Iryna Kolosenko

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.

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.

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.

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

Abstract 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. Graphical abstract Plk1‐targeting RNAi prodrugs (siRNNs) enter T‐ALL patient cells. Inside cells cytoplasmic thioesterases cleave the phosphotriester siRNNs into phosphodiester siRNAs. The Plk1 siRNA is loaded into RISC followed by Plk1 mRNA cleavage. Plk1 silencing results in G2 arrest and apoptosis. Figure. No Caption available.

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.

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.

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

HIGHLIGHTSTruncated variants of STAT3 allowed the development of conventional STAT3 thermal stability assays.Thermal denaturing of STAT3 can be tracked using Sypro OrangeTM fluorescence, tryptophan fluorescence or light scattering.Novel thermal stability assays confirm that peptide STAT3 inhibitors bind specifically to the STAT3 SH2 domain.Thermal stability assays show that reported small molecule STAT3 inhibitors may not specifically bind the STAT3 SH2 domain.High‐throughput STAT3 thermal stability assays could help to accelerate the development of new STAT3 inhibitors. ABSTRACT 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.

A drug screening assay on cancer cells chronically adapted to acidosis

BackgroundDrug 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.MethodsHere, 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.ResultsThe 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.ConclusionsOur study provides an example and a tool to identify anticancer drugs targeting acid-adapted cancer cells.

Abstract 3999: Spontaneous time-dependent accumulation of interferon-regulatory factor 9 induces chemo-resistance in colon carcinoma cells independently of STAT1 signaling.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC Spontaneous time-dependent accumulation of interferon-regulatory factor 9 induces chemo-resistance in colon carcinoma cells independently of STAT1 signaling Iryna Kolosenko1, Marten Fryknas2, Hanif Rassoolzadeh1, Paola Pellegrini1, Slavica Brnjic1, Per Johnsson1, Giuseppe Di Lernia1, Dan Grander1, Rolf Larsson2, Katja Pokrovskaja Tamm1, Stig Linder1, Angelo De Milito1 1 Cancer Center Karolinska, Dep. Oncology-Pathology, Karolinska Institute, Stockholm, Sweden 2 Division of Clinical Pharmacology, Department of Medical Sciences, Uppsala University Hospital, 751 85 Uppsala, Sweden Solid tumors contain microenvironments characterized by hypoxia and limited availability of nutrients. Such metabolically compromised environments show an increased level of resistance to many chemotherapeutic agents. Multicellular tumor spheroids (MCS) contain hypoxic and nutrient-starved cell populations and provide a model to study multicellular therapy-resistance. We here used 3D cultures of HCT116 colon cancer cells as a model to identify factors related to multicellular drug-resistance. Microarray analysis revealed a dramatic increase in the expression of interferon-alpha (IFNα)-regulated genes in the 3D as compared to 2D cultures. This finding is of significance since an IFNα-related gene signature has previously been associated with therapy resistance in several human cancers. IFNα is known to regulate gene transcription through transcription complex ISGF3 consisting of STAT1, STAT2 and IRF9, as well as through STAT1 homodimers. We found that IRF9, but not STAT1, was consistently induced in colon cancer cell lines grown in 3D as compared to the 2D cultures. Upregulation of IFN-induced genes was observed during crowding of monolayer HCT116 cultures, providing both mechanistic insight as well as a convenient experimental model. STAT1 knock-down experiments showed that induction of IRF9 occurred independently of STAT1. Interestingly, over-expression of IRF9 rendered monolayer 2D HCT116 cells significantly more resistant to cisplatin, docetaxel and etoposide. Vice versa, inhibition of IRF9 expression significantly increased drug-sensitivity of monolayer HCT116 cells to cisplatin. Our data further support an association of IFNα-related gene signature and drug resistance and suggest an important role for the STAT1-independent IRF9 upregulation associated with therapy resistance in cancer. Citation Format: Iryna Kolosenko, Marten Fryknas, Hanif Rassoolzadeh, Paola Pellegrini, Slavica Brnjic, Per Johnsson, Giuseppe Di Lernia, Dan Grander, Rolf Larsson, Katja Pokrovskaja, Stig Linder, Angelo De Milito De Milito. Spontaneous time-dependent accumulation of interferon-regulatory factor 9 induces chemo-resistance in colon carcinoma cells independently of STAT1 signaling. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3999. doi:10.1158/1538-7445.AM2013-3999