Glutaminase is essential for the growth of triple-negative breast cancer cells with a deregulated glutamine metabolism pathway and its suppression synergizes with mTOR inhibition

<div><p>Tumor cells display fundamental changes in metabolism and nutrient uptake in order to utilize additional nutrient sources to meet their enhanced bioenergetic requirements. Glutamine (Gln) is one such nutrient that is rapidly taken up by tumor cells to fulfill this increased metabolic demand. A vital step in the catabolism of glutamine is its conversion to glutamate by the mitochondrial enzyme glutaminase (GLS). This study has identified GLS a potential therapeutic target in breast cancer, specifically in the basal subtype that exhibits a deregulated glutaminolysis pathway. Using inducible shRNA mediated gene knockdown, we discovered that loss of GLS function in triple-negative breast cancer (TNBC) cell lines with a deregulated glutaminolysis pathway led to profound tumor growth inhibition <i>in vitro</i> and <i>in vivo</i>. GLS knockdown had no effect on growth and metabolite levels in non-TNBC cell lines. We rescued the anti-tumor effect of GLS knockdown using shRNA resistant cDNAs encoding both GLS isoforms and by addition of an α-ketoglutarate (αKG) analog thus confirming the critical role of GLS in TNBC. Pharmacological inhibition of GLS with the small molecule inhibitor CB-839 reduced cell growth and led to a decrease in mammalian target of rapamycin (mTOR) activity and an increase in the stress response pathway driven by activating transcription factor 4 (ATF4). Finally, we found that GLS inhibition synergizes with mTOR inhibition, which introduces the possibility of a novel therapeutic strategy for TNBC. Our study revealed that GLS is essential for the survival of TNBC with a deregulated glutaminolysis pathway. The synergistic activity of GLS and mTOR inhibitors in TNBC cell lines suggests therapeutic potential of this combination for the treatment of vulnerable subpopulations of TNBC.</p></div

MDA-MB-231 breast cancer cell line requires GLS for growth and survival <i>in vitro</i>.

<p>Two inducible forms of GLS shRNAs, sh1 and sh2 were introduced into MDA-MB-231 cells. GLS knockdown was induced upon doxycycline addition. Panel A. Knockdown of <i>GLS</i> transcripts as measured by qRT-PCR. Panel B. Depiction of GLS protein knockdown by immunoblot analysis (Ab used-H00002744-A01, Novus Biologicals LLC and 19958-1-AP, Proteintech Group Inc.) at 72 hours post-doxycycline induction. Panel C. Incorporation of carbons from U¹³- C₅ labeled L-glutamine into other non-essential amino acids as measured by mass spectrometry analysis after 24 hours of doxycycline induction. Data are normalized to a no doxycycline level and represented with a red dotted line indicating 100%. Panel D. Mass spectrometry analysis of TCA cycle intermediates upon GLS knockdown in MDA-MB-231 cells. Panel E. Relative proliferation of MDA-MB-231 cells upon GLS knockdown. Proliferation was measured using the CellTiter-GLO assay at the indicated time points post doxycycline induction. CellTiter-GLO values are plotted on the y-axis. The histogram plots describe the average and standard deviation of triplicate samples. Panel F. GLS knockdown induces apoptosis in MDA-MB-231 cells. 72 hours after doxycycline induction, cells were stained with Annexin V and 7-AAD and analyzed by flow cytometry for quantitation of apoptosis as illustrated by the FACS plots.</p

GLS is essential for the growth of TNBC tumor xenografts.

<p>Panels A (i-v) and D represent the growth curves of tumor xenografts for the doxycycline induced GLS shRNA stable MDA-MB-231 (i-iii) and Sum-159PT (iv and v) cell lines. The black line represents tumor growth in mice fed on a diet without doxycycline while the colored line represents tumor growth in mice fed on a diet with doxycycline. Panel B: qRTPCR analysis of <i>GLS</i> gene expression in MDA-MB-231 (left) and SUM-159PT GLS shRNA stable xenografts (right) confirming knockdown upon doxycycline induction. Panel C: Mass spectrometry analysis of glutamate to glutamine levels in MDA-MB-231 and SUM-159PT tumors. Data are normalized to no doxycycline levels. The histogram plots show the average and standard deviation of caliper measurements from 5 mice in each group. Panel D: Tumor growth depicting genetic rescue of GLS knockdown in doxycycline induced MDA- MB-231 GLS shRNA stable xenograft.</p

Single isoforms do not rescue GLS knockdown phenotype.

<p>Expression of either shRNA resistant KGA (Panel A) or GAC (Panel B) isoforms in MDA-MB-231. Doxycycline induction of GLS shRNA 1 and 2 effectively reduced endogenous levels of KGA and GAC isoforms. However, attempts to rescue the knockdown phenotype were unsuccessful when only an individual shRNA resistant isoform was introduced. Immunoblot analysis was performed 72 hours post doxycycline induction. Ctrlsh,GLS sh1, and GLS sh2 refer to cell lines with stably integrated Null shRNA or GLS shRNAs TRCN0000051135 and TRCN0000051136, respectively. Protein expression of shRNA resistant clones by immunoblot analysis using isoform specific antibodies, metabolite analysis by mass spectrometry and relative proliferation as measured by the CellTiter-GLO assay are displayed.</p

GLS is essential for intermediary metabolism in MDA-MB-231.

<p>Diagram representation of labelled ¹³C (red dots) and unlabeled ¹²C (black dots) from glutamine (Panel A) and glucose (Panel B) through the first TCA cycle is shown. Incorporation of the labelled carbon into TCA cycle intermediates upon GLS knockdown are shown in the bottom panels. Data acquired in triplicate are normalized to no doxycycline levels. Decrease in ¹³C₂ labelled OAA seen with glucose labelling and GLS knockdown are indicated by the circles drawn.</p

Combination index obtained for MDA-MB-231 and SUM-159PT treated with CB-839 and AZD8055.

<p>‘F’ denotes effective fraction of CB-839 from the combination. ‘Ki’ denotes the calculated combination index. N1, N2, and N3 denote samples obtained from three independent experiments.</p

Growth inhibition activity of CB-839 across a panel of breast cancer cell lines.

<p>Cells were cultured in ATCC recommended medium and treated with varying concentrations of CB-839 to generate a 10- point dose response curve. Cell growth was measured after 72 hours incubation with CB-839 using the CellTiter-GLO assay. Maximum concentration of CB-839 used was 10μM. Cell lines refractory to compound treatment are plotted with an IC50 of 10μM.</p

Basal breast cancers enrich for deregulation of glutamine metabolism pathway.

<p>Panel A. Scatter plot describing the relative gene expression levels of <i>GLS</i> and <i>GLUL</i> among breast cancers in the TCGA database. Data are plotted as log₂ fold change around the mean of all breast cancer samples. Panel B. A total of 15 breast cancer cell lines were cultured in ATCC recommended media. Intracellular glutamine and glutamate levels were determined by mass spectrometry analysis. Data are plotted as a ratio of glutamate to glutamine levels and sorted from the highest to lowest values.</p

GLS inhibition activates the ISR pathway, inhibits the mTOR pathway, and synergizes with the mTOR inhibitor AZD8055.

<p>Panel A. Responders MDA-MB-231 and SUM-159PT and non-responder MDA-MB-453 cell lines were treated with increasing concentrations of CB-839 for 24 hours. Cell lysates were analyzed by immunoblot analysis with antibodies as indicated. Panel B. qRT-PCR analysis of MDA-MB-231 (top) and SUM-159PT (bottom) cells treated with varying concentrations of CB-839 or in the absence of glutamine in culture medium. Targets of ATF4 transcription factor as described were analyzed 24 hours after compound treatment. Data was acquired in triplicate and normalized to DMSO treated cells. Panel C. SUM-159PT xenograft lysates were analyzed by immunoblot analysis with antibodies as indicated. Each lane corresponds to xenografts from individual mice, 5 mice per treatment group. Panel D. Isobologram representing combined treatment of the GLS inhibitor CB-839 and mTOR inhibitor AZD8055 in MDA-MB-231 and SUM-159PT. The reference line joining the point (0,1) on the y-axis to the point (1,0) on the x-axis is a marker for additivity. The Ki values calculated from the combination rays in the experiment are found below the additivity line indicating synergy between the two compounds. Three Ki measurements corresponding to three independent experiments are plotted individually for each ray. The numbers in the plot represent the effective fraction of CB-839 in each combination ray.</p

Assessment of GLS requirement for growth in multiple breast cancer cells.

<p>Two inducible GLS specific shRNAs, sh1 and sh2 were introduced in SUM-159PT (Panel A), SKBR3 (Panel B), MDA-MB-453 (Panel C) and 9 additional breast cancer lines (Panel D). Knockdown of <i>GLS</i> upon doxycycline induction was confirmed by qRT-PCR analysis (left). Cell proliferation as function of ATP generation was measured using the CellTiter-GLO assay (right).</p