Acquired Resistance to HER2-Targeted Therapies Creates Vulnerability to ATP Synthase Inhibition.

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 in vivo. 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. SIGNIFICANCE: These findings implicate ATP synthase as a novel potential target for tumors resistant to HER2-targeted therapies.

Structural Basis for KDM5A Histone Lysine Demethylase Inhibition by Diverse Compounds.

The KDM5/JARID1 family of Fe(II)- and α-ketoglutarate-dependent demethylases removes methyl groups from methylated lysine 4 of histone H3. Accumulating evidence supports a role for KDM5 family members as oncogenic drivers. We compare the in vitro inhibitory properties and binding affinity of ten diverse compounds with all four family members, and present the crystal structures of the KDM5A-linked Jumonji domain in complex with eight of these inhibitors in the presence of Mn(II). All eight inhibitors structurally examined occupy the binding site of α-ketoglutarate, but differ in their specific binding interactions, including the number of ligands involved in metal coordination. We also observed inhibitor-induced conformational changes in KDM5A, particularly those residues involved in the binding of α-ketoglutarate, the anticipated peptide substrate, and intramolecular interactions. We discuss how particular chemical moieties contribute to inhibitor potency and suggest strategies that might be utilized in the successful design of selective and potent epigenetic inhibitors.

An easy and efficient inducible CRISPR/Cas9 platform with improved specificity for multiple gene targeting.

The CRISPR/Cas9 system is a powerful genome editing tool and has been widely used for biomedical research. However, many challenges, such as off-target effects and lack of easy solutions for multiplex targeting, are still limiting its applications. To overcome these challenges, we first developed a highly efficient doxycycline-inducible Cas9-EGFP vector. This vector allowed us to track the cells for uniform temporal control and efficient gene disruption, even in a polyclonal setting. Furthermore, the inducible CRISPR/Cas9 system dramatically decreased off-target effects with a pulse exposure of the genome to the Cas9/sgRNA complex. To target multiple genes simultaneously, we established simple one-step cloning approaches for expression of multiple sgRNAs with improved vectors. By combining our inducible and multiplex genome editing approaches, we were able to simultaneously delete Lysine Demethylase (KDM) 5A, 5B and 5C efficiently in vitro and in vivo This user friendly and highly efficient toolbox provides a solution for easy genome editing with tight temporal control, minimal off-target effects and multiplex targeting.

Screen-identified selective inhibitor of lysine demethylase 5A blocks cancer cell growth and drug resistance.

Lysine demethylase 5A (KDM5A/RBP2/JARID1A) is a histone lysine demethylase that is overexpressed in several human cancers including lung, gastric, breast and liver cancers. It plays key roles in important cancer processes including tumorigenesis, metastasis, and drug tolerance, making it a potential cancer therapeutic target. Chemical tools to analyze KDM5A demethylase activity are extremely limited as available inhibitors are not specific for KDM5A. Here, we characterized KDM5A using a homogeneous luminescence-based assay and conducted a screen of about 9,000 small molecules for inhibitors. From this screen, we identified several 3-thio-1,2,4-triazole compounds that inhibited KDM5A with low µM in vitro IC50 values. Importantly, these compounds showed great specificity and did not inhibit its close homologue KDM5B (PLU1/JARID1B) or the related H3K27 demethylases KDM6A (UTX) and KDM6B (JMJD3). One compound, named YUKA1, was able to increase H3K4me3 levels in human cells and selectively inhibit the proliferation of cancer cells whose growth depends on KDM5A. As KDM5A was shown to mediate drug tolerance, we investigated the ability of YUKA1 to prevent drug tolerance in EGFR-mutant lung cancer cells treated with gefitinib and HER2+ breast cancer cells treated with trastuzumab. Remarkably, this compound hindered the emergence of drug-tolerant cells, highlighting the critical role of KDM5A demethylase activity in drug resistance. The small molecules presented here are excellent tool compounds for further study of KDM5A's demethylase activity and its contributions to cancer.

High-throughput screening to identify inhibitors of lysine demethylases.

Lysine demethylases (KDMs) are epigenetic regulators whose dysfunction is implicated in the pathology of many human diseases including various types of cancer, inflammation and X-linked intellectual disability. Particular demethylases have been identified as promising therapeutic targets, and tremendous efforts are being devoted toward developing suitable small-molecule inhibitors for clinical and research use. Several High-throughput screening strategies have been developed to screen for small-molecule inhibitors of KDMs, each with advantages and disadvantages in terms of time, cost, effort, reliability and sensitivity. In this Special Report, we review and evaluate the High-throughput screening methods utilized for discovery of novel small-molecule KDM inhibitors.

Acquired resistance of EGFR-mutant lung adenocarcinomas to afatinib plus cetuximab is associated with activation of mTORC1.

Patients with EGFR-mutant lung adenocarcinomas (LUADs) who initially respond to first-generation tyrosine kinase inhibitors (TKIs) develop resistance to these drugs. A combination of the irreversible TKI afatinib and the EGFR antibody cetuximab can be used to overcome resistance to first-generation TKIs; however, resistance to this drug combination eventually emerges. We identified activation of the mTORC1 signaling pathway as a mechanism of resistance to dual inhibition of EGFR in mouse models. The addition of rapamycin reversed resistance in vivo. Analysis of afatinib-plus-cetuximab-resistant biopsy specimens revealed the presence of genomic alterations in genes that modulate mTORC1 signaling, including NF2 and TSC1. These findings pinpoint enhanced mTORC1 activation as a mechanism of resistance to afatinib plus cetuximab and identify genomic mechanisms that lead to activation of this pathway, revealing a potential therapeutic strategy for treating patients with resistance to these drugs.

Differential effects of AKT1(p.E17K) expression on human mammary luminal epithelial and myoepithelial cells.

Recently, we identified a somatic mutation in AKT1, which results in a glutamic acid to lysine substitution (p.Glu17Lys or E17K). E17K mutations appear almost exclusively in breast cancers of luminal origin. Cellular models involving cell lines such as human mammary epithelial and MCF10 are model systems that upon transformation lead to rare forms of human breast cancer. Hence, we studied the effects of E17K using a clinically pertinent luminal cell line model while providing evidence to explain why E17K mutations do not occur in the mammary myoepithelium. Thus the purpose of our study was to perform a functional and differential proteomics study to assess the role of AKT1(E17K) in the development of breast cancer. We used a set of genetically matched nontumorigenic and tumorigenic mammary luminal and myoepithelial cells. We demonstrated that in myoepithelial cells, expression of E17K inhibited growth, migration, and protein synthesis compared with wild-type AKT1. In luminal cells, E17K enhanced cell survival and migration, possibly offering a selective advantage in this type of cell. However, antineoplastic effects of E17K in luminal cells, such as inhibition of growth and protein synthesis, may ultimately be associated with favorable prognosis. Our study illustrates the importance of cellular context in determining phenotypic effects of putative oncogenic mutations.

DNA methylation in multiple myeloma is weakly associated with gene transcription.

Previous studies have now demonstrated that both genic and global hypomethylation characterizes the multiple myeloma (MM) epigenome. Whether these methylation changes are associated with global and corresponding increases (or decreases) in transcriptional activity are poorly understood. The purpose of our current study was to correlate DNA methylation levels in MM to gene expression. We analyzed matching datasets generated by the GoldenGate methylation BeadArray and Affymetrix gene expression platforms in 193 MM samples. We subsequently utilized two independent statistical approaches to identify methylation-expression correlations. In the first approach, we used a linear correlation parameter by computing a Pearson correlation coefficient. In the second approach, we discretized samples into low and high methylation groups and then compared the gene expression differences between the groups. Only methylation of 2.1% and 25.3% of CpG sites on the methylation array correlated to gene expression by Pearson correlation or the discretization method, respectively. Among the genes with methylation-expression correlations were IGF1R, DLC1, p16, and IL17RB. In conclusion, DNA methylation may directly regulate relatively few genes and suggests that additional studies are needed to determine the effects of genome-wide methylation changes in MM.