Bortezomib resistance in mantle cell lymphoma is associated with plasmacytic differentiation

Abstract

Bortezomib induces remissions in 30%-50% of patients with relapsed mantle cell lymphoma (MCL). Conversely, more than half of patients' tumors are intrinsically resistant to bortezomib. The molecular mechanism of resistance has not been defined. We generated a model of bortezomib-adapted subclones of the MCL cell lines JEKO and HBL2 that were 40- to 80-fold less sensitive to bortezomib than the parental cells. Acquisition of bortezomib resistance was gradual and reversible. Bortezomib-adapted subclones showed increased proteasome activity and tolerated lower proteasome capacity than the parental lines. Using gene expression profiling, we discovered that bortezomib resistance was associated with plasmacytic differentiation, including up-regulation of IRF4 and CD38 and expression of CD138. In contrast to plasma cells, plasmacytic MCL cells did not increase immunoglobulin secretion. Intrinsically bortezomib-resistant MCL cell lines and primary tumor cells from MCL patients with inferior clinical response to bortezomib also expressed plasmacytic features. Knockdown of IRF4 was toxic for the subset of MCL cells with plasmacytic differentiation, but only slightly sensitized cells to bortezomib. We conclude that plasmacytic differentiation in the absence of an increased secretory load can enable cells to withstand the stress of proteasome inhibition. Expression of CD38 and IRF4 could serve as markers of bortezomib resistance in MCL. This study has been registered at https://ichgcp.net/clinical-trials-registry/NCT00131976" title="See in ClinicalTrials.gov">NCT00131976.

Figures

Figure 1

Bortezomib-adapted cell lines have higher proteasome activity and tolerate lower chymotrypsin-like proteasome activity than PT cells. (A) Dose response to bortezomib (Bzm) in PT and BR cell lines. Viability by MTT assay at 48 hours normalized to the carrier control is shown. (B) Reversibility of bortezomib resistance in BR cell lines cultured in the absence of bortezomib. IC50 values were determined as shown in panel A at the indicated time points after bortezomib washout. (C) Baseline proteasome activity in PT and BR cell lines. Chymotrypsin activity was measured at 3 different time points over a 1-month period and data are normalized to the average activity in the corresponding PT cell line. Comparison was by Student t test: *P < .05; **P < .01. Trypsin and caspase-like activities were measured in duplicate and are shown for comparison. (D) Correlation between cell viability by MTT assay and chymotrypsin-like proteasome activity (arbitrary units) in PT and BR cell lines treated with increasing concentrations of bortezomib for 48 hours.

Figure 2

Bortezomib-adapted cell lines display a plasmacytic phenotype. (A-C) Gene expression signatures of plasma-cell differentiation distinguish bortezomib-adapted HBL2-BR (A, C) and JEKO-BR cell lines (B) from the respective PT cell lines. The leading edge of each gene set is displayed in a heat map. Changes in the expression of selected B-cell and plasma-cell surface markers were confirmed by flow cytometry. (D) Early differentiation genes variably expressed between the PT and BR cell lines. Of the 50 genes described in the signature, 43 were expressed in the MCL cell lines. Shown are genes with at least a 1.2-fold change in expression: down-regulation for genes expressed in B cells and up-regulation for genes expressed in plasma cells. (E) BLIMP and IRF4 expression in nuclear fractions from PT and BR cell lines assessed by Western blot using TBP as a loading control. (F) HBL2-BR cells were maintained in culture with (HBL2-BR) or without 100nM bortezomib (HBL2-BR Bzm washout) for 2 months. The expression of B-cell and plasma-cell surface markers assessed by flow cytometry in these and the PT cell line (HBL2-PT) are shown.

Figure 3

Intrinsically bortezomib-resistant MCL cell lines show plasmacytic features. (A) Dose response of bortezomib (Bzm) in JEKO, HBL2, MINO, and REC1 PT cell lines at 48 hours. The MTT assay was used as readout of viability. (B) Expression of the plasma-cell surface markers CD38 and CD138 measured by flow cytometry. (C) IRF4 expression assessed by Western blot in nuclear fractions using TBP as loading control, and by flow cytometry using the same antibody conjugated to Alexa 647. (D) Analysis of XBP1 mRNA expression by RT-PCR. The active, spliced form (XBP-1s) is shorter and runs faster on the gel than the inactive, unspliced form (XBP-1u). JEKO cells treated with thapsigargin (2μM) for 6 hours were used as the positive control (+) and glyceraldehyde-3-phosphate dehydrogenase (GADPH) for the loading control. (E) Detection of κ and λ immunoglobulins by sandwich ELISA in media containing 50 ng/mL of polyclonal human immunoglobulin G and in supernatants of MCL cell lines cultured for 24 hours at 1 × 106 cells/mL. The absorbance at 450 nm is shown for triplicates ± SD.

Figure 4

Plasma-cell differentiation and bortezomib sensitivity. HBL2-PT cells were treated for the times indicated with CpG ODN 2006. (A) Expression of CD19, CD138, and IRF4 was measured by flow cytometry. Triplicates and SD from 3 independent experiments are shown. (B) Detection of λ light chain by sandwich ELISA represented as the ratio between CpG-stimulated and unstimulated cells. Cell death in cells treated with 2.5nM bortezomib for 20 hours in the presence or absence of CpG was measured by flow cytometry using MitoTracker, and is shown as the ratio between CpG-stimulated and unstimulated cells. A representative experiment of 3 is shown. (C) Induction of ER-resident chaperones assessed by Western blotting in RIPA lysates using γ-tubulin for loading normalization. Densitometry measurements are given for the ratio between CpG-stimulated and unstimulated cells.

Figure 5

Addiction to IRF4 in MCL cells. (A) The MCL (JEKO and REC1), MM (H929), and GCB-DLBCL (OCI-LY19) cell lines were retrovirally transfected with a doxycycline-inducible, IRF4-targeting shRNA plasmid containing GFP. shRNA expression was induced by the addition of doxycycline (50 ng/mL) starting on day 0. The percentage of GFP+ cells was monitored by flow cytometry as a measure of live cells expressing shIRF4, and normalized to baseline (day 0). Four days after the addition of doxycycline, IRF4 knockdown was assessed by Western blot, normalized to TBP, and expressed as the ratio between cells transduced with shcontrol and shIRF4. (B) Expression of IRF4 coding region (open symbols) or the corresponding empty vector (black symbols) in REC1 (squares) and H929 (triangles) cells also transfected with shIRF4. Induction of shRNA expression and monitoring of transfected cells was as in (A). (C) REC1 cells expressing shcontrol or shIRF4 were selected with puromycin and induced for 4 days with doxycycline. After 24 hours with increasing bortezomib (Bzm) doses, cytotoxicity was analyzed by MTT.

Figure 6

CD38 and IRF4 expression in primary MCL cells is correlated with clinical response to bortezomib. (A) Reduction in the ALC on day 21 of one cycle of bortezomib (Bzm) therapy. (B) IRF4 protein expression in leukemic tumor cells from MCL patients assessed by Western blotting and normalized to TBP. Average of 2 independent determinations is shown. Statistical significance was evaluated using the Mann-Whitney nonparametric U test. (C) Mean fluorescence intensity (MFI) of CD38 surface expression was measured on CD19-gated cells using flow cytometry. Statistical significance was evaluated using the Mann-Whitney nonparametric U test. (D) Pearson correlation between IRF4 expression and CD38 surface expression in MCL patients.

Figure 7

A model for bortezomib resistance in MCL. Bortezomib sensitivity is shown as a function of secretory load relative to the capacity of a cell to deal with protein load. Bortezomib-resistant MCL cells progressing in differentiation through the GC acquire a plasmacytic phenotype that increases their capacity to deal with the protein load. In the absence of increased protein synthesis, this confers a survival advantage during proteasome inhibition. In fully differentiated plasma cells, the secretory load increases and sensitizes these cells to bortezomib.