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. 2020 Oct;13(10):100828.
doi: 10.1016/j.tranon.2020.100828. Epub 2020 Jul 8.

Combined blockade of EGFR and glutamine metabolism in preclinical models of colorectal cancer

Allison S Cohen  1 , Ling Geng  1 , Ping Zhao  1 , Allie Fu  1 , Michael L Schulte  2 , Ramona Graves-Deal  3 , M Kay Washington  4 , Jordan Berlin  5 , Robert J Coffey  6 , H Charles Manning  7
Affiliations

Combined blockade of EGFR and glutamine metabolism in preclinical models of colorectal cancer

Allison S Cohen et al. Transl Oncol. 2020 Oct.

Abstract

Improving response to epidermal growth factor receptor (EGFR)-targeted therapies in patients with advanced wild-type (WT) RAS colorectal cancer (CRC) remains an unmet need. In this preclinical work, we evaluated a new therapeutic combination aimed at enhancing efficacy by targeting cancer cell metabolism in concert with EGFR. We hypothesized that combined blockade of glutamine metabolism and EGFR represents a promising treatment approach by targeting both the "fuel" and "signaling" components that these tumors need to survive. To explore this hypothesis, we combined CB-839, an inhibitor of glutaminase 1 (GLS1), the mitochondrial enzyme responsible for catalyzing conversion of glutamine to glutamate, with cetuximab, an EGFR-targeted monoclonal antibody in preclinical models of CRC. 2D and 3D in vitro assays were executed following treatment with either single agent or combination therapy. The combination of cetuximab with CB-839 resulted in reduced cell viability and demonstrated synergism in several cell lines. In vivo efficacy experiments were performed in cell-line xenograft models propagated in athymic nude mice. Tumor volumes were measured followed by immunohistochemical (IHC) analysis of proliferation (Ki67), mechanistic target of rapamycin (mTOR) signaling (pS6), and multiple mechanisms of cell death to annotate molecular determinants of response. In vivo, a significant reduction in tumor growth and reduced Ki67 and pS6 IHC staining were observed with combination therapy, which was accompanied by increased apoptosis and/or necrosis. The combination showed efficacy in cetuximab-sensitive as well as resistant models. In conclusion, this therapeutic combination represents a promising new precision medicine approach for patients with refractory metastatic WT RAS CRC.

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Conflict of interest statement

Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Figures

Fig. 1
Fig. 1
Glutamine (Gln) and EGFR cooperate to promote growth and proliferation. Gln “fuels” the citric acid cycle (CAC) as required for signal transduction-mediated growth and proliferation. Glutamine transport is mediated through solute carrier transporters including ASCT2 (SLC1A5), a key Gln transporter in CRC, and xCT (SLC7A11), an exchanger of glutamine-derived glutamate (Glu) and cystine. Intracellular Gln is metabolized by mitochondrial enzymes, glutaminase 1 and 2 (GLS1/2), to Glu, which fuels the CAC and contributes to redox balance via glutathione biosynthesis, a process that requires exchange via xCT (SLC7A11). This work explored the combination of an EGFR neutralizing mAb, cetuximab, with GLS1 inhibition using CB-839 to enhance the anti-tumor efficacy of cetuximab.
Fig. 2
Fig. 2
Combined CB-839/anti-EGFR mAb therapy results in cooperative efficacy in 2D culture in vitro. CRC cell lines were propagated in 2D culture. Cells were treated with either cetuximab (black), CB-839 (white), or cetuximab and CB-839 (grey). Cell viability was determined using MultiTox Glo cytotoxicity assay. Shown is the percentage change in live cells from the fluorescence assay normalized to vehicle control. CRC cell lines propagated in 2D culture exhibit reduced viability with combined therapy compared to single agents (relative to vehicle control). Error bars represent ± standard deviation (SD) (n = 3 or 4 technical replicates). Combination indices were determined and are indicated for each cell line by the following symbols: * synergism, ^ additive, and” not available.
Fig. 3
Fig. 3
Combined CB-839/anti-EGFR mAb therapy results in cooperative efficacy in 3D culture in vitro. CRC cell lines were grown in 3D collagen culture. Cells were treated with either cetuximab (black), CB-839 (white), or cetuximab and CB-839 (grey). The number of colonies were counted. Shown is the percentage change in colony number normalized to vehicle control. Combination therapy results in decreased colonies in a 3D culture model compared to single agents (relative to vehicle control). Error bars represent ± SD (n = 2–3 biological replicates and 3 technical replicates).
Fig. 4
Fig. 4
Combined CB-839/anti-EGFR mAb therapy overcomes intrinsic cetuximab-resistance in SW48 xenografts in vivo. (A) Mice with SW48 cell line xenografts, a model of intrinsic cetuximab resistance, were treated with either vehicle (black), CB-839 (red), cetuximab (blue) or cetuximab and CB-839 (purple) for 30 days and tumor volumes were monitored (n = 5 mice/group). (B) Representative images of mice treated with cetuximab alone (left) or cetuximab in combination with CB-839 (right). Mice progressed on single agent CB-839 or cetuximab yet exhibit reduced tumor volume with combination cetuximab and CB-839. (C) Immunohistochemistry (IHC) was performed on tumors from each of the treatment groups to evaluate Ki67 and pS6 expression. Shown are representative images (40×). The number of positive cells were counted in regions of interest on images from Ki67 IHC (D) and pS6 IHC (E) (n = 3–15 fields of view, at least 3 animals/treatment group). Reduced Ki67 and pS6 were observed when treated with CB-839 in combination with cetuximab compared to cetuximab or CB-839 alone. (F) Representative H&E images (0.46×) of tumor tissues from each of the treatment groups. (G) IHC was performed on tumors from each of the treatment groups to evaluate cleaved caspase-3 expression. Shown are representative images (20×). (H) The number of positive cells were counted in regions of interest on images from cleaved caspase-3 IHC (n = 3–15 fields of view, at least 3 animals/treatment group). Error bars represent ± SD. Statistical significance is defined as follows: *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
Fig. 5
Fig. 5
Combined CB-839/anti-EGFR mAb therapy overcomes acquired cetuximab-resistance in HCA-7 CC-CR xenografts in vivo. (A) Mice with HCA-7 CC-CR cell line xenografts, a model of acquired cetuximab-resistance, were treated with either vehicle (black), CB-839 (red), cetuximab (blue) or cetuximab and CB-839 (purple) for 21 days. At this time point, treatment was stopped (dotted line). Tumor volumes were monitored to day 57 (n = 8 mice/group). Mice progressed on single agent CB-839 or cetuximab yet exhibit reduced tumor volume with combination cetuximab and CB-839. (B) IHC was performed on tumors from each of the treatment groups to evaluate Ki67 and pS6 expression. Shown are representative images (40×). The number of positive cells were counted in regions of interest on images from Ki67 IHC (C) and pS6 IHC (D) (n = 3–15 fields of view, at least 3 animals/treatment group). Reduced Ki67 and pS6 were observed when treated with CB-839 in combination with cetuximab compared to cetuximab or CB-839 alone. (E) Representative H&E images (0.46×) of tumor tissues from each of the treatment groups. (F) IHC was performed on tumors from each of the treatment groups to evaluate cleaved caspase-3 expression. Shown are representative images (20×). (G) The number of positive cells were counted in regions of interest on images from cleaved caspase-3 IHC (n = 3–15 fields of view, at least 3 animals/treatment group). Error bars represent ± SD. Statistical significance is defined as follows: #p = 0.0574, *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
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References

    1. Siegel R.L., Miller K.D., Jemal A. Cancer statistics, 2018. CA Cancer J. Clin. 2018;68(1):7–30. doi: 10.3322/caac.21442. 29313949 - DOI - PubMed
    1. Moriarity A., O'Sullivan J., Kennedy J., Mehigan B., McCormick P. Current targeted therapies in the treatment of advanced colorectal cancer: a review. Ther Adv Med Oncol. 2016;8(4):276–293. doi: 10.1177/1758834016646734. (PubMed PMID: 27482287; PMCID: PMC4952023) - DOI - PMC - PubMed
    1. Yewale C., Baradia D., Vhora I., Patil S., Misra A. Epidermal growth factor receptor targeting in cancer: a review of trends and strategies. Biomaterials. 2013;34(34):8690–8707. doi: 10.1016/j.biomaterials.2013.07.100. (PubMed PMID: 23953842) - DOI - PubMed
    1. Bertotti A., Papp E., Jones S., Adleff V., Anagnostou V., Lupo B. The genomic landscape of response to EGFR blockade in colorectal cancer. Nature. 2015;526(7572):263–267. doi: 10.1038/nature14969. (PubMed PMID: 26416732; PMCID: PMC4878148) - DOI - PMC - PubMed
    1. Diaz L.A., Jr., Williams R.T., Wu J., Kinde I., Hecht J.R., Berlin J. The molecular evolution of acquired resistance to targeted EGFR blockade in colorectal cancers. Nature. 2012;486(7404):537–540. doi: 10.1038/nature11219. (PubMed PMID: 22722843; PMCID: PMC3436069) - DOI - PMC - PubMed