Marginal zone lymphomas (MZLs) are characterized by an indolent course, but relapses/progressions are common and might heavily impact patients' survival. Chimeric antigen receptor (CAR) T cells revolutionized the treatment of several relapsing/refractory B cell malignancies but their success in MZL is still limited and none of the currently available products has been approved so far for this indication. Little is known about the reasons behind the limited efficacy of CAR T cells against MZL. In this work we investigated the contribution of CD19 levels in MZL resistance to CAR T cells and we tested a strategy to increase sensitivity to CAR T cells through pharmacological modulation of CD19 expression.

mCherry/Luciferase-transduced MZL cell lines (VL51, HAIR-M, ESKOL, HC1, SSK41, Karpas1718) were co-cultured with untransduced T (UT) cells or CD19.CD28z CAR T cells at an E:T ratio of 1:1 adjusted to transduction efficacy and tumor burden at different time points was quantified by bioluminescence (BLI). Chronic BTK inhibition was obtained by constantly exposing VL51 to ibrutinib (20 uM) for more than 8 weeks. Epigenetic regulation of CD19 gene expression was analyzed using H3K4me3 CHIPseq and ATACseq.

We first employed a panel of 6 MZL cell lines to test their sensitivity to CD19 CAR T cells. After 3 days of co-culture, all cell lines displayed high sensitivity to CAR T cells with the only exception of VL51 cells that were totally resistant (Figure 1A). VL51 cells displayed the lowest CD19 expression (Figure 1B). Transduction of VL51 cells with truncated CD19 restored their sensitivity to CAR T cells demonstrating that low CD19 expression was their mechanism of resistance. We observed that prolonged BTK inhibition with ibrutinib led to CD19 upregulation on VL51 (Figure 1C). Higher H3K4me3 and higher chromatin accessibility at the CD19 gene transcription starting site were revealed by CHIPseq and ATACseq respectively, pointing to an epigenetic modulation of CD19 expression. According to high CD19 expression, ibrutinib-treated VL51 cells displayed high sensitivity to CAR T cell mediated tumor growth inhibition (Figure 1D).

CD19 modulation by BTK inhibition increased the sensitivity to CAR T cells in a CD19low MZL model. These data support exploring combination therapies for CAR T cell treatment of MZL.