Júlia Carabia

Targeting the proliferative and chemoresistant compartment in chronic lymphocytic leukemia by inhibiting survivin protein

Chronic lymphocytic leukemia (CLL) cells located in proliferation centers are constantly stimulated by accessory cells, which provide them with survival and proliferative signals and mediate chemotherapy resistance. Herein, we designed an experimental strategy with the aim of mimicking the microenvironment found in the proliferative centers to specifically target actively proliferating CLL cells. For this, we co-cultured CLL cells and bone marrow stromal cells with concomitant CD40 and Toll-like receptor 9 stimulation. This co-culture system induced proliferation, cell-cycle entry and marked resistance to treatment with fludarabine and bendamustine. Proliferating CLL cells clustered together showed a typical morphology of activated B cells and expressed survivin protein, a member of the inhibitor of apoptosis family that is mainly expressed by CLL cells in the proliferation centers. With the aim of specifically targeting actively proliferating and chemoresistant CLL cells, we investigated the effects of treatment with YM155, a small-molecule survivin inhibitor. YM155 treatment suppressed the co-culture-induced survivin expression and that was sufficient to inhibit proliferation and effectively induce apoptosis particularly in the proliferative subset of CLL cells. Interestingly, sensitivity to YM155 was independent from common prognostic markers, including 17p13.1 deletion. Altogether, these findings provide a rationale for clinical development of YM155 in CLL.

ZAP-70 Promotes the Infiltration of Malignant B-Lymphocytes into the Bone Marrow by Enhancing Signaling and Migration after CXCR4 Stimulation

ZAP-70 in chronic lymphocytic leukemia (CLL) is associated with enhanced response to microenvironmental stimuli. We analyzed the functional consequences of ZAP-70 ectopic expression in malignant B-cells in a xenograft mouse model of disseminated B-cell leukemia. Mice injected with B-cells expressing ZAP-70 showed a prominently higher infiltration of the bone marrow. In vitro analysis of the response of malignant B-cells to CXCL12, the main attracting chemokine regulating trafficking of lymphocytes to the bone marrow, or to bone marrow stromal cells, revealed that ZAP-70 induces an increased response in terms of signaling and migration. These effects are probably mediated by direct participation of ZAP-70 in CXCL12-CXCR4 signaling since CXCR4 stimulation led to activation of ZAP-70 and downstream signaling pathways, such as MAPK and Akt, whereas ZAP-70 did not alter the expression of the CXCR4 receptor. In addition, subclones of primary CLL cells with high expression of ZAP-70 also showed increased migrative capacity toward CXCL12. Neutralization of CXCR4 with a monoclonal antibody resulted in impaired in vitro responses to CXCL12 and bone marrow stromal cells. We conclude that ZAP-70 enhances the migration of malignant B-cells into the supportive microenvironment found in the bone marrow mainly by enhancing signaling and migration after CXCR4 stimulation.

Inhibition of BCR signaling using the Syk inhibitor TAK-659 prevents stroma-mediated signaling in chronic lymphocytic leukemia cells.

Proliferation and survival of chronic lymphocytic leukemia (CLL) cells depend on microenvironmental signals coming from lymphoid organs. One of the key players involved in the crosstalk between CLL cells and the microenvironment is the B-cell receptor (BCR). Syk protein, a tyrosine kinase essential for BCR signaling, is therefore a rational candidate for targeted therapy in CLL. Against this background, we tested the efficacy of the highly specific Syk inhibitor TAK-659 in suppressing the favorable signaling derived from the microenvironment. To ex vivo mimic the microenvironment found in the proliferation centers, we co-cultured primary CLL cells with BM stromal cells (BMSC), CD40L and CpG ODN along with BCR stimulation. In this setting, TAK-659 inhibited the microenvironment-induced activation of Syk and downstream signaling molecules, without inhibiting the protein homologue ZAP-70 in T cells. Importantly, the pro-survival, proliferative, chemoresistant and activation effects promoted by the microenvironment were abrogated by TAK-659, which furthermore blocked CLL cell migration toward BMSC, CXCL12, and CXCL13. Combination of TAK-659 with other BCR inhibitors showed synergistic effect in inducing apoptosis, and the sequential addition of TAK-659 in ibrutinib-treated CLL cells induced significantly higher cytotoxicity. These findings provide a strong rationale for the clinical development of TAK-659 in CLL.

Microenvironment regulates the expression of miR-21 and tumor suppressor genes PTEN, PIAS3 and PDCD4 through ZAP-70 in chronic lymphocytic leukemia

Chronic lymphocytic leukemia (CLL) cells are highly dependent on microenvironment, being the BCR pathway one key player in this crosstalk. Among proteins participating, ZAP-70 enhances response to microenvironmental stimuli. MicroRNA-21 (miR-21) is overexpressed in diverse neoplasias including CLL, where it has been associated to refractoriness to fludarabine and to shorter time to progression and survival. To further elucidate the role of ZAP-70 in the biology of CLL, we studied its involvement in miR-21 regulation. MiR-21 expression was higher in CLL cells with high ZAP-70. Ectopic expression of ZAP-70 induced transcription of miR-21 via MAPK and STAT3, which subsequently induced downregulation of tumor suppressors targeted by miR-21. The co-culture of primary CLL cells mimicking the microenvironment induced ZAP-70 and miR-21 expression, as well as downregulation of miR-21 targets. Interestingly, the increase in miR-21 after co-culture was significantly impaired by ibrutinib, indicating that the BCR signaling pathway is involved in its regulation. Finally, survival of CLL cells induced by the co-culture correlated with miR-21 upregulation. In conclusion, stimuli from the microenvironment regulate miR-21 and its targeted tumor suppressor genes via a signaling pathway involving ZAP-70, thus contributing to the cytoprotection offered by the microenvironment particularly observed in CLL cells expressing ZAP-70.

Abstract 4568: Reversal of immune tolerance and increased anti tumoral immune response in a mouse model of CNS B cell lymphoma after combined XPO1 and BCR inhibition

Primary CNS lymphoma (PCNSL) is an aggressive non-Hodgkin lymphoma usually classified as ABC-DLBCL. Selinexor inactivates XPO-1 protein and induces anti-tumor effects mainly due to forced nuclear retention of tumor suppressors. It has shown excellent brain penetrance and promising results in glioblastoma and can inhibit both BCR and NF-kB signaling in malignant B-cells. Herein, we assessed the role of XPO-1 inhibition in intracerebral xenograft murine models. Proliferation and survival analysis in DLBCL cell lines showed that cell of origin does not influence sensitivity to selinexor and a strong synergy with ibrutinib mainly in ABC-DLBCL. We then established an orthotopic xenograft model of PCNSL by stereotactic injection of OCI-Ly10 cells transfected with luciferase into the brain parenchyma of nude athymic mice. Mice were treated with 5mg/kg oral selinexor or vehicle three times a week and bioluminiscence was assessed twice a week. Selinexor significantly blocked tumoral growth with differences starting as soon as 12 days after treatment. Also, selinexor improved mice survival (median: 48 days vs. 34 days). Next, we evaluated the potential synergy between ibrutinib and selinexor in vivo. For that mice were assigned into one of the following groups: selinexor (5mg/kg twice a week), ibrutinib (25mg/kg daily), combination or vehicle. The combination of both drugs significantly increased survival compared to both treatments as single agent, whereas there was no significant difference between ibrutinib and selinexor alone. Tumoral growth was equally blocked by all treatments. We next evaluated the innate immune response to the lymphoma by flow cytometry. Remarkably, CNS lymphomas were infiltrated by PD-1 positive M2 macrophages, as well as NK cells. Treatment with selinexor was able to decrease the proportion of M2-protumoral macrophages by half whereas the combination induced a 3-fold reduction (V: 22.72%+/-1.6, S: 12.29%+/-1.38, C: 7.1%+/-1.19). As a consequence, the proportion of M1 macrophages increased (V: 70.22%+/-1.8, S: 82.4%+/-1.7, C: 86.59%+/-1.5). In turn, ibrutinib was the main responsible for downregulation of PD-L1 in malignant cells (V: 3.7%+/-0.68, I: 0.6%+/-0.19, C: 0.19%+/- 0.08). Only when both treatments were combined there was significant downregulation of PD1 expression in M2 (V: 37.6%+/-5.5, C: 24.61%+/- 2.2) and a downregulation in PD1/SIRPα double positive M2 (V: 14.4%+-/1.44, C: 9.16%+/- 0.78). Selinexor and combination were able to significantly increase the proportion of NK cells (V:4.8%+/-0.21, S:9.7%+/-1.16, C:11.54%+/-2.02) and downregulate their PD1 expression (V:8.2%+/-2.7, S:3.7%+/-0.9, C: 2.8%+/-1.06). These results provide pre-clinical evidence for the development of selinexor and ibrutinib combination as new therapeutic option for PCNSL or DLBCL with CNS involvement. Citation Format: Marta Crespo, Isabel Jimenez, Julia Carabia, Sabela Bobillo, Pau Abrisqueta, Carles Palacio, Juan Camilo Nieto, Joan Boix, Cecilia del Carmen Carpio, Josep Castellvi, Joan Seoane, Francesc Bosch. Reversal of immune tolerance and increased anti tumoral immune response in a mouse model of CNS B cell lymphoma after combined XPO1 and BCR inhibition [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4568.