Unlike normal cells, transformed cells are unable to grow when methionine in the growth media is restricted. Reversion to methionine independence is a rare event in transformed and malignant cells. Methionine-independent revertants provide an excellent system to identify metabolic signatures and molecular characteristics associated with methionine dependency of transformed cells. Revertants maintain the genetic background and general growth behavior of the parental cell line, except that they proliferate under methionine restriction such as in methionine-free media supplemented with homocysteine. Here we describe a general approach to generate methionine-independent revertants using the example of the triple-negative breast cancer cell line MDA-MB-468. To validate and characterize reversion we describe assays to evaluate cell proliferation and anchorage-independent growth in soft agar.

Methionine and homocysteine are metabolites in the transmethylation pathway leading to synthesis of the methyl-donor S-adenosylmethionine (SAM). Most cancer cells stop proliferating during methionine stress conditions, when methionine is replaced in the growth media by its immediate metabolic precursor homocysteine (Met-Hcy+). Non-transformed cells proliferate in Met-Hcy+ media, making the methionine metabolic requirement of cancer cells an attractive target for therapy, yet there is relatively little known about the molecular mechanisms governing the methionine stress response in cancer cells. To study this phenomenon in breast cancer cells, we selected methionine-independent-resistant cell lines derived from MDAMB468 breast cancer cells. Resistant cells grew normally in Met-Hcy+ media, whereas their parental MDAMB468 cells rapidly arrest in the G 1 phase. Remarkably, supplementing Met-Hcy+ growth media with S-adenosylmethionine suppressed the cell proliferation defects, indicating that methionine stress is a consequence of SAM limitation rather than low amino acid concentrations. Accordingly, mTORC1 activity, the primary effector responding to amino acid limitation, remained high. However, we found that levels of the replication factor Cdc6 decreased and pre-replication complexes were destabilized in methionine-stressed MDAMB468 but not resistant cells. Our study characterizes metabolite requirements and cell cycle responses that occur during methionine stress in breast cancer cells and helps explain the metabolic uniqueness of cancer cells.

Work in yeast models has benefitted tremendously from the insertion of epitope or fluorescence tags at the native gene locus to study protein function and behavior under physiological conditions. In contrast, work in mammalian cells largely relies on overexpression of tagged proteins because high-quality antibodies are only available for a fraction of the mammalian proteome. CRISPR/Cas9-mediated genome editing has recently emerged as a powerful genome-modifying tool that can also be exploited to insert various tags and fluorophores at gene loci to study the physiological behavior of proteins in most organisms, including mammals. Here we describe a versatile toolset for rapid tagging of endogenous proteins. The strategy utilizes CRISPR/Cas9 and microhomology-mediated end joining repair for efficient tagging. We provide tools to insert 3×HA, His₆FLAG, His₆-Biotin-TEV-RGSHis₆, mCherry, GFP, and the auxin-inducible degron tag for compound-induced protein depletion. This approach and the developed tools should greatly facilitate functional analysis of proteins in their native environment.

Methionine (Met) is an essential amino acid and critical precursor to the cellular methyl donor S-adenosylmethionine. Unlike nontransformed cells, cancer cells have a unique metabolic requirement for Met and are unable to proliferate in growth media where Met is replaced with its metabolic precursor, homocysteine. This metabolic vulnerability is common among cancer cells regardless of tissue origin and is known as "methionine dependence", "methionine stress sensitivity", or the Hoffman effect. The response of lipids to Met stress, however, is not well-understood. Using mass spectroscopy, label-free vibrational microscopy, and next-generation sequencing, we characterize the response of lipids to Met stress in the triple-negative breast cancer cell line MDA-MB-468 and its Met stress insensitive derivative, MDA-MB-468res-R8. Lipidome analysis identified an immediate, global decrease in lipid abundances with the exception of triglycerides and an increase in lipid droplets in response to Met stress specifically in MDA-MB-468 cells. Furthermore, specific gene expression changes were observed as a secondary response to Met stress in MDA-MB-468, resulting in a downregulation of fatty acid metabolic genes and an upregulation of genes in the unfolded protein response pathway. We conclude that the extensive changes in lipid abundance during Met stress is a direct consequence of the modified metabolic profile previously described in Met stress-sensitive cells. The changes in lipid abundance likely results in changes in membrane composition inducing the unfolded protein response we observe.

The tumour suppressor p53 is the most frequently mutated gene in human cancer. Reactivation of mutant p53 by small molecules is an exciting potential cancer therapy. Although several compounds restore wild-type function to mutant p53, their binding sites and mechanisms of action are elusive. Here computational methods identify a transiently open binding pocket between loop L1 and sheet S3 of the p53 core domain. Mutation of residue Cys124, located at the centre of the pocket, abolishes p53 reactivation of mutant R175H by PRIMA-1, a known reactivation compound. Ensemble-based virtual screening against this newly revealed pocket selects stictic acid as a potential p53 reactivation compound. In human osteosarcoma cells, stictic acid exhibits dose-dependent reactivation of p21 expression for mutant R175H more strongly than does PRIMA-1. These results indicate the L1/S3 pocket as a target for pharmaceutical reactivation of p53 mutants.About 40% of human cancers carry missense mutations in the tumour suppressor protein p53. Here the authors identify a transiently open pocket in the protein, and by targeting a small molecule to it, partially restore mutant p53 tumour suppressor activity.

The tumor suppressor p53 is the most frequently mutated protein in human cancer. The majority of these mutations are missense mutations in the DNA binding domain of p53. Restoring p53 tumor suppressor function could have a major impact on the therapy for a wide range of cancers. Here we report a virtual screening approach that identified several small molecules with p53 reactivation activities. The UCI-LC0023 compound series was studied in detail and was shown to bind p53, induce a conformational change in mutant p53, restore the ability of p53 hotspot mutants to associate with chromatin, reestablish sequence-specific DNA binding of a p53 mutant in a reconstituted in vitro system, induce p53-dependent transcription programs, and prevent progression of tumors carrying mutant p53, but not p53^null or p53^WT alleles. Our study demonstrates feasibility of a computation-guided approach to identify small molecule corrector drugs for p53 hotspot mutations.