Figure 6. CCND1 mutations in primary MCL.

(A) Sequencing chromatograms of CCND1 point mutations in MCL samples. WT and mutated CCND1 alleles on residue cysteine-47 (yellow bar) from MCL tumors 10, 11, 12 and 47 are shown. For MCL 47, complementary sequence from the reverse primer is shown. (B) CCND1 expression in MCL tumors. Lysates (10 μg per lane) from indicated MCL tumors were immunoblotted with indicated antibodies. Peripheral blood mononuclear cells (PBMC) were used as negative control for CCND1 expression. Bar graphs show relative densitometric values of CCND1 expression from the immunoblot after normalization to GAPDH loading control. Red arrows highlight the CCND1 mutated samples. (C) Analysis of CCND1 truncated 3′UTR transcript in MCL tumors. The relative abundance between the full-length and truncated 3′UTR was determined by using proximal and distal primers (see primer locations in the diagram of the CCND1 mRNA transcript). (D) CCND1 stability in MCL tumors. Indicated MCL tumors were treated with 10 μM of cyclohexamide (CHX) for indicated times and 10 μg of cell lysates were prepared for immunoblot analysis with indicated antibodies. Bar graphs show relative densitometric values of CCND1 expression from corresponding blots after normalization to GAPDH. (E) Sensitivity of MCL tumors to ibrutinib. Indicated MCL tumors were treated with indicated doses of ibrutinib for three days and metabolically active cells were assessed by CellTiter-Glo Luminescent assay (Promega). Shown are fractions of luminescence signals from metabolically active cells normalized to untreated samples. Line graphs show mean values from three independent experiments. Error bars, S.E.M. IC₅₀ values were calculated by GraphPad Prism 7 software. The table summarizes the ibrutinib response with respect to CCND1 mutation status or protein stability. Correlation of either mutation status or protein stability with ibrutinib sensitivity was analyzed by the two-sided Fisher's exact test.