Mariusz Milik

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.

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 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 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 2160: Development of selective MELK kinase inhibitors for cancer treatments.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC Maternal embryonic leucine zipper kinase (MELK) is an atypical member of the AMPK family of serine-threonine kinases that has been implicated in stem cell renewal, cell cycle progression, cytokinesis, mRNA splicing and apoptosis. MELK activity is correlated with its phosphorylation level, is cell cycle dependent and maximal during mitosis although direct upstream regulators of MELK kinase activity are unknown. This kinase is highly expressed in several types of solid cancers: colon, breast, ovary, lung, and brain and shows relatively low expression levels in normal tissues. MELK overexpression in patient-derived tumors strongly correlates with poor prognosis in glioblastoma and breast cancer. Moreover, siRNA mediated knockdown of MELK kinase significantly inhibits growth of tumor cell lines both in vitro and in vivo. Therefore, MELK kinase is an emerging and interesting target of significant potential for therapeutic intervention in cancer. In this study, we are reporting results for a series of new MELK kinase inhibitors that were developed at Selvita. Newly synthesized derivatives exert good selectivity and potency in MELK inhibitions with the low sub-micromolar range. Anticancer effects of these compounds were investigated in several cancer cell lines of solid tumor origin where the compounds were shown to induce cell death with low micromolar ED50 values. Compounds were also analyzed for their effects on cell death, proliferation, apoptosis, cell cycle parameters and ADME properties. Taken altogether, the presented data supports our rationale of using inhibitors of MELK kinases as a novel approach for the cancer therapy, especially for the treatment of glioblastoma and breast cancer. Citation Format: Piotr Kowalczyk, Paulina Wegrzyn, Przemyslaw Zawadzki, Edyta Palacz, Ewa Trebacz, Katarzyna Wiklik, Mariusz Milik, Adrian Zarebski, Karolina Krawczynska, Krzysztof Brzozka. Development of selective MELK kinase inhibitors for cancer treatments. [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 2160. doi:10.1158/1538-7445.AM2013-2160

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.

Abstract 3245: Identification of potent, dual PIM/FLT3 kinase inhibitors for AML treatment.

Acute myeloid leukemia (AML) is a highly heterogeneous disease with numerous signaling pathways that contribute to its pathogenesis. Advances in our understanding of molecular mechanisms of AML pathogenesis and prognosis so far did not translate into significant clinical improvements. AML is still the highest unmet medical need within hematological malignancies area, particularly in case of the elderly patients. FLT3 inhibitors were investigated in the recent years as possible therapeutic agents, however to date the clinical trials of FLT3 inhibitors have yielded disappointing results. On the other hand, PIM kinases have been identified in the last years as critical downstream components of FLT3 signaling, especially in the case of oncogenic FLT3 mutants. PIM kinases are being overexpressed in a range of hematopoietic malignancies and solid cancers and the overexpression of PIMs is associated with a poor prognosis and decreased survival of patients suffering from cancer. In many cases also, PIM overexpression was associated with development of drug resistance. As dual and simultaneous inhibition of various pathway components is an emerging therapeutic idea, exemplified by several compounds in development for cancer treatment, we have developed a series of dual PIM/FLT3 small molecule inhibitors to investigate this concept. Similarly to examples of small molecule inhibitors from the JAK/STAT or PI3K/AKT pathways, where compounds are being developed as a way to improve efficacy, resistance development and overcoming the negative feedback loops, often seen after single target inhibition, we have observed increased potency of compounds developed in this series of dual PIM/FLT3 inhibitors. Synthesized inhibitors showed higher activity towards mutated FLT3 (FLT3-ITD and other FLT3 mutants) than wild type kinase and the selectivity profile on a panel of 450 kinases was comparable to best clinical examples of kinase inhibitors. In contrast to selective PIM inhibitors, the treatment with dual PIM/FLT3 inhibitors showed potent apoptosis induction as a results of Erk and S6 phosphorylation inhibition. The in vitro activity in FTL3-ITD positive cells was also confirmed in vivo in a PK/PD xenograft experiment, where sustained biomarker inhibition was observed already after single compounds administration. Oral administration of dual PIM/FLT3 inhibitors led to potent effect in vivo and in certain cases also to remissions in subcutaneous xenograft models. Observed activity profile and synergistic effects observed with other targeted therapies and standard of care compounds, makes dual PIM/FLT3 inhibitors an exciting approach for treatment of FLT3-mutant positive AML patients with high chances of clinical success. Citation Format: Wojciech Czardybon, Michal Galezowski, Renata Windak, Magdalena Salwinska, Izabela Dolata, Ewa Trebacz, Radoslaw Obuchowicz, Pawel Guzik, Magdalena Zawadzka, Ewelina Wincza, Katarzyna Wiklik, Mariusz Milik, Malgorzata Zurawska, Karolina Krawczynska, Krzysztof Brzozka. Identification of potent, dual PIM/FLT3 kinase inhibitors for AML 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 3245. doi:10.1158/1538-7445.AM2013-3245

Abstract 3845: Antitumor activity of SEL120: An orally available dual inhibitors of Haspin/CDK9, for standalone and combination therapy with AuroraB inhibitors in solid tumors and hematopoietic malignancies

The mitotic machinery is a validated target for potential drug therapies in cancer. However, anti-mitotic agents are not without complications from various side effects, notably neurological and hematological toxicities. There is much interest in identifying new drug targets in mitosis which could lead to safer and more efficacious treatments of cancer. Recently, Haspin has been identified as another important kinase involved in mitosis. Similar to other mitotic kinases such as Polo-like kinase-1 and the Aurora kinases, inhibition of Haspin represents a novel approach in anti-mitotic cancer therapeutics. In this study we report the development of SEL120, series of novel ATP competitive inhibitors of Haspin kinase. These compounds have binding affinities towards Haspin kinase in the low nM range. Several compounds in the series exerted significant activity on CDK9 (selectively over other CDK kinases), a part of positive transcription elongation factor b (P-TEFb) involved in the expression of survival proteins during progression of cancer. SEL120 inhibited proliferation and clonogenic survival of a number of tumor cell lines with particularly good cytostatic activity in colon, lung (NSCLS) and B cell lymphoma cell lines. The only substrate of Haspin reported in literature to date is histone H3. During mitosis Haspin targets a single site in this protein, namely threonine at position T3 (H3T3ph). By using siRNA, we have confirmed that Thr3 phosphorylation of histone H3 could be completely repressed by the knockdown of Haspin in HCT116 colon and A549 (NSCLC) lung cancer cell lines. We also consistently observed decreased phosphorylation of histone H3 (Thr3) in both synchronized and asynchronous cell lines treated with SEL120 inhibitor. In addition SEL120-1 treatment decreased levels of CDK9 biomarkers- phosphorylation of pol II CTD (Ser2) and expression of the pro-survival protein Mcl-1 Treatment with SEL120 consistently resulted in mitotic cell cycle arrest in A549 cell line confirming inhibition of Haspin as the mechanism of action. Remarkably, inhibitory activity on CDK9 correlated with levels of cell death. Furthermore, co-administration of SEL120 with an AuroraB inhibitors, resulted in strong synergistic cytostatic effects. Treatment with both compounds increased the number of cells arrested in mitosis, notably without any signs of polyploidy typically observed after inhibition of AuroraB. Oral administration of SEL120 (25mg/kg BID) revealed excellent potency in colon and B-cell lymphoma xenograft models. Analysis of pharamcokinetic, ADMET and histopathological parameters afforded encouraging results towards potential development of new therapeutics emerging from our SEL120 program. 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 3845. doi:1538-7445.AM2012-3845

Abstract B242: Antitumor activity of SEL120: An orally available small-molecule inhibitor of Haspin kinase for standalone and combination therapy with AuroraB inhibitors in colorectal cancer.

The mitotic machinery is a validated target for potential drug therapies in cancer. Anti-mitotic drugs like vinca alkaloids and taxanes interfering with microtubules are important classes of chemotherapeutical agents. However, these agents are not without complications from various side effects, notably neurological and hematological toxicities. There is much interest in identifying new drug targets in mitosis which could lead to safer and more efficacious treatments of cancer. Recently, Haspin has been identified as another important kinase involved in mitosis. Similar to other mitotic kinases such as Polo-like kinase-1 and the Aurora kinases, inhibition of Haspin represents a novel approach in anti-mitotic cancer therapeutics. In this study we report the development of SEL120, a novel ATP competitive inhibitor of Haspin kinase. These small-molecule compounds have binding affinities towards Haspin kinase in the low nM range. SEL120 inhibited proliferation and clonogenic survival of a number of tumor cell lines with particularly good cytostatic activity in colon, lung (NSCLS) and B cell lymphoma cell lines. The only substrate of Haspin reported in literature to date is histone H3. During mitosis Haspin targets a single site in this protein, namely threonine at position T3 (H3T3ph). By using siRNA, we have confirmed that Thr3 phosphorylation of histone H3 could be completely repressed by the knockdown of Haspin in HCT116 colon and A549 (NSCLC) lung cancer cell lines. We also consistently observed decreased phosphorylation of histone H3 (Thr3) in both synchronized and asynchronous cell lines treated with SEL120 inhibitor. Haspin-depleted cells were arrested in the prometaphase as a result of chromosome alignment defects and activation of the mitotic checkpoint. Treatment with SEL120 consistently resulted in mitotic cell cycle arrest in HCT116 and A549 cell lines confirming inhibition of Haspin as the mechanism of action. Furthermore, co-administration of SEL120 with an AuroraB inhibitor, AZD1152, resulted in strong synergistic cytostatic effects. Treatment with both compounds increased the number of cells arrested in mitosis, notably without any signs of polyploidy (cells with >4n) typically observed after inhibition of AuroraB. Oral administration of SEL120 (25mg/kg BID) revealed excellent potency in the HCT116 xenograft model; observed tumor growth inhibition was over 80%. Analysis of pharamcokinetic, ADMET and histopathological parameters afforded encouraging results towards potential development of new therapeutics emerging from our SEL120 program. Presented data validates Haspin as a promising target for anticancer treatments, particularly for colon cancer, lung cancer and lymphomas. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr B242.