maria Yanez-M

CD9 inhibition reveals a functional connection of extracellular vesicle secretion with mitophagy in melanoma cells

Tetraspanins are often used as Extracellular Vesicle (EV) detection markers because of their abundance on these secreted vesicles. However, data on their function on EV biogenesis are controversial and compensatory mechanisms often occur upon gene deletion. To overcome this handicap, we have compared the effects of tetraspanin CD9 gene deletion with those elicited by cytopermeable peptides with blocking properties against tetraspanin CD9. Both CD9 peptide or gene deletion reduced the number of early endosomes. CD9 peptide induced an increase in lysosome numbers, while CD9 deletion augmented the number of MVB and EV secretion, probably because of compensatory CD63 expression upregulation. In vivo, CD9 peptide delayed primary tumour cell growth and reduced metastasis size. These effects on cell proliferation were shown to be concomitant with an impairment in mitochondrial quality control. CD9 KO cells were able to compensate the mitochondrial malfunction by increasing total mitochondrial mass reducing mitophagy. Our data thus provide the first evidence for a functional connection of tetraspanin CD9 with mitophagy in melanoma cells.Ministerio Espanol de Economia y Competitividad (MINECO), grant from the Fundacion Ramon Areces and Leonardo Grant from fBBVA to Maria Yanez-M

Results of an early access treatment protocol of daratumumab monotherapy in spanish patients with relapsed or refractory multiple myeloma

Daratumumab is a human CD38-targeted monoclonal antibody approved as monotherapy for heavily pretreated relapsed and refractory multiple myeloma. We report findings for the Spanish cohort of an open-label treatment protocol that provided early access to daratumumab monotherapy and collected safety and patient-reported outcomes data for patients with relapsed or refractory multiple myeloma. At 15 centers across Spain, intravenous daratumumab (16mg/kg) was administered to 73 patients who had ≥3 prior lines of therapy, including a proteasome inhibitor and an immunomodulatory drug, or who were double refractory to both. The median duration of daratumumab treatment was 3.3 (range: 0.03–13.17) months, with a median number of 12 (range: 1–25) infusions. Grade 3/4 treatment-emergent adverse events were reported in 74% of patients and included lymphopenia (28.8%), thrombocytopenia (27.4%), neutropenia (21.9%), leukopenia (19.2%), and anemia (15.1%). Common (>5%) serious treatmentemergent adverse events included respiratory tract infection (9.6%), general physical health deterioration (6.8%), and back pain (5.5%). Infusion-related reactions occurred in 45% of patients. The median change from baseline in all domains of the EQ-5D-5L and EORTC QLQ-C30 was mostly 0. A total of 18 (24.7%) patients achieved a partial response or better, with 10 (13.7%) patients achieving a very good partial response or better. Median progression-free survival was 3.98 months. The results of this early access treatment protocol are consistent with previously reported trials of daratumumab monotherapy and confirm its safety and antitumoral efficacy in Spanish patients with heavily treated relapsed or refractory multiple myeloma