The mechanisms by which cells adapt to proteotoxic stress are largely unknown, but are key to understanding how tumor cells, particularly in vivo, are largely resistant to proteasome inhibitors. Analysis of cancer cell lines, mouse xenografts and patient-derived tumor samples all showed an association between mitochondrial metabolism and proteasome inhibitor sensitivity. When cells were forced to use oxidative phosphorylation rather than glycolysis, they became proteasome-inhibitor resistant. This mitochondrial state, however, creates a unique vulnerability: sensitivity to the small molecule compound elesclomol. Genome-wide CRISPR-Cas9 screening showed that a single gene, encoding the mitochondrial reductase FDX1, could rescue elesclomol-induced cell death. Enzymatic function and nuclear-magnetic-resonance-based analyses further showed that FDX1 is the direct target of elesclomol, which promotes a unique form of copper-dependent cell death. These studies explain a fundamental mechanism by which cells adapt to proteotoxic stress and suggest strategies to mitigate proteasome inhibitor resistance.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Genome-scale CRISPR-Cas9 viability screens performed in cancer cell lines provide a systematic approach to identify cancer dependencies and new therapeutic targets. As multiple large-scale screens become available, a formal assessment of the reproducibility of these experiments becomes necessary. We analyze data from recently published pan-cancer CRISPR-Cas9 screens performed at the Broad and Sanger Institutes. Despite significant differences in experimental protocols and reagents, we find that the screen results are highly concordant across multiple metrics with both common and specific dependencies jointly identified across the two studies. Furthermore, robust biomarkers of gene dependency found in one data set are recovered in the other. Through further analysis and replication experiments at each institute, we show that batch effects are driven principally by two key experimental parameters: the reagent library and the assay length. These results indicate that the Broad and Sanger CRISPR-Cas9 viability screens yield robust and reproducible findings.

Aurora kinases B and C (AURKB/AURKC) are activated by binding to the C-terminal domain of INCENP. Full activation requires phosphorylation of two serine residues of INCENP that are conserved through evolution, although the mechanism of this activation has not been explained. Here we present crystal structures of the fully active complex of AURKC bound to INCENP, consisting of phosphorylated, activated, AURKC and INCENP phosphorylated on its TSS motif, revealing the structural and biochemical mechanism of synergistic activation of AURKC:INCENP. The structures show that TSS motif phosphorylation stabilises the kinase activation loop of AURKC. The TSS motif phosphorylations alter the substrate-binding surface consistent with a mechanism of altered kinase substrate selectivity and stabilisation of the protein complex against unfolding. We also analyse the binding of the most specific available AURKB inhibitor, BRD-7880, and demonstrate that the well-known Aurora kinase inhibitor VX-680 disrupts binding of the phosphorylated INCENP TSS motif.

Large panels of comprehensively characterized human cancer models, including the Cancer Cell Line Encyclopedia (CCLE), have provided a rigorous framework with which to study genetic variants, candidate targets, and small-molecule and biological therapeutics and to identify new marker-driven cancer dependencies. To improve our understanding of the molecular features that contribute to cancer phenotypes, including drug responses, here we have expanded the characterizations of cancer cell lines to include genetic, RNA splicing, DNA methylation, histone H3 modification, microRNA expression and reverse-phase protein array data for 1,072 cell lines from individuals of various lineages and ethnicities. Integration of these data with functional characterizations such as drug-sensitivity, short hairpin RNA knockdown and CRISPR-Cas9 knockout data reveals potential targets for cancer drugs and associated biomarkers. Together, this dataset and an accompanying public data portal provide a resource for the acceleration of cancer research using model cancer cell lines.

Despite the success of approved systemic therapies for estrogen receptor alpha (ER)-positive breast cancer, drug resistance remains common. We hypothesized that secreted factors from the human tumor microenvironment could modulate drug resistance. We previously screened a library of 297 recombinant-secreted microenvironmental proteins for the ability to confer resistance to the anti-estrogen fulvestrant in 2 ER(+) breast cancer cell lines. Herein, we considered whether factors that enhanced drug sensitivity could be repurposed as therapeutics and provide leads for drug development. Screening data revealed bone morphogenic protein (BMP)4 as a factor that inhibited cell growth and synergized with approved anti-estrogens and cyclin-dependent kinase 4/6 inhibitors (CDK4/6i). BMP4-mediated growth inhibition was dependent on type I receptor activin receptor-like kinase (ALK)3-dependent phosphorylation (P) of mothers against decapentaplegic homolog (SMAD/P-SMAD)1 and 5, which could be reversed by BMP receptor inhibitors and ALK3 knockdown. The primary effect of BMP4 on cell fate was cell-cycle arrest, in which RNA sequencing, immunoblot analysis, and RNA interference revealed to be dependent on p21(WAF1/Cip1) upregulation. BMP4 also enhanced sensitivity to approved inhibitors of mammalian target of rapamycin complex 1 and CDK4/6 via ALK3-mediated P-SMAD1/5 and p21 upregulation in anti-estrogen-resistant cells. Patients bearing primary ER(+) breast tumors, exhibiting a transcriptomic signature of BMP4 signaling, had improved disease outcome following adjuvant treatment with anti-estrogen therapy, independently of age, tumor grade, and tumor stage. Furthermore, a transcriptomic signature of BMP4 signaling was predictive of an improved biologic response to the CDK4/6i palbociclib, in combination with an aromatase inhibitor in primary tumors. These findings highlight BMP4 and its downstream pathway activation as a therapeutic opportunity in ER(+) breast cancer.-Shee, K., Jiang, A., Varn, F. S., Liu, S., Traphagen, N. A., Owens, P., Ma, C. X., Hoog, J., Cheng, C., Golub, T. R., Straussman, R., Miller, T. W. Cytokine sensitivity screening highlights BMP4 pathway signaling as a therapeutic opportunity in ER(+) breast cancer.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Intracellular accumulation of misfolded proteins causes toxic proteinopathies, diseases without targeted therapies. Mucin 1 kidney disease (MKD) results from a frameshift mutation in the MUC1 gene (MUC1-fs). Here, we show that MKD is a toxic proteinopathy. Intracellular MUC1-fs accumulation activated the ATF6 unfolded protein response (UPR) branch. We identified BRD4780, a small molecule that clears MUC1-fs from patient cells, from kidneys of knockin mice and from patient kidney organoids. MUC1-fs is trapped in TMED9 cargo receptor-containing vesicles of the early secretory pathway. BRD4780 binds TMED9, releases MUC1-fs, and re-routes it for lysosomal degradation, an effect phenocopied by TMED9 deletion. Our findings reveal BRD4780 as a promising lead for the treatment of MKD and other toxic proteinopathies. Generally, we elucidate a novel mechanism for the entrapment of misfolded proteins by cargo receptors and a strategy for their release and anterograde trafficking to the lysosome.

During cancer therapy, tumor heterogeneity can drive the evolution of multiple tumor subclones harboring unique resistance mechanisms in an individual patient(1-3). Previous case reports and small case series have suggested that liquid biopsy (specifically, cell-free DNA (cfDNA)) may better capture the heterogeneity of acquired resistance(4-8). However, the effectiveness of cfDNA versus standard single-lesion tumor biopsies has not been directly compared in larger-scale prospective cohorts of patients following progression on targeted therapy. Here, in a prospective cohort of 42 patients with molecularly defined gastrointestinal cancers and acquired resistance to targeted therapy, direct comparison of postprogression cfDNA versus tumor biopsy revealed that cfDNA more frequently identified clinically relevant resistance alterations and multiple resistance mechanisms, detecting resistance alterations not found in the matched tumor biopsy in 78% of cases. Whole-exome sequencing of serial cfDNA, tumor biopsies and rapid autopsy specimens elucidated substantial geographic and evolutionary differences across lesions. Our data suggest that acquired resistance is frequently characterized by profound tumor heterogeneity, and that the emergence of multiple resistance alterations in an individual patient may represent the 'rule' rather than the 'exception'. These findings have profound therapeutic implications and highlight the potential advantages of cfDNA over tissue biopsy in the setting of acquired resistance.

Cancer is often seen as a disease of mutations and chromosomal abnormalities. However, some cancers, including pediatric rhabdoid tumors (RTs), lack recurrent alterations targetable by current drugs and need alternative, informed therapeutic options. To nominate potential targets, we performed a high-throughput small-molecule screen complemented by a genome-scale CRISPR-Cas9 gene-knockout screen in a large number of RT and control cell lines. These approaches converged to reveal several receptor tyrosine kinases (RTKs) as therapeutic targets, with RTK inhibition effective in suppressing RT cell growth in vitro and against a xenograft model in vivo. RT cell lines highly express and activate (phosphorylate) different RTKs, creating dependency without mutation or amplification. Downstream of RTK signaling, we identified PTPN11, encoding the pro-growth signaling protein SHP2, as a shared dependency across all RT cell lines. This study demonstrates that large-scale perturbational screening can uncover vulnerabilities in cancers with "quiet" genomes.