Agnieszka Dreas

Mitogen-activated protein kinase (MAPK) interacting kinases 1 and 2 (MNK1 and MNK2) as targets for cancer therapy: recent progress in the development of MNK inhibitors.

MAP kinase-interacting kinases (MNK1 and MNK2) are often activated downstream of ERK and p38 MAPK in the MAP kinase family. The role of MNKs in the development and progression of solid tumors and hematological malignancies has been widely discussed, particularly in the context of cap dependent translation, regulated by phosphorylation of eIF4E. MNK/eIF4E axis is involved in the expression of pro angiogenic, antiapoptotic, cell cycle, and motility proteins, such as MCL1, VEGF, MMP3, SNAIL, SMAD2, β-catenin or cyclin D1, and is essential during Ras and c Myc-induced transformation. MNK1/2 emerged as eligible targets for drug discovery in oncology, based on the antitumor effects observed in genetic knockout and RNA interference experiments and at the same time lack of adverse effects in dual knockout animals. There is a high interest in the development of pharmacological inhibitors of MNK1/2 as not only tools for further basic research studies but also potential drugs in diseases characterized by deregulated translation. Unfortunately, the role of MNK1/2 in cancer still remains elusive due to the absence of potent and selective probes. Moreover, in many instances, hypotheses have been built reliant upon unspecific MNK1/2 inhibitors such as CGP57380 or cercosporamide. Lately, the first two clinical programs targeting MNKs in oncology have been revealed (eFT508 and BAY 1143269), although several other MNK programs are currently running at the preclinical stage. This review aims to provide an overview of recent progress in the development of MNK inhibitors.

SEL120-34A is a novel CDK8 inhibitor active in AML cells with high levels of serine phosphorylation of STAT1 and STAT5 transactivation domains

Inhibition of oncogenic transcriptional programs is a promising therapeutic strategy. A substituted tricyclic benzimidazole, SEL120-34A, is a novel inhibitor of Cyclin-dependent kinase 8 (CDK8), which regulates transcription by associating with the Mediator complex. X-ray crystallography has shown SEL120-34A to be a type I inhibitor forming halogen bonds with the proteins hinge region and hydrophobic complementarities within its front pocket. SEL120-34A inhibits phosphorylation of STAT1 S727 and STAT5 S726 in cancer cells in vitro. Consistently, regulation of STATs- and NUP98-HOXA9- dependent transcription has been observed as a dominant mechanism of action in vivo. Treatment with the compound resulted in a differential efficacy on AML cells with elevated STAT5 S726 levels and stem cell characteristics. In contrast, resistant cells were negative for activated STAT5 and revealed lineage commitment. In vivo efficacy in xenotransplanted AML models correlated with significant repression of STAT5 S726. Favorable pharmacokinetics, confirmed safety and in vivo efficacy provide a rationale for the further clinical development of SEL120-34A as a personalized therapeutic approach in AML.

MNK1/2 inhibition limits oncogenicity and metastasis of KIT-mutant melanoma

Melanoma can be stratified into unique subtypes based on distinct pathologies. The acral/mucosal melanoma subtype is characterized by aberrant and constitutive activation of the proto-oncogene receptor tyrosine kinase C-KIT, which drives tumorigenesis. Treatment of these melanoma patients with C-KIT inhibitors has proven challenging, prompting us to investigate the downstream effectors of the C-KIT receptor. We determined that C-KIT stimulates MAP kinase–interacting serine/threonine kinases 1 and 2 (MNK1/2), which phosphorylate eukaryotic translation initiation factor 4E (eIF4E) and render it oncogenic. Depletion of MNK1/2 in melanoma cells with oncogenic C-KIT inhibited cell migration and mRNA translation of the transcriptional repressor SNAI1 and the cell cycle gene CCNE1. This suggested that blocking MNK1/2 activity may inhibit tumor progression, at least in part, by blocking translation initiation of mRNAs encoding cell migration proteins. Moreover, we developed an MNK1/2 inhibitor (SEL201), and found that SEL201-treated KIT-mutant melanoma cells had lower oncogenicity and reduced metastatic ability. Clinically, tumors from melanoma patients harboring KIT mutations displayed a marked increase in MNK1 and phospho-eIF4E. Thus, our studies indicate that blocking MNK1/2 exerts potent antimelanoma effects and support blocking MNK1/2 as a potential strategy to treat patients positive for KIT mutations.

Abstract 442: Small molecule inhibitors of SHMT1/2 validate serine metabolism as a target in the treatment of c-Myc positive solid tumors

Many oncogenes modulate metabolic pathways and altered metabolism is one of the hallmarks of cancer. In order to sustain proliferation, cell growth and adopt to a very specific tumor microenvironment, cancer cells have to undergo metabolic reprogramming. Increased uptake of glucose, consumed in anaerobic manner, allows to maintain essential bioenergetic and biosynthetic pathways. Various reports indicated that many cancers cells are crucially dependent on serine, which could be either imported or synthesized by the serine synthesis pathway (SSP) branched from glycolysis. Serine can be converted to glycine by Serine Hydroxymethyltransferases isoforms 1 and 2 (SHMT1/2), which provide also carbon for the folate cycle. There is a growing interest in targeting SSP and SHMT1/2 have been proposed as druggable targets for the treatment of various cancers. One of the major challenges is validation of these concepts by high quality pharmacological and genetic tools, particularly in the context of high tumor heterogeneity, artificial tissue culture conditions and many branching points of tumor metabolism, which eventually result in acquired resistance. We have identified and characterized a series of sub micromolar dual SHMT1/2 inhibitors. Affinity of these compounds to protein targets has been confirmed in biochemical and binding assays and further corroborated by X-ray crystallography studies. In order to confirm efficacy of these compounds in cancer cells, both sensitive and resistant cells to the inhibition of SSP have been identified by using conditioned serine and glycine depleted media. Moreover functional roles of both paralogs: SHMT1, which fuels cytoplasmic folate cycle and SHMT2, which is responsible for the mitochondrial branch, were further confirmed by gene knockdown studies. Cell lines resistant to depletion of serine in cell culture media were characterized by elevated levels of proteins involved in the synthesis of serine, namely PHGDH, PSAT1, SHMT2, c-Myc amplification and increased 13C flux from glucose to serine and glycine. Metabolic flux analysis further indicated that treatment with presented SHMT1/2 inhibitors effectively blocked the production of glycine from glucose and serine in cancer cells. Viability studies confirmed anti-cancer efficacy of SHMT1/2 inhibitors at concentrations consistent with metabolic flux studies in the same cells. Moreover rescue experiments with media supplemented with glycine and formate, which is a crucial intermediate between mitochondrial and cytoplasmic branches of folate cycle, were sufficient to reduce activity of SHMT1/2 inhibitors. Finally synergistic studies with antifolates provided an insight how efficacy of SHMT1/2 could be exploited therapeutically also in rational combinations with approved drugs. Citation Format: Tomasz Rzymski, Anna Wrobel, Karolina Pyziak, Agnieszka Sroka, Marta Sowinska, Agnieszka Dreas, Marcin Krol, Pawel Guzik, Agnieszka Adamus, Agnieszka Przybylowicz, Katarzyna Hamara, Magdalena Sieprawska-Lupa, Artur Biela, Krzysztof Brzozka. Small molecule inhibitors of SHMT1/2 validate serine metabolism as a target in the treatment of c-Myc positive solid tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 442. doi:10.1158/1538-7445.AM2017-442

Abstract 2174: Inhibition of CDK8 kinase with SEL120-34A allows for a personalized approach in AML

Inhibition of oncogenic transcriptional programs is recognized to be a promising therapeutic strategy. SEL120-34A is a novel inhibitor of Cyclin- dependent kinase 8 (CDK8), which regulates transcription by associating with the Mediator complex. SEL120-34A interacts with the ATP binding site of CDK8 in type I inhibitor fashion and forms several types of interactions, including halogen bonds with the protein’s hinge region and hydrophobic complementarities within its front pocket. Although the compound was only modestly active in solid tumor cell lines, it repressed phosphorylation of STAT5 Ser726 and could differentially inhibit viability of AML and ALL cell lines in vitro and in vivo, along with other type I CDK8 inhibitors. Transcriptomic analysis identified major transcriptional programs altered in responder cell lines, which strongly indicated that apart from repression of survival pathways, CDK8 inhibitors could induce differentiation in cell lines with leukemia stem cells characteristics. Further studies on a large panels of responder and non-responder cell lines identified robust biomarkers which could be used with high confidence for stratification and personalized approach in CDK8-dependent AML cases. Favorable pharmacokinetics, confirmed safety and in vivo efficacy in leukemia models provide the rationale for further clinical development of SEL120-34A. Citation Format: Tomasz Rzymski, Michal Mikula, Eliza Zylkiewicz, Agnieszka Dreas, Katarzyna Wiklik, Aniela Golas, Katarzyna Wojcik, Magdalena Masiejczyk, Iga Dudzicz, Katarzyna Kucwaj, Malgorzata Statkiewicz, Krzysztof Goryca, Aleksandra Grochowska, Aleksandra Cabaj, Jerzy Ostrowski, Urszula Kukliniska, Krzysztof Brzozka. Inhibition of CDK8 kinase with SEL120-34A allows for a personalized approach in AML [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2174. doi:10.1158/1538-7445.AM2017-2174

Abstract C194: Repression of tumor survival pathways by novel and selective inhibitors of MNK1 and MNK2 kinases in cancer

Herewith, we report development of small molecule inhibitors of MNK1 and MNK2 kinases and their cellular activity. MNK1 and 2 are MAP kinase-interacting kinases are activated by RAS and MAPK signaling pathways, and are involved in regulation of translation. Both kinases phosphorylate translation initiation factor eIF4e on a conserved serine 209. eIF4E can contribute to the oncogenic transformation both in vitro and in vivo and is highly expressed in diverse types of cancer. Interestingly, mice that lack both Mnk1 and Mnk2 do not have any apparent phenotype. Recently first dual MNK1/MNK2 inhibitors have entered clinical trials as a combinational therapy with docetaxel in NSCLC. SEL201 is a series of small molecule inhibitors which inhibit activity of both MNK1 and MNK2 in a low nM range and high selectivity confirmed in kinome panels. Analysis of SEL201 cellular activity indicated potent inhibition of eIF4e Ser209 in vitro in cancer cells and in vivo after oral administration in xenograft tumors. Repressed phosphorylation of eIF4e resulted in impaired translation of several proteins involved in metastasis and activation of immune cells. High potency, selectivity and favorable ADME/PK profile indicates that SEL201 inhibitors would be useful tools in probing molecular consequences of eIF4e Ser209 inhibition in cancer cells. SEL201 in vitro and in vivo activities on viability and metastasis will be presented in cellular and in vivo models of solid tumors and hematological malignancies. SEL201 series is further developed as a cancer therapy with a good therapeutic window. Citation Format: Tomasz Rzymski, Agnieszka Dreas, Ewelina Wincza, Charles-Henry Fabritius, Urszula Kulesza, Katarzyna Kucwaj- Brysz, Mariusz Milik, Aniela Golas, Renata Windak, Eliza Żylkiewicz, Anna Wrobel, Maciej Sulkowski, Krzysztof Brzozka. Repression of tumor survival pathways by novel and selective inhibitors of MNK1 and MNK2 kinases in cancer. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr C194.

Abstract 1663: Selective CDK8 inhibitor SEL120-34A alters expression of interferon-related DNA damage resistance signature genes in colorectal cancer

Proceedings: AACR 106th Annual Meeting 2015; April 18-22, 2015; Philadelphia, PA CDK8 (cyclin-dependent kinase 8) is a kinase component of a multi - protein Mediator complex, involved in transcription control. Several studies indicated that high overexpression and activity of CDK8 could be a driver of malignant progression in colorectal cancer (CRC). Herewith we present molecular insights into mechanism of action of SEL120-34A - a selective small molecule inhibitor of CDK8 kinase. Biochemical and binding studies indicated that SEL120-34A selectively binds and inhibits enzymatic activity of CDK8 in the low nM range. Recently CDK8 has been described as a regulator of STAT1 activity in NK cells where by phosphorylating STAT1 serine 727 (Ser727) influences a possible immunoescape mechanism in various cancers. Consistently, SEL120-34A and other recently reported selective CDK8 inhibitors could repress phosphorylation of STAT1 at a Ser727 at low nM concentrations in cancer cells without any significant changes on tyrosine sites directly regulated by JAK kinases. SEL120-34A inhibited expression of several STAT1 dependent genes in CRC cell lines, stimulated by various cytokines and growth factors. These results were further corroborated with specific CDK8 siRNA knockdown experiments and chromatin immunoprecipitation studies showing CDK8 occupancy on promoters of SEL120-34A regulated genes. In order to better characterize in vivo mechanism of action, mice bearing HCT116 and Colo205 xenograft tumors were treated with SEL120-34A and gene expression changes were measured with microarrays in excised tumors. In animals treated with the CDK8 inhibitor a dose dependent repression of STAT1 Ser727 was observed. The functional analyses of significantly (adj. p. value < 0.05) altered genes with Gene Ontology revealed that those with reduced expression belong to interferon I pathway and type I interferon-mediated signaling pathway terms. This subset of STAT regulated genes was further characterized as an interferon-related DNA damage resistance signature (IRDS) - a prosurvival pathway which correlated strongly with resistance to radiation and chemotherapy in various tumors. Consistently, SEL120-34A has shown very potent cytotoxic synergy with standard of care drugs in CRC, particularly in cells stimulated with interferons. Taken together, for the first time we have shown that selective CDK8 inhibitors are potent regulators of STAT related - IRDS signaling pathway in vitro and in vivo. In addition to previously reported stand-alone efficacy of CDK8 inhibitors in vivo, we provide also a combination treatment rationale for CRC. Citation Format: Tomasz Rzymski, Michal Mikula, Malgorzata Szajewska-Skuta, Eliza Zylkiewicz, Łukasz Sapala, Izabela Dolata, Agata Kitlinska, Krzysztof Goryca, Aleksandra Grochowska, Aleksandra Cabaj, Agnieszka Dreas, Katarzyna Kucwaj, Artur Bialas, Adam Radzimierski, Aniela Golas, Renata Windak, Jerzy Ostrowski, Krzysztof Brzozka. Selective CDK8 inhibitor SEL120-34A alters expression of interferon-related DNA damage resistance signature genes in colorectal cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1663. doi:10.1158/1538-7445.AM2015-1663

Abstract 755: Repression of tumor survival pathways by novel and selective inhibitors of MNK1 and MNK2 kinases in glioblastoma and colorectal cancer

Herewith, we report development of small molecule inhibitors of MNK1 and MNK2 kinases and their cellular activity. MNK1 and 2 are MAP kinase-interacting kinases that are activated by RAS and MAPK signaling pathways and are involved in regulation of translation. Both kinases phosphorylate translation initiation factor eIF4e on a conserved serine 209 residue. eIF4E can contribute to the oncogenic transformation both in vitro and in vivo and is highly expressed in various tumor types. Interestingly, mice that lack both MNK1 and MNK2 do not have any apparent phenotype, which is promising for the therapeutic window of MNK1/2 inhibitors. SEL201 is a series of small molecule inhibitors which inhibit activity of both MNK1 and MNK2 in a low nM range. Selected compounds were tested on the kinome panels and indicated MNK1 and MNK2 as primary kinase targets. SEL201 compounds caused dose dependent inhibition of phosphorylation of eIF4e at Ser209 in various cancer cell lines at concentrations Citation Format: Tomasz Rzymski, Malgorzata Szajewska-Skuta, Adrian Zarebski, Kamil Sitarz, Lukasz Sapala, Malgorzata Zurawska, Magdalena Salwinska, Renata Windak, Ewa Trebacz, Joanna Daniel-Wojcik, Radoslaw Obuchowicz, Bozena Winnik, Ewelina Wincza, Urszula Kulesza, Katarzyna Kucwaj-Borysz, Mariusz Milik, Agnieszka Dreas, Krzysztof Brzozka. Repression of tumor survival pathways by novel and selective inhibitors of MNK1 and MNK2 kinases in glioblastoma and colorectal cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 755. doi:10.1158/1538-7445.AM2014-755

Abstract 696: Development of selective CDK8 inhibitors for colorectal cancer and mantle cell lymphoma treatment.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC CDK8 is a kinase component of the mediator complex which functions as a bridge between a basal transcriptional machinery and specific transcription factors. CDK8 is amplified and differentially expressed in colorectal cancer and in certain hematological malignancies such as mantle cell lymphomas. Cells that express elevated CDK8 levels are highly dependent on its expression for proliferation. Here we report development of first-in-class selective inhibitors CDK8. Compounds from the SEL120 series have binding affinities towards CDK8 in the low nM range. Results from the kinome panel indicated that selectivity of SEL120 compounds was comparable with some of the most selective clinical kinase inhibitors. SEL120 compounds reduced viability of mantle cell lymphoma and colorectal cancer cell lines, with particularly good activity in cell lines overexpressing CDK8 and with G13D mutation in KRAS. Slightly lower sensitivity was observed for cells with mutated P53 and other mutations in KRAS/BRAF pathway. In contrast to pan-CDK inhibitors with main target activity on CDK9, treatment with SEL120 compounds did not repress phosphorylation of PolII and did not cause global transcriptional shutdown. Selective inhibition of CDK8 was sufficient to inhibit both paracrine and autocrine activities of cancer cells and stimulated normal cells. Production of proinflammatory cytokines, such as IL6 was repressed by SEL120 compounds in normal and cancer cells stimulated by sub-optimal doses of chemotherapeutics. SEL120 also reduced both murine and human IL6 in blood of mice bearing human xenograft models. Oral administration of SEL120 revealed favorable pharmacokinetics profile and strong, dose dependent potency in colon cancer mouse xenograft models. Presented data validate inhibition of CDK8 as a promising strategy for anticancer treatment, particularly for CRC and mantle cell lymphomas resistant to current treatments. Citation Format: Tomasz Rzymski, Adrian Zarebski, Renata Windak, Karolina Krawczynska, Ewa Trebacz, Agnieszka Dreas, Katarzyna Kucwaj, Karolina Osowska, Marek Cholody, Paulina Szczepanska, Jakub Woyciechowski, Radoslaw Obuchowicz, Magdalena Salwinska, Joanna Fogt, Malgorzata Zurawska, Arkadiusz Bialas, Katarzyna Wiklik, Mariusz Milik, Angelo Sanzone, Adam Radzimierski, Krzysztof Brzozka. Development of selective CDK8 inhibitors for colorectal cancer and mantle cell lymphoma treatment. [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 696. doi:10.1158/1538-7445.AM2013-696

Abstract A49: Repression of tumor survival pathways by novel, selective inhibitors of MNK1 and MNK2 kinases

MNK1 and MNK2 are MAP kinase-interacting kinases involved in regulation of translation. Both kinases phosphorylate translation initiation factor eIF4E on a conserved serine 209. eIF4E can contribute to the oncogenic transformation both in vitro and in vivo and is highly expressed in diverse types of cancer. Interestingly, mice that lack both MNK1 and MNK2 do not have any apparent phenotype and therefore represent interesting possibility to develop targeted and safe anticancer therapies. Herewith, we report the development of first selective small molecule inhibitors of MNK1 and MNK2 kinases and their in vitro cellular activity. Selvita developed a series of small molecule type I, ATP-competitive inhibitors targeting both MNK1 and MNK2 with mid nM activity range. Selected compounds were tested on a panel of 456 kinases and showed very high selectivity. Additionally, in cellular models such as serum stimulated SW480 cells, synthesized MNK1/2 inhibitors caused dose dependent inhibition of phosphorylation of eIF4e at Ser209 in line with the kinase activity profile. The observed cellular activity on biomarker inhibition was more potent than observed for reported in the literature MNK1/2 inhibitors such as cercosporamide and CGP57380. High activity of MNK1/2 inhibitors on in vitro biomarkers correlated with efficacy on cancer cells challenged with various stress conditions, typical for tumor microenvironment. These initial findings confirm that selective inhibition of MNK1/2 may repress major tumor survival pathways induced under stress and support further development of this class of compounds as a novel anticancer therapy with a promising therapeutic window. Citation Format: Tomasz Rzymski, Agnieszka Dreas, Mariusz Milik, Katarzyna Kucwaj, Adrian Zarebski, Malgorzata Szajewska Skuta, Anna Cierpich, Charles Fabritius, Krzysztof Brzozka. Repression of tumor survival pathways by novel, selective inhibitors of MNK1 and MNK2 kinases. [abstract]. In: Proceedings of the Third AACR International Conference on Frontiers in Basic Cancer Research; Sep 18-22, 2013; National Harbor, MD. Philadelphia (PA): AACR; Cancer Res 2013;73(19 Suppl):Abstract nr A49.