PS1321 INHIBITION OF NOTCH SIGNALING RESTORES SENSITIVITY TO IMMUNOCHEMOTHERAPY IN AN IN VITRO MODEL OF ACQUIRED RESISTANCE TO R‐CHOP

Abstract

Background:Diffuse large B‐cell lymphoma (DLBCL) accounts for almost 40% of all lymphomas. One‐third of DLBCL patients either initially fail to respond to Rituximab plus anthracycline based therapy CHOP (R‐CHOP) or suffer a relapse (i.e. acquired resistance). The Notch pathway is involved in cell proliferation, differentiation and survival in a wide range of cancers.Aims:We investigated the role of Notch signaling in an in vitro model of acquired resistance to R‐CHOP treatment.Methods:We developed an in vitro model of acquired R‐CHOP resistance using different DLBCL cell lines (SU‐DHL‐4, SU‐DHL‐10 for GCB‐like subtype; RIVA and U2938 for ABC‐like subtype). Using gene expression microarrays, we screened parental and R‐CHOP resistant cell lines to identify putative genes responsible for acquired resistance. Notch1/2 and JAG1 expression levels were validated by qPCR and flow cytometry in R‐CHOP resistant U2932 cells. We treated U2932 cells with a neutralising anti‐JAG1 antibody (clone J1–65D, 10ug/ml) or with the γ‐secretase inhibitor dibenzazepine (DBZ, 100 nM). We then evaluated cell viability after 72 h of R‐CHOP treatment at 400 ng/ml (corresponding to the GI50 of the resistant clones).Results:Our microarray analysis showed many genes were differentially expressed. We focused particularly on the U2932 cell line, where overexpression of NOTCH1, NOTCH2 and JAG1 were independently confirmed by qPCR and flow cytometry. We evaluated the viability of U2932 after 72 h treatment with R‐CHOP and we found that the treatment of the resistant clones with DBZ or anti‐JAG1 restored sensitivity to RCHOP, compared to DMSO (vehicle) or IgG controls.Summary/Conclusion:We have demonstrated that blocking of Notch signaling, either by preventing JAG1 ligand binding or impeding the cleavage of the intracellular Notch domain, can restore sensitivity to R‐CHOP at levels comparable to the parental control. These preliminary results may suggest Notch signaling as new therapeutic target for resistant DLBCL.