Search

Fms-like tyrosine kinase 3 internal tandem duplication (FLT3-ITD) is one of the most common genetic lesions in acute myeloid leukemia patients (AML). Although FLT3 tyrosine kinase inhibitors initially exhibit clinical activity, resistance to treatment inevitably occurs within months. PIM kinases are thought to be major drivers of the resistance phenotype and their inhibition in relapsed samples restores cell sensitivity to FLT3 inhibitors. Thus, simultaneous PIM and FLT3 inhibition represents a promising strategy in AML therapy. For such reasons, we have developed SEL24-B489 - a potent, dual PIM and FLT3-ITD inhibitor. SEL24-B489 exhibited significantly broader on-target activity in AML cell lines and primary AML blasts than selective FLT3-ITD or PIM inhibitors. SEL24-B489 also demonstrated marked activity in cells bearing FLT3 tyrosine kinase domain (TKD) mutations that lead to FLT3 inhibitor resistance. Moreover, SEL24-B489 inhibited the growth of a broad panel of AML cell lines in xenograft models with a clear pharmacodynamic-pharmacokinetic relationship. Taken together, our data highlight the unique dual activity of the SEL24-B489 that abrogates the activity of signaling circuits involved in proliferation, inhibition of apoptosis and protein translation/metabolism. These results underscore the therapeutic potential of the dual PIM/FLT3-ITD inhibitor for the treatment of AML.

// 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.

Accumulating evidence highlights an important role of type I interferon response in the immune surveillance mechanisms. IFNβ release by antigen-presenting cells promotes spontaneous anti-tumor CD8+ T cell priming being largely dependent on activation of Stimulator of Interferon Genes (STING). STING agonists promote regression of established tumors and generation of long-term immunologic memory in preclinical animal models. Herein we report the discovery of potent and selective, first-in-class non-nucleotide, non-macrocyclic, small molecule direct STING agonists with molecular weight below 500, structurally unrelated to known cyclic dinucleotide chemotypes with potential for systemic administration. Activation of STING pathway was monitored in THP-1 Dual reporter monocytic cell line as well as peripheral blood mononuclear cells (PBMC) or antigen presenting cells from human and mouse origin. Surface expression of the antigen-presenting cell maturation markers i.e. CD80, CD86, CD83 and HLA-DR was assessed by flow cytometry. Binding affinity was confirmed by three independent assays. RNA sequencing was performed on total RNA isolated from THP-1 cells and PBMC isolated from 2 healthy human donors. Direct binding to both mouse and human STING protein of Selvita agonists have been confirmed in biophysical binding assays (FTS, MST and FP) and by crystallography studies. The compounds have fine-tunable ADME properties with good solubility, permeability and human plasma stability. They selectively activates STING-dependent signaling in both THP-1 reporter assays and in primary cells of human and mouse origin. In addition, RNA sequencing data confirmed selectivity of the Selvita compounds. In vitro functional assays demonstrated their ability to induce cytokine responses (IFNβ, TNFα) in a panel of human peripheral blood mononuclear cell (PBMC), human monocyte derived macrophage (HMDM) and human dendritic cells samples with various STING haplotypes including refractory alleles. Additionally, the compounds efficiently induced cytokine release in mouse bone marrow-derived macrophages and dendritic cells. Pro-inflammatory cytokine profile was accompanied by up-regulation of the maturation markers, i.e. CD80, CD86, CD83 and HLA-DR, on the surface of human antigen presenting cells. These data demonstrate potent, novel, next-generation small molecule STING agonists activating STING-dependent signaling in both mouse and human immune cells to promote potential antitumor immunity. The compounds show good selectivity and in vitro ADME properties enabling further development for systemic administration as a single agent or in combinatory immunotherapies for cancer treatment. Citation Format: Monika Dobrzańska, Stefan Chmielewski, Magdalena Zawadzka, Jolanta Mazurek, Karolina Gluza, Katarzyna Wójcik-Jaszczyńska, Maciej Kujawa, Grzegorz Topolnicki, Grzegorz Ćwiertnia, Aleksandra Poczkaj, Izabela Dolata, Magdalena Mroczkowska, Agnieszka Gibas, Marcin Leś, Sylwia Sudoł, Adam Radzimierski, Kinga Michalik, Magdalena Sieprawska-Lupa, Katarzyna Banaszak, Katarzyna Wiklik, Federico Malusa, Michał Combik, Karolina Wiatrowska, Agnieszka Adamus, Lukasz Dudek, Jose Alvarez, Charles Fabritius, Anna Rajda, Maciej Rogacki, Faustyna Gajdosz, Peter Littlewood, Luigi Stasi, Krzysztof Brzózka. Discovery and characterization of next-generation small molecule direct STING agonists [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4983.

Despite huge effort spent on understanding the pathogenesis of acute myeloid leukemia (AML), current standards of care are still based on the same chemotherapy agents as two decades ago - combinational treatment of cytarabine with an anthracycline. Fms-like tyrosine kinase 3 internal tandem duplication (FLT3-ITD) is one of the most common genetic lesions in AML. Although FLT3 inhibitors initially exhibit clinical activity, resistance to treatment inevitably occurs within months. PIM kinases are thought to be major drivers of the resistance phenotype and their inhibition in relapsed samples restores cell sensitivity to FLT3 inhibitors. Thus, simultaneous PIM and FLT3 inhibition represents a promising strategy in AML therapy. Selvita has developed a potent and selective first-in-class, dual PIM/FLT3 kinase inhibitor, the SEL24-B489 compound, and profiled its activity for in vitro and in vivo AML models showing significantly broader anti-tumor activity of SEL24-B489 than selective FLT3-ITD or PIM inhibitors. We compared SEL24-B489 head-to-head with a selective PIM inhibitor (AZD1208) and a selective FLT3-ITD inhibitor (AC220) in a panel of AML cell lines with FLT3-ITD or unmutated kinase (FLT3-WT) as well as peripheral AML cells and CD34+ bone marrow blasts. SEL24-B489 exhibited a significantly broader activity, irrespective of FLT3 status, than either of the selective inhibitors. Since PIM kinases have emerged as important mediators of FLT3-inhibitor resistance, we hypothesized that the dual specificity of SEL24-B489 might overcome the phenotype of resistance. We utilized previously developed MOLM-14 cells transduced with either FLT3-WT or FLT3 alleles containing TKD point mutations to show that neither of the these mutations decreased the cellular sensitivity to SEL24-B489. Higher cellular activity and biomarker response of SEL24-B489 than competitive inhibitors was shown by inhibition of specific biomarkers such as S6 and STAT5 phosphorylation at nanomolar concentrations in both FLT3-ITD positive and FLT3-WT cell lines in vitro. We have also demonstrated SEL24-B489 superior potency of SEL24-B489 in xenograft models in vivo. Consistent with the experiments in vitro showing marked synergy between SEL24-B489 and AraC, a combination of these agents resulted in almost completely blocked tumor growth in vivo. Most importantly, SEL24-B489 has been selected as a clinical candidate and is currently in phase I clinical trials. Citation Format: Wojciech Czardybon, Renata Windak, Aniela Gołas, Michał Gałęzowski, Aleksandra Sabiniarz, Izabela Dolata, Magdalena Salwińska, Paweł Guzik, Magdalena Zawadzka, Ewelina Gabor-Worwa, Bożena Winnik, Małgorzata Żurawska, Ewa Kolasińska, Ewelina Wincza, Marta Bugaj, Monika Danielewicz, Grzegorz Dubin, Ewa Jabłońska, Maciej Szydłowski, Tomasz Sewastianik, Bartosz Puła, Anna Szumera-Ciećkiewicz, Monika Prochorec-Sobieszek, Elżbieta Mądro, Ewa Lech-Marańda, Krzysztof Warzocha, Jerome Tamburini, Przemysław Juszczyński, Krzysztof Brzózka. Development of a potent, dual pan-PIM/FLT3 inhibitor for the treatment of heme malignancies [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 4087. doi:10.1158/1538-7445.AM2017-4087

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, Michał Mikula, Małgorzata Szajewska-Skuta, Eliza Zyłkiewicz, Łukasz Sapała, Izabela Dolata, Agata Kitlińska, Krzysztof Goryca, Aleksandra Grochowska, Aleksandra Cabaj, Agnieszka Dreas, Katarzyna Kucwaj, Artur Białas, Adam Radzimierski, Aniela Gołas, Renata Windak, Jerzy Ostrowski, Krzysztof Brzózka. 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

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