Pan-RAF:MEK Molecular Glues Take Center Stage.

In this issue, Ryan and colleagues describe the preclinical development of a pan-RAF:MEK molecular glue with superior efficacy, brain penetrance, and tolerability in xenograft models of Ras/Raf/MAPK pathway-driven tumors. See related article by Ryan et al., p. 1190 (1).

High-Complexity shRNA Libraries and PI3 Kinase Inhibition in Cancer: High-Fidelity Synthetic Lethality Predictions.

Deregulated signal transduction is a cancer hallmark, and its complexity and interconnectivity imply that combination therapy should be considered, but large data volumes that cover the complexity are required in user-friendly ways. Here, we present a searchable database resource of synthetic lethality with a PI3 kinase signal transduction inhibitor by performing a saturation screen with an ultra-complex shRNA library containing 30 independent shRNAs per gene target. We focus on Ras-PI3 kinase signaling with T cell leukemia as a screening platform for multiple clinical and experimental reasons. Our resource predicts multiple combination-based therapies with high fidelity, ten of which we confirmed with small molecule inhibitors. Included are biochemical assays, as well as the IPI145 (duvelisib) inhibitor. We uncover the mechanism of synergy between the PI3 kinase inhibitor GDC0941 (pictilisib) and the tubulin inhibitor vincristine and demonstrate broad synergy in 28 cell lines of 5 cancer types and efficacy in preclinical leukemia mouse trials.

Drug-tolerant persister cancer cells are vulnerable to GPX4 inhibition.

Acquired drug resistance prevents cancer therapies from achieving stable and complete responses. Emerging evidence implicates a key role for non-mutational drug resistance mechanisms underlying the survival of residual cancer 'persister' cells. The persister cell pool constitutes a reservoir from which drug-resistant tumours may emerge. Targeting persister cells therefore presents a therapeutic opportunity to impede tumour relapse. We previously found that cancer cells in a high mesenchymal therapy-resistant cell state are dependent on the lipid hydroperoxidase GPX4 for survival. Here we show that a similar therapy-resistant cell state underlies the behaviour of persister cells derived from a wide range of cancers and drug treatments. Consequently, we demonstrate that persister cells acquire a dependency on GPX4. Loss of GPX4 function results in selective persister cell ferroptotic death in vitro and prevents tumour relapse in mice. These findings suggest that targeting of GPX4 may represent a therapeutic strategy to prevent acquired drug resistance.

Drug Resistance to EGFR Inhibitors in Lung Cancer.

BACKGROUND: The discovery of mutations in epidermal growth factor receptor (EGFR) has dramatically changed the treatment of patients with non-small-cell lung cancer (NSCLC), the leading cause of cancer deaths worldwide. EGFR-targeted therapies show considerable promise, but drug resistance has become a substantial issue. METHODS: We reviewed the literature to provide an overview of the drug resistance to EGFR tyrosine kinase inhibitors (TKIs) in NSCLC. RESULTS: The mechanisms causing primary, acquired and persistent drug resistance to TKIs vary. Researchers and clinicians, who have used study findings to develop more effective therapeutic approaches, have found that the sequential use of single agents presents a formidable challenge, suggesting that multidrug combinations must be considered. CONCLUSIONS: In the era of precision medicine, oncologists should promptly obtain an accurate diagnosis of drug resistance in each patient to be able to design the most relevant combination therapy to overcome patient-specific drug resistance.

AKT inactivation causes persistent drug tolerance to EGFR inhibitors.

Drug resistance is a major obstacle to the success of EGFR-targeted therapy. We recently studied the mechanism by which a small subset of EGFR mutant lung cancer cells remains viable after EGFR inhibition. We found that this drug-tolerant subpopulation develops because EGFR inhibition prevents AKT activity and thus inactivates Ets-1 function. In this article, we discuss how changes in intrinsic cell signaling after EGFR inhibition open a new avenue to drug resistance in NSCLCs, and comment on combined TKI and MEK inhibitor treatment to reduce the probability of emergent resistance to EGFR TKIs.

Epigenetic and transcriptional determinants of the human breast.

While significant effort has been dedicated to the characterization of epigenetic changes associated with prenatal differentiation, relatively little is known about the epigenetic changes that accompany post-natal differentiation where fully functional differentiated cell types with limited lifespans arise. Here we sought to address this gap by generating epigenomic and transcriptional profiles from primary human breast cell types isolated from disease-free human subjects. From these data we define a comprehensive human breast transcriptional network, including a set of myoepithelial- and luminal epithelial-specific intronic retention events. Intersection of epigenetic states with RNA expression from distinct breast epithelium lineages demonstrates that mCpG provides a stable record of exonic and intronic usage, whereas H3K36me3 is dynamic. We find a striking asymmetry in epigenomic reprogramming between luminal and myoepithelial cell types, with the genomes of luminal cells harbouring more than twice the number of hypomethylated enhancer elements compared with myoepithelial cells.

Identification of palmitoylated mitochondrial proteins using a bio-orthogonal azido-palmitate analogue.

Increased levels of circulating saturated free fatty acids, such as palmitate, have been implicated in the etiology of type II diabetes and cancer. In addition to being a constituent of glycerolipids and a source of energy, palmitate also covalently attaches to numerous cellular proteins via a process named palmitoylation. Recognized for its roles in membrane tethering, cellular signaling, and protein trafficking, palmitoylation is also emerging as a potential regulator of metabolism. Indeed, we showed previously that the acylation of two mitochondrial proteins at their active site cysteine residues result in their inhibition. Herein, we sought to identify other palmitoylated proteins in mitochondria using a nonradioactive bio-orthogonal azido-palmitate analog that can be selectively derivatized with various tagged triarylphosphines. Our results show that, like palmitate, incorporation of azido-palmitate occurred on mitochondrial proteins via thioester bonds at sites that could be competed out by palmitoyl-CoA. Using this method, we identified 21 putative palmitoylated proteins in the rat liver mitochondrial matrix, a compartment not recognized for its content in palmitoylated proteins, and confirmed the palmitoylation of newly identified mitochondrial 3-hydroxy-3-methylglutaryl-CoA synthase. We postulate that covalent modification and perhaps inhibition of various mitochondrial enzymes by palmitoyl-CoA could lead to the metabolic impairments found in obesity-related diseases.

Nonacid cleavable detergents applied to MALDI mass spectrometry profiling of whole cells.

Although cleavable detergents were first synthesized a number of years ago, they have only recently been successfully applied to problems involving biological molecules. Recent reports have demonstrated that these compounds are useful for applications involving both 2D PAGE and mass spectrometry. However, most cleavable surfactants have utilized acid-labile functional groups to affect cleavage. In applications where extreme pH is required, acid cleavable detergents have limited usefulness. We report the synthesis of fluoride cleavable silane compounds and photolabile cinnamate esters as cleavable detergents having alternative cleavage chemistries than previously reported cleavable detergents. These compounds were applied to whole cell analysis using MALDI mass spectrometry, and it was demonstrated that their use results in an increase in the number of proteins analyzed by increasing protein solubility.