CRISPR activation screens identify the SWI/SNF ATPases as suppressors of ferroptosis.

Ferroptosis is an iron-dependent cell death mechanism characterized by the accumulation of toxic lipid peroxides and cell membrane rupture. GPX4 (glutathione peroxidase 4) prevents ferroptosis by reducing these lipid peroxides into lipid alcohols. Ferroptosis induction by GPX4 inhibition has emerged as a vulnerability of cancer cells, highlighting the need to identify ferroptosis regulators that may be exploited therapeutically. Through genome-wide CRISPR activation screens, we identify the SWI/SNF (switch/sucrose non-fermentable) ATPases BRM (SMARCA2) and BRG1 (SMARCA4) as ferroptosis suppressors. Mechanistically, they bind to and increase chromatin accessibility at NRF2 target loci, thus boosting NRF2 transcriptional output to counter lipid peroxidation and confer resistance to GPX4 inhibition. We further demonstrate that the BRM/BRG1 ferroptosis connection can be leveraged to enhance the paralog dependency of BRG1 mutant cancer cells on BRM. Our data reveal ferroptosis induction as a potential avenue for broadening the efficacy of BRM degraders/inhibitors and define a specific genetic context for exploiting GPX4 dependency.

Functionalized Lineage Tracing Can Enable the Development of Homogenization-Based Therapeutic Strategies in Cancer.

The therapeutic landscape across many cancers has dramatically improved since the introduction of potent targeted agents and immunotherapy. Nonetheless, success of these approaches is too often challenged by the emergence of therapeutic resistance, fueled by intratumoral heterogeneity and the immense evolutionary capacity inherent to cancers. To date, therapeutic strategies have attempted to outpace the evolutionary tempo of cancer but frequently fail, resulting in lack of tumor response and/or relapse. This realization motivates the development of novel therapeutic approaches which constrain evolutionary capacity by reducing the degree of intratumoral heterogeneity prior to treatment. Systematic development of such approaches first requires the ability to comprehensively characterize heterogeneous populations over the course of a perturbation, such as cancer treatment. Within this context, recent advances in functionalized lineage tracing approaches now afford the opportunity to efficiently measure multimodal features of clones within a tumor at single cell resolution, enabling the linkage of these features to clonal fitness over the course of tumor progression and treatment. Collectively, these measurements provide insights into the dynamic and heterogeneous nature of tumors and can thus guide the design of homogenization strategies which aim to funnel heterogeneous cancer cells into known, targetable phenotypic states. We anticipate the development of homogenization therapeutic strategies to better allow for cancer eradication and improved clinical outcomes.

DNA interstrand cross-links induced by the major oxidative adenine lesion 7,8-dihydro-8-oxoadenine.

Oxidative damage to DNA generates 7,8-dihydro-8-oxoguanine (oxoG) and 7,8-dihydro-8-oxoadenine (oxoA) as two major lesions. Despite the comparable prevalence of these lesions, the biological effects of oxoA remain poorly characterized. Here we report the discovery of a class of DNA interstrand cross-links (ICLs) involving oxidized nucleobases. Under oxidative conditions, oxoA, but not oxoG, readily reacts with an opposite base to produce ICLs, highlighting a latent alkylating nature of oxoA. Reactive halogen species, one-electron oxidants, and the myeloperoxidase/H2O2/Cl- system induce oxoA ICLs, suggesting that oxoA-mediated cross-links may arise endogenously. Nucleobase analog studies suggest C2-oxoA is covalently linked to N2-guanine and N3-adenine for the oxoA-G and oxoA-A ICLs, respectively. The oxoA ICLs presumably form via the oxidative activation of oxoA followed by the nucleophilic attack by an opposite base. Our findings provide insights into oxoA-mediated mutagenesis and contribute towards investigations of oxidative stress-induced ICLs and oxoA-based latent alkylating agents.

Structural insights into the promutagenic bypass of the major cisplatin-induced DNA lesion.

The cisplatin-1,2-d(GpG) (Pt-GG) intrastrand cross-link is the predominant DNA lesion generated by cisplatin. Cisplatin has been shown to predominantly induce G to T mutations and Pt-GG permits significant misincorporation of dATP by human DNA polymerase ß (polß). In agreement, polß overexpression, which is frequently observed in cancer cells, is linked to cisplatin resistance and a mutator phenotype. However, the structural basis for the misincorporation of dATP opposite Pt-GG is unknown. Here, we report the first structures of a DNA polymerase inaccurately bypassing Pt-GG. We solved two structures of polß misincorporating dATP opposite the 5'-dG of Pt-GG in the presence of Mg2+ or Mn2+. The Mg2+-bound structure exhibits a sub-optimal conformation for catalysis, while the Mn2+-bound structure is in a catalytically more favorable semi-closed conformation. In both structures, dATP does not form a coplanar base pairing with Pt-GG. In the polß active site, the syn-dATP opposite Pt-GG appears to be stabilized by protein templating and pi stacking interactions, which resembles the polß-mediated dATP incorporation opposite an abasic site. Overall, our results suggest that the templating Pt-GG in the polß active site behaves like an abasic site, promoting the insertion of dATP in a non-instructional manner.

Caught with One's Zinc Fingers in the Genome Integrity Cookie Jar.

Zinc finger (ZnF) domains are present in at least 5% of human proteins. First characterized as binding to DNA, ZnFs display extraordinary binding plasticity and can bind to RNA, lipids, proteins, and protein post-translational modifications (PTMs). The diverse binding properties of ZnFs have made their functional characterization challenging. While once confined to large and poorly characterized protein families, proteomic, cellular, and molecular studies have begun to shed light on their involvement as protectors of the genome. We focus here on the emergent roles of ZnF domain-containing proteins in promoting genome integrity, including their involvement in telomere maintenance and DNA repair. These findings have highlighted the need for further characterization of ZnF proteins, which can reveal the functions of this large gene class in normal cell function and human diseases, including those involving genome instability such as aging and cancer.

Reversal of DNA damage induced Topoisomerase 2 DNA-protein crosslinks by Tdp2.

Mammalian Tyrosyl-DNA phosphodiesterase 2 (Tdp2) reverses Topoisomerase 2 (Top2) DNA-protein crosslinks triggered by Top2 engagement of DNA damage or poisoning by anticancer drugs. Tdp2 deficiencies are linked to neurological disease and cellular sensitivity to Top2 poisons. Herein, we report X-ray crystal structures of ligand-free Tdp2 and Tdp2-DNA complexes with alkylated and abasic DNA that unveil a dynamic Tdp2 active site lid and deep substrate binding trench well-suited for engaging the diverse DNA damage triggers of abortive Top2 reactions. Modeling of a proposed Tdp2 reaction coordinate, combined with mutagenesis and biochemical studies support a single Mg(2+)-ion mechanism assisted by a phosphotyrosyl-arginine cation-π interface. We further identify a Tdp2 active site SNP that ablates Tdp2 Mg(2+) binding and catalytic activity, impairs Tdp2 mediated NHEJ of tyrosine blocked termini, and renders cells sensitive to the anticancer agent etoposide. Collectively, our results provide a structural mechanism for Tdp2 engagement of heterogeneous DNA damage that causes Top2 poisoning, and indicate that evaluation of Tdp2 status may be an important personalized medicine biomarker informing on individual sensitivities to chemotherapeutic Top2 poisons.

Hyperglycemia stimulates p62/PKCζ interaction, which mediates NF-κB activation, increased Nox4 expression, and inflammatory cytokine activation in vascular smooth muscle.

Hyperglycemia leads to vascular smooth muscle cell (VSMC) dedifferentiation and enhances responses to IGF-I. Prior studies showed that hyperglycemia stimulated NADPH oxidase 4 (Nox4) synthesis, and IGF-I facilitated its recruitment to a signaling complex where it oxidized src, leading to AKT and MAPK activation. To determine the mechanism that led to these changes, we analyzed the roles of p62 (sequestrosome1) and PKCζ. Hyperglycemia induced a 4.9 ± 1.0-fold increase in p62/PKCζ association, and disruption of PKCζ/p62 using a peptide inhibitor or p62 knockdown reduced PKCζ activation (78 ± 6%). 3-Phosphoinoside-dependent protein kinase 1 was also recruited to the p62 complex and directly phosphorylated PKCζ, leading to its activation (3.1 ± 0.4-fold). Subsequently, activated PKCζ phosphorylated p65 rel, which led to increased Nox4 synthesis. Studies in diabetic mice confirmed these findings (6.0 ± 0.4-fold increase in p62/PKCζ) and their disruption of attenuated Nox4 synthesis (76 ± 9% reduction). PKCζ/p62 activation stimulated inflammatory cytokine production and enhanced IGF-I-stimulated VSMC proliferation. These results define the molecular mechanism by which PKCζ is activated in response to hyperglycemia and suggest that this could be a mechanism by which other stimuli such as cytokines or metabolic stress function to stimulate NF-κB activation, thereby altering VSMC sensitivity to IGF-I.