Targeting the crosstalk between tumor cells and microenvironment: a new therapeutic approach for the treatment of lymphoid neoplasms

Abstract

[eng] In this thesis we have focused on two novel therapeutic strategies that target the crosstalk between the tumor cells and the microenvironment. Chronic lymphocytic leukemia (CLL) remains an incurable disease where high CD38 expression is associated with poor prognosis and identifies cells that are prone to proliferate. CD38 cooperates in migration, adhesion and invasion through its molecular association with CXCR4, MMP9 and CD49d. The human anti-CD38 monoclonal antibody daratumumab has shown efficient cell killing and a good safety profile in clinical trials in multiple myeloma. In this thesis, we demonstrate that daratumumab also exerts significant cytotoxicity against patient-derived CLL cells, via ADCC and ADCP in vitro and in vivo. Furthermore, daratumumab interferes with CD38 signaling and reduces CLL cell adhesion, migration and homing. Moreover, daratumumab shows therapeutic activity in two mouse models. Thus, daratumumab improves overall survival in a systemic CD38+ MEC2 cell line mouse model and reduces tumor burden in CLL-patient derived xenografts. These results provide scientific rationale for the clinical development of daratumumab in poor prognosis CD38+CLL. Despite unprecedented activity of ibrutinib in mantle cell lymphoma (MCL), acquired or induced resistances appear in association with dismal prognosis. CC-292 (spebrutinib) is a novel, highly specific covalent BTK inhibitor that binds irreversibly to the Cys481 in the BTK active site, with demonstrated preclinical activity in several B-cell malignancies, and initial encouraging results in a phase 1 trial in relapsed/refractory chronic lymphocytic leukemia. We have characterized CC-292 activity in a panel of MCL cell lines (n=5) and primary cases (n=11) with different mutational status of NF-κB pathway genes. CC-292 shows a similar anti-proliferative profile than ibrutinib, conditioned by the existence of activating mutations in alternative NF-κB pathway genes such as TRAF2/3 and BIRC3. Noteworthy, CC-292 blocks B-cell receptor activation, independently of the presence of mutations on TRAF2/3 or BIRC3 genes. However, CC-292 interferes with CXCL12-induced migration mostly in MCL cells without activation of alternative NF-κB pathway. In sensitive cell lines, CC-292 activity was significantly enhanced by co-treatment with the immunomodulatory agent lenalidomide, maintaining its efficacy in co-culture with mesenchymal stromal cells (MSC). For MCL cases bearing mutations in the alternative NF-κB pathway, inhibitors of NIK, a central kinase promoting processing of p100 to p52, engaged cytostatic and cytotoxic responses in MCL cell lines and primary cultures. This effect was maintained in MSC-MCL co-cultures and was further enhanced by CC-292 leading to a complete impairment of NF-κB pathway. These substantial effects of CC-292 on MCL, especially in combination regimens, warrant further investigation in the clinical setting. All in all, the findings of the present thesis support the importance of developing strategies to target the microenvironment and its interaction with neoplastic cells.

[spa] En esta tesis nos hemos centrado en dos estrategias terapéuticas novedosas que interfieren en la comunicación entre las células tumorales y el microambiente. Nuestro trabajo con Daratumumab, un anticuerpo monoclonal humano anti-CD38, sugiere que el DARA puede ser un agente terapéutico prometedor para el tratamiento de pacientes de LLC CD38+. DARA ejerce sus efectos principalmente a través de ADCC/ADCP y también a través de la inhibición de la migración y la adhesión de las células tumorales. La inhibición de la migración de las células tumorales a tejidos linfoides puede ser clínicamente relevante, ya que hay una creciente evidencia que indica que las interacciones tumor-microambiente pueden desempeñar un papel importante en la resistencia a los fármacos y contribuir a los fracasos clínicos. DARA también demostró una importante actividad in vivo en modelos inmunocomprometidos de ratón de LLC. Los resultados que hemos obtenido indican que el CC-292 es eficaz en el bloqueo de la señalización del BCR y la migración inducida por la quimiocina CXCL12 de células de LCM. Además, el CC-292 inhibe la activación celular inducida por α-IgM, evaluada por la expresión en membrana de CD69 y CD86, y la secreción de CCL3/CCL4. Se sabe que el bloqueo de la proteína BTK inhibe la vía clásica de NF-κB. Un estudio reciente descubrió que las líneas celulares de LCM resistentes al inhibidor de BTK ibrutinib, presentaban activación de la vía alternativa de NF-κB y que podían ser sensibles a los inhibidores de esta vía, tales como los nuevos inhibidores de NIK. En este sentido, hemos confirmado que el CC-292 inhibe la vía NF-κ B en aquellas líneas celulares sensibles al CC-292 pero no en las resistentes. Con el fin de aumentar el efecto antitumoral del CC-292 en las líneas resistentes, el CC-292 se combinó con dos inhibidores de NIK. Los resultados sugieren que las líneas celulares resistentes al CC-292 son sensibles a estos inhibidores y muestran una potencial sinergia con el CC-292