Aggressive B-cell Lymphoma with MYC/TP53 Dual Alterations Displays Distinct Clinicopathobiological Features and Response to Novel Targeted Agents

Abstract

Diffuse large B-cell lymphoma (DLBCL) is the major type of aggressive B-cell lymphoma. High-grade B-cell lymphoma (HGBCL) with MYC/BCL2 double-hit (DH) represents a distinct entity with dismal prognosis after standard immunochemotherapy in the current WHO lymphoma classification. However, whether TP53 mutation synergizes with MYC abnormalities (MYC rearrangement and/or Myc protein overexpression) contributing to HGBCL-like biology and prognosis is not well investigated. In this study, patients with DLBCL with MYC/TP53 abnormalities demonstrated poor clinical outcome, high-grade morphology, and distinct gene expression signatures. To identify more effective therapies for this distinctive DLBCL subset, novel MYC/TP53/BCL-2-targeted agents were investigated in DLBCL cells with MYC/TP53 dual alterations or HGBCL-MYC/BCL2-DH. A BET inhibitor INCB057643 effectively inhibited cell viability and induced apoptosis in DLBCL/HGBCL cells regardless of MYC/BCL2/TP53 status. Combining INCB057643 with a MDM2-p53 inhibitor DS3032b significantly enhanced the cytotoxic effects in HGBCL-DH without TP53 mutation, while combining with the BCL-2 inhibitor venetoclax displayed potent therapeutic synergy in DLBCL/HGBCL cells with and without concurrent TP53 mutation. Reverse-phase protein arrays revealed the synergistic molecular actions by INCB057643, DS3032b and venetoclax to induce cell-cycle arrest and apoptosis and to inhibit AKT/MEK/ERK/mTOR pathways, as well as potential drug resistance mechanisms mediated by upregulation of Mcl-1 and RAS/RAF/MEK/ERK pathways. In summary, these findings support subclassification of DLBCL/HGBCL with dual MYC/TP53 alterations, which demonstrates distinct pathobiologic features and dismal survival with standard therapy, therefore requiring additional targeted therapies. IMPLICATIONS: The clinical and pharmacologic studies suggest recognizing DLBCL with concomitant TP53 mutation and MYC abnormalities as a distinctive entity necessary for precision oncology practice. VISUAL OVERVIEW: http://mcr.aacrjournals.org/content/molcanres/19/2/249/F1.large.jpg.

Figure 1.

Prognostic effects of TP53 mutation (Mut-TP53) in patients with and without Myc overexpression (Myc^high). Myc^high and Mut-TP53 dual abnormality was associated with significantly inferior OS and PFS in overall cohort (A) and the ABC (B) and GCB (C) subtypes.

Figure 2.

Prognostic effects of TP53 mutation (Mut-TP53) and p53 overexpression (p53^high) in patients with MYC rearrangement (MYC-R). A, Patients with DLBCL with concurrent MYC-R and p53^high displayed poor OS and PFS but showed little additive negative effect to that of single MYC-R or p53^high abnormality. B, Concurrent Mut-TP53 and MYC-R synergistically worsened the OS and PFS in DLBCL. C and D, TP53 mutation had significantly adverse impact independent of MYC-R status.

Figure 3.

Cytotoxicity and proteomic effects of a BET inhibitor INCB057643 in DLBCL/HGBCL cell lines with MYC rearrangement or Myc overexpression and with or without TP53 mutation. A total of 8 DLBCL/HGBCL cell lines were selected for study, including 4 Wt-TP53 (OCI-LY10, OCI-LY19, MCA, RC) and 4 Mut-TP53 (GR, HBL1, MZ, and TMD8) cell lines. A, Cell viability of lymphoma cells was significantly inhibited after exposure with INCB057643 for 72 hours using CellTiter-Glo 2.0 Assay. B, IC₅₀ values of INCB057643 in 8 DLBCL/HGBCL cell lines as calculated by GraphPad Prism 8. C, Percentage of apoptotic cells by Annexin V/PI double staining flow cytometry analysis after treatment with INCB057643 for 48 hours. Values indicate mean ± SD for at least three independent experiments performed in triplicate. D, Heatmaps illustrating significantly increased or decreased protein expression measured by the RPPA assays after INCB057643 treatment (2.5 μmol/L for 24 hours) in RC and TMD8 cell lines. E, Venn diagrams (right) illustrate the common and unique modulations in three evaluated cell lines (OCI-LY19, RC, and TMD8). RPPA assays were performed for three independent experiments.

Figure 4.

Cotreatment of INCB057643 with DS3032b or venetoclax showed significant and synergistic cytotoxicity on HGBCL-DH cells with Wt-TP53. OCI-LY10, OCI-LY19, MCA, and RC all are HGBCL cells with concurrent MYC-R, BCL2-R, and Wt-TP53 (A) DS3032b combined with INCB057643 induced remarkable cell viability loss in OCI-LY10, OCI-LY19, MCA, and RC cells. B, INCB057643 and DS3032b combination induced higher percentage of apoptosis than single-agent measured in all cells. C, Combined venetoclax and INCB057643 synergistically inhibited cell viability. D, The combination index (CI) of INCB057643 and venetoclax was calculated by CompuSyn software according to the Chou–Talalay method, where CI < 1 indicates synergy. Cell viability was assessed by CellTiter-Glo 2.0 Assay at 72 hours. Apoptosis was determined by Annexin V/PI staining at 48 hours. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001; ns, not significant. Values indicate mean ± SD for at least three independent experiments performed in triplicate.

Figure 5.

Combination therapy with INCB057643 and venetoclax in DLBCL/HGBCL cells with concurrent MYC alterations and Mut-TP53. A, Cotreatment with venetoclax and INCB057643 synergistically inhibited cell viability in four DLBCL/HGBCL cell lines with concurrent TP53 mutations and MYC-R (MZ and TMD8) or Myc^high (GR and HBL1). B, The combination index (CI) of INCB057643 and venetoclax was calculated by CompuSyn software according to the Chou–Talalay method, where CI < 1 indicates synergy, = 1 is additive, and > 1 is antagonistic effect. C, Apoptosis in GR, HBL1, MZ, and TMD8 was measured by Annexin V/PI staining after treatment with venetoclax, INCB057643 and the combination for 48 hours at designated concentration. The data are presented as mean ± SD for at least three independent experiments performed in triplicate. “ns” indicates not significant; *, P < 0.05; **, P < 0.01; ****, P < 0.0001. D, Western blot analysis to validate RPPA results, including proteins with roles in mitosis and cell growth (Myc, Aurora-A, PLK1, and CDC2), apoptotic indicator cleaved PARP, caspase-3, p53, and p53-targeted p21. β-Actin was probed as loading control. Four DLBCL/HGBCL cell lines with mutant TP53 were exposed to single-agent or combined venetoclax (3.125 nmol/L for GR; 6.25 nmol/L for HBL1; 25 nmol/L for MZ; and 3.125 nmol/L for TMD8) and INCB057643 (2.5 μmol/L for GR; 1.25 μmol/L for HBL1, MZ, and TMD8) treatment for 24 hours. Combined INCB057643 and venetoclax treatment decreased expression of Myc, Aurora-A, and the phosphorylation of downstream targets CDC2 and PLK1, and increased cleaved PARP and p21 expression.