Background: BRD4 functions as an important epigenetic regulator of transcription of pathways commonly involved in oncogenesis, including cell cycle progression and apoptosis. Inhibition of BRD4 with small molecule inhibitors has demonstrated activity in various hematologic and solid malignancies. While downregulation of c-myc following BRD4 inhibition has been described, the exact mechanism of action of BRD4 inhibitors across B cell malignancies remains unclear. Here we demonstrate the efficacy of PLX51107 across a spectrum of B cell malignancies and the signaling pathways involved.
Methods: A panel of 22 human multiple myeloma (MM) cell lines and 56 B cell lymphoma (BCL) cell line, were evaluated for the effects of PLX51107 on cell proliferation after a 5 day exposure to the drug. Sensitive NCI-H929, MM.1S, KMS-12BM and resistant RPMI, EJM, L363 - MM cell lines were selected for further mechanism of action (MOA) studies; along with BCL sensitive DoHH2- DLBCL GCB subtype, Ly3- DLBCL ABC subtype, MINO-mantle cell, 2A8-Burkitt. Matched resistant cell lines SUDHL-8, U2932, GRANTA, DB were also selected for the MOA studies. All MOA assays were performed by treating the MM cells at a dose of 150nM PLX51107 except the BCL cells which were exposed to 300nM. Cell cycle analysis and induction of apoptosis was performed by FACS after propidium iodide or Annexin V FITC staining respectively on cells treated for 72 hours. Cellular senescencewas determined by measurement of β-galactosidase activity in cells treated for 7 days (BCL) or 5 days (MM). Western blot analyses for c-myc, p-IRAK, p-Iκβα, p65 and p-MAPK Erk1/2 were performed at 4 and 24 hours post treatment. DMSO treatment was included as a control in the MOA experiments.
Results: PLX51107 potently inhibited the in vitro proliferation and survival of a variety of B cell malignancies, including GCB and ABC subtype DLBCL, mantle cell lymphoma, Burkitt lymphoma, and multiple myeloma cell lines.Cell cycle arrest in G1 phasewas observed in 3/4 sensitive BCL, Ly3, 2A8 and MINO and 2/3 sensitive MM, NCI-H2929, MM1.S. Increased apoptosis was demonstrated in the sensitive cell lines 2A8, MINO (BCL) and NCI-H2929, KMS12BM (MM). Three sensitive BCL (DoHH2, Ly3, 2A8) and 2 sensitive MM (KMS12BM, MM1.S) displayed premature senescence. Interestingly, DoHH2 (DLBCL-GCB), Ly3 (DLBCL-ABC) and the MM cell line MM1.S, all of which were growth inhibited by PLX51107 treatment, did not demonstrate an increase in apoptosis and all were found to have the greatest degree of premature senescence. Our data indicate that these differential mechanistic findings do not correspond with a known underlying predictive biomarker. In contrast to the resistant cell lines, all sensitive lines demonstrated marked downregulation of c-myc expression after PLX51107 treatment. Downregulation of p-IRAK, a signaling intermediary of the MyD88-dependent pathway, was also observed in all sensitive BCL cell lines, while no corresponding change of this marker was observed in the sensitive MM cell lines. P-MAPK Erk 1/ 2 was downregulated in all sensitive BCL cell lines, but only 1 of 3 sensitive MM cell lines. Expression of p-Iκβα, a regulator of the NFκʙ pathway, was unchanged in all sensitive BCL cell lines but was downregulated in all 3 sensitive MM cell lines.
Discussion: PLX51107 demonstrates in vitro efficacy through differential mechanisms across a maturational spectrum of B-cell malignancies. Treatment of BCL and MM cell cultures results in downregulation of c-myc in sensitive cell lines, consistent with findings from previous BRD4 inhibitors. We demonstrate the involvement of novel signaling pathways with PLX51107 treatment that vary based on the maturational stage of the malignant B cell. These data suggests treatment with PLX51107 affects the MyD88-dependent toll-like receptor pathway in the BCL cell cultures, while the NFκʙ pathway is changed in MM cell cultures. In conclusion, these results demonstrate that PLX51107 has distinct mechanisms of action beyond downregulation of the c-myc transcriptional program and is a promising targeted therapeutic agent across a broad range of clinically and histologically diverse B-cell neoplasms.
Powell:Plexxikon: Employment. Slamon:Pfizer: Equity Ownership, Research Funding; BioMarin: Membership on an entity's Board of Directors or advisory committees, Research Funding. Larson:BMS: Consultancy.
Asterisk with author names denotes non-ASH members.