Abstract P1-08-01: Signaling consequences and rational therapeutic combinations with glutaminase inhibitor, CB-839, in basal breast cancer
Authors: Dennison Jennifer B , Ju Zhenlin , Gordon Mills B 
Published: May 2015

Abstract
Purpose: Proliferation rates of basal breast cancers are dependent on glutamine metabolism as previously demonstrated by glutamine withdrawal or glutaminase inhibition by CB-839 in cell line models. Early and late adaptive signaling and metabolic responses to glutamine stress may enable cells to proliferate or evade cell death. We hypothesized that inhibition of these pathways would be synergistic in combination with glutaminase inhibition by CB-839.
Methods: Protein signaling responses in HCC1806 cells (basal A breast cancer) were quantified using reverse phase protein arrays (RPPA) at early (4 h) and late (24 h) time points in response to low glucose or low glutamine concentrations as well as treatment with CB-839, a selective glutaminase inhibitor. Signaling pathways evaluated included mTORC1/2, PI3K/AKT, MAPK, and AMPK. ADP and ATP were quantified by ion exchange chromatography. Drug combinations predicted by RPPA were evaluated at therapeutically relevant concentrations using proliferation or apoptosis assays after 72 h of continuous treatment.
Results: In agreement with previous reports of glutamine withdrawal, low glutamine and glutaminase inhibition resulted in inhibition of proliferation and early inhibition of mTORC1 signaling including pmTOR, pP70S6K, p4EBP1, and pS6. In contrast, for cells incubated in low glucose, proliferation and mTOR signaling were maintained at early time points despite robust pAMPK/pACC signaling and increased ADP/ATP ratios (2-orders of magnitude higher than baseline); mTOR signaling was only inhibited after 24 h when ATP concentrations were reduced. Bioenergetic stress as detected by pAMPK and pACC was not observed with glutaminase inhibition – ATP concentrations were increased by CB-839 treatment. Importantly, 2-deoxyglucose, a compound used to reduce ATP levels and activate AMPK signaling, was antagonistic in combination with CB-839. In contrast, hydroxychloroquine, an inhibitor of autophagy activated by mTOR inhibition, was synergistic with CB-839.
Consistent with mTORC1-mediated negative feedback loops, CB-839 treatment also increased AKT and ERK signaling. Combinations of CB-839 with AKT or MEK inhibitors (AZD5363 and GSK1120212B) were at synergistic and additive, respectively, for cell proliferation. The MEK inhibitor in combination with CB-839 resulted in supra-additive effects using apoptosis as an endpoint.
Conclusion: Glutaminase inhibition by CB-839 acutely inhibited mTORC1 signaling independently of AMPK signaling in HCC1806 cells. MEK and AKT signaling increased, likely in response to negative feedback loops with mTORC1. Combinatorial approaches with CB-839 targeting these compensation pathways resulted in increased responses supporting further preclinical and clinical studies.
Citation Format: Jennifer B Dennison, Zhenlin Ju, Mills B Gordon. Signaling consequences and rational therapeutic combinations with glutaminase inhibitor, CB-839, in basal breast cancer [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P1-08-01.