CORACLE (COVID-19 liteRAture CompiLEr): A platform for efficient tracking and extraction of SARS-CoV-2 and COVID-19 literature, with examples from post-COVID with respiratory involvement.

Background: During the COVID-19 pandemic a need to process large volumes of publications emerged. As the pandemic is winding down, the clinicians encountered a novel syndrome - Post-acute Sequelae of COVID-19 (PASC) - that affects over 10 % of those who contract SARS-CoV-2 and presents a significant challenge in the medical field. The continuous influx of publications underscores a need for efficient tools for navigating the literature. Objectives: We aimed to develop an application which will allow monitoring and categorizing COVID-19-related literature through building publication networks and medical subject headings (MeSH) maps to identify key publications and networks. Methods: We introduce CORACLE (COVID-19 liteRAture CompiLEr), an innovative web application designed to analyse COVID-19-related scientific articles and to identify research trends. CORACLE features three primary interfaces: The "Search" interface, which displays research trends and citation links; the "Citation Map" interface, allowing users to create tailored citation networks from PubMed Identifiers (PMIDs) to uncover common references among selected articles; and the "MeSH" interface, highlighting current MeSH trends and their associations. Results: CORACLE leverages PubMed data to categorize literature on COVID-19 and PASC, aiding in the identification of relevant research publication hubs. Using lung function in PASC patients as a search example, we demonstrate how to identify and visualize the interactions between the relevant publications. Conclusion: CORACLE is an effective tool for the extraction and analysis of literature. Its functionalities, including the MeSH trends and customizable citation mapping, facilitate the discovery of emerging trends in COVID-19 and PASC research.

PLK1 as a cooperating partner for BCL2-mediated antiapoptotic program in leukemia.

The deregulation of BCL2 family proteins plays a crucial role in leukemia development. Therefore, pharmacological inhibition of this family of proteins is becoming a prevalent treatment method. However, due to the emergence of primary and acquired resistance, efficacy is compromised in clinical or preclinical settings. We developed a drug sensitivity prediction model utilizing a deep tabular learning algorithm for the assessment of venetoclax sensitivity in T-cell acute lymphoblastic leukemia (T-ALL) patient samples. Through analysis of predicted venetoclax-sensitive and resistant samples, PLK1 was identified as a cooperating partner for the BCL2-mediated antiapoptotic program. This finding was substantiated by additional data obtained through phosphoproteomics and high-throughput kinase screening. Concurrent treatment using venetoclax with PLK1-specific inhibitors and PLK1 knockdown demonstrated a greater therapeutic effect on T-ALL cell lines, patient-derived xenografts, and engrafted mice compared with using each treatment separately. Mechanistically, the attenuation of PLK1 enhanced BCL2 inhibitor sensitivity through upregulation of BCL2L13 and PMAIP1 expression. Collectively, these findings underscore the dependency of T-ALL on PLK1 and postulate a plausible regulatory mechanism.

Thermal proteome profiling identifies PIP4K2A and ZADH2 as off-targets of Polo-like kinase 1 inhibitor volasertib.

Polo-like kinase 1 (PLK1) is an important cell cycle kinase and an attractive target for anticancer treatments. An ATP-competitive small molecular PLK1 inhibitor, volasertib, has reached phase III in clinical trials in patients with refractory acute myeloid leukemia as a combination treatment with cytarabine. However, severe side effects limited its use. The origin of the side effects is unclear and might be due to insufficient specificity of the drug. Thus, identifying potential off-targets to volasertib is important for future clinical trials and for the development of more specific drugs. In this study, we used thermal proteome profiling (TPP) to identify proteome-wide targets of volasertib. Apart from PLK1 and proteins regulated by PLK1, we identified about 200 potential volasertib off-targets. Comparison of this result with the mass-spectrometry analysis of volasertib-treated cells showed that phosphatidylinositol phosphate and prostaglandin metabolism pathways are affected by volasertib. We confirmed that PIP4K2A and ZADH2-marker proteins for these pathways-are, indeed, stabilized by volasertib. PIP4K2A, however, was not affected by another PLK1 inhibitor onvansertib, suggesting that PIP4K2A is a true off-target of volasertib. Inhibition of these proteins is known to impact both the immune response and fatty acid metabolism and could explain some of the side effects seen in volasertib-treated patients.

RNAi prodrugs decrease elevated mRNA levels of Polo-like kinase 1 in ex vivo cultured primary cells from pediatric acute myeloid leukemia patients.

Polo-like kinase 1 (Plk1) is an important regulator of the cell cycle and it is frequently overexpressed in cancer cells. Several small molecule inhibitors have been developed to target Plk1 and some of them have reached clinical trials in adults with acute myeloid leukemia (AML). Pediatric AML patients have a poor prognosis and survivors suffer from long-term side effects. As adult AML cells have an elevated expression of Plk1, AML is a disease candidate for Plk1 inhibition. However, the relative success of clinical trials have been hampered by adverse reactions. Herein, PLK1-targeting RNA interference (RNAi) prodrugs that enter cells without a transfection reagent are used to target PLK1 selectively in primary cells from pediatric AML patients. We show that PLK1 and PLK4 mRNA expression are significantly higher in pediatric AML patients when compared to healthy donors and that PLK1 is downregulated by on average 50% using RNAi prodrugs without a significant effect on other PLK family members. In addition, the RNAi prodrug-induced decrease in PLK1 can be used to potentiate the effect of cytarabine. In summary, PLK1-targeting RNAi prodrugs can decrease the elevated levels of PLK1 in primary cells from pediatric AML patients and sensitize pediatric AML cells to chemotherapeutics.

Targeting Plk1 with siRNNs in primary cells from pediatric B-cell acute lymphoblastic leukemia patients.

B-cell acute lymphoblastic leukemia (B-ALL) accounts for nearly one fifth of all childhood cancers and current challenges in B-ALL treatment include resistance, relapse and late-onset side effects due to the chemotherapy. To overcome these hurdles, novel therapies need to be investigated. One promising target is Polo-like kinase 1 (Plk1), a key regulator of the cell cycle. In this study, the Plk family expression is investigated in primary peripheral blood and bone marrow mononuclear cells from ten pediatric B-ALL patients. For the first time, short interfering RiboNucleic Neutrals (siRNNs) that enter cells without a transfection reagent are used to target Plk1 mRNA in primary cells from pediatric B-ALL patients. Our results show that the expression of Plk1 and Plk4 is significantly higher in pediatric B-ALL patients compared to healthy donors. Moreover, treatment of primary peripheral blood and bone marrow mononuclear cells from pediatric B-ALL patients, cultured ex vivo, with Plk1-targeting siRNNs results in cleavage of Plk1 mRNA. Importantly, the Plk1 knockdown is specific and does not affect other Plk members in contrast to many small molecule Plk1 inhibitors. Thus, Plk1 is a potential therapeutic target in pediatric B-ALL and selective targeting of Plk1 can be achieved by the use of siRNNs.

Designing siRNA and Evaluating Its Effect on RNA Targets Using qPCR and Western Blot.

The discovery of the RNA interference (RNAi) pathway followed by the usage of synthetic short-interfering RNAs (siRNA) has contributed greatly to the understanding of gene function. Carefully designed siRNAs can considerably improve siRNA specificity leading to more accurate and efficient gene silencing. Evaluation of gene knockdown is vital for optimization of siRNA efficacy. Here we describe the fundamental principles of siRNA design and strategies for evaluating gene knockdown.

STAT3 is activated in multicellular spheroids of colon carcinoma cells and mediates expression of IRF9 and interferon stimulated genes.

Three-dimensional cell cultures, such as multicellular spheroids (MCS), reflect the in vivo architecture of solid tumours and multicellular drug resistance. We previously identified interferon regulatory factor 9 (IRF9) to be responsible for the up-regulation of a subset of interferon (IFN)-stimulated genes (ISGs) in MCS of colon carcinoma cells. This set of ISGs closely resembled a previously identified IFN-related DNA-damage resistance signature (IRDS) that was correlated to resistance to chemo- and radiotherapy. In this study we found that transcription factor STAT3 is activated upstream of IRF9 and binds to the IRF9 promoter in MCS of HCT116 colorectal carcinoma cells. Transferring conditioned media (CM) from high cell density conditions to non-confluent cells resulted in STAT3 activation and increased expression of IRF9 and a panel of IRDS genes, also observed in MCS, suggesting the involvement of a soluble factor. Furthermore, we identified gp130/JAK signalling to be responsible for STAT3 activation, IRF9, and IRDS gene expression in MCS and by CM. Our data suggests a novel mechanism where STAT3 is activated in high cell density conditions resulting in increased expression of IRF9 and, in turn, IRDS genes, underlining a mechanism by which drug resistance is regulated.

Polo-like kinases and acute leukemia.

Acute leukemia is a common malignancy among children and adults worldwide and many patients suffer from chronic health issues using current therapeutic approaches. Therefore, there is a great need for the development of novel and more specific therapies with fewer side effects. The family of Polo-like kinases (Plks) is a group of five serine/threonine kinases that play an important role in cell cycle regulation and are critical targets for therapeutic invention. Plk1 and Plk4 are novel targets for cancer therapy as leukemic cells often express higher levels than normal cells. In contrast, Plk2 and Plk3 are considered to be tumor suppressors. Several small molecule inhibitors have been developed for targeting Plk1 inhibition. Despite reaching phase III clinical trials, one of the ATP-competitive Plk1 inhibitor, volasertib, did not induce an objective clinical response and even caused lethal side effects in some patients. In order to improve the specificity of the Plk1 inhibitors and reduce off-target side effects, novel RNA interference (RNAi)-based therapies have been developed. In this review, we summarize the mechanisms of action of the Plk family members in acute leukemia, describe preclinical studies and clinical trials involving Plk-targeting drugs and discuss novel approaches in Plk targeting.

A drug screening assay on cancer cells chronically adapted to acidosis.

BACKGROUND: Drug screening for the identification of compounds with anticancer activity is commonly performed using cell lines cultured under normal oxygen pressure and physiological pH. However, solid tumors are characterized by a microenvironment with limited access to nutrients, reduced oxygen supply and acidosis. Tumor hypoxia and acidosis have been identified as important drivers of malignant progression and contribute to multicellular resistance to different forms of therapy. Tumor acidosis represents an important mechanism mediating drug resistance thus the identification of drugs active on acid-adapted cells may improve the efficacy of cancer therapy. METHODS: Here, we characterized human colon carcinoma cells (HCT116) chronically adapted to grow at pH 6.8 and used them to screen the Prestwick drug library for cytotoxic compounds. Analysis of gene expression profiles in parental and low pH-adapted cells showed several differences relating to cell cycle, metabolism and autophagy. RESULTS: The screen led to the identification of several compounds which were further selected for their preferential cytotoxicity towards acid-adapted cells. Amongst 11 confirmed hits, we primarily focused our investigation on the benzoporphyrin derivative Verteporfin (VP). VP significantly reduced viability in low pH-adapted HCT116 cells as compared to parental HCT116 cells and normal immortalized epithelial cells. The cytotoxic activity of VP was enhanced by light activation and acidic pH culture conditions, likely via increased acid-dependent drug uptake. VP displayed the unique property to cause light-dependent cross-linking of proteins and resulted in accumulation of polyubiquitinated proteins without inducing inhibition of the proteasome. CONCLUSIONS: Our study provides an example and a tool to identify anticancer drugs targeting acid-adapted cancer cells.

STAT3 differential scanning fluorimetry and differential scanning light scattering assays: Addressing a missing link in the characterization of STAT3 inhibitor interactions.

STAT3 protein is an established target for the development of new cancer therapeutic agents. Despite lacking a traditional binding site for small molecule inhibitors, many STAT3 inhibitors have been identified and explored for their anti-cancer activity. Because STAT3 signaling is mediated by protein-protein interactions, indirect methods are often employed to determine if proposed STAT3 inhibitors bind to STAT3 protein. While established STAT3 inhibition assays (such as the fluorescence polarization assay, electrophoretic mobility shift assay and ELISAs) have been used to identify novel inhibitors of STAT3 signaling, methods that directly assess STAT3 protein-inhibitor interactions could facilitate the development of novel inhibitors. In this context, we herein report new STAT3 binding assays based on differential scanning fluorimetry (DSF) and differential scanning light scattering (DSLS) to characterize interactions between STAT3 protein and inhibitors. Several peptide and small molecule STAT3 inhibitors have been evaluated, and new insight into how these compounds may interact with STAT3 is provided.

RNAi prodrugs targeting Plk1 induce specific gene silencing in primary cells from pediatric T-acute lymphoblastic leukemia patients.

Epidemiological studies of childhood leukemia survivors reveal an alarmingly high incidence of chronic health disabilities after treatment, therefore, more specific therapies need to be developed. Polo-like kinase 1 (Plk1) is a key player in mitosis and a target for drug development as it is upregulated in multiple cancer types. Small molecules targeting Plk1 are mainly ATP-competitors and, therefore, are known to elicit side effects due to lack of specificity. RNA interference (RNAi) is known for its high catalytic activity and target selectivity; however, the biggest barrier for its introduction into clinical use is its delivery. RNAi prodrugs are modified, self-delivering short interfering Ribonucleic Neutrals (siRNNs), cleaved by cytoplasmic enzymes into short interfering Ribonucleic Acids (siRNAs) once inside cells. In this study we aimed to investigate the potential of siRNNs as therapeutic tools in T-acute lymphoblastic leukemia (T-ALL) using T-ALL cell lines and patient-derived samples. We demonstrate for the first time that RNAi prodrugs (siRNNs) targeting Plk1, can enter pediatric T-ALL patient cells without a transfection reagent and induce Plk1 knockdown on both protein and mRNA levels resulting in G2/M-arrest and apoptosis. We also show that siRNNs targeting Plk1 generate less toxicity in normal cells compared to the small molecule Plk1 inhibitor, BI6727, suggesting a potentially good therapeutic index.

Identification of novel small molecules that inhibit STAT3-dependent transcription and function.

Activation of Signal Transducer and Activator of Transcription 3 (STAT3) has been linked to several processes that are critical for oncogenic transformation, cancer progression, cancer cell proliferation, survival, drug resistance and metastasis. Inhibition of STAT3 signaling has shown a striking ability to inhibit cancer cell growth and therefore, STAT3 has become a promising target for anti-cancer drug development. The aim of this study was to identify novel inhibitors of STAT-dependent gene transcription. A cellular reporter-based system for monitoring STAT3 transcriptional activity was developed which was suitable for high-throughput screening (Z' = 0,8). This system was used to screen a library of 28,000 compounds (the ENAMINE Drug-Like Diversity Set). Following counter-screenings and toxicity studies, we identified four hit compounds that were subjected to detailed biological characterization. Of the four hits, KI16 stood out as the most promising compound, inhibiting STAT3 phosphorylation and transcriptional activity in response to IL6 stimulation. In silico docking studies showed that KI16 had favorable interactions with the STAT3 SH2 domain, however, no inhibitory activity could be observed in the STAT3 fluorescence polarization assay. KI16 inhibited cell viability preferentially in STAT3-dependent cell lines. Taken together, using a targeted, cell-based approach, novel inhibitors of STAT-driven transcriptional activity were discovered which are interesting leads to pursue further for the development of anti-cancer therapeutic agents.

Therapeutic implications of tumor interstitial acidification.

Interstitial acidification is a hallmark of solid tumor tissues resulting from the combination of different factors, including cellular buffering systems, defective tissue perfusion and high rates of cellular metabolism. Besides contributing to tumor pathogenesis and promoting tumor progression, tumor acidosis constitutes an important intrinsic and extrinsic mechanism modulating therapy sensitivity and drug resistance. In fact, pharmacological properties of anticancer drugs can be affected not only by tissue structure and organization but also by the distribution of the interstitial tumor pH. The acidic tumor environment is believed to create a chemical barrier that limits the effects and activity of many anticancer drugs. In this review article we will discuss the general protumorigenic effects of acidosis, the role of tumor acidosis in the modulation of therapeutic efficacy and potential strategies to overcome pH-dependent therapy-resistance.

Cell crowding induces interferon regulatory factor 9, which confers resistance to chemotherapeutic drugs.

The mechanism of multicellular drug resistance, defined as the reduced efficacy of chemotherapeutic drugs in solid tumors is incompletely understood. Here we report that colon carcinoma cells cultured as 3D microtissues (spheroids) display dramatic increases in the expression of a subset of type I interferon-(IFN)-stimulated genes (ISGs). A similar gene signature was associated previously with resistance to radiation and chemotherapy, prompting us to examine the underlying biological mechanisms. Analysis of spheroids formed by different tumor cell lines and studies using knock-down of gene expression showed that cell crowding leads to the induction of IFN regulatory factor-9 (IRF9) which together with STAT2 and independently of IFNs, is necessary for ISG upregulation. Increased expression of IRF9 alone was sufficient to induce the ISG subset in monolayer cells and to confer increased resistance to clinically used cytotoxic drugs. Our data reveal a novel mechanism of regulation of a subset of ISGs, leading to drug resistance in solid tumors.

An activated JAK/STAT3 pathway and CD45 expression are associated with sensitivity to Hsp90 inhibitors in multiple myeloma.

The molecular chaperone Hsp90 is required to maintain the activity of many signaling proteins, including members of the JAK/STAT and the PI3K pathways. Inhibitors of Hsp90 (Hsp90-Is) demonstrated varying activity against multiple myeloma (MM) in clinical trials. We aimed to determine which signaling pathways that account for the differential sensitivity to the Hsp90-I 17DMAG on a panel of MM cell lines and freshly obtained MM cells. Three CD45(+) cell lines with an activated JAK/STAT3 pathway were sensitive to 17DMAG and underwent prominent apoptosis upon treatment, while the majority of CD45(-) cell lines, that were dependent on the activated PI3K pathway, were more resistant to the drug. Culturing the most resistant cell line, LP1, in the presence of IL-6 resulted in up-regulation of CD45 and pSTAT3, and sensitized to 17DMAG-induced apoptosis, primarily in the induced CD45(+) sub-population of cells. The high CD45 expressers among primary myeloma cells also expressed significantly higher levels of pSTAT3, as compared to the low CD45 expressers. Ex vivo treatment of primary myeloma cells with 17DMAG resulted in a stronger caspase3 activation in tumor samples with the prevalence of high CD45 expressers. STAT3 activity was efficiently inhibited by Hsp90-Is in both cell lines and primary cells suggesting an importance of STAT3 inactivation for the pro-apoptotic effects of HSP90-Is. Indeed, over-expression of STAT3C, a variant with an increased DNA binding activity, in U266 cells protected them from 17DMAG-induced cell death. The down-regulation of the STAT3 target gene Mcl-1 at both the mRNA and protein levels following 17DMAG treatment was significantly attenuated in STAT3C-expressing cells, and transient over-expression of Mcl-1 protected U266 cells from 17DMAG-induced cell death. The finding that CD45(+) MM cells with an IL-6-activated JAK/STAT3 pathway are particularly sensitive to Hsp90-Is as compared to the low CD45 expressers may provide a rational basis for selection of MM patients amenable to Hsp90-I treatment.

miR-185 and miR-133b deregulation is associated with overall survival and metastasis in colorectal cancer.

Colorectal cancer (CRC) is one of the most common and deadly forms of cancer. Despite improved treatment modalities, post-operative recurrence and metastasis remain the major problems for extending patient survival after surgery. This highlights the need to search for biomarkers for prognostication and treatment stratification of colorectal cancer patients. In this study, we applied the SYBR-green quantitative PCR-based array approach to screen for differentially expressed miRNAs between patients with short (<50 months, range 10-33 months) and long survival (≥ 50 months, range 50-152 months). The selected candidate prognostic miRNAs were validated in a cohort of 50 CRC patients by TaqMan quantitative PCR. We found that high expression of miR-185 and low expression of miR-133b were correlated with poor survival (p=0.001 and 0.028, respectively) and metastasis (p=0.007 and 0.036, respectively) in colorectal cancer. Our findings suggest the potential prognostic values of these miRNAs for predicting clinical outcome after surgery.

Activation of STAT1 is required for interferon-alpha-mediated cell death.

Interferon-alpha (IFNα)-induced cell death of tumor cells is likely mediated through several signaling pathways. We previously demonstrated that blocking the activation of phosphoinositide-3-kinase, PI3K, or mammalian target of rapamycin, mTOR, partially inhibited apoptosis induced by IFNα. Here, we postulate using pharmacological inhibition and dominant negative mutants that activation of signal transducer and activator of transcription-1, STAT1, is also required for the cell death induced by IFNα. Inhibition of STAT1 tyrosine phosphorylation and DNA binding by a naturally occurring rotenoid deguelin also rescued U266 myeloma cell lines from IFNα-induced apoptosis. Deguelin had no effect on upstream Jak kinases or STAT2 phosphorylation suggesting the involvement of a yet unknown mechanism. Inhibition of STAT1 tyrosine phosphorylation and activity was independent of the known effects of deguelin on PI3K, Akt or mTOR as shown using selective pharmacological inhibitors against these kinases. The combination of deguelin and PI3K or mTOR antagonists further inhibited apoptosis suggesting that both the Jak-STAT and the PI3K/mTOR pathways contribute to the induction of apoptosis by IFNα in these cells. Over-expression of STAT1-Y701A or K410/413A mutants in Rhek-1 keratinocytes largely inhibited apoptosis further supporting the importance of STAT1 phosphorylation and activity for IFNα-induced cell death. Thus, at least two signaling pathways, one of which requires STAT1 activation, cooperate to mediate IFNα-induced apoptosis.