CD20 is a direct regulator of B-cell receptor signaling in the microenvironment of chronic lymphocytic leukemia

Kód výsledku v IS VaVaI

<a href="https://www.isvavai.cz/riv?ss=detail&h=RIV%2F65269705%3A_____%2F18%3A00069441" target="_blank" >RIV/65269705:_____/18:00069441 - isvavai.cz</a>

Nalezeny alternativní kódy

RIV/00216224:14740/18:00104060

Výsledek na webu

<a href="https://library.ehaweb.org/eha/2018/stockholm/214654/gabriela.pavlasova.cd20.is.a.direct.regulator.of.b-cell.receptor.signaling.in.html?f=listing%3D0%2Abrowseby%3D8%2Asortby%3D2%2Ace_id%3D1346%2Atopic%3D1574%2Aot_id%3D19041" target="_blank" >https://library.ehaweb.org/eha/2018/stockholm/214654/gabriela.pavlasova.cd20.is.a.direct.regulator.of.b-cell.receptor.signaling.in.html?f=listing%3D0%2Abrowseby%3D8%2Asortby%3D2%2Ace_id%3D1346%2Atopic%3D1574%2Aot_id%3D19041</a>

DOI - Digital Object Identifier

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Jazyk výsledku

angličtina

Název v původním jazyce

CD20 is a direct regulator of B-cell receptor signaling in the microenvironment of chronic lymphocytic leukemia

Popis výsledku v původním jazyce

Background: The biological function of CD20 and the reasons for the impressive activity of anti-CD20 antibodies remain elusive. We have previously shown that CLL cells which have recently exited the lymph node microenvironment (CXCR4dimCD5bright cells) express higher CD20 levels. This is due to its direct up-regulation by CXCR4/SDF1 axis (Pavlasova et al., Blood, 2016). Aims: We hypothesized that CD20 expression is of direct functional importance for B-cell receptor (BCR) signaling as CLL cells with high CD20 express also higher cell surface IgM levels and this was coupled with higher responsiveness to BCR-crosslinking (P&lt;0.0001). Methods: CLL cells were obtained from untreated CLL patients or patients undergoing FCR therapy and analyzed for BCR signaling capacity and rituximab effect. Results: CD20 silencing using CD20 siRNA in B cells revealed that low levels of CD20 impair the phosphorylation of BCR/PI3K-associated molecules (LYN, SYK, ERK, GAB1) after BCR-crosslinking and BCR-induced calcium flux (P&lt;0.05). This also impaired LYN kinase phosphorylation, which is the first kinase phosphorylated in response to BCR ligation. This suggests that CD20 up-regulation in the microenvironment is required for proper activity of BCR and that CD20 is very &quot;intimately&quot; connected with BCR. Interestingly, the CD20 effect on phosphorylation of BCR-associated kinases was not entirely dependent on its known calcium-channel function (Bubien et al., JCB, 1993), as the effect of CD20 silencing was present also in calcium-free media. This suggests that CD20 has also other functions, such as a putative docking site for BCR-associated molecules. To investigate the relevance of CD20 in primary CLL cells in vivo we also analyzed proteins involved in BCR signaling pathway and cell activation in CXCR4/CD5 CLL subpopulations. The CD20brightCXCR4dimCD5bright subpopulation (represents cells that recently exited the lymph nodes) had higher levels of pAKT/pERK/pCD79a (P&lt;0.001) and a gene expression signature enriched for genes involved in BCR and MAPK signaling, migration and actin cytoskeleton organization (P&lt;0.0001). The overall activated status of CD20brightCXCR4dimCD5bright cells is also evidenced by enrichment in proliferative Ki67-positive cells (P&lt;0.0001). We further hypothesized that the higher CD20 levels on CXCR4dimCD5bright cells make them the primary target of rituximab, since certain rituximab-mediated effects depend on CD20 levels. Indeed, rituximab was in vivo ~10-fold more efficient in eliminating BCR signaling proficient CD20brightCXCR4dimCD5bright CLL cells than CD20dimCXCR4brightCD5dim cells in FCR treated patients (day 0 vs 1 and 3; P&lt;0.0001). Conclusion: Altogether, we described for the first time a direct role of CD20 in BCR signaling and its contribution to the aggressiveness of an intra-clonal CLL cell subpopulation. Additionally, we showed that this BCR signaling proficient CD20brightCXCR4dimCD5bright intra-clonal CLL cell subpopulation is preferentially eliminated by rituximab in vivo, which might partially account for the success of anti-CD20 antibodies.

Název v anglickém jazyce

CD20 is a direct regulator of B-cell receptor signaling in the microenvironment of chronic lymphocytic leukemia

Popis výsledku anglicky

Background: The biological function of CD20 and the reasons for the impressive activity of anti-CD20 antibodies remain elusive. We have previously shown that CLL cells which have recently exited the lymph node microenvironment (CXCR4dimCD5bright cells) express higher CD20 levels. This is due to its direct up-regulation by CXCR4/SDF1 axis (Pavlasova et al., Blood, 2016). Aims: We hypothesized that CD20 expression is of direct functional importance for B-cell receptor (BCR) signaling as CLL cells with high CD20 express also higher cell surface IgM levels and this was coupled with higher responsiveness to BCR-crosslinking (P&lt;0.0001). Methods: CLL cells were obtained from untreated CLL patients or patients undergoing FCR therapy and analyzed for BCR signaling capacity and rituximab effect. Results: CD20 silencing using CD20 siRNA in B cells revealed that low levels of CD20 impair the phosphorylation of BCR/PI3K-associated molecules (LYN, SYK, ERK, GAB1) after BCR-crosslinking and BCR-induced calcium flux (P&lt;0.05). This also impaired LYN kinase phosphorylation, which is the first kinase phosphorylated in response to BCR ligation. This suggests that CD20 up-regulation in the microenvironment is required for proper activity of BCR and that CD20 is very &quot;intimately&quot; connected with BCR. Interestingly, the CD20 effect on phosphorylation of BCR-associated kinases was not entirely dependent on its known calcium-channel function (Bubien et al., JCB, 1993), as the effect of CD20 silencing was present also in calcium-free media. This suggests that CD20 has also other functions, such as a putative docking site for BCR-associated molecules. To investigate the relevance of CD20 in primary CLL cells in vivo we also analyzed proteins involved in BCR signaling pathway and cell activation in CXCR4/CD5 CLL subpopulations. The CD20brightCXCR4dimCD5bright subpopulation (represents cells that recently exited the lymph nodes) had higher levels of pAKT/pERK/pCD79a (P&lt;0.001) and a gene expression signature enriched for genes involved in BCR and MAPK signaling, migration and actin cytoskeleton organization (P&lt;0.0001). The overall activated status of CD20brightCXCR4dimCD5bright cells is also evidenced by enrichment in proliferative Ki67-positive cells (P&lt;0.0001). We further hypothesized that the higher CD20 levels on CXCR4dimCD5bright cells make them the primary target of rituximab, since certain rituximab-mediated effects depend on CD20 levels. Indeed, rituximab was in vivo ~10-fold more efficient in eliminating BCR signaling proficient CD20brightCXCR4dimCD5bright CLL cells than CD20dimCXCR4brightCD5dim cells in FCR treated patients (day 0 vs 1 and 3; P&lt;0.0001). Conclusion: Altogether, we described for the first time a direct role of CD20 in BCR signaling and its contribution to the aggressiveness of an intra-clonal CLL cell subpopulation. Additionally, we showed that this BCR signaling proficient CD20brightCXCR4dimCD5bright intra-clonal CLL cell subpopulation is preferentially eliminated by rituximab in vivo, which might partially account for the success of anti-CD20 antibodies.

Druh

O - Ostatní výsledky

CEP obor

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OECD FORD obor

10606 - Microbiology

Projekt

<a href="/cs/project/NV16-29622A" target="_blank" >NV16-29622A: VLIV TERAPEUTICKÉ INHIBICE BCR SIGNALIZACE NA GENOVOU EXPRESI U B BUNĚČNÝCH MALIGNIT A JEJÍ PROGNOSTICKÝ A PREDIKTIVNÍ VÝZNAM</a><br>

Návaznosti

P - Projekt vyzkumu a vyvoje financovany z verejnych zdroju (s odkazem do CEP)<br>I - Institucionalni podpora na dlouhodoby koncepcni rozvoj vyzkumne organizace

Rok uplatnění

2018

Kód důvěrnosti údajů

S - Úplné a pravdivé údaje o projektu nepodléhají ochraně podle zvláštních právních předpisů