Abstract
In B-chronic lymphocytic leukemia (B-CLL), the interaction between leukemic cells and the microenvironment promotes tumor cell survival. The Bruton’s tyrosine kinase (BTK) inhibitor ibrutinib is one of the first-in-class molecules for the treatment of B-CLL patients; however, the emerging mechanisms of resistance to ibrutinib call for new therapeutic strategies. The purpose of the current study was to investigate the ability of ibrutinib plus the MDM2-inhibitor nutlin-3 to counteract the tumor microenvironment protective effect. We observed that primary B-CLL cells cultivated in microenvironment mimicking conditions were protected from apoptosis by the up-regulation of c-MYC and of p53. In the same setting, combined treatments with ibrutinib plus nutlin-3 led to significantly higher levels of apoptosis compared to the single treatments, counteracting the c-MYC up-regulation. Moreover, the combination induced high p53 levels and a significant dissipation of the mitochondrial membrane potential, together with BAX cleavage in the more active p18 form and phospho-BAD down-regulation, that are key components of the mitochondrial apoptotic pathway, enhancing the apoptosis level. Our findings propose a new therapeutic strategy to overcome the tumor microenvironment protection involved in B-CLL resistance to drugs, with possible clinical implications also for other hematologic and solid tumors for which ibrutinib is considered a therapeutic option.
Highlights
The existence of a tumor microenvironment that allows tumor cells to prevent apoptosis or evade immune system control is a crucial feature of cancer, and it is significant for B-chronic lymphocytic leukemia (B-CLL)
We previously demonstrated that the combination of ibrutinib with MDM2-inhibitors synergized in promoting apoptosis in B-CLL cell lines carrying wild-type p53 as well as 17p13 deletion and/or TP53 mutations [16], and that ibrutinib combined with gamma-secretase inhibitors exhibited enhanced cytotoxicity that was coupled with the down-regulation of the c-MYC oncogene [8]
Since the sensitivity of B-CLL to treatments is heavily reduced by the microenvironment within the lymphoid organs, in the present study we have analyzed primary cells isolated from 26 B-CLL affected patients (Table 1) co-cultured in the presence of stroma or stimulated with CpG-ODN and CD40L, to mimic the lymph node environment in vitro
Summary
The existence of a tumor microenvironment that allows tumor cells to prevent apoptosis or evade immune system control is a crucial feature of cancer, and it is significant for B-chronic lymphocytic leukemia (B-CLL). The crosstalk between CLL cells and bystander cells involves receptor–ligand bindings, release of soluble signals (e.g., chemokines) and the engagement of adhesion molecules on cell membrane surfaces, leading to several events regulating cell cycle, apoptosis, metabolism, proliferation, and migration of leukemic cells [2,3] Overall, this condition results in a protective milieu that offers to CLL cells a protection from both the natural immune response as well as the pharmacological treatments. In microenvironment mimicking conditions, based on co-culture with fibroblasts or mesenchymal stromal cells and/or stimulation with soluble molecules, such as CD40L and IL4 associated to CpG-oligodeoxynucleotides (CpG-ODNs), several authors have demonstrated the activation of survival pathways, including the B-cell receptor (BCR) pathway, leading to the up-regulation of the key proteins MCL-1 and BCL-XL [4,5,6,7] In this context, our group preliminarily observed the activation of NOTCH1 and C-MYC pathways in p53 wild-type B-CLL primary cells grown in co-culture with stromal cells [8]
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