Overexpression of CDCA2 in Diffuse Large B-Cell Lymphoma Promotes Cell Proliferation and Bortezomib Sensitivity

Abstract

Numerous clinical trials have attempted to improve first-line R-CHOP treatment of diffuse large B-cell lymphoma (DLBCL) through the addition or substitution of drugs. The REMoDL-B trial, testing the addition of bortezomib (RB-CHOP), revealed that ABC and molecular high-grade DLBCL patients benefit from bortezomib. The aim of this study was to achieve a better understanding of the bortezomib response in DLBCL through a functional investigation of clinically identified markers. A retrospective analysis of transcriptional and clinical data from the REMoDL-B trial was conducted to identify genes associated with bortezomib response, identifying CDCA2. DLBCL patients with high expression of CDCA2 had a superior survival outcome when treated with RB-CHOP in comparison to R-CHOP, whereas no difference in outcome was observed for patients with low CDCA2. Moreover, CDCA2 was found to be overexpressed in DLBCL compared to non-malignant tissue, and to have higher levels in GCB and MYC/BCL2 double-expressor patients. Functional in vitro and in vivo studies revealed that knockout of CDCA2 decreased DLBCL cell proliferation and a bortezomib dose-response analysis showed less sensitivity in CDCA2 knockout cells compared to control cells. This study shows that DLBCL patients with high CDCA2 expression benefitted from the addition of bortezomib to R-CHOP and functional studies documented a direct impact of CDCA2 on the bortezomib response in DLBCL cells.

Keywords: CDCA2; CRISPR; DLBCL; RB-CHOP; bortezomib; diffuse large B-cell lymphoma; treatment response.

Figure 1

CDCA2 expression. (A) CDCA2 mRNA expression in non-malignant lymph node (LN) and tonsil (TN) tissues, diagnostic DLBCL samples, and DLBCL cell lines. (B) CDCA2 expression in ABC/GCB classified patients of the REMoDL-B cohort. (C) CDCA2 expression in MYC/BCL2 double- and non-double-expressor patients of the REMoDL-B cohort. (D) CDCA2 expression in normal B-cell subsets FACS sorted from healthy tonsils. Wilcoxon test. * p  ≤  0.05; ** p  ≤  0.01; *** p  ≤  0.001; **** p  ≤  0.0001. ABC, activated B-cell-like; CB, centroblast; CC, centrocyte; GCB, germinal-center B-cell-like; M, memory; N, naïve; PB, plasmablast.

Figure 2

Survival analysis. Kaplan–Meier plots presenting the overall survival of DLBCL patients from the REMoDL-B cohort. Patients grouped by median split of CDCA2 mRNA expression irrespective of treatment regimen. (A) Patients with low CDCA2 expression. (B) Patients with high CDCA2 expression. (C) Patients treated with R-CHOP. (D) Patients treated with RB-CHOP.

Figure 3

Functional investigation of CDCA2. CDCA2 knockout cells were generated by RNP nucleofection (KO-1 and KO-2) and by lentiviral transduction (−/− #cloneID). (A) Knockouts validated by indel analysis and Western blotting. (B) Proliferation examined by MTS presented relative to SCR. (C) Flow cytometry assessment of cell cycle phases. Percentages of cells in the G1-phase are depicted. (D) Mean fluorescence intensity (MFI) of phosphorylated AKT (pAKT, Ser473), normalized to AKT levels. (E) Dot plot of tumor sizes (mm) for each OCILY7 xenograft mice measured every 3rd-4th day. (F) Survival (%) in days from time of injection for xenografts. (G) Ki-67 intensity normalized to DAPI. Mean value of 10 images per sample. Values are presented as mean ± standard deviation. * p  ≤  0.05; ** p  ≤  0.01; *** p  ≤  0.001; **** p  ≤  0.0001.

Figure 4

Drug response analysis of CDCA2-KO cells. (A–C) Bortezomib dose–response screens. Cell viability assessed by MTS and presented relative to the untreated control. (D,E) Apoptosis examined by flow cytometry upon bortezomib exposure. Apoptotic cells (Apop+) including early and late apoptotic stages presented relative to the untreated control. (F) CHOP and B-CHOP dose–response analysis in OCILY7. Cell viability assessed by MTS and presented relative to untreated control. KO-1 and KO-2 are heterogenous cell populations created by RNP nucleofection. −/− #cloneID represents monoclonal populations established by lentiviral transduction followed by single cell expansion. Values are presented as mean ± standard deviation. * p  ≤  0.05; ** p  ≤  0.01; *** p  ≤  0.001; **** p  ≤  0.0001.

Figure 5

(A) Proteasomal activity of SCR and CDCA2-KO OCILY7 cells upon exposure to 3.5 ng/mL bortezomib for 5 h. Caspase, chymotrypsin, and trypsin activity presented as luminescence read-out of bortezomib cells relative to untreated cells. (B) Proliferation of SCR and CDCA2-KO OCILY7 cells examined by MTS. Cells were exposed to saline or 10 nM LY294002 for 48 h. Data presented relative to SCR in untreated condition. (C) Combinatory dose–response analysis of bortezomib and LY294002 (10 nM). Cell viability assessed by MTS and presented relative to untreated. −/− #cloneID represents monoclonal populations established by lentiviral transduction followed by single cell expansion. Values are presented as mean ± standard deviation. *** p  ≤  0.001; **** p  ≤  0.0001.