No abstract

Acquired resistance to HER2-targeted therapies occurs frequently in HER2 þ breast tumors and new strategies for overcoming resistance are needed. Here, we report that resistance to trastuzumab is reversible, as resistant cells regained sensitivity to the drug after being cultured in drug-free media. RNA-sequencing analysis showed that cells resistant to trastuzumab or trastuzumab þ pertuzumab in combination increased expression of oxidative phosphorylation pathway genes. Despite minimal changes in mitochondrial respiration, these cells exhibited increased expression of ATP synthase genes and selective dependency on ATP synthase function. Resistant cells were sensitive to inhibition of ATP synthase by oligomycin A, and knockdown of ATP5J or ATP5B, components of ATP synthase complex, rendered resistant cells responsive to a low dose of trastuzumab. Furthermore, combining ATP synthase inhibitor oligomycin A with trastuzumab led to regression of trastuzumab-resistant tumors in vivo. In conclusion, we identify a novel vulnerability of cells with acquired resistance to HER2targeted antibody therapies and reveal a new therapeutic strategy to overcome resistance.Significance: These findings implicate ATP synthase as a novel potential target for tumors resistant to HER2-targeted therapies.

No abstract

Tumors acquire alterations in oncogenes and tumor suppressor genes in an adaptive walk through the fitness landscape of tumorigenesis. However, the features of this landscape remain poorly understood and cannot be revealed by human cancer genotyping alone. Here, we use a multiplexed, autochthonous mouse platform to model and quantify the initiation and growth of more than one hundred genotypes of lung tumors across four oncogenic contexts: KRAS G12D, KRAS G12C, BRAF V600E, and EGFR L858R. The resulting fitness landscape is rugged (the effect of tumor suppressor inactivation often switches between beneficial and deleterious depending on the oncogenic context), shows no evidence of diminishing-returns epistasis within variants of the same oncogene, and is inconsistent with expectations of a simple linear signaling relationship among these three oncogenes. Our findings suggest that tumor suppressor effects are strongly context-specific, which limits the set of evolutionary paths that can be taken through the fitness landscape.

<p>Supplementary Figure 1: Generation and characterization of resistant cell pools. Supplementary Figure 2: Copy number assessment of receptor tyrosine kinases in resistant cell pools. Supplementary Figure 3: Trastuzumab resistance is reversible. Supplementary Figure 4: Drug resistant cells are more sensitive to OXPHOS inhibitors than parental cells. Supplementary Figure 5: Drug resistant cells have similar mitochondrial DNA copy numbers and ATP levels as the parental cells. Supplementary Figure 6: Glycolysis in parental BT474 versus trastuzumab resistant cells. Supplementary Figure 7: ATP synthase gene expression in BT474, SKBR3 cells and their derived trastuzumab-resistant cells. Supplementary Figure 8: Cumulative survival of breast cancer (all, basal, luminal) patients with patients divided in half by ATP5B (left) or ATP5A1 (right) mRNA level. Supplementary Figure 9: ATP5B protein expression correlation with survival in HER2+, trastuzumab treated patients from HeCOG 10/05. Supplementary Figure 10: ATP5B is required for the maintenance of resistance. Supplementary Figure 11: BT-TR2 tumor morphology and proliferation. Supplementary Table 1: Sequences of primers and plasmid inserts. Supplementary Methods Supplementary References</p>

<div>Abstract<p>Acquired resistance to HER2-targeted therapies occurs frequently in HER2⁺ breast tumors and new strategies for overcoming resistance are needed. Here, we report that resistance to trastuzumab is reversible, as resistant cells regained sensitivity to the drug after being cultured in drug-free media. RNA-sequencing analysis showed that cells resistant to trastuzumab or trastuzumab + pertuzumab in combination increased expression of oxidative phosphorylation pathway genes. Despite minimal changes in mitochondrial respiration, these cells exhibited increased expression of ATP synthase genes and selective dependency on ATP synthase function. Resistant cells were sensitive to inhibition of ATP synthase by oligomycin A, and knockdown of ATP5J or ATP5B, components of ATP synthase complex, rendered resistant cells responsive to a low dose of trastuzumab. Furthermore, combining ATP synthase inhibitor oligomycin A with trastuzumab led to regression of trastuzumab-resistant tumors <i>in vivo</i>. In conclusion, we identify a novel vulnerability of cells with acquired resistance to HER2-targeted antibody therapies and reveal a new therapeutic strategy to overcome resistance.</p>Significance:<p>These findings implicate ATP synthase as a novel potential target for tumors resistant to HER2-targeted therapies.</p></div>

<p>Supplementary Figure 1: Generation and characterization of resistant cell pools. Supplementary Figure 2: Copy number assessment of receptor tyrosine kinases in resistant cell pools. Supplementary Figure 3: Trastuzumab resistance is reversible. Supplementary Figure 4: Drug resistant cells are more sensitive to OXPHOS inhibitors than parental cells. Supplementary Figure 5: Drug resistant cells have similar mitochondrial DNA copy numbers and ATP levels as the parental cells. Supplementary Figure 6: Glycolysis in parental BT474 versus trastuzumab resistant cells. Supplementary Figure 7: ATP synthase gene expression in BT474, SKBR3 cells and their derived trastuzumab-resistant cells. Supplementary Figure 8: Cumulative survival of breast cancer (all, basal, luminal) patients with patients divided in half by ATP5B (left) or ATP5A1 (right) mRNA level. Supplementary Figure 9: ATP5B protein expression correlation with survival in HER2+, trastuzumab treated patients from HeCOG 10/05. Supplementary Figure 10: ATP5B is required for the maintenance of resistance. Supplementary Figure 11: BT-TR2 tumor morphology and proliferation. Supplementary Table 1: Sequences of primers and plasmid inserts. Supplementary Methods Supplementary References</p>

Drug resistant mutations that arise in therapeutic targets often limit clinical responses. However, the discovery of such mutations has historically been performed one gene or mutation at a time, often over decades of experimental and clinical testing, limiting our understanding of conserved mechanisms of drug resistance. We hypothesized that deep mutational scanning of canonical kinases may expedite this process and identify novel conserved elements that cause drug resistance when mutated (similar to the well-studied “Gatekeeper” residue). To test this, we generated cDNA-expression libraries containing all possible amino acid substitutions in CDK6, CDK4, ERK2, and EGFR. We screened each library against clinically utilized, ATP-competitive small molecule inhibitors. We then mapped the phenotypic data for over 40,000 missense mutations onto the aligned crystal structures of each protein and searched for shared structural attributes associated with drug resistance. This analysis revealed 4 equivalent amino acid sites whose mutation conferred drug resistance to ATP-competitive inhibitors in all of our screens: the Gatekeeper residue, as well as three uncharacterized residues. One of these sites, which we have termed the “Keymaster”, was additionally found to cause resistance in published data sets of sub-saturation BRAF, HER2, BCR-ABL, and MEK1 mutagenesis screens against their respective inhibitors. We confirmed that drug resistant phenotypes are caused by these alterations utilizing growth assays and protein target phosphorylation detection assays. Mechanistically, we show preliminary evidence that Keymaster-mutant proteins are competent for drug binding, but may display elevated basal activity. Consistent with our findings, we additionally identified mutations at Keymaster residues in reported patient tumors in a number of oncogene kinases, suggesting that Keymaster mutations could be drivers of tumorigenesis, as well as drug resistance. These efforts may prove useful for characterizing somatic kinase mutations of unknown function, designing next-generation therapeutics and deepening our understanding of kinase regulation. Citation Format: Nicole S. Persky, Desiree Hernandez, Jonathon Cordova, Amanda Walker, Lisa Brenan, Federica Piccioni, Sasha Pantel, Yenarae Lee, Amy Goodale, Xiaoping Yang, Yoichiro Mitsuishi, Mariana Do Carmo, Cong Zhu, Aleksandr Andreev, David E. Root, Cory M. Johannessen. Massively parallel identification of conserved drug resistant mutations in kinases [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1815.