Abstract P1-08-07: A novel pharmacodynamic assay to measure glutaminase inhibition following oral administration of CB-839 in triple negative breast cancer biopsies
Authors: MacKinnon Andy , Bennett Mark , Emberley Ethan , Gross Mathew , Janes Julie , Lewis Evan , Pan Alison , Rodriguez Mirna , Shwonek Peter , Wang Taotao , Yang Jinfu , Zhao Frances , Parlati Francesco 
Published: May 2015

Abstract
Triple negative breast cancer (TNBC) cell lines are highly dependent on glutamine (Gln) for growth and survival. A critical step in Gln utilization is its conversation to glutamate (Glu) by the mitochondrial enzyme glutaminase (GLS). CB-839 is a potent inhibitor of GLS that has anti-proliferative activity in TNBC cell lines and antitumor activity in TNBC xenograft models (Gross et al., Mol. Cancer Ther. 13:890). Across a panel of breast cancer cell lines derived from both receptor positive and TNBC tumors, sensitivity to CB-839 was associated with (i) elevated GLS expression, (ii) elevated GLS activity, and (iii) the TNBC subtype. Importantly, many of the determinants of CB-839 sensitivity in cell lines are also present in primary tumor samples, including high mRNA and protein expression of GLS and a high Glu to Gln ratio in TNBC tumors as compared to receptor positive tumors. These observations motivate the Phase 1 clinical study of CB-839 in TNBC patients. To aid in the selection of a recommended Phase 2 dose, we sought to develop a pharmacodynamic (PD) assay to directly measure the GLS activity in breast tumor lysates in order to determine the extent of GLS inhibition in tumor biopsies from CB-839 treated patients.
To develop a robust PD assay, we first identified conditions that maintain the GLS:CB-839 inhibitory complex during preparation of lysates from CB-839 treated samples. High concentrations of KCl (150 mM) and low concentrations of K-phosphate (15 mM) in the lysis buffer, as well as maintaining the lysate at a low temperature stabilized the inhibited complex. Following gel filtration of the lysate to remove unbound CB-839 and exchange the buffer, GLS activity was immediately measured with a coupled enzyme assay. The GLS activity measured at this step reflects the residual activity present in a sample that was exposed to CB-839. To quantify the amount of total GLS present in the sample, we incubated the same lysate for 3 hours at room temperature under conditions of low KCl and high phosphate to allow the the GLS:CB-839 complex to fully dissociate prior to measuring activity. This assay format allows quantitation of the % GLS inhibition from a single tumor lysate sample and eliminates the requirement for multiple biopsies as well as any assay variability due to tumor heterogeneity.
We utilized this tumor PD assay to determine the plasma drug levels required for maximal tumor GLS inhibition in a preclinical TNBC model. Mice bearing HCC1806 TNBC tumors were first treated with a range of CB-839 doses. Four hours after oral administration, a 10 mg/kg dose of CB-839 resulted in &gt;90% inhibition of tumor GLS. CB-839 plasma concentrations of 100 nM corresponded to 50% inhibition of tumor GLS, while maximal inhibition occurred at plasma concentrations ≥300 nM. In xenograft studies, maximal anti-tumor efficacy was achieved with BID dosing at 200 mg/kg, a dose and schedule that resulted in trough plasma levels of CB-839 of ≥300 nM and sustained GLS inhibition in tumors. As part of an ongoing Phase 1 trial, this assay will be utilized to monitor tumor PD responses in TNBC patients undergoing treatment.
Citation Format: Andy MacKinnon, Mark Bennett, Ethan Emberley, Mathew Gross, Julie Janes, Evan Lewis, Alison Pan, Mirna Rodriguez, Peter Shwonek, Taotao Wang, Jinfu Yang, Frances Zhao, Francesco Parlati. A novel pharmacodynamic assay to measure glutaminase inhibition following oral administration of CB-839 in triple negative breast cancer biopsies [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 P1-08-07.