Abstract 2526: The Bcl-2 family proteins in CLL: Effects by the cytogenetics, microenvironments and therapy

Abstract

Abstract The balance between the pro- and anti-apoptotic members of the Bcl-2 family proteins governs the fate of CLL cells and determines their response to therapeutics. Thus, we have developed an assay to quantify the Bcl-2 family proteins at the molecular level and express them as molecules/pg total protein. This set of proteins represents the 10 major Bcl-2 members in CLL: Bcl-2, Bcl-XL, Mcl-1, BIM, BID, BMF, BAX, BAK, PUMA and Noxa. This analysis enabled us to evaluation of the interplay of these proteins and how they respond to cytogenetic aberrations, drug treatment or environmental changes. In 36 CLL samples that represent 6 districted cytogenetic features: normal cytogenetics, del17p, del11q, del13q14, 11q/13q double deletion, and trisomy 12, we found that the major anti-apoptotic proteins in CLL is Bcl-2, that averaged >9,000 molecules/pg of total protein. Mcl-1 was expressed at less than one tenth of Bcl-2 and Bcl-XL was expressed less than one percent of Bcl-2 molecules. The major pro-apoptotic proteins were Bax and Puma which were expressed at similar levels to Bcl-2. Among the cytogenetic groups, there were significant increases of Bcl-2 in the 11q/13q group whereas Mcl-1 was increased in 11q and trisomy 12 groups compared to samples with normal cytogenetic characteristics. Next, we screened CLL response to different microenvironments by incubating primary CLL cells in vitro in media supplemented with either fetal bovine serum (FBS), patient autologous plasma, a layer of StromaNKtert cells representing the stroma cell support in the bone marrow, a B-cell activating media (BCA) that consisted of anti-CD40, IL-4 and anti-IgM to mimic the lymphoid microenvironment, or oligodeoxynucleotide (ODN) plus IL-15 to represent the T-cell independent stimulation in the lymph nodes. Compared to FBS, homologous patient plasma moderately promoted CLL viability. However, stroma cells, BCA media as well ODN+IL-15 greatly induced levels of Mcl-1 and Bcl-XL and were more effective in promoting CLL survival. For the pro-apoptotic proteins, BMF was induced by BCA media, and PUMA, NOXA and BID were greatly induced by ODN+IL-15. We also examined the changes in Bcl-2 family proteins in response to therapeutic agents with different mechanisms of action, such as the CDK9 inhibitor fadraciclib, the Bcl-2 antagonist venetoclax, the Mcl-1 inhibitors AZD5991 and S63845, the proteasome inhibitor carfilzomib, the BTK inhibitor ibrutinib and the splicing inhibitor herboxidiene. Fadraciclib and herboxidiene were effective in reducing Mcl-1 to induce apoptosis. While both the Mcl-1 inhibitors and carfilzomib increased the Mcl-1 level, and carfilzomib increased Noxa that counteracted its effect on Mcl-1. Puma was reduced by most of the drugs except ibrutinib. Thus, the systematic analysis of the Bcl-2 family proteins provide insights on how the interplay of members of this important family affects the destiny of the CLL cells. Citation Format: Rong Chen, Ping Xiong, Chaomei Liu, Yuling Chen, Yingjun Jiang, Varsha Gandhi, Nitin Jain, William Wierda, William Plunkett. The Bcl-2 family proteins in CLL: Effects by the cytogenetics, microenvironments and therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2526.