Predictive biomarkers for Bcl2-inhibitor use in neuroblastoma

Apoptosis commitment is regulated by the interaction of Bcl2-family proteins at the mitochondria. Survival proteins such as Bcl2 and Mcl1 bind stress-activated BH3 proteins (like Bim, Puma and Bid), to prevent them from activating the obligate executioner proteins Bak and Bax and provides a survival advantage. Such apoptosis evasion is a cancer hallmark. Neuroblastoma (NB) is a highly lethal childhood tumor in which therapy resistance contributes to treatment failure. We used mitochondrial profiling and co-IP to define NB survival dependencies and develop biomarkers to predict response to Bcl2-inhibitors (BCL2i) such as Venetoclax as well as newer Mcl1 inhibitors (MCL1i). Mitochondrial profiling was used to interrogate isolated mitochondria with diverse BH3-only proteins to define their stress response “set-point” and infer their survival protein dependency. NBs can be classified as Bcl2- or Mcl1-dependent. The former have Bim sequestered by Bcl2 (confirmed by coIP) and a dominant mitochondrial response to BikBH3, the latter have Bim sequestered by Mcl1 (Bim:Mcl1 by coIP) and a dominant NoxaBH3 response. While Bcl2 and Mcl1 proteins are frequently co-expressed, a dominant survival protein is identified functionally. Bcl2-dependent NBs are highly sensitive to BCL2i’s (Venetoclax and Navitoclax) with an IC50 2 uM). Surprisingly, no NB was sensitive to the MCL1i, S63845, as monotherapy, including the Mcl1-dependent subset. However, S63845 markedly sensitizes Mcl1-dependent NBs to BCL2i’s (shifting IC50>1 log in all tested). Ongoing mechanistic work tests a hierarchical model in which Bcl2 can sequester Bim displaced from Mcl1 (by an MCL1i), sensitizing a previously Mcl1-dependent NB to a BCL2i. In contrast, Bim displaced from Bcl2 by a BCL2i (in a Bcl2-dependent NB) does not bind Mcl1 but ostensibly interacts with Bak/Bax to induce death. To measure Bim:Bcl2 and Bim:Mcl1 as a predictive biomarker we are developing proximity ligation assays (PLAs) for use with FFPE-tumor slides. NB xenografts of defined survival dependency are being used to optimize these assays. Heterogeneous survival dependencies define NB sensitivity to Bcl2-family inhibitors. We propose a hierarchical binding model where BCL2i monotherapy sensitivity is defined by Bim:Bcl2 binding (Bcl2-dependency). Mcl1-dependent NBs are resistant to MCL1i’s, yet by displacing Bim these agents restore sensitivity to BCL2i’s. Lastly, development of clinically-relevant biomarker assays to define Bim binding will allow stratification of patients in clinical trials with Bcl2-family inhibitors. Citation Format: Claudia Zapata, Jorida Coku, Kangning Liu, Annette Vu, Michael Goutnik, C. Patrick Reynolds, Kelly Goldsmith, Michael Hogarty. Predictive biomarkers for Bcl2-inhibitor use in neuroblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2504.