ICAP-1 regula la adhesión celular mediada por VLA-4 y el desarrollo del sistema inmunitario. Caracterización de relaciones entre VLA-4 y resistencia a bortezomib en mieloma múltiple

La integrina α4β1 desempeña importantes funciones durante la migración linfocitaria hacia sitios de inflamación y en diferentes etapas de la hematopoyesis. La actividad de esta integrina está regulada por proteínas intracelulares que se asocian al dominio citoplasmático de la subunidad β1. Mientras que se han realizado numerosos estudios acerca de los reguladores positivos de las integrinas tales como talina y kindlin, aquellas proteínas que regulan negativamente su activación han sido menos caracterizadas, como es el caso de ICAP-1. Puesto que α4β1 se expresa en linfocitos, hemos estudiado el efecto de la falta de ICAP-1 en la adhesión dependiente de α4β1 en estas células, así como en la diferenciación de células del sistema inmunitario en el timo, bazo y médula ósea (MO). Otra de las funciones conocidas de α4β1 es su participación en la interacción entre células de mieloma múltiple (MM) y la microvasculatura de la MO y células del estroma medular. Esta interacción favorece la proliferación, supervivencia y resistencia a quimioterapia de las células de MM, promoviendo la colonización de la MO y la progresión de la enfermedad. Debido a que ICAP-1 se postula como regulador negativo de la activación de integrinas β1, es importante estudiar su papel en la adhesión de células de MM dependiente de α4β1. El Bortezomib (BTZ) es un inhibidor del proteasoma ampliamente utilizado en el tratamiento frente al MM. Desafortunadamente, muchos pacientes acaban desarrollando resistencia al BTZ, por lo que es crucial investigar los mecanismos que utilizan las células de MM para sobrevivir al tratamiento con este agente, incluyendo una posible relación entre el BTZ y la integrina α4β1..

ICAP-1 loss impairs CD8+ thymocyte development and leads to reduced marginal zone B cells in mice

ICAP-1 regulates β1-integrin activation and cell adhesion. Here, we used ICAP-1-null mice to study ICAP-1 potential involvement during immune cell development and function. Integrin α4β1-dependent adhesion was comparable between ICAP-1-null and control thymocytes, but lack of ICAP-1 caused a defective single-positive (SP) CD8+ cell generation, thus, unveiling an ICAP-1 involvement in SP thymocyte development. ICAP-1 bears a nuclear localization signal and we found it displayed a strong nuclear distribution in thymocytes. Interestingly, there was a direct correlation between the lack of ICAP-1 and reduced levels in SP CD8+ thymocytes of Runx3, a transcription factor required for CD8+ thymocyte generation. In the spleen, ICAP-1 was found evenly distributed between cytoplasm and nuclear fractions, and ICAP-1–/– spleen T and B cells displayed upregulation of α4β1-mediated adhesion, indicating that ICAP-1 negatively controls their attachment. Furthermore, CD3+- and CD19+-selected spleen cells from ICAP-1-null mice showed reduced proliferation in response to T- and B-cell stimuli, respectively. Finally, loss of ICAP-1 caused a remarkable decrease in marginal zone B- cell frequencies and a moderate increase in follicular B cells. Together, these data unravel an ICAP-1 involvement in the generation of SP CD8+ thymocytes and in the control of marginal zone B-cell numbers

ICAP-1 regula la adhesión celular mediada por VLA-4 y el desarrollo del sistema inmunitario.

252 p.-91 fig.-7 tab.[EN]The integrin α4β1 plays important roles during lymphocyte migration to sites of inflammation and at distinct steps in hematopoiesis. The activity of this integrin is regulated by intracellular proteins which associate with the β1 subunit cytoplasmic domain. While much is known on positive regulators such as talin and kindlin, less is known about those which negatively regulate integrin activation, such as ICAP-1. As α4β1 is expressed on lymphocytes, we have studied the effects of the lack of ICAP-1 on α4β1-dependent lymphocyte adhesion and on immune cell differentiation in thymus, spleen and bone marrow (BM). α4β1 also mediates the interaction between multiple myeloma (MM) cells and the BM microvasculature, as well as with BM stromal cells. This interaction favours MM cell proliferation, survival and resistance to chemotherapy, leading to BM colonization and disease progression. Given the potential role of ICAP-1 as negative regulator of β1 integrin activation, it is important to study its role on α4β1-dependent MM cell adhesion. Bortezomib (BTZ) is a proteasome inhibitor widely used in the treatment against MM. Unfortunately, many patients develop resistance to BTZ, and therefore is crucial to investigate the mechanisms that the MM cells use to bypass this inhibitor, including the possible relationships between BTZ and α4β1. In the first objective of this Doctoral Thesis we have characterized the ICAP-1 implication in α4β1-mediated lymphocyte adhesion, and in the development and maturation of cells of the immune system. In addition, we have analyzed the α4β1-dependent MM cell adhesion to the BM microvasculature.To address the first part of this objective, we used mice lacking ICAP-1. CD3 and -CD19 splenic cells from ICAP-1 mice displayed higher adhesion levels to α4β1 ligands than ICAP-1 cells. ICAP-1-deficient mice showed a specific reduction of TCRαβ hiCD8 CD69 cells in comparison with control mice, suggesting an involvement of ICAP-1 in thymocyte positive selection. The decreased CD8 T cell frequency was also evident in spleen and lymph nodes from ICAP-1 mice. Contrary to spleen cells, there were no differences in adhesion to CS-1/FN between thymocytes from ICAP-1 and ICAP-1 mice. Interestingly, we observed a preferential distribution of ICAP-1 in the nucleus of control thymocytes, suggesting that ICAP-1 function in these cells might be more relevant in the nucleus than in the cytosol. Analyzing regulatory elements of the thymocyte development program, we found that lack of ICAP-1 caused a specific reduction in the expression of Runx3, an important transcription factor controlling the generation of CD8 cells in thymus. We did not detect alterations in stem and progenitor cells frequencies in BM from ICAP-1 mice relative to control counterparts, and B lymphopoiesis was neither affected. Of note, we observed a reduction of marginal zone B cells and an increase in follicular B cells in spleen from ICAP-1 mice comparing to control mice. These data suggest that ICAP-1 might have a role as modulator of B cell trafficking in spleen, thus regulating B cell maturation. In the second part of this objective we demonstrated that ICAP-1 is expressed in MM bone marrow primary samples, and that silencing ICAP-1 in MM cell lines led to enhanced α4β1-dependent adhesion together with increased integrin activation. Furthermore, intravital microscopy assays revealed that ICAP-1 knockdown led to upregulated MM cell adhesion to the BM microvasculature. The second objective consisted in the characterization of functional relationships between MM cell resistance to BTZ and the expression and function of the α4β1 integrin. To this purpose we have used two MM cell lines resistant to BTZ. We found that the expression of α4β1 in these BTZ-resistant cells was higher than that of the original parental cells, which resulted in increased α4β1-dependent cell adhesion and enhanced cell infiltration in the BM of immunodeficient mice, which caused disease progression. A partial involvement of NF-κB and FOXO3a in enhanced α4 expression in the resistant cells was found. Upregulation of α4β1 expression in BTZ-resistant cells was not a general effect, as it was not observed in BTZ-resistant cells which served as models for acute lymphoblastic leukemia T and B cell lymphoma.[ES]La integrina α4β1 desempeña importantes funciones durante la migración linfocitaria hacia sitios de inflamación y en diferentes etapas de la hematopoyesis. La actividad de esta integrina está regulada por proteínas intracelulares que se asocian al dominio citoplasmático de la subunidad β1. Mientras que se han realizado numerosos estudios acerca de los reguladores positivos de las integrinas tales como talina y kindlin, aquellas proteínas que regulan negativamente su activación han sido menos caracterizadas, como es el caso de ICAP-1. Puesto que α4β1 se expresa en linfocitos, hemos estudiado el efecto de la falta de ICAP-1 en la adhesión dependiente de α4β1 en estas células, así como en la diferenciación de células del sistema inmunitario en el timo, bazo y médula ósea (MO). Otra de las funciones conocidas de α4β1 es su participación en la interacción entre células de mieloma múltiple (MM) y la microvasculatura de la MO y células del estroma medular. Esta interacción favorece la proliferación, supervivencia y resistencia a quimioterapia de las células de MM, promoviendo la colonización de la MO y la progresión de la enfermedad. Debido a que ICAP-1 se postula como regulador negativo de la activación de integrinas β1, es importante estudiar su papel en la adhesión de células de MM dependiente de α4β1. El Bortezomib (BTZ) es un inhibidor del proteasoma ampliamente utilizado en el tratamiento frente al MM. Desafortunadamente, muchos pacientes acaban desarrollando resistencia al BTZ, por lo que es crucial investigar los mecanismos que utilizan las células de MM para sobrevivir al tratamiento con este agente, incluyendo una posible relación entre el BTZ y la integrina α4β1. En el primer objetivo de esta Tesis Doctoral hemos caracterizado la implicación de ICAP-1 en la adhesión linfocitaria dependiente de α4β1, y en el desarrollo y maduración de células del sistema inmunitario. A su vez, hemos analizado la regulación por ICAP-1 de la adhesión de células de MM a la microvasculatura de la MO a través de α4β1.Para abordar la primera parte de este objetivo hemos utilizado ratones deficientes en ICAP-1. Esplenocitos CD3 y CD19 procedentes de ratones ICAP-1 mostraron niveles mayores de adhesión a ligandos de α4β1 que las células ICAP-1 . Detectamos una reducción hi específica de la población de células TCRαβ CD8 CD69 en muestras de timos de ratones ICAP-1 en comparación con muestras ICAP-1 , sugiriendo un posible papel de ICAP-1 en la selección positiva de los timocitos. El descenso de la frecuencia de células T CD8 también fue notorio en bazo y nódulos linfáticos de ratones ICAP-1 . Contrariamente a lo observado para los esplenocitos, no se encontraron diferencias en los niveles de adhesión a CS-1/FN entre timocitos ICAP-1 y ICAP-1 . Notablemente, detectamos una distribución preferencial de ICAP-1 en el núcleo de los timocitos, sugiriendo que la función desempeñada por esta proteína en estas células podría ser más relevante en el núcleo que en el citoplasma. Analizamos elementos reguladores del programa de desarrollo por el que los timocitos se determinan hacia los distintos linajes de células T, y encontramos que la falta de ICAP-1 causaba una reducción específica de la expresión de Runx3, un factor de transcripción crítico para la generación de células CD8 en el timo. No detectamos alteraciones en la frecuencia de células madre y progenitoras presentes en la MO de ratones ICAP-1 , ni en la linfopoyesis de células B. El análisis de los bazos de ratones ICAP-1 mostró una reducción de las células B de la zona marginal (MZ) y un incremento de las células B foliculares (FO), en comparación con los bazos de ratones control. Estos datos sugieren la posibilidad de que ICAP-1 actúe como modulador del tráfico de células B en bazo, por lo que pudiera regular la maduración de las células B. En la segunda parte de este objetivo silenciamos ICAP-1 en líneas celulares de MM, lo que supuso un incremento de la adhesión dependiente de α4β1 junto con una mayor activación de la integrina. Ensayos de microscopía intravital revelaron que la reducción de la expresión de ICAP-1 correlacionaba con un aumento de la adhesión de las células de MM a la microvasculatura de la MO. El segundo objetivo de esta Tesis consistió en la caracterización de relaciones funcionales entre la resistencia de células de MM a BTZ y la expresión y función de α4β1. Para desarrollar este trabajo hemos utilizado líneas celulares de MM resistentes a BTZ. Hemos observado que la expresión de α4β1 en estas células resistentes era mayor que la de las células parentales, lo que se tradujo en un aumento en los niveles de adhesión mediada por esta integrina y en una mayor infiltración de la MO de ratones inmunodeficientes, contribuyendo al progreso de la enfermedad. Los factores de transcripción NF-κB y FOXO3a están involucrados en el aumento de la expresión de α4 en las poblaciones celulares de MM resistentes a BTZ. El incremento de expresión de α4β1 en las células de MM resistentes a BTZ no resultó ser un efecto extensible a otras neoplasias hematológicas, ya que no se observó en células resistentes a BTZ utilizadas como modelos de leucemia linfoblástica aguda de células T y linfoma de células B del manto. Nuestros resultados indican que ICAP-1 está implicado en el desarrollo de células T CD8 en el timo, regulando la expresión de Runx3 y modulando la selección positiva de los timocitos. ICAP-1 controla negativamente la adhesión dependiente de α4β1 de células T y B del bazo, y regula el tráfico de células B en este órgano afectando a la frecuencia de células MZ y FO. Demostramos asimismo que ICAP-1 regula negativamente la adhesión de células de MM mediada por α4β1. Las células de MM resistentes a BTZ muestran mayor expresión de α4β1 en comparación a las células parentales, lo que causa un aumento de la adhesión mediada por esta integrina, así como una mayor infiltración de la MO y progresión del mieloma. Proponemos a NF-κB y FOXO3a como reguladores del aumento de la expresión de α4 en células de MM resistentes a BTZ.Financiación del proyecto (SAF2014-53059-R) del MINECOPeer reviewe

Running head: Myeloma resistance to bortezomib and α4β1 expression and function

36 p.-6 fig.The interaction of multiple myeloma (MM) cells with the bone marrow (BM) microenvironment promotes MM cell retention, survival and resistance to different anti‐MM agents, including proteasome inhibitors (PIs) such as bortezomib (BTZ). The α4β1 integrin is a main adhesion receptor mediating MM cell‐stroma interactions and MM cell survival, and its expression and function are downregulated by BTZ, leading to inhibition of cell adhesion‐mediated drug resistance (CAM‐DR) and MM cell apoptosis. Whether decreased α4β1 expression and activity is maintained or recovered upon development of resistance to BTZ represents an important question, as a potential rescue of α4β1 function could boost MM cell survival and disease progression. Using BTZ‐resistant MM cells, we found that they not only rescue their α4β1 expression, but its levels were higher than in parental cells. Increased α4β1 expression in resistant cells correlated with enhanced α4β1‐mediated cell lodging in the BM, and with disease progression. BTZ‐resistant MM cells displayed enhanced NF‐κB pathway activation relative to parental counterparts, which contributed to upregulated α4 expression and to α4β1‐dependent MM cell adhesion. These data emphasize the upregulation of α4β1 expression and function as a key event during resistance to BTZ in MM, which might indirectly contribute to stabilize this resistance, as stronger MM cell attachment to BM stroma will regain CAM‐DR and MM cell growth and survival. Finally, we found a strong correlation between high ITGB1 (integrin β1) expression in MM and poor progression‐free survival (PFS) and overall survival (OS) during treatment of MM patients with BTZ and IMIDs, and combination of high ITGB1 levels and presence of the high‐risk genetic factor amp1q causes low PFS and OS. These results unravel a novel prognostic value for ITGB1 in myeloma.This work was supported by grants SAF2014-53059-R and SAF2017-85146-R from the Ministerio de Ciencia, Innovación y Universidades (MCIU) to JT; SAF2017-89672-R from MCIU to FM; by the Research Institute Hospital 12 de Octubre (i+12) and grants from Instituto de Salud Carlos III (ISCIII) and CIBERONC to JML; by grants from ISCIII PI16/02024,PI17/00701 and PI19/01352, TRASCAN (EPICA and Immunocell), Fundació La Marató de TV3, the Accelerator award CRUK/AIRC/AECC joint funder-partnership, CIBERONC(CB16/12/00489) and co-financed with FEDER funds MINECO Explora (RTHALMY),Gobierno de Navarra, Departamento de Salud 40/2016 and Departamento de Industria (Proyecto Estrategico, Reto Genomica, DIANA) and Fundación Ramón Areces (PREMAMM) to FP and XA The study was also supported by the Multiple Myeloma Research Foundation Networks of excellence, the International Myeloma Foundation (Brian van Novis), and the Qatar National Research Fund award 7-916-3-237.Peer reviewe

In vivo adhesion of malignant B cells to bone marrow microvasculature is regulated by α4β1 cytoplasmic-binding proteins

40 p.-7 fig. Martínez-Moreno, Mónica et al.Multiple myeloma (MM) and chronic lymphocytic leukemia (CLL) cells must attach to the bone marrow (BM) microvasculature before lodging in the BM microenvironment. Using intravital microscopy (IVM) of the BM calvariae we demonstrate that the α4β1 integrin is required for MM and CLL cell firm arrest onto the BM microvasculature, while endothelial P-selectin and E-selectin mediate cell rolling. Talin, kindlin-3 and ICAP-1 are β1-integrin-binding partners that regulate β1-mediated cell adhesion. We show that talin and kindlin-3 cooperatively stimulate high affinity and strength of α4β1-dependent MM and CLL cell attachment, whereas ICAP-1 negatively regulates this adhesion. A functional connection between talin/kindlin-3 and Rac1 was found to be required for MM cell attachment mediated by α4β1. Importantly, IVM analyses with talin- and kindlin-3-silenced MM cells indicate that these proteins are needed for cell arrest on the BM microvasculature. Instead, MM cell arrest is repressed by ICAP-1. Moreover, MM cells silenced for talin and kindlin-3, and cultured on α4β1 ligands showed higher susceptibility to bortezomib-mediated cell apoptosis. Our results highlight the requirement of α4β1 and selectins for the in vivo attachment of MM and CLL cells to the BM microvasculature, and indicate that talin, kindlin-3 and ICAP-1 differentially control physiological adhesion by regulating α4β1 activity.This work was supported by the following grants from the Ministerio de Economía y Competitividad (Spain): SAF2011-24022 to J.T; SAF2012-31613 to A.G-P; SAF2012-31142 and SAF2013-49662-EXP to A.H; PI13/01454 to P.S-M; RD12/0036/0061 to J.T. and A.G-P; RD12/0036/0058 to N.C.G. The work was also funded by grant P2010/BMD-2314 from the Comunidad de Madrid to A.G-P., J.T., P. S-M and A.H., and partially supported by a CRIS foundation grant to fight cancer to J.M-L. N.C.G. is also funded by a grant from the Asociación Española Contra el Cáncer (GCB120981SAN), and from the Gerencia Regional de Salud, Junta de Castilla y León (GRS-702/A/11).Peer reviewe

Lateral mobility and nanoscale spatial arrangement of chemokine-activated α4β1 integrins on T cells

11p.-8 fig.Chemokine stimulation of integrin α4β1-dependent T lymphocyte adhesion is a key step during lymphocyte trafficking. A central question regarding α4β1 function is how its lateral mobility and organization influence its affinity and avidity following cell stimulation with chemokines and/or ligands. Using single particle tracking and super-resolution imaging approaches, we explored the lateral mobility and spatial arrangement of individual α4β1 integrins on T cells exposed to different activating stimuli. We show that CXCL12 stimulation leads to rapid and transient α4β1 activation, measured by induction of the activation epitope recognized by the HUTS-21 anti-β1 antibody and by increased talin-β1 association. CXCL12-dependent α4β1 activation directly correlated with restricted lateral diffusion and integrin immobilization. Moreover, co-stimulation by CXCL12 together with soluble VCAM-1 potentiated integrin immobilization with a five-fold increase in immobile integrins as compared to unstimulated conditions. Our data indicate that docking by talin of the chemokine-activated α4β1 to the actin cytoskeleton favors integrin immobilization, which likely facilitates ligand interaction and increased adhesiveness. Super-resolution imaging showed that the nanoscale organization of high-affinity α4β1 remains unaffected following chemokine and/or ligand addition. Instead, newly activated α4β1 integrins organize on the cell membrane as independent units without joining pre-established integrin sites to contribute to cluster formation. Altogether, our results provide a rationale to understand how the spatiotemporal organization of activated α4β1 integrins regulates T lymphocyte adhesion.This work was supported by Erasmus Mundus Doctorate Program Europhotonics Grant 159224-1-2009-1-FR-ERA MUNDUS-EMJD), Spanish Ministry of Economy and Competitiveness “Severo Ochoa” Programme for Centres of Excellence in R&D Grants SEV-2015–0522, FIS2014 –5617-R, SAF2014–53059-R, and RD12/0036/0061; Fundacio Privada CELLEX (Barcelona);HFSP Grant GA RGP0027/2012; and LaserLab Europe 4 Grant GA 654148.Peer reviewe

Regulation of miRNA expression by α4β1 integrin‐dependent multiple myeloma cell adhesion

Abstract The α4β1 integrin regulates the trafficking of multiple myeloma (MM) cells and contributes to MM disease progression. MicroRNAs (miRNAs) can have both tumor suppressor and oncogenic roles and thus are key controllers of tumor evolution, and have been associated with different phases of MM pathogenesis. Using small RNAseq analysis, we show here that α4β1‐dependent MM cell adhesion regulates the expression of forty different miRNAs, therefore expanding our current view of the α4β1 involvement in MM cell biology. Specific upregulation of miR‐324‐5p and miR‐331‐3p in cells attached to α4β1 ligands was confirmed upon silencing the α4 integrin subunit, and their increased levels found to be dependent on Erk1/2‐ and PI3K‐Akt‐, but not Src‐dependent signaling. Enhanced miR‐324‐5p expression upon α4β1‐mediated MM cell adhesion aimed the hedgehog (Hh) component SMO, revealing that the miR‐324‐5p‐SMO module represents a α4β1‐regulated pathway that could control Hh‐dependent cellular responses in myeloma. Our results open new therapy research avenues around the α4β1 contribution to MM progression that deserve to be investigated

Positive and negative regulation by SLP-76/ADAP and Pyk2 of chemokine-stimulated T-lymphocyte adhesion mediated by integrin α4β1

Stimulation by chemokines of integrin α4β1-dependent T-lymphocyte adhesion is a crucial step for lymphocyte trafficking. The adaptor Vav1 is required for chemokine-activated T-cell adhesion mediated by α4β1. Conceivably, proteins associating with Vav1 could potentially modulate this adhesion. Correlating with activation by the chemokine CXCL12 of T-lymphocyte attachment to α4β1 ligands, a transient stimulation in the association of Vav1 with SLP-76, Pyk2, and ADAP was observed. Using T-cells depleted for SLP-76, ADAP, or Pyk2, or expressing Pyk2 kinase-inactive forms, we show that SLP-76 and ADAP stimulate chemokine-activated, α4β1-mediated adhesion, whereas Pyk2 opposes T-cell attachment. While CXCL12-promoted generation of high-affinity α4β1 is independent of SLP-76, ADAP, and Pyk2, the strength of α4β1-VCAM-1 interaction and cell spreading on VCAM-1 are targets of regulation by these three proteins. GTPase assays, expression of activated or dominant-negative Rac1, or combined ADAP and Pyk2 silencing indicated that Rac1 activation by CXCL12 is a common mediator response in SLP-76-, ADAP-, and Pyk2-regulated cell adhesion involving α4β1. Our data strongly suggest that chemokine-stimulated associations between Vav1, SLP-76, and ADAP facilitate Rac1 activation and α4β1-mediated adhesion, whereas Pyk2 opposes this adhesion by limiting Rac1 activation.This work was supported by grants SAF2011-24022 from Ministerio de Economía y Competitividad, RD12/0036/0061, and S2010/BMD-2314 from Comunidad de Madrid to J.T.Peer Reviewe