Correction: AI-Assisted chemical probe discovery for the understudied Calcium-Calmodulin Dependent Kinase, PNCK.

[This corrects the article DOI: 10.1371/journal.pcbi.1010263.].

AI-Assisted chemical probe discovery for the understudied Calcium-Calmodulin Dependent Kinase, PNCK.

PNCK, or CAMK1b, is an understudied kinase of the calcium-calmodulin dependent kinase family which recently has been identified as a marker of cancer progression and survival in several large-scale multi-omics studies. The biology of PNCK and its relation to oncogenesis has also begun to be elucidated, with data suggesting various roles in DNA damage response, cell cycle control, apoptosis and HIF-1-alpha related pathways. To further explore PNCK as a clinical target, potent small-molecule molecular probes must be developed. Currently, there are no targeted small molecule inhibitors in pre-clinical or clinical studies for the CAMK family. Additionally, there exists no experimentally derived crystal structure for PNCK. We herein report a three-pronged chemical probe discovery campaign which utilized homology modeling, machine learning, virtual screening and molecular dynamics to identify small molecules with low-micromolar potency against PNCK activity from commercially available compound libraries. We report the discovery of a hit-series for the first targeted effort towards discovering PNCK inhibitors that will serve as the starting point for future medicinal chemistry efforts for hit-to-lead optimization of potent chemical probes.

Innate immune signaling drives late cardiac toxicity following DNA-damaging cancer therapies.

Late cardiac toxicity is a potentially lethal complication of cancer therapy, yet the pathogenic mechanism remains largely unknown, and few treatment options exist. Here we report DNA-damaging agents such as radiation and anthracycline chemotherapies inducing delayed cardiac inflammation following therapy due to activation of cGAS- and STING-dependent type I interferon signaling. Genetic ablation of cGAS-STING signaling in mice inhibits DNA damage-induced cardiac inflammation, rescues late cardiac functional decline, and prevents death from cardiac events. Treatment with a STING antagonist suppresses cardiac interferon signaling following DNA-damaging therapies and effectively mitigates cardiac toxicity. These results identify a therapeutically targetable, pathogenic mechanism for one of the most vexing treatment-related toxicities in cancer survivors.

Cellular and molecular effects of PNCK, a non-canonical kinase target in renal cell carcinoma.

Renal cell carcinoma (RCC) is a fatal disease when advanced. While immunotherapy and tyrosine kinase inhibitor-based combinations are associated with improved survival, the majority of patients eventually succumb to the disease. Through a comprehensive pan-cancer, pan-kinome analysis of the Cancer Genome Atlas (TCGA), pregnancy-upregulated non-ubiquitous calcium-calmodulin-dependent kinase (PNCK), was identified as the most differentially overexpressed kinase in RCC. PNCK overexpression correlated with tumor stage, grade and poor survival. PNCK overexpression in RCC cells was associated with increased CREB phosphorylation, increased cell proliferation, and cell cycle progression. PNCK down-regulation, conversely, was associated with the opposite, in addition to increased apoptosis. Pathway analyses in PNCK knockdown cells showed significant down-regulation of hypoxia and angiogenesis pathways, as well as the modulation of the cell cycle, DNA damage, and apoptosis pathways. These results demonstrate for the first time the biological role of PNCK, an understudied kinase, in RCC and validate PNCK as a druggable target.

The C-terminus of Gain-of-Function Mutant p53 R273H Is Required for Association with PARP1 and Poly-ADP-Ribose.

The TP53 gene is mutated in 80% of triple-negative breast cancers. Cells that harbor the hot-spot p53 gene mutation R273H produce an oncogenic mutant p53 (mtp53) that enhances cell proliferative and metastatic properties. The enhanced activities of mtp53 are collectively referred to as gain-of-function (GOF), and may include transcription-independent chromatin-based activities shared with wild-type p53 (wtp53) such as association with replicating DNA and DNA replication associated proteins like PARP1. However, how mtp53 upregulates cell proliferation is not well understood. wtp53 interacts with PARP1 using a portion of its C-terminus. The wtp53 oligomerization and far C-terminal domain (CTD) located within the C-terminus constitute putative GOF-associated domains, because mtp53 R273H expressing breast cancer cells lacking both domains manifest slow proliferation phenotypes. We addressed if the C-terminal region of mtp53 R273H is important for chromatin interaction and breast cancer cell proliferation using CRISPR-Cas9 mutated MDA-MB-468 cells endogenously expressing mtp53 R273H C-terminal deleted isoforms (R273HΔ381-388 and R273HΔ347-393). The mtp53 R273HΔ347-393 lacks the CTD and a portion of the oligomerization domain. We observed that cells harboring mtp53 R273HΔ347-393 (compared with mtp53 R273H full-length) manifest a significant reduction in chromatin, PARP1, poly-ADP-ribose (PAR), and replicating DNA binding. These cells also exhibited impaired response to hydroxyurea replicative stress, decreased sensitivity to the PARP-trapping drug combination temozolomide-talazoparib, and increased phosphorylated 53BP1 foci, suggesting reduced Okazaki fragment processing. IMPLICATIONS: The C-terminal region of mtp53 confers GOF activity that mediates mtp53-PARP1 and PAR interactions assisting DNA replication, thus implicating new biomarkers for PARP inhibitor therapy.

In vivo antitumor activity by dual stromal and tumor-targeted oncolytic measles viruses.

The tumor stroma acts as a barrier that limits the efficacy of systemically administered oncolytic viruses (OV). We previously demonstrated that stromal-selective, retargeted oncolytic measles viruses (MVs) delay in vivo tumor progression. To further characterize the contribution of stromal targeting to MV's overall in vivo efficacy in an experimental cancer model, a dual targeted oncolytic measles virus (MV-CD46-muPA) able to simultaneously infect murine stromal (via murine uPAR) and human cancer (via CD46) cells was developed. MV-CD46-muPA infected, replicated, and induced cytotoxicity in both murine and human cancer cells. Viral infection was successfully transferred from stromal to tumor cells in vitro, leading to tumor cell oncolysis. Systemic administration of MV-CD46-muPA led to improved antitumor effects in colon (HT-29) cancer xenografts compared to vehicle or CD46 only targeted MVs. These effects were associated with improved tumor viral deposition, increased apoptosis, and decreases in murine stromal endothelial cells and fibroblasts. MV-CD46-muPA modulated cell cycle, survival, proliferation, and metabolic pathways, as determined by functional proteomic analysis of treated tumors. The above findings further validate the concept that dual stromal and tumor cell viral targeting enhances the therapeutic effects of systemically administered OVs and support further preclinical and clinical development of stromal directed virotherapies.

Molecular Effects of Stromal-Selective Targeting by uPAR-Retargeted Oncolytic Virus in Breast Cancer.

The tumor microenvironment (TME) is a relevant target for novel biological therapies. MV-m-uPA and MV-h-uPA are fully retargeted, species-specific, oncolytic measles viruses (MV) directed against murine or human urokinase receptor (PLAUR/uPAR), expressed in tumor and stromal cells. The effects of stromal-selective targeting by uPAR-retargeted MVs were investigated. In vitro infection, virus-induced GFP expression, and cytotoxicity by MV-h-uPA and MV-m-uPA were demonstrated in human and murine cancer cells and cancer-associated fibroblasts in a species-specific manner. In a murine fibroblast/human breast cancer 3D coculture model, selective fibroblast targeting by MV-m-uPA inhibited breast cancer cell growth. Systemic administration of murine-specific MV-m-uPA in mice bearing human MDA-MB-231 xenografts was associated with a significant delay in tumor progression and improved survival compared with controls. Experiments comparing tumor (MV-h-uPA) versus stromal (MV-m-uPA) versus combined virus targeting showed that tumor and stromal targeting was associated with improved tumor control over the other groups. Correlative studies confirmed in vivo viral targeting of tumor stroma by MV-m-uPA, increased apoptosis, and virus-induced differential regulation of murine stromal genes associated with inflammatory, angiogenesis, and survival pathways, as well as indirect regulation of human cancer pathways, indicating viral-induced modulation of tumor-stroma interactions. These data demonstrate the feasibility of stromal-selective targeting by an oncolytic MV, virus-induced modulation of tumor-stroma pathways, and subsequent tumor growth delay. These findings further validate the critical role of stromal uPAR in cancer progression and the potential of oncolytic viruses as antistromal agents.Implications: The current report demonstrates for the first time the biological, in vitro, and in vivo antitumor and molecular effects of stromal selective targeting by an oncolytic virus. Mol Cancer Res; 15(10); 1410-20. ©2017 AACR.

Hypoxic resistance of KRAS mutant tumor cells to 3-Bromopyruvate is counteracted by Prima-1 and reversed by N-acetylcysteine.

BACKGROUND: The metabolic inhibitor 3-bromopyruvate (3-BrPA) is a promising anti-cancer alkylating agent, shown to inhibit growth of some colorectal carcinoma with KRAS mutation. Recently, we demonstrated increased resistance to 3-BrPA in wt p53 tumor cells compared to those with p53 silencing or mutation. Since hypoxic microenvironments select for tumor cells with diminished therapeutic response, we investigated whether hypoxia unequally increases resistance to 3-BrPA in wt p53 MelJuso melanoma harbouring (Q61L)-mutant NRAS and wt BRAF, C8161 melanoma with (G12D)-mutant KRAS (G464E)-mutant BRAF, and A549 lung carcinoma with a KRAS (G12S)-mutation. Since hypoxia increases the toxicity of the p53 activator, Prima-1 against breast cancer cells irrespective of their p53 status, we also investigated whether Prima-1 reversed hypoxic resistance to 3-BrPA. RESULTS: In contrast to the high susceptibility of hypoxic mutant NRAS MelJuso cells to 3-BrPA or Prima-1, KRAS mutant C8161 and A549 cells revealed hypoxic resistance to 3-BrPA counteracted by Prima-1. In A549 cells, Prima-1 increased p21CDKN1mRNA, and reciprocally inhibited mRNA expression of the SLC2A1-GLUT1 glucose transporter-1 and ALDH1A1, gene linked to detoxification and stem cell properties. 3-BrPA lowered CAIX and VEGF mRNA expression. Death from joint Prima-1 and 3-BrPA treatment in KRAS mutant A549 and C8161 cells seemed mediated by potentiating oxidative stress, since it was antagonized by the anti-oxidant and glutathione precursor N-acetylcysteine. CONCLUSIONS: This report is the first to show that Prima-1 kills hypoxic wt p53 KRAS-mutant cells resistant to 3-BrPA, partly by decreasing GLUT-1 expression and exacerbating pro-oxidant stress.

Influence of charge and metal coordination of meso-substituted porphyrins on bacterial photoinactivation.

The photodynamic effect of meso-substituted porphyrins with different charges and metal ions: meso-tetraphenylporphyrin tetrasulfonate 1, its nickel 2 and zinc complexes 3; meso-tetranaphthylporphyrin tetrasulfonate 4, and its zinc complex Zn 5; and tetra piridyl ethylacetate porphirins 6 and their nickel 7 and zinc 8 complexes, were synthesized and studied their antimicrobial activity against Escherichia coli. Fluorescence quantum yields (ΦF) were measured in water using reference TPPS4, obtaining higher values for complexes 3 and 4. The singlet oxygen ΦΔ were measured using histidine as trapping singlet oxygen and Rose Bengal as a reference standard. Complexes 1, 2 and 6 have the highest quantum yields of singlet oxygen formation, showing no relation with the peripheral charges and efficiency as Type II photosensitizers. Meanwhile complexes 3, 8 and 4 were the most efficient in producing radical species, determined with their reaction with NADH. The photoinduced antibacterial activity of complex was investigated at different concentrations of the photosensitizers with an irradiation time of 30 min. The higher antibacterial activities were obtained for the complexes 1-3 that are those with greater production of ROS and minor structural deformations. Complexes 7 and 8 had moderate activity, while 4-6 a low activity. Thus, in this work demonstrates that the production of ROS and structural deformations due to peripheral substituents and metal coordination, influence the activity of the complexes studied. Therefore, is important to perform comprehensive study physics and structurally when predicting or explain such activity.