Studying the role of Bcl2 and myc in the pathogenesis of diffuse large b cell lymphoma

Abstract

Approximately 30% of patients with diffuse large B-cell lymphoma (DLBCL) co-express BCL2 and MYC, known as double-expressor lymphomas (DEL). These lymphomas associate with poorer prognosis and response to standard-of-care R-CHOP, and a preferred therapeutic alternative has not been identified yet. There are very interesting ongoing clinical trials to evaluate the potential of venetoclax to inhibit the anti-apoptotic protein BCL2 in high grade B cell lymphomas. Additionally, there are investigations ongoing trying to find a MYC inhibitor that can be efficiently used in the clinic without secondary effects. Here, we aimed to provide complementary preclinical in vivo evidences for these investigations, modelling this DEL-scenario in new mouse models and demonstrating that combination of anti-CD20 with BCL2 or MYC specific inhibitors can improve long-term survival in BCL2/MYC-expressor DLBCL mice. For this, we first generated and characterized three multi-transgenic mouse models and further demonstrated that it is possible to successfully recapitulate the complex progression and tumor microenvironment of DEL-DLBCL in the murine setting, recapitulating the classical genetic alterations of these patients by conditional mutagenesis at early stages of the germinal center reaction. We then demonstrated that lymphomas in these mice rapidly acquire aberrant BCL2/MYC co-expression and impair apoptosis during NF-κB-driven malignant transformation of germinal center B cells, hindering DLBCL cells sensitive to inhibition of BCL2 (with venetoclax) or MYC (with MYCi975). Then, we provided in vivo evidences that combination of venetoclax with anti-CD20-based immunotherapy can result in synergistic anti-lymphoma effects and extended overall survival of mice. Not only we demonstrated specific cell killing of BCL2/MYC co-expressing lymphoma cells in response to the combination treatment; but also, we revealed, for the first time in DLBCL, that venetoclax can promote the enrichment of activated intratumoral effector/effector memory CD8+ T cells, exhibiting additional immunomodulatory potential in the DEL-DLBCL tumor microenvironment. In addition, we demonstrated that acceleration of lymphomagenesis was evidenced when MYC expression was enforced from early stages of germinal center reaction. However, this lymphomagenesis was accompanied by the appearance of non-B-cells gastrointestinal tumors, possibly driven by the leakage of MYC expression, hence, resulting resistant to the conventional anti-CD20 immunotherapeutic regimen and to antibiotic treatment, impairing overall survival. Altogether, our results strongly support pre-clinical proof-of-concept and rationale for incorporating venetoclax to anti-CD20-based treatments to improve the outcome of aggressive DEL-DLBCL, and provide evidences for future combinations with specific MYC inhibitors, either incorporating them to the actual gold-standard R-CHOP or by dual targeting the Achilles heels of tumor cells through combined BCL2 and MYC specific inhibition. These preclinical combinations could either be performed in our murine models or in the novel 3D culture technique that we have successfully implemented here, which allows the long-term culture of lymphoma cells with its tumor microenvironment in the form of tumor spheroids and could serve as an ex vivo platform for future screening of novel therapeutic strategies in DLBCL.