Crosstalk and complexity within signaling pathways and their perturbation by oncogenes limit component-by-component approaches to understanding human disease. Network analysis of how normal and ...oncogenic signaling can be rewired by drugs may provide opportunities to target tumors with high specificity and efficacy. Using targeted inhibition of oncogenic signaling pathways, combined with DNA-damaging chemotherapy, we report that time-staggered EGFR inhibition, but not simultaneous coadministration, dramatically sensitizes a subset of triple-negative breast cancer cells to genotoxic drugs. Systems-level analysis—using high-density time-dependent measurements of signaling networks, gene expression profiles, and cell phenotypic responses in combination with mathematical modeling—revealed an approach for altering the intrinsic state of the cell through dynamic rewiring of oncogenic signaling pathways. This process converts these cells to a less tumorigenic state that is more susceptible to DNA damage-induced cell death by reactivation of an extrinsic apoptotic pathway whose function is suppressed in the oncogene-addicted state.
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► The efficacy of combination treatments for cancer depends on drug order and timing ► Dynamic rewiring of signaling networks by drugs can provide therapeutic benefit ► Sustained EGFR inhibition sensitizes triple-negative breast cancer cells to DNA damage ► EGFR activity, but not EGFR expression, is a biomarker of response to this treatment
The effectiveness of combination therapy for triple-negative breast cancer may be increased by delivering the drugs sequentially. Inhibiting EGFR dynamically rewires tumor cell signaling networks to enhance subsequent susceptibility to DNA-damaging agents, an effect not seen when the drugs are applied simultaneously.
Genetic approaches to cancer research have dramatically advanced our understanding of the pathophysiology of this disease, leading to similar genetics-based approaches for precision therapy, which ...have been less successful. Reconfiguring and adapting the types of technologies that underlie genetic research to dissect tumor cell signaling in clinical samples may offer an alternative road forward.
A prothrombotic coagulopathy is commonly found in critically ill COVID-19 patients with acute respiratory distress syndrome (ARDS). A unique feature of COVID-19 respiratory failure is a relatively ...preserved lung compliance and high Alveolar-arterial oxygen gradient, with pathology reports consistently demonstrating diffuse pulmonary microthrombi on autopsy, all consistent with a vascular occlusive etiology of respiratory failure rather than the more classic findings of low-compliance in ARDS. The COVID-19 pandemic is overwhelming the world's medical care capacity with unprecedented needs for mechanical ventilators and high rates of mortality once patients progress to needing mechanical ventilation, and in many environments including in parts of the United States the medical capacity is being exhausted. Fibrinolytic therapy has previously been used in a Phase 1 clinical trial that led to reduced mortality and marked improvements in oxygenation. Here we report a series of three patients with severe COVID-19 respiratory failure who were treated with tissue plasminogen activator. All three patients had a temporally related improvement in their respiratory status, with one of them being a durable response.
Coordinated progression through the cell cycle is a complex challenge for eukaryotic cells. Following genotoxic stress, diverse molecular signals must be integrated to establish checkpoints specific ...for each cell cycle stage, allowing time for various types of DNA repair. Phospho-Ser/Thr-binding domains have emerged as crucial regulators of cell cycle progression and DNA damage signalling. Such domains include 14-3-3 proteins, WW domains, Polo-box domains (in PLK1), WD40 repeats (including those in the E3 ligase SCF(βTrCP)), BRCT domains (including those in BRCA1) and FHA domains (such as in CHK2 and MDC1). Progress has been made in our understanding of the motif (or motifs) that these phospho-Ser/Thr-binding domains connect with on their targets and how these interactions influence the cell cycle and DNA damage response.
DNA-damage signaling utilizes a multitude of posttranslational modifiers as molecular switches to regulate cell-cycle checkpoints, DNA repair, cellular senescence, and apoptosis. Here we show that ...RNF8, a FHA/RING domain-containing protein, plays a critical role in the early DNA-damage response. We have solved the X-ray crystal structure of the FHA domain structure at 1.35 Å. We have shown that RNF8 facilitates the accumulation of checkpoint mediator proteins BRCA1 and 53BP1 to the damaged chromatin, on one hand through the phospho-dependent FHA domain-mediated binding of RNF8 to MDC1, on the other hand via its role in ubiquitylating H2AX and possibly other substrates at damage sites. Moreover, RNF8-depleted cells displayed a defective G2/M checkpoint and increased IR sensitivity. Together, our study implicates RNF8 as a novel DNA-damage-responsive protein that integrates protein phosphorylation and ubiquitylation signaling and plays a critical role in the cellular response to genotoxic stress.
DNA double-strand breaks (DSB) are repaired by multiple distinct pathways, with outcomes ranging from error-free repair to mutagenesis and genomic loss. DSB-repair pathway cross-talk and compensation ...is incompletely understood, despite its importance for genomic stability, oncogenesis, and genome editing using CRISPR/Cas9. To address this, we constructed and validated three fluorescent Cas9-based reporters, named DSB-Spectrum, that simultaneously quantify the contribution of multiple DNA repair pathways at a DSB. DSB-Spectrum reporters distinguish between DSB-repair by error-free canonical non-homologous end-joining (c-NHEJ) versus homologous recombination (HR; reporter 1), mutagenic repair versus HR (reporter 2), and mutagenic end-joining versus single strand annealing (SSA) versus HR (reporter 3). Using these reporters, we show that inhibiting the c-NHEJ factor DNA-PKcs increases repair by HR, but also substantially increases mutagenic SSA. Our data indicate that SSA-mediated DSB-repair also occurs at endogenous genomic loci, driven by Alu elements or homologous gene regions. Finally, we demonstrate that long-range end-resection factors DNA2 and Exo1 promote SSA and reduce HR, when both pathways compete for the same substrate. These new Cas9-based DSB-Spectrum reporters facilitate the comprehensive analysis of repair pathway crosstalk and DSB-repair outcome.
The energy-sensing AMP-activated protein kinase (AMPK) is activated by low nutrient levels. Functions of AMPK, other than its role in cellular metabolism, are just beginning to emerge. Here we use a ...chemical genetics screen to identify direct substrates of AMPK in human cells. We find that AMPK phosphorylates 28 previously unidentified substrates, several of which are involved in mitosis and cytokinesis. We identify the residues phosphorylated by AMPK in vivo in several substrates, including protein phosphatase 1 regulatory subunit 12C (PPP1R12C) and p21-activated protein kinase (PAK2). AMPK-induced phosphorylation is necessary for PPP1R12C interaction with 14-3-3 and phosphorylation of myosin regulatory light chain. Both AMPK activity and PPP1R12C phosphorylation are increased in mitotic cells and are important for mitosis completion. These findings suggest that AMPK coordinates nutrient status with mitosis completion, which may be critical for the organism's response to low nutrients during development, or in adult stem and cancer cells.
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► The energy-sensing kinase AMPK directly phosphorylates proteins involved in mitosis ► The phosphatase regulatory subunit PPP1R12C and the kinase PAK2 are AMPK substrates ► AMPK activity and PPP1R12C phosphorylation are particularly high in mitosis ► Altering AMPK activity or PPP1R12C phosphorylation impairs proper mitotic completion
Effective treatment for glioblastoma (GBM) is limited by the presence of the blood-brain barrier (BBB) and rapid resistance to single agent therapies. To address these issues, we developed a ...transferrin-functionalized nanoparticle (Tf-NP) that can deliver dual combination therapies. Using intravital imaging, we show the ability of Tf-NPs to traverse intact BBB in mice as well as achieve direct tumor binding in two intracranial orthotopic models of GBM. Treatment of tumor-bearing mice with Tf-NPs loaded with temozolomide and the bromodomain inhibitor JQ1 leads to increased DNA damage and apoptosis that correlates with a 1.5- to 2-fold decrease in tumor burden and corresponding increase in survival compared to equivalent free-drug dosing. Immunocompetent mice treated with Tf-NP-loaded drugs also show protection from the effects of systemic drug toxicity, demonstrating the preclinical potential of this nanoscale platform to deliver novel combination therapies to gliomas and other central nervous system tumors.
Proper chromatin function and maintenance of genomic stability depends on spatiotemporal coordination between the transcription and replication machinery. Loss of this coordination can lead to DNA ...damage from increased transcription-replication collision events. We report that deregulated transcription following BRD4 loss in cancer cells leads to the accumulation of RNA:DNA hybrids (R-loops) and collisions with the replication machinery causing replication stress and DNA damage. Whole genome BRD4 and γH2AX ChIP-Seq with R-loop IP qPCR reveals that BRD4 inhibition leads to accumulation of R-loops and DNA damage at a subset of known BDR4, JMJD6, and CHD4 co-regulated genes. Interference with BRD4 function causes transcriptional downregulation of the DNA damage response protein TopBP1, resulting in failure to activate the ATR-Chk1 pathway despite increased replication stress, leading to apoptotic cell death in S-phase and mitotic catastrophe. These findings demonstrate that inhibition of BRD4 induces transcription-replication conflicts, DNA damage, and cell death in oncogenic cells.
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