Tomasz Rzymski

The Role of ATF4 Stabilization and Autophagy in Resistance of Breast Cancer Cells Treated with Bortezomib

The ubiquitin-proteasome system plays a key regulatory role in cellular homeostasis. The inhibition of the 26S proteasome by Bortezomib leads to the accumulation of misfolded proteins, resulting in endoplasmic reticulum stress followed by a coordinated cellular response called unfolded protein response (UPR). Endoplasmic reticulum stress is also a potent inducer of macroautophagy. Bortezomib is a selective and potent inhibitor of the 26S proteasome and is approved for the treatment of multiple myeloma. Clinical trials with Bortezomib have shown promising results for some types of cancers, but not for some others, including those of the breast. In this study, we show that Bortezomib induces the UPR and autophagy in MCF7 breast cancer cells. Surprisingly, Bortezomib did not induce phosphorylation of PERK, a key initial step of the UPR. We show that induction of autophagy by Bortezomib is dependent on the proteasomal stabilisation of ATF4 and up-regulation of LC3B by ATF4. We show that ATF4 and LC3B play a critical role in activating autophagy and protecting cells from Bortezomib-induced cell death. Our experiments also reveal that HDAC6 knockdown results in decreased LC3B protein and reduced autophagy. Our work shows that the induction of autophagy through ATF4 may be an important resistance mechanism to Bortezomib treatment in breast cancer, and targeting autophagy may represent a novel approach to sensitize breast cancers to Bortezomib.

CDK8 kinase--An emerging target in targeted cancer therapy.

Cyclin-dependent kinase (CDK) inhibitors have been developed as potential anticancer therapeutics and several nonselective compounds are currently in advanced clinical trials. This review is focused on the key biological roles of CDK8 kinase, which provide a proof-of-principle for continued efforts toward effective cancer treatment, targeting activity of this CDK family member. Among currently identified kinase inhibitors, several displayed significant selectivity for CDK8 and notably the effectiveness in targeting cancer specific gene expression programs. Structural features of CDK8 and available ligands were discussed from a perspective of the rational drug design process. Current state of the art confirms that further development of CDK8 inhibitors will translate into targeted therapies in oncology. This article is part of a Special Issue entitled:Inhibitors of Protein Kinases.

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.

Abstract 2826: Preclinical development of a Pim kinase inhibitor for cancer treatment

Pim kinases were identified as the crucial downstream effectors various important oncogenes like Jak, FLT3 or Ras kinases. Multiple targets phosphorylated by Pim kinases, such as 4EBP1, p21Waf, p27KIP1, c-Myc or CXCR4 play important roles in intracellular signaling and contribute to pathways involved in cell survival, proliferation, stress response and cellular motility. Moreover, the significance and relevance of Pim kinases as valid therapeutic targets is further confirmed by their expression levels in variety of cancer types, especially in various types of lymphomas, leukemias or myelomas. For example Pim kinase overexpression contributes to the development of diffuse large B cell lymphoma, mantle cell lymphoma, B-cell chronic lymphocytic leukemia and FLT3-mediated acute myelogenous leukemia. As members of the family, Pim-1, Pim-2 and Pim-3 were shown to be prevent apoptosis, promote protein translation and cell survival thereby enhancing proliferation of malignant cells, Pim kinases emerged as a novel and interesting target with significant potential for therapeutic intervention in cancer. In the current study we are reporting the results of a second generation small molecule Pim kinase inhibitors that were developed by Selvita and results for the best characterized potent Pim kinase inhibitor developed so far. The small molecule inhibitors exert high potency in vitro both on all three Pim kinases as well as on a large panel of cancer cell lines. Mechanism of action for selected compounds was confirmed both in vitro in variety of cell lines and in vivo in xenograft models by downregulation of c-Myc and 4EBP1 phosphorylation inhibition. Selected compounds were tested in a range of combinations with standard anti-cancer therapeutics in both leukemias and solid tumor cell lines and showed potent synergistic effects. Strong synergies were observed particularly in combination with targeted therapies like the PI3K/Akt pathway inhibitors or Jak kinase inhibitors. Data will be presented on in vivo efficacy of lead compounds examined in xenograft models. Additionally, the results of safety assessment of the lead compound will be presented revealing preferable safety profile with no effect on ion channel mediated cardiotoxicity and favorable pharmacokinetic profile. Taken together, presented data will further support the rationale of using Pim kinase inhibition as a novel approach to standalone cancer therapy, and in combination with other targeted and cytotoxic therapies, especially to overcome developing drug resistance. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2826. doi:1538-7445.AM2012-2826

Abstract 5063: Epigenetic modulators show differential activity on lung adenocarcinoma cells with loss-of-function mutations of SWI/SNF protein SMARCA4

SWI/SNF is a multiprotein chromatin remodeler with ATP-dependent activities leading to selective gene expression, DNA repair, recombination and replication. Various sequencing efforts indicated that nearly 20% of cancers bear mutations in at least one subunit of the complex. One of the crucial regulators of the complex is SMARCA4, a member of SWI/SNF family of helicases with ATPase activities, which are thought to regulate transcription of certain genes by altering the chromatin structure. SMARCA4 is mutated in virtually all cases of small cell carcinoma of the ovary and SMARCA4 is fourth the most frequently mutated gene in lung adenocarcinoma. High occurrence of inactivating mutations prompted several screenings projects focused on synthetic lethality interactions with other proteins, which led to the identification of SMARCA2 as an essential gene in SMARCA4 mutated cancers. This vulnerability could be potentially exploited therapeutically and several groups managed to identify potent ligands of SMARCA2 bromodomain. Surprisingly these molecules were inactive in SMARCA4 mutant cells, however additional studies indicated that ATPase rather than bromodomain is a target for novel compounds with anticancer activities. Overall, these results revealed functional complexity of SMARCA2 and SMARCA4 in cancer cells. In order to characterize molecular consequences of SMARCA2 silencing in SMARCA4 mutant lung adenocarcinoma cells, we have carried out a series of gene knockdown experiments, followed by transcriptional profiling by RNAseq and analysis of posttranslational histone modifications. These studies indicated rapid and irreversible loss of viability in SMARCA4 mutant cells after SMARCA2 gene silencing. Interestingly double SMARCA2/SMARCA4 knockdown in SMARCA4 WT cells has not resulted in lowered viability. Transcriptional profiling of SMARCA2 knockdown in SMARCA4 mutated cells revealed broad, predominantly repressory effects on gene expression levels. Gene set enrichment analysis showed significant inhibitory effects of SMARCA2 knockdown, particularly on transcripts which could be induced after reintroduction of SMARCA4. Furthermore MS/MS analysis indicated global rearrangements in histone epigenetic marks. These results prompted us to test activity of small-molecule epigenetic modulators, including DNA Methyltransferases (DNMTs), Histone Acetyltransferases (HATs), Histone Deacetylases (HDACs), Histone Methyltransferases (HMTs), Histone Demethylases (HDMs) and a distinct set of chromatin readers, bromodomains. Differential activity of these compounds provides a strong basis for novel strategies for the treatment of cancers with loss-of-function mutations of SMARCA4. Citation Format: Tomasz Rzymski, Anna Wrobel, Michal Mikula, Karolina Pyziak, Anna Bartosik, Agnieszka Sroka, Agnieszka Paziewska, Aleksandra Grochowska, Malgorzata Statkiewicz, Katarzyna Paczkowska, Michalina Dabrowska, Jerzy Ostrowski, Krzysztof Brzozka. Epigenetic modulators show differential activity on lung adenocarcinoma cells with loss-of-function mutations of SWI/SNF protein SMARCA4 [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 5063. doi:10.1158/1538-7445.AM2017-5063

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