A comprehensive AI-driven analysis of large-scale omic datasets reveals novel dual-purpose targets for the treatment of cancer and aging.

As aging and tumorigenesis are tightly interconnected biological processes, targeting their common underlying driving pathways may induce dual-purpose anti-aging and anti-cancer effects. Our transcriptomic analyses of 16,740 healthy samples demonstrated tissue-specific age-associated gene expression, with most tumor suppressor genes downregulated during aging. Furthermore, a large-scale pan-cancer analysis of 11 solid tumor types (11,303 cases and 4431 control samples) revealed that many cellular processes, such as protein localization, DNA replication, DNA repair, cell cycle, and RNA metabolism, were upregulated in cancer but downregulated in healthy aging tissues, whereas pathways regulating cellular senescence were upregulated in both aging and cancer. Common cancer targets were identified by the AI-driven target discovery platform-PandaOmics. Age-associated cancer targets were selected and further classified into four groups based on their reported roles in lifespan. Among the 51 identified age-associated cancer targets with anti-aging experimental evidence, 22 were proposed as dual-purpose targets for anti-aging and anti-cancer treatment with the same therapeutic direction. Among age-associated cancer targets without known lifespan-regulating activity, 23 genes were selected based on predicted dual-purpose properties. Knockdown of histone demethylase KDM1A, one of these unexplored candidates, significantly extended lifespan in Caenorhabditis elegans. Given KDM1A's anti-cancer activities reported in both preclinical and clinical studies, our findings propose KDM1A as a promising dual-purpose target. This is the first study utilizing an innovative AI-driven approach to identify dual-purpose target candidates for anti-aging and anti-cancer treatment, supporting the value of AI-assisted target identification for drug discovery.

Identification of dual-purpose therapeutic targets implicated in aging and glioblastoma multiforme using PandaOmics - an AI-enabled biological target discovery platform.

Glioblastoma Multiforme (GBM) is the most aggressive and most common primary malignant brain tumor. The age of GBM patients is considered as one of the disease's negative prognostic factors and the mean age of diagnosis is 62 years. A promising approach to preventing both GBM and aging is to identify new potential therapeutic targets that are associated with both conditions as concurrent drivers. In this work, we present a multi-angled approach of identifying targets, which takes into account not only the disease-related genes but also the ones important in aging. For this purpose, we developed three strategies of target identification using the results of correlation analysis augmented with survival data, differences in expression levels and previously published information of aging-related genes. Several studies have recently validated the robustness and applicability of AI-driven computational methods for target identification in both cancer and aging-related diseases. Therefore, we leveraged the AI predictive power of the PandaOmics TargetID engine in order to rank the resulting target hypotheses and prioritize the most promising therapeutic gene targets. We propose cyclic nucleotide gated channel subunit alpha 3 (CNGA3), glutamate dehydrogenase 1 (GLUD1) and sirtuin 1 (SIRT1) as potential novel dual-purpose therapeutic targets to treat aging and GBM.

AlphaFold accelerates artificial intelligence powered drug discovery: efficient discovery of a novel CDK20 small molecule inhibitor.

The application of artificial intelligence (AI) has been considered a revolutionary change in drug discovery and development. In 2020, the AlphaFold computer program predicted protein structures for the whole human genome, which has been considered a remarkable breakthrough in both AI applications and structural biology. Despite the varying confidence levels, these predicted structures could still significantly contribute to structure-based drug design of novel targets, especially the ones with no or limited structural information. In this work, we successfully applied AlphaFold to our end-to-end AI-powered drug discovery engines, including a biocomputational platform PandaOmics and a generative chemistry platform Chemistry42. A novel hit molecule against a novel target without an experimental structure was identified, starting from target selection towards hit identification, in a cost- and time-efficient manner. PandaOmics provided the protein of interest for the treatment of hepatocellular carcinoma (HCC) and Chemistry42 generated the molecules based on the structure predicted by AlphaFold, and the selected molecules were synthesized and tested in biological assays. Through this approach, we identified a small molecule hit compound for cyclin-dependent kinase 20 (CDK20) with a binding constant Kd value of 9.2 ± 0.5 µM (n = 3) within 30 days from target selection and after only synthesizing 7 compounds. Based on the available data, a second round of AI-powered compound generation was conducted and through this, a more potent hit molecule, ISM042-2-048, was discovered with an average Kd value of 566.7 ± 256.2 nM (n = 3). Compound ISM042-2-048 also showed good CDK20 inhibitory activity with an IC50 value of 33.4 ± 22.6 nM (n = 3). In addition, ISM042-2-048 demonstrated selective anti-proliferation activity in an HCC cell line with CDK20 overexpression, Huh7, with an IC50 of 208.7 ± 3.3 nM, compared to a counter screen cell line HEK293 (IC50 = 1706.7 ± 670.0 nM). This work is the first demonstration of applying AlphaFold to the hit identification process in drug discovery.

High-confidence cancer patient stratification through multiomics investigation of DNA repair disorders.

Multiple cancer types have limited targeted therapeutic options, in part due to incomplete understanding of the molecular processes underlying tumorigenesis and significant intra- and inter-tumor heterogeneity. Identification of novel molecular biomarkers stratifying cancer patients with different survival outcomes may provide new opportunities for target discovery and subsequent development of tailored therapies. Here, we applied the artificial intelligence-driven PandaOmics platform ( https://pandaomics.com/ ) to explore gene expression changes in rare DNA repair-deficient disorders and identify novel cancer targets. Our analysis revealed that CEP135, a scaffolding protein associated with early centriole biogenesis, is commonly downregulated in DNA repair diseases with high cancer predisposition. Further screening of survival data in 33 cancers available at TCGA database identified sarcoma as a cancer type where lower survival was significantly associated with high CEP135 expression. Stratification of cancer patients based on CEP135 expression enabled us to examine therapeutic targets that could be used for the improvement of existing therapies against sarcoma. The latter was based on application of the PandaOmics target-ID algorithm coupled with in vitro studies that revealed polo-like kinase 1 (PLK1) as a potential therapeutic candidate in sarcoma patients with high CEP135 levels and poor survival. While further target validation is required, this study demonstrated the potential of in silico-based studies for a rapid biomarker discovery and target characterization.

Effector T cell responses unleashed by regulatory T cell ablation exacerbate oral squamous cell carcinoma.

Immune suppression by CD4+FOXP3+ regulatory T (Treg) cells and tumor infiltration by CD8+ effector T cells represent two major factors impacting response to cancer immunotherapy. Using deconvolution-based transcriptional profiling of human papilloma virus (HPV)-negative oral squamous cell carcinomas (OSCCs) and other solid cancers, we demonstrate that the density of Treg cells does not correlate with that of CD8+ T cells in many tumors, revealing polarized clusters enriched for either CD8+ T cells or CD4+ Treg and conventional T cells. In a mouse model of carcinogen-induced OSCC characterized by CD4+ T cell enrichment, late-stage Treg cell ablation triggers increased densities of both CD4+ and CD8+ effector T cells within oral lesions. Notably, this intervention does not induce tumor regression but instead induces rapid emergence of invasive OSCCs via an effector T cell-dependent process. Thus, induction of a T cell-inflamed phenotype via therapeutic manipulation of Treg cells may trigger unexpected tumor-promoting effects in OSCC.

Correction: Targeting focal adhesion kinase overcomes erlotinib resistance in smoke induced lung cancer by altering phosphorylation of epidermal growth factor receptor.

[This corrects the article DOI: 10.18632/oncoscience.395.].

Doublecortin-Like Kinase 1 (DCLK1) Is a Novel NOTCH Pathway Signaling Regulator in Head and Neck Squamous Cell Carcinoma.

Despite recent advancements, the 5 year survival of head and neck squamous cell carcinoma (HNSCC) hovers at 60%. DCLK1 has been shown to regulate epithelial-to-mesenchymal transition as well as serving as a cancer stem cell marker in colon, pancreatic and renal cancer. Although it was reported that DCLK1 is associated with poor prognosis in oropharyngeal cancers, very little is known about the molecular characterization of DCLK1 in HNSCC. In this study, we performed a comprehensive transcriptome-based computational analysis on hundreds of HNSCC patients from TCGA and GEO databases, and found that DCLK1 expression positively correlates with NOTCH signaling pathway activation. Since NOTCH signaling has a recognized role in HNSCC tumorigenesis, we next performed a series of in vitro experiments in a collection of HNSCC cell lines to investigate the role of DCLK1 in NOTCH pathway regulation. Our analyses revealed that DCLK1 inhibition, using either a pharmacological inhibitor or siRNA, resulted in substantially decreased proliferation, invasion, migration, and colony formation. Furthermore, these effects paralleled downregulation of active NOTCH1, and its downstream effectors, HEY1, HES1 and HES5, whereas overexpression of DCLK1 in normal keratinocytes, lead to an upregulation of NOTCH signaling associated with increased proliferation. Analysis of 233 primary and 40 recurrent HNSCC cancer biopsies revealed that high DCLK1 expression was associated with poor prognosis and showed a trend towards higher active NOTCH1 expression in tumors with elevated DCLK1. Our results demonstrate the novel role of DCLK1 as a regulator of NOTCH signaling network and suggest its potential as a therapeutic target in HNSCC.

Radioprotectors.org: an open database of known and predicted radioprotectors.

The search for radioprotectors is an ambitious goal with many practical applications. Particularly, the improvement of human radioresistance for space is an important task, which comes into view with the recent successes in the space industry. Currently, all radioprotective drugs can be divided into two large groups differing in their effectiveness depending on the type of exposure. The first of these is radioprotectors, highly effective for pulsed, and some types of relatively short exposure to irradiation. The second group consists of long-acting radioprotectors. These drugs are effective for prolonged and fractionated irradiation. They also protect against impulse exposure to ionizing radiation, but to a lesser extent than short-acting radioprotectors. Creating a database on radioprotectors is a necessity dictated by the modern development of science and technology. We have created an open database, Radioprotectors.org, containing an up-to-date list of substances with proven radioprotective properties. All radioprotectors are annotated with relevant chemical and biological information, including transcriptomic data, and can be filtered according to their properties. Additionally, the performed transcriptomics analysis has revealed specific transcriptomic profiles of radioprotectors, which should facilitate the search for potent radioprotectors.

PIM1 kinase promotes gallbladder cancer cell proliferation via inhibition of proline-rich Akt substrate of 40 kDa (PRAS40).

Gallbladder cancer (GBC) is a rare malignancy, associated with poor disease prognosis with a 5-year survival of only 20%. This has been attributed to late presentation of the disease, lack of early diagnostic markers and limited efficacy of therapeutic interventions. Elucidation of molecular events in GBC can contribute to better management of the disease by aiding in the identification of therapeutic targets. To identify aberrantly activated signaling events in GBC, tandem mass tag-based quantitative phosphoproteomic analysis of five GBC cell lines was carried out. Proline-rich Akt substrate 40 kDa (PRAS40) was one of the proteins found to be hyperphosphorylated in all the invasive GBC cell lines. Tissue microarray-based immunohistochemical labeling of phospho-PRAS40 (T246) revealed moderate to strong staining in 77% of the primary gallbladder adenocarcinoma cases. Regulation of PRAS40 activity by inhibiting its upstream kinase PIM1 resulted in a significant decrease in cell proliferation, colony forming and invasive ability of GBC cells. Our results support the role of PRAS40 phosphorylation in GBC cell survival and aggressiveness. This study also elucidates phospho-PRAS40 as a clinical marker in GBC and the role of PIM1 as a therapeutic target in GBC.

Targeting focal adhesion kinase overcomes erlotinib resistance in smoke induced lung cancer by altering phosphorylation of epidermal growth factor receptor.

EGFR-based targeted therapies have shown limited success in smokers. Identification of alternate signaling mechanism(s) leading to TKI resistance in smokers is critically important. We observed increased resistance to erlotinib in H358 NSCLC (non-small cell lung carcinoma) cells chronically exposed to cigarette smoke (H358-S) compared to parental cells. SILAC-based mass-spectrometry approach was used to study altered signaling in H358-S cell line. Importantly, among the top phosphosites in H358-S cells we observed hyperphosphorylation of EGFR (Y1197) and non-receptor tyrosine kinase FAK (Y576/577). Supporting these observations, a transcriptomic-based pathway activation analysis of TCGA NSCLC datasets revealed that FAK and EGFR internalization pathways were significantly upregulated in smoking patients, compared to the never-smokers and were associated with elevated PI3K signaling and lower level of caspase cascade and E-cadherin pathways activation. We show that inhibition of FAK led to decreased cellular proliferation and invasive ability of the smoke-exposed cells, and restored their dependency on EGFR signaling. Our data suggests that activation of focal adhesion pathway significantly contributes to erlotinib resistance, and that FAK is a potential therapeutic target for management of erlotinib resistance in smoke-induced NSCLC.

Vive la radiorésistance!: converging research in radiobiology and biogerontology to enhance human radioresistance for deep space exploration and colonization.

While many efforts have been made to pave the way toward human space colonization, little consideration has been given to the methods of protecting spacefarers against harsh cosmic and local radioactive environments and the high costs associated with protection from the deleterious physiological effects of exposure to high-Linear energy transfer (high-LET) radiation. Herein, we lay the foundations of a roadmap toward enhancing human radioresistance for the purposes of deep space colonization and exploration. We outline future research directions toward the goal of enhancing human radioresistance, including upregulation of endogenous repair and radioprotective mechanisms, possible leeways into gene therapy in order to enhance radioresistance via the translation of exogenous and engineered DNA repair and radioprotective mechanisms, the substitution of organic molecules with fortified isoforms, and methods of slowing metabolic activity while preserving cognitive function. We conclude by presenting the known associations between radioresistance and longevity, and articulating the position that enhancing human radioresistance is likely to extend the healthspan of human spacefarers as well.

Bifunctional immune checkpoint-targeted antibody-ligand traps that simultaneously disable TGFß enhance the efficacy of cancer immunotherapy.

A majority of cancers fail to respond to immunotherapy with antibodies targeting immune checkpoints, such as cytotoxic T-lymphocyte antigen-4 (CTLA-4) or programmed death-1 (PD-1)/PD-1 ligand (PD-L1). Cancers frequently express transforming growth factor-ß (TGFß), which drives immune dysfunction in the tumor microenvironment by inducing regulatory T cells (Tregs) and inhibiting CD8+ and TH1 cells. To address this therapeutic challenge, we invent bifunctional antibody-ligand traps (Y-traps) comprising an antibody targeting CTLA-4 or PD-L1 fused to a TGFß receptor II ectodomain sequence that simultaneously disables autocrine/paracrine TGFß in the target cell microenvironment (a-CTLA4-TGFßRIIecd and a-PDL1-TGFßRIIecd). a-CTLA4-TGFßRIIecd is more effective in reducing tumor-infiltrating Tregs and inhibiting tumor progression compared with CTLA-4 antibody (Ipilimumab). Likewise, a-PDL1-TGFßRIIecd exhibits superior antitumor efficacy compared with PD-L1 antibodies (Atezolizumab or Avelumab). Our data demonstrate that Y-traps counteract TGFß-mediated differentiation of Tregs and immune tolerance, thereby providing a potentially more effective immunotherapeutic strategy against cancers that are resistant to current immune checkpoint inhibitors.

Residual γH2AX foci induced by low dose x-ray radiation in bone marrow mesenchymal stem cells do not cause accelerated senescence in the progeny of irradiated cells.

Mechanisms underlying the effects of low-dose ionizing radiation (IR) exposure (10-100 mGy) remain unknown. Here we present a comparative study of early (less than 24h) and delayed (up to 11 post-irradiation passages) radiation effects caused by low (80 mGy) vs intermediate (1000 mGy) dose X-ray exposure in cultured human bone marrow mesenchymal stem cells (MSCs). We show that γН2АХ foci induced by an intermediate dose returned back to the control value by 24 h post-irradiation. In contrast, low-dose irradiation resulted in residual γН2АХ foci still present at 24 h. Notably, these low dose induced residual γН2АХ foci were not co-localized with рАТМ foci and were observed predominantly in the proliferating Кi67 positive (Кi67+) cells. The number of γН2АХ foci and the fraction of nonproliferating (Кi67-) and senescent (SA-ß-gal+) cells measured at passage 11 were increased in cultures exposed to an intermediate dose compared to unirradiated controls. These delayed effects were not seen in the progeny of cells that were irradiated with low-dose X-rays, although such exposure resulted in residual γН2АХ foci in directly irradiated cells. Taken together, our results support the hypothesis that the low-dose IR induced residual γH2AÐ¥ foci do not play a role in delayed irradiation consequences, associated with cellular senescence in cultured MSCs.

γH2AX, 53BP1 and Rad51 protein foci changes in mesenchymal stem cells during prolonged X-ray irradiation.

At high exposure levels ionizing radiation is a carcinogen. Little is known about how human stem cells, which are known to contribute to tumorigenesis, respond to prolonged radiation exposures. We studied formation of DNA double strand breaks, accessed as γH2AX and 53BP1 foci, in human mesenchymal stem cells (MSCs) exposed to either acute (5400 mGy/h) or prolonged (270 mGy/h) X-irradiation. We show a linear γH2AX and 53BP1 dose response for acute exposures. In contrast, prolonged exposure resulted in a dose-response curve that had an initial linear portion followed by a plateau. Analysis of Rad51 foci, as a marker of homologous recombination, in cells exposed to prolonged irradiation revealed a threshold in a dose response. Using Ki67 as a marker of proliferating cells, we show no difference in the γH2AX distribution in proliferating vs. quiescent cells. However, Rad51 foci were found almost exclusively in proliferating cells. Concurrent increases in the fraction of S/G2 cells were detected in cells exposed to prolonged irradiation by scoring CENPF-positive cells. Our data suggest that prolonged exposure of MSCs to ionizing radiation leads to cell cycle redistribution and associated activation of homologous recombination. Also, proliferation status may significantly affect the biological outcome, since homologous repair is not activated in resting MSCs.

Accumulation of spontaneous γH2AX foci in long-term cultured mesenchymal stromal cells.

Expansion of mesenchymal stromal/stem cells (MSCs) used in clinical practices may be associated with accumulation of genetic instability. Understanding temporal and mechanistic aspects of this process is important for improving stem cell therapy protocols. We used γH2AX foci as a marker of a genetic instability event and quantified it in MSCs that undergone various numbers of passage (3-22). We found that γH2AX foci numbers increased in cells of late passages, with a sharp increase at passage 16-18. By measuring in parallel foci of ATM phosphorylated at Ser-1981 and their co-localization with γaH2AX foci, along with differentiating cells into proliferating and resting by using a Ki67 marker, we conclude that the sharp increase in γH2AX foci numbers was ATM-independent and happened predominantly in proliferating cells. At the same time, gradual and moderate increase in γH2AX foci with passage number seen in both resting and proliferating cells may represent a slow, DNA double-strand break related component of the accumulation of genetic instability in MSCs. Our results provide important information on selecting appropriate passage numbers exceeding which would be associated with substantial risks to a patient-recipient, both with respect to therapeutic efficiency and side-effects related to potential neoplastic transformations due to genetic instability acquired by MSCs during expansion.

In silico Pathway Activation Network Decomposition Analysis (iPANDA) as a method for biomarker development.

Signalling pathway activation analysis is a powerful approach for extracting biologically relevant features from large-scale transcriptomic and proteomic data. However, modern pathway-based methods often fail to provide stable pathway signatures of a specific phenotype or reliable disease biomarkers. In the present study, we introduce the in silico Pathway Activation Network Decomposition Analysis (iPANDA) as a scalable robust method for biomarker identification using gene expression data. The iPANDA method combines precalculated gene coexpression data with gene importance factors based on the degree of differential gene expression and pathway topology decomposition for obtaining pathway activation scores. Using Microarray Analysis Quality Control (MAQC) data sets and pretreatment data on Taxol-based neoadjuvant breast cancer therapy from multiple sources, we demonstrate that iPANDA provides significant noise reduction in transcriptomic data and identifies highly robust sets of biologically relevant pathway signatures. We successfully apply iPANDA for stratifying breast cancer patients according to their sensitivity to neoadjuvant therapy.

Activation of homologous recombination DNA repair in human skin fibroblasts continuously exposed to X-ray radiation.

Molecular and cellular responses to protracted ionizing radiation exposures are poorly understood. Using immunofluorescence microscopy, we studied the kinetics of DNA repair foci formation in normal human fibroblasts exposed to X-rays at a dose rate of 4.5 mGy/min for up to 6 h. We showed that both the number of γH2AX foci and their integral fluorescence intensity grew linearly with time of irradiation up to 2 h. A plateau was observed between 2 and 6 h of exposure, indicating a state of balance between formation and repair of DNA double-strand breaks. In contrast, the number and intensity of foci formed by homologous recombination protein RAD51 demonstrated a continuous increase during 6 h of irradiation. We further showed that the enhancement of the homologous recombination repair was not due to redistribution of cell cycle phases. Our results indicate that continuous irradiation of normal human cells triggers DNA repair responses that are different from those elicited after acute irradiation. The observed activation of the error-free homologous recombination DNA double-strand break repair pathway suggests compensatory adaptive mechanisms that may help alleviate long-term biological consequences and could potentially be utilized both in radiation protection and medical practices.

Changes in the number of double-strand DNA breaks in Chinese hamster V79 cells exposed to γ-radiation with different dose rates.

A comparative investigation of the induction of double-strand DNA breaks (DSBs) in the Chinese hamster V79 cells by γ-radiation at dose rates of 1, 10 and 400 mGy/min (doses ranged from 0.36 to 4.32 Gy) was performed. The acute radiation exposure at a dose rate of 400 mGy/min resulted in the linear dose-dependent increase of the γ-H2AX foci formation. The dose-response curve for the acute exposure was well described by a linear function y = 1.22 + 19.7x, where "y" is an average number of γ-H2AX foci per a cell and "x" is the absorbed dose (Gy). The dose rate reduction down to 10 mGy/min lead to a decreased number of γ-H2AX foci, as well as to a change of the dose-response relationship. Thus, the foci number up to 1.44 Gy increased and reached the "plateau" area between 1.44 and 4.32 Gy. There was only a slight increase of the γ-H2AX foci number (up to 7) in cells after the protracted exposure (up to 72 h) to ionizing radiation at a dose rate of 1 mGy/min. Similar effects of the varying dose rates were obtained when DNA damage was assessed using the comet assay. In general, our results show that the reduction of the radiation dose rate resulted in a significant decrease of DSBs per cell per an absorbed dose.