Lymphoid-like Environment, Which Promotes Proliferation and Induces Resistance to BH3-Mimetics, Is Counteracted By Obinutuzumab in MCL: Biological Rationale for the Oasis Clinical Trial
Authors: Chiron David , Bellanger Celine , Papin Antonin , Tessoulin Benoit , Dousset Christelle , Maiga Sophie , Moreau Anne , Esbelin Julie , Trichet Valerie , Chen-Kiang Selina , Moreau Philippe , Touzeau Cyrille , Le Gouill Steven , Amiot Martine , Pellat-Deceunynck Catherine 
Published: November 2019

Abstract
Mantle cell lymphoma (MCL) accumulates in lymphoid organs but disseminates early on in extranodal tissues. However, there is relatively little information regarding the nature of surrounding cells and soluble factors and the resulting molecular regulations induced in MCL. Further investigations that integrate the key role of the microenvironment are now needed to overcome tumor drug resistance in protective niches. The benefits of such an approach have been recently reinforced by studies reporting that selective inhibition of BTK or PI3Kδ, critical kinases of the BCR and CXCR4 pathways, prevents the homing of MCL, leading to peripheral lymphocytosis. However, the consequences of cell egress in the peripheral blood on proliferation and survival are still unclear and need further investigations.
In spite of the significant level of the proliferation index Ki67 in secondary lymphoid organs and the elevated percentage of circulating malignant cells, we did not detect any proliferating peripheral blood MCL cells, which were arrested in the G1-phase of the cell cycle. The proliferation remains located in lymphoid organs only, suggesting a major role of the tumor ecosystem. In situ, lymphoma B-cells are in close contact with cells of mesenchymal or immune origin. To determine whether stromal (hMSC) or lymphoid-like (CD40L) interactions could support survival and proliferation of MCL, primary circulating MCL cells from 21 patients were cocultured several weeks ex vivo. In all samples tested we showed that CD40L signal, but not hMSC, induced cell-cycle progression, which was amplified by a MCL-specific cytokine cocktail, defined by assessment of cytokine receptor expression in situ. Using this system, we were able to maintain proliferation of primary MCL cells for several weeks, and most MCL samples remained dependent on the extrinsic signal over time.
Of major interest, using RNA-seq, we showed that 70% of genes induced ex vivo are expressed in lymph nodes, confirming the relevance of the coculture. We further observed that our model recapitulated in situ MCL molecular signatures such as proliferation, BCR/NFkB and survival signatures.
Among the genes related to survival, coculture resulted in anti- and pro-apoptotic Bcl-2 family member modulation. We demonstrated that an anti-apoptotic Bcl-xL protein increase was associated with a striking downregulation of pro-apoptotic Bim, Bax and Bak. Of note, this unbalanced regulation seemed to be specific to MCL cells as it did not occur in naïve CD5+ B cells or memory B cells. We provided evidences that Bcl-xL upregulation was responsible for loss of mitochondrial priming and drug resistance. Using BH3-profiling, we showed a dynamic sequestration of the BH3-only activator Bim by Bcl-xL proteins at the mitochondrial level. In addition, we showed that the mitochondrial priming directly correlated with the sensitivity toward BH3-mimetics (venetoclax) and alkylating drugs (bendamustine). Our results thus highlight molecular basis for microenvironment-dependent modulation of the mitochondrial priming and consequent drug resistance in primary MCL cells.
Thus, whereas venetoclax is highly efficient in inducing apoptosis in peripheral blood MCL cells, tumor cells protected into their niches could be resistant and in fine induce relapse. Using our ex vivo model, we further demonstrated that, in rational combinations, anti-CD20 antibody (obinutuzumab) can suppress survival and overcomes drug resistance to venetoclax exploiting NFkB/Bcl-xL-specific dependence of primary MCL cells. Moreover, ibrutinib, which mediates indirect Bcl-xL down-modulation upon BTK-dependent lymphocytosis, could also increase in vivo venetoclax efficacy. Our ongoing OAsIs Trial for MCL patients (obinutuzumab, ibrutinib and venetoclax, NTC#02558816) will rapidly determine in vivoefficacy.
In summary, we reported here the development of a reproducible ex vivo coculture model for primary MCL cells. This model has provided new insights into microenvironment-dependent proliferation and Bcl-2 family regulation, which are central components of survival and drug resistance. Our increased understanding of intrinsic abnormalities, the development of highly selective inhibitors and integration of the microenvironment offer new opportunities to design mechanism-based strategies that should overcome drug resistance in MCL and potentially other B cell malignancies

Disclosures
Moreau: Janssen: Honoraria, Speakers Bureau; Amgen: Honoraria; Bristol-Myers Squibb: Honoraria; Celgene: Honoraria; Novartis: Honoraria; Takeda: Honoraria.