Diverse rescue potencies of p53 mutations to ATO are predetermined by intrinsic mutational properties.

Tumor suppressor p53 is inactivated by thousands of heterogeneous mutations in cancer, but their individual druggability remains largely elusive. Here, we evaluated 800 common p53 mutants for their rescue potencies by the representative generic rescue compound arsenic trioxide (ATO) in terms of transactivation activity, cell growth inhibition, and mouse tumor-suppressive activities. The rescue potencies were mainly determined by the solvent accessibility of the mutated residue, a key factor determining whether a mutation is a structural one, and the temperature sensitivity, the ability to reassemble the wild-type DNA binding surface at a low temperature, of the mutant protein. A total of 390 p53 mutants were rescued to varying degrees and thus were termed as type 1, type 2a, and type 2b mutations, depending on the degree to which they were rescued. The 33 type 1 mutations were rescued to amounts comparable to the wild type. In PDX mouse trials, ATO preferentially inhibited growth of tumors harboring type 1 and type 2a mutants. In an ATO clinical trial, we report the first-in-human mutant p53 reactivation in a patient harboring the type 1 V272M mutant. In 47 cell lines derived from 10 cancer types, ATO preferentially and effectively rescued type 1 and type 2a mutants, supporting the broad applicability of ATO in rescuing mutant p53. Our study provides the scientific and clinical communities with a resource of the druggabilities of numerous p53 mutations (www.rescuep53.net) and proposes a conceptual p53-targeting strategy based on individual mutant alleles rather than mutation type.

Developmental arsenic exposure impairs cognition, directly targets DNMT3A, and reduces DNA methylation.

Developmental arsenic exposure has been associated with cognitive deficits in epidemiological studies, but the underlying mechanisms remain poorly understood. Here, we establish a mouse model of developmental arsenic exposure exhibiting deficits of recognition and spatial memory in the offspring. These deficits are associated with genome-wide DNA hypomethylation and abnormal expression of cognition-related genes in the hippocampus. Arsenic atoms directly bind to the cysteine-rich ADD domain of DNA methyltransferase 3A (DNMT3A), triggering ubiquitin- and proteasome-mediated degradation of DNMT3A in different cellular contexts. DNMT3A degradation leads to genome-wide DNA hypomethylation in mouse embryonic fibroblasts but not in non-embryonic cell lines. Treatment with metformin, a first-line antidiabetic agent reported to increase DNA methylation, ameliorates the behavioral deficits and normalizes the aberrant expression of cognition-related genes and DNA methylation in the hippocampus of arsenic-exposed offspring. Our study establishes a DNA hypomethylation effect of developmental arsenic exposure and proposes a potential treatment against cognitive deficits in the offspring of pregnant women in arsenic-contaminated areas.

Repurposing antiparasitic antimonials to noncovalently rescue temperature-sensitive p53 mutations.

The tumor suppressor p53 is inactivated by over hundreds of heterogenous mutations in cancer. Here, we purposefully selected phenotypically reversible temperature-sensitive (TS) p53 mutations for pharmacological rescue with thermostability as the compound-screening readout. This rational screening identified antiparasitic drug potassium antimony tartrate (PAT) as an agent that can thermostabilize the representative TS mutant p53-V272M via noncovalent binding. PAT met the three basic criteria for a targeted drug: availability of a co-crystal structure, compatible structure-activity relationship, and intracellular target specificity, consequently exhibiting antitumor activity in a xenograft mouse model. At the antimony dose in clinical antiparasitic therapy, PAT effectively and specifically rescued p53-V272M in patient-derived primary leukemia cells in single-cell RNA sequencing. Further scanning of 815 frequent p53-missense mutations identified 65 potential PAT-treatable mutations, most of which were temperature sensitive. These results lay the groundwork for repurposing noncovalent antiparasitic antimonials for precisely treating cancers with the 65 p53 mutations.

A Prognostic Model for Acute Myeloid Leukemia Based on IL-2/STAT5 Pathway-Related Genes.

Accurate prognostic stratification of patients can provide guidance for personalized therapy. Many prognostic models for acute myeloid leukemia (AML) have been reported, but most have considerable inaccuracies due to contained variables with insufficient capacity of predicting survival and lack of adequate verification. Here, 235 genes strongly related to survival in AML were systematically identified through univariate Cox regression analysis of eight independent AML datasets. Pathway enrichment analysis of these 235 genes revealed that the IL-2/STAT5 signaling pathway was the most highly enriched. Through Cox proportional-hazards regression model and stepwise algorithm, we constructed a six-gene STAT5-associated signature based on the most robustly survival-related genes related to the IL-2/STAT5 signaling pathway. Good prognostic performance was observed in the training cohort (GSE37642-GPL96), and the signature was validated in seven other validation cohorts. As an independent prognostic factor, the STAT5-associated signature was positively correlated with patient age and ELN2017 risk levels. An integrated score based on these three prognostic factors had higher prognostic accuracy than the ELN2017 risk category. Characterization of immune cell infiltration indicated that impaired B-cell adaptive immunity, immunosuppressive effects, serious infection, and weakened anti-inflammatory function tended to accompany high-risk patients. Analysis of in-house clinical samples revealed that the STAT5-assocaited signature risk scores of AML patients were significantly higher than those of healthy people. Five chemotherapeutic drugs that were effective in these high-risk patients were screened in silico. Among the five drugs, MS.275, a known HDAC inhibitor, selectively suppressed the proliferation of cancer cells with high STAT5 phosphorylation levels in vitro. Taken together, the data indicate that the STAT5-associated signature is a reliable prognostic model that can be used to optimize prognostic stratification and guide personalized AML treatments.

Structural basis for activation and allosteric modulation of full-length calcium-sensing receptor.

Calcium-sensing receptor (CaSR) is a class C G protein-coupled receptor (GPCR) that plays an important role in calcium homeostasis and parathyroid hormone secretion. Here, we present multiple cryo-electron microscopy structures of full-length CaSR in distinct ligand-bound states. Ligands (Ca2+ and l-tryptophan) bind to the extracellular domain of CaSR and induce large-scale conformational changes, leading to the closure of two heptahelical transmembrane domains (7TMDs) for activation. The positive modulator (evocalcet) and the negative allosteric modulator (NPS-2143) occupy the similar binding pocket in 7TMD. The binding of NPS-2143 causes a considerable rearrangement of two 7TMDs, forming an inactivated TM6/TM6 interface. Moreover, a total of 305 disease-causing missense mutations of CaSR have been mapped to the structure in the active state, creating hotspot maps of five clinical endocrine disorders. Our results provide a structural framework for understanding the activation, allosteric modulation mechanism, and disease therapy for class C GPCRs.

Arsenic Trioxide Rescues Structural p53 Mutations through a Cryptic Allosteric Site.

TP53 is the most frequently mutated gene in cancer, yet these mutations remain therapeutically non-actionable. Major challenges in drugging p53 mutations include heterogeneous mechanisms of inactivation and the absence of broadly applicable allosteric sites. Here we report the identification of small molecules, including arsenic trioxide (ATO), an established agent in treating acute promyelocytic leukemia, as cysteine-reactive compounds that rescue structural p53 mutations. Crystal structures of arsenic-bound p53 mutants reveal a cryptic allosteric site involving three arsenic-coordinating cysteines within the DNA-binding domain, distal to the zinc-binding site. Arsenic binding stabilizes the DNA-binding loop-sheet-helix motif alongside the overall ß-sandwich fold, endowing p53 mutants with thermostability and transcriptional activity. In cellular and mouse xenograft models, ATO reactivates mutant p53 for tumor suppression. Investigation of the 25 most frequent p53 mutations informs patient stratification for clinical exploration. Our results provide a mechanistic basis for repurposing ATO to target p53 mutations for widely applicable yet personalized cancer therapies.

Dimerization of MICU Proteins Controls Ca2+ Influx through the Mitochondrial Ca2+ Uniporter.

The mitochondrial Ca2+ uniporter complex (MCUC) is responsible for Ca2+ influx into the mitochondrial matrix, playing critical roles in various mitochondrial functions. Eukaryotic MCUC consists of multiple subunits, and its Ca2+ influx activity is controlled by regulatory subunits, including mitochondrial Ca2+ uptake 1 (MICU1) and its paralogs (MICU2 and MICU3). However, the underlying mechanism remains unclear. Here, we determined multiple crystal structures of MICU2 and MICU3 from Homo sapiens. Our data demonstrate that distinct MICU protein N-domains determine the specific type of MICU dimers that perform the opposing roles in mitochondrial Ca2+ uptake at low cytosolic Ca2+ levels. In contrast, at high cytosolic Ca2+ levels, all MICU proteins undergo dimer rearrangement induced by Ca2+ binding, which releases the suppression of the MCUC pore-forming subunit and promotes the influx of large amounts of Ca2+. Altogether, our results elucidate the delicate mechanism of mitochondrial Ca2+ uptake regulation by MICU proteins.

CaM/BAG5/Hsc70 signaling complex dynamically regulates leaf senescence.

Calcium signaling plays an essential role in plant cell physiology, and chaperone-mediated protein folding directly regulates plant programmed cell death. The Arabidopsis thaliana protein AtBAG5 (Bcl-2-associated athanogene 5) is unique in that it contains both a BAG domain capable of binding Hsc70 (Heat shock cognate protein 70) and a characteristic IQ motif that is specific for Ca(2+)-free CaM (Calmodulin) binding and hence acts as a hub linking calcium signaling and the chaperone system. Here, we determined crystal structures of AtBAG5 alone and in complex with Ca(2+)-free CaM. Structural and biochemical studies revealed that Ca(2+)-free CaM and Hsc70 bind AtBAG5 independently, whereas Ca(2+)-saturated CaM and Hsc70 bind AtBAG5 with negative cooperativity. Further in vivo studies confirmed that AtBAG5 localizes to mitochondria and that its overexpression leads to leaf senescence symptoms including decreased chlorophyll retention and massive ROS production in dark-induced plants. Mutants interfering the CaM/AtBAG5/Hsc70 complex formation leads to different phenotype of leaf senescence. Collectively, we propose that the CaM/AtBAG5/Hsc70 signaling complex plays an important role in regulating plant senescence.

Crystallographic analysis of the Arabidopsis thaliana BAG5-calmodulin protein complex.

Arabidopsis thaliana BAG5 (AtBAG5) belongs to the plant BAG (Bcl-2-associated athanogene) family that performs diverse functions ranging from growth and development to abiotic stress and senescence. BAG family members can act as nucleotide-exchange factors for heat-shock protein 70 (Hsp70) through binding of their evolutionarily conserved BAG domains to the Hsp70 ATPase domain, and thus may be involved in the regulation of chaperone-mediated protein folding in plants. AtBAG5 is distinguished from other family members by the presence of a unique IQ motif adjacent to the BAG domain; this motif is specific for calmodulin (CaM) binding, indicating a potential role in the plant calcium signalling pathway. To provide a better understanding of the IQ motif-mediated interaction between AtBAG5 and CaM, the two proteins were expressed and purified separately and then co-crystallized together. Diffraction-quality crystals of the complex were grown using the sitting-drop vapour-diffusion technique from a condition consisting of 0.1 M Tris-HCl pH 8.5, 2.5 M ammonium sulfate. The crystals belonged to space group P2(1)2(1)2(1), with unit-cell parameters a = 64.56, b = 74.89, c = 117.09 Å. X-ray diffraction data were recorded to a resolution of 2.5 Šfrom a single crystal using synchrotron radiation. Assuming the presence of two molecules in the asymmetric unit, a Matthews coefficient of 2.44 Å(3) Da(-1) was calculated, corresponding to a solvent content of approximately 50%.