Selective Aurora A-TPX2 Interaction Inhibitors Have Efficacy as Targeted Antimitotic Agents.

Aurora A kinase, a cell division regulator, is frequently overexpressed in various cancers, provoking genome instability and resistance to antimitotic chemotherapy. Localization and enzymatic activity of Aurora A are regulated by its interaction with the spindle assembly factor TPX2. We have used fragment-based, structure-guided lead discovery to develop small molecule inhibitors of the Aurora A-TPX2 protein-protein interaction (PPI).

A glycolytic metabolite bypasses "two-hit" tumor suppression by BRCA2.

Knudson's "two-hit" paradigm posits that carcinogenesis requires inactivation of both copies of an autosomal tumor suppressor gene. Here, we report that the glycolytic metabolite methylglyoxal (MGO) transiently bypasses Knudson's paradigm by inactivating the breast cancer suppressor protein BRCA2 to elicit a cancer-associated, mutational single-base substitution (SBS) signature in nonmalignant mammary cells or patient-derived organoids. Germline monoallelic BRCA2 mutations predispose to these changes.

Cabergoline as a Novel Strategy for Post-Pregnancy Breast Cancer Prevention in Mice and Human.

Post-pregnancy breast cancer often carries a poor prognosis, posing a major clinical challenge. The increasing trend of later-life pregnancies exacerbates this risk, highlighting the need for effective chemoprevention strategies. Current options, limited to selective estrogen receptor modulators, aromatase inhibitors, or surgical procedures, offer limited efficacy and considerable side effects.

A transcriptional response to replication stress selectively expands a subset of Brca2-mutant mammary epithelial cells.

Germline BRCA2 mutation carriers frequently develop luminal-like breast cancers, but it remains unclear how BRCA2 mutations affect mammary epithelial subpopulations. Here, we report that monoallelic Brca2 mammary organoids subjected to replication stress activate a transcriptional response that selectively expands Brca2 luminal cells lacking hormone receptor expression (HR-). While CyTOF analyses reveal comparable epithelial compositions among wildtype and Brca2 mammary glands, Brca2 HR- luminal cells exhibit greater organoid formation and preferentially survive and expand under replication stress.

Ketogenic diet promotes tumor ferroptosis but induces relative corticosterone deficiency that accelerates cachexia.

Glucose dependency of cancer cells can be targeted with a high-fat, low-carbohydrate ketogenic diet (KD). However, in IL-6-producing cancers, suppression of the hepatic ketogenic potential hinders the utilization of KD as energy for the organism. In IL-6-associated murine models of cancer cachexia, we describe delayed tumor growth but accelerated cachexia onset and shortened survival in mice fed KD.

Domain-specific p53 mutants activate EGFR by distinct mechanisms exposing tissue-independent therapeutic vulnerabilities.

Mis-sense mutations affecting TP53 promote carcinogenesis both by inactivating tumor suppression, and by conferring pro-carcinogenic activities. We report here that p53 DNA-binding domain (DBD) and transactivation domain (TAD) mis-sense mutants unexpectedly activate pro-carcinogenic epidermal growth factor receptor (EGFR) signaling via distinct, previously unrecognized molecular mechanisms. DBD- and TAD-specific TP53 mutants exhibited different cellular localization and induced distinct gene expression profiles.

Ketogenic diet promotes tumor ferroptosis but induces relative corticosterone deficiency that accelerates cachexia.

The dependency of cancer cells on glucose can be targeted with high-fat low-carbohydrate ketogenic diet (KD). However, hepatic ketogenesis is suppressed in IL-6 producing cancers, which prevents the utilization of this nutrient source as energy for the organism. In two IL-6 associated murine models of cancer cachexia we describe delayed tumor growth but accelerated onset of cancer cachexia and shortened survival when mice are fed KD.

A fragment-based approach leading to the discovery of inhibitors of CK2α with a novel mechanism of action.

CK2 is a ubiquitous protein kinase with an anti-apoptotic role and is found to be overexpressed in multiple cancer types. To this end, the inhibition of CK2 is of great interest with regard to the development of novel anti-cancer therapeutics. ATP-site inhibition of CK2 is possible; however, this typically results in poor selectivity due to the highly conserved nature of the catalytic site amongst kinases.

A mitochondrial response to oxidative stress mediated by unscheduled RNA-DNA hybrids (R-loops).

How oxidative stress promotes aging-related human diseases like cancer and neurodegeneration remains unclear. Here, we discuss the origins and implications of an oxidative-stress response recently reported to destabilize the mitochondrial (mt) genome via unscheduled RNA/DNA hybrid (R-loop) accumulation, by impairing the recruitment of RNAseH1 to the regulatory regions of mtDNA.

Rapid recruitment of p53 to DNA damage sites directs DNA repair choice and integrity.

SignificanceOur work focuses on the critical longstanding question of the nontranscriptional role of p53 in tumor suppression. We demonstrate here that poly(ADP-ribose) polymerase (PARP)-dependent modification of p53 enables rapid recruitment of p53 to damage sites, where it in turn directs early repair pathway selection. Specifically, p53-mediated recruitment of 53BP1 at early time points promotes nonhomologous end joining over the more error-prone microhomology end-joining.

Early Neutrophilia Marked by Aerobic Glycolysis Sustains Host Metabolism and Delays Cancer Cachexia.

An elevated neutrophil-lymphocyte ratio negatively predicts the outcome of patients with cancer and is associated with cachexia, the terminal wasting syndrome. Here, using murine model systems of colorectal and pancreatic cancer we show that neutrophilia in the circulation and multiple organs, accompanied by extramedullary hematopoiesis, is an early event during cancer progression. Transcriptomic and metabolic assessment reveals that neutrophils in tumor-bearing animals utilize aerobic glycolysis, similar to cancer cells.

BRCA2 deficiency reveals that oxidative stress impairs RNaseH1 function to cripple mitochondrial DNA maintenance.

Oxidative stress is a ubiquitous cellular challenge implicated in aging, neurodegeneration, and cancer. By studying pathogenic mutations in the tumor suppressor BRCA2, we identify a general mechanism by which oxidative stress restricts mitochondrial (mt)DNA replication. BRCA2 inactivation induces R-loop accumulation in the mtDNA regulatory region and diminishes mtDNA replication initiation.

Target identification for small-molecule discovery in the FOXO3a tumor-suppressor pathway using a biodiverse peptide library.

Genetic screening technologies to identify and validate macromolecular interactions (MMIs) essential for complex pathways remain an important unmet need for systems biology and therapeutics development. Here, we use a library of peptides from diverse prokaryal genomes to screen MMIs promoting the nuclear relocalization of Forkhead Box O3 (FOXO3a), a tumor suppressor more frequently inactivated by post-translational modification than mutation. A hit peptide engages the 14-3-3 family of signal regulators through a phosphorylation-dependent interaction, modulates FOXO3a-mediated transcription, and suppresses cancer cell growth.

Cancer-causing BRCA2 missense mutations disrupt an intracellular protein assembly mechanism to disable genome maintenance.

Cancer-causing missense mutations in the 3418 amino acid BRCA2 breast and ovarian cancer suppressor protein frequently affect a short (∼340 residue) segment in its carboxyl-terminal domain (DBD). Here, we identify a shared molecular mechanism underlying their pathogenicity. Pathogenic BRCA2 missense mutations cluster in the DBD's helical domain (HD) and OB1-fold motifs, which engage the partner protein DSS1.

A small-molecule inhibitor of the BRCA2-RAD51 interaction modulates RAD51 assembly and potentiates DNA damage-induced cell death.

BRCA2 controls RAD51 recombinase during homologous DNA recombination (HDR) through eight evolutionarily conserved BRC repeats, which individually engage RAD51 via the motif Phe-x-x-Ala. Using structure-guided molecular design, templated on a monomeric thermostable chimera between human RAD51 and archaeal RadA, we identify CAM833, a 529 Da orthosteric inhibitor of RAD51:BRC with a K of 366 nM. The quinoline of CAM833 occupies a hotspot, the Phe-binding pocket on RAD51 and the methyl of the substituted α-methylbenzyl group occupies the Ala-binding pocket.

A kinase-independent function for AURORA-A in replisome assembly during DNA replication initiation.

The catalytic activity of human AURORA-A kinase (AURKA) regulates mitotic progression, and its frequent overexpression in major forms of epithelial cancer is associated with aneuploidy and carcinogenesis. Here, we report an unexpected, kinase-independent function for AURKA in DNA replication initiation whose inhibition through a class of allosteric inhibitors opens avenues for cancer therapy. We show that genetic depletion of AURKA, or its inhibition by allosteric but not catalytic inhibitors, blocks the G1-S cell cycle transition.

Author Correction: A systems view of spliceosomal assembly and branchpoints with iCLIP.

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

Pulsatile MAPK Signaling Modulates p53 Activity to Control Cell Fate Decisions at the G2 Checkpoint for DNA Damage.

Cell-autonomous changes in p53 expression govern the duration and outcome of cell-cycle arrest at the G2 checkpoint for DNA damage. Here, we report that mitogen-activated protein kinase (MAPK) signaling integrates extracellular cues with p53 dynamics to determine cell fate at the G2 checkpoint. Optogenetic tools and quantitative cell biochemistry reveal transient oscillations in MAPK activity dependent on ataxia-telangiectasia-mutated kinase after DNA damage.

Development of a Novel Cell-Permeable Protein-Protein Interaction Inhibitor for the Polo-box Domain of Polo-like Kinase 1.

Polo-like kinase 1 (PLK1) is a key regulator of mitosis and a recognized drug target for cancer therapy. Inhibiting the polo-box domain of PLK1 offers potential advantages of increased selectivity and subsequently reduced toxicity compared with targeting the kinase domain. However, many if not all existing polo-box domain inhibitors have been shown to be unsuitable for further development.

A cryptic hydrophobic pocket in the polo-box domain of the polo-like kinase PLK1 regulates substrate recognition and mitotic chromosome segregation.

The human polo-like kinase PLK1 coordinates mitotic chromosome segregation by phosphorylating multiple chromatin- and kinetochore-binding proteins. How PLK1 activity is directed to specific substrates via phosphopeptide recognition by its carboxyl-terminal polo-box domain (PBD) is poorly understood. Here, we combine molecular, structural and chemical biology to identify a determinant for PLK1 substrate recognition that is essential for proper chromosome segregation.

A systems view of spliceosomal assembly and branchpoints with iCLIP.

Studies of spliceosomal interactions are challenging due to their dynamic nature. Here we used spliceosome iCLIP, which immunoprecipitates SmB along with small nuclear ribonucleoprotein particles and auxiliary RNA binding proteins, to map spliceosome engagement with pre-messenger RNAs in human cell lines. This revealed seven peaks of spliceosomal crosslinking around branchpoints (BPs) and splice sites.

ZNF281 is recruited on DNA breaks to facilitate DNA repair by non-homologous end joining.

Efficient repair of DNA double-strand breaks (DSBs) is of critical importance for cell survival. Although non-homologous end joining (NHEJ) is the most used DSBs repair pathway in the cells, how NHEJ factors are sequentially recruited to damaged chromatin remains unclear. Here, we identify a novel role for the zinc-finger protein ZNF281 in participating in the ordered recruitment of the NHEJ repair factor XRCC4 at damage sites.

Fast single-cell biochemistry: theory, open source microscopy and applications.

Fluorescence lifetime sensing enables researchers to probe the physicochemical environment of a fluorophore providing a window through which we can observe the complex molecular make-up of the cell. Fluorescence lifetime imaging microscopy (FLIM) quantifies and maps cell biochemistry, a complex ensemble of dynamic processes. Unfortunately, typical high-resolution FLIM systems exhibit rather limited acquisition speeds, often insufficient to capture the time evolution of biochemical processes in living cells.