Abstract 3164: Identifying Bcl-2 family protein dependencies in tumors using dimerization specific antibody biomarkers as a method for predicting response to apoptosis inducing therapies
Authors: Karakas Bahriye , Jean Sae Rin , Narovlyansky Max , Kumar Sonia , Gillies Stephen , Krajewski Stan , Lim Bora , Cardone Michael 
Published: August 2019

Here, we present a novel method for detecting heterodimer complexes consisting of anti-apoptotic and pro-apoptotic Bcl-2 family proteins as indicators of cancer cell apoptotic priming state. The readout of Bcl-2 family complex-specific biomarkers is seen to identify specific survival dependencies in leukemia and breast cancer cells suggesting utility for guiding cancer treatments.

Methods: Engineered immunogens that recapitulate conformation-specific epitopes induced during binding of the Bcl-xL/Bim protein complex were made. Mice were immunized with selected immunogen and the resulting monoclonal antibodies that exclusively bound to the conformation-induced epitope were identified. One Heterodimer Specific Bcl-xL Bim (HSBXB) was chosen. The selective binding of HSBXB was confirmed using ELISA, FP (fluorescence polarization), IF (immunofluorescence) in Bim or Bcl-xL knockdowns, Bcl-xL knock out cell line, as well as immunohistochemistry (IHC) in formalin-fixed paraffin-embedded (FFPE) patient tissue. Correlation of HSBXB measurements to BH3 profiling readouts from the Bcl-xL restricted Hrk BH3 containing peptide was explored, including shifts in signals after treatment with Bcl-xL-specific BH3 mimetic compounds.

Results: We have demonstrated the dependency of the HSBXB signal on both Bcl-xL and Bim protein levels in in vitro and ex vivo experiments. Multiplexed two-color IHC staining in cell blocks comprised of breast cancer and AML cell lines showed that the measurement of the ratio of heterodimer (Bcl-xL/Bim) to unbound protein (Bcl-xL) signal ([HSBXB]/[Bcl-xL]) aligned with flow-based BH3 profiling readouts. The [HSBXB]/[Bcl-xL] readout was also taken in AML and CLL patient samples using flow cytometry. A strong correlation (p=0.003) was seen between the [HSBXB]/[Bcl-xL] ratio and the Hrk readout of BH3 profiling assay. The [HSBXB]/[Bcl-xL] readout also shifted following treatment of cells with Bcl-xL specific BH3 mimetics. Further, the dynamic range of the [HSBXB]/[Bcl-xL] signal ratio was demonstrated across triple-negative breast cancer FFPE samples. The [HSBXB]/[Bcl-xL] readout potentially provides a functional biomarker for cancer cell sensitivity to a wide range of Bcl-xL-targeted compounds in solid tumors.

Conclusion: This work provides a basis for further studies into the Bcl-xL dependence as a predictive biomarker for Bcl-xL targeted therapies, and/or therapies that are resistant in Bcl-xL dependent cells. IHC based measurement of a heterodimer complex offers easier access in comparison to other assay platforms, thus harbors a potential for use in clinical settings. Additional antibodies targeting Bcl-2 family complexes as predictive biomarkers for Mcl-1 dependence in solid and liquid tumors have been identified.

Citation Format: Bahriye Karakas, Sae Rin Jean, Max Narovlyansky, Sonia Kumar, Stephen Gillies, Stan Krajewski, Bora Lim, Michael Cardone. Identifying Bcl-2 family protein dependencies in tumors using dimerization specific antibody biomarkers as a method for predicting response to apoptosis inducing therapies [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 3164.