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// Tomasz Rzymski 1, * , Michal Mikula 2, * , Eliza Żylkiewicz 1 , Agnieszka Dreas 1 , Katarzyna Wiklik 1 , Aniela Golas 1 , Katarzyna Wojcik 1 , Magdalena Masiejczyk 1 , Anna Wrobel 1 , Izabela Dolata 1 , Agata Kitlinska 1 , Malgorzata Statkiewicz 2 , Urszula Kuklinska 2 , Krzysztof Goryca 2 , Łukasz Sapala 1 , Aleksandra Grochowska 3 , Aleksandra Cabaj 2, 4 , Malgorzata Szajewska-Skuta 1 , Ewelina Gabor-Worwa 1 , Katarzyna Kucwaj 1 , Arkadiusz Bialas 1 , Adam Radzimierski 1 , Michal Combik 1 , Jakub Woyciechowski 1 , Maciej Mikulski 1 , Renata Windak 1 , Jerzy Ostrowski 2, 3 and Krzysztof Brzozka 1 1 R&D Department, Selvita S.A., Krakow, Poland 2 Department of Genetics, Maria Sklodowska-Curie Memorial Cancer Center, Warsaw, Poland 3 Department of Gastroenterology, Hepatology and Clinical Oncology, Medical Center for Postgraduate Education, Warsaw, Poland 4 Laboratory of Bioinformatics, Nencki Institute of Experimental Biology, Warsaw, Poland * These authors have contributed equally to this work Correspondence to: Krzysztof Brzozka, email: krzysztof.brzozka@selvita.com Keywords: CDK8 mediator, kinase inhibitor, STAT5, AML, leukemia stem cells Received: August 04, 2016 Accepted: March 09, 2017 Published: April 04, 2017 ABSTRACT 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 protein’s 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.

PIM kinases represent an emerging therapeutic target in multiple hematological malignancies, as exemplified by currently ongoing phase I clinical trials by Astra Zeneca (AZD1208) and Novartis (LGH447) in acute myeloid leukemia and multiple myeloma. Selvita has developed a potent and selective dual PIM/FLT3 mutant kinase inhibitor - SEL24-B489 showing high inhibitory activity on all three PIM kinase isoforms and FLT3 kinase mutants. We have previously reported that PIM kinases are important downstream effectors of FLT3 signaling and play a crucial role in cell survival and inhibition of apoptosis upon expression. Due to heterogeneous nature of AML, dual inhibition of FLT3 mutant kinase and PIM kinases led to improved efficacy of our compound in comparison to selective inhibitors of either PIM of FLT3 kinases. Herewith, we would like to report further progress of characterizing the B489 inhibitor beyond AML. We assessed PIM kinase expression levels in a panel of lymphoid malignancies and found that PIM1 and PIM2 exhibit high expression levels in a fraction of mantle cell lymphoma (MCL), diffuse large-B-cell lymphoma (DLBCL), follicular lymphoma (FL), Hodgkin's lymphoma (HL), chronic lymphocytic leukemia (CLL) and mucosa associated lymphoid tissue-type (MALT) lymphoma cell lines and primary tumors . High levels of PIM kinases were associated with certain established adverse prognostic factors and clinical outcome of the patients and correlated with aggressiveness of the disease in some of these tumors. Inhibition of PIM kinases with tool inhibitors was shown to influence cellular proliferation and, translational inhibition of 4EBP1 as reported in the literature. In addition, SEL24-B489 inhibited NFκB activity and decreased CXCR4 expression. Comparison of SEL24-B489 to competitive PIM inhibitors revealed higher cellular activity and biomarker response, as shown by inhibition of phospho-S6 phosphorylation in sub-microM concentrations. The presented data will further validate SEL24-B489 as a successful example of rational drug design and present a promising therapeutic approach in multiple hematological malignancies, both stand alone and in combination with standard of care and targeted therapies in clinical development. Citation Format: Wojciech Czardybon, Renata Windak, Izabela Dolata, Magdalena Salwińska, Maciej Szydlowski, Tomasz Sewastianik, Emilia Białopiotrowicz, Elżbieta Mądro, Ewa Lech-Marańda, Bożena K. Budziszewska, Katarzyna Borg, Przemysław Juszczynski, Krzysztof D. Brzózka. Preclinical characterization of SEL24-B489, a dual PIM/FLT3 inhibitor for the treatment of hematological malignancies. [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 1749. doi:10.1158/1538-7445.AM2014-1749

Despite an evolving understanding of acute myeloid leukemia (AML), a group of heterogeneous diseases with a common feature of abnormal levels of myeloblasts in bone marrow and in circulation, the current standard of care including chemotherapy and allogeneic hematopoietic stem cell transplantation results in less than 40 % cure rate with little progress over the last decades. One of the most extensively investigated approaches in targeted therapy of AML are FLT3 inhibitors that address the population of patients harboring FLT3 mutations that result in high relapse rate and decreased overall survival when compared to patients lacking such mutations. Therefore inhibition of the constitutively active mutants of FLT3 is a promising therapeutic approach. Unfortunately, the clinical development of selective or multitargeted FLT3 inhibitors turned out to result in rapid, but very transient responses followed by disease progression. One of the main reasons for development of resistance was selection of clones harboring both ITD and tyrosine kinase mutations (TKD). In parallel, the role of PIM kinases, especially PIM1 in FLT3 mediated leukemogenesis attracted attention of the pharmaceutical industry with currently two phase I programs targeting PIM kinases in hematological malignancies. PIM kinases are important downstream effectors of FLT3 signaling and play a crucial role in cell survival and inhibition of apoptosis upon expression. For that reason, PIM kinases represent an emerging therapeutic target class in AML with promising preliminary data from clinical trials. Due to the heterogeneous nature of AML, dual inhibition of FLT3 mutant kinase and PIM kinases could lead to improved efficacy and constitute a promising approach to overcome rapid resistance development to targeted therapies. Selvita has developed a potent and selective dual PIM/FLT3 mutant kinase inhibitor - SEL24-B489 which is highly active in in vitro and in vivo AML models. The compound shows high inhibitory activity against mutated FLT3 (FLT3-ITD and TKD mutations such as D835H, D835Y, N841I) and all three PIM kinase isoforms, comparable to activity of selective inhibitors of FLT3 (A220) and PIM (AZD1208). A head to head comparison of SEL24-B489 in cellular models reveals high activity across tested cellular AML models and biomarker inhibition in line with the expected kinase activity profile. Most importantly, SEL24-B489 showed strong synergistic effect in combination with the current standard of care in AML - cytarabine, and with other targeted inhibitors in advanced clinical development. The activity of B489 in vivo in xenograft models of AML and lymphomas after oral administration was higher than compared to selective PIM inhibitor and led to remissions in certain models. SEL24-B489 is a successful example of rational drug design and is currently in preclinical development. It represents a promising therapeutic approach which addresses alternative survival pathways downstream of FLT3 in addition to the activity on resistant FLT3 mutant kinases, which hopefully will translate into improved survival of AML patients in clinical trials. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):C257. Citation Format: Wojciech Czardybon, Michal Galezowski, Pawel Guzik, Magdalena Zawadzka, Renata Windak, Izabela Dolata, Magdalena Salwińska, Radoslaw Obuchowicz, Krzysztof D. Brzózka. Preclinical development of a potent dual PIM/FLT3 mutant kinase inhibitor for the treatment of AML and other hematological malignancies. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr C257.

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 Salwińska, Izabela Dolata, Ewa Trebacz, Radosław Obuchowicz, Pawel Guzik, Magdalena Zawadzka, Ewelina Wincza, Katarzyna Wiklik, Mariusz Milik, Malgorzata Zurawska, Karolina Krawczynska, Krzysztof Brzózka. 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