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The mitogen-activated protein kinase (MAPK)-interacting kinases 1 and 2 (MNKs 1/2) and their downstream target eIF4E, play a role in oncogenic transformation, progression and metastasis. These results provided rationale for development of first MNKs inhibitors, currently in clinical trials for cancer treatment. Inhibitors of the MNKs/eIF4E pathway are also proposed as treatment strategy for inflammatory conditions. Here we present results of optimization of indazole-pyridinone derived MNK1/2 inhibitors among which compounds 24 and 26, selective and metabolically stable derivatives. Both compounds decreased levels of eIF4E Ser206 phosphorylation (pSer209-eIF4E) in MOLM16 cell line. When administered in mice compounds 24 and 26 significantly improved survival rates of animals in the endotoxin lethal dose challenge model, with concomitant reduction of proinflammatory cytokine levels – TNFα and IL-6 in serum. Identified MNK1/2 inhibitors represent a novel class of immunomodulatory compounds with a potential for the treatment of inflammatory diseases including sepsis.

Background Stimulator of Interferon Genes (STING) is a major player in the activation of robust innate immune response leading to initiation and enhancement of tumor-specific adaptive immunity. Several clinical and pre-clinical programs have shown that activation of the STING pathway triggers immune-mediated antitumor response. Although vast majority of programs focus on development of analogues of the endogenous STING ligands, their chemical nature and stability often limit their use to local administration. Herein, we present recent results from the development of our selective non-nucleotide, non-macrocyclic, small molecule direct STING agonists, suitable for systemic administration, characterized by improved activity in human immune cells. Methods Binding to recombinant STING protein was examined using FTS, MST, FP and crystallography studies. Phenotypic screen was performed in THP-1 Dual reporter cells. Mouse bone marrow-derived dendritic cells (BMDC) were obtained from C57BL/6 mice and differentiated with mIL-4 and mGM-CSF. STING agonists were administered into BALB/c mice and cytokine release was measured in plasma. Additionally, mice were inoculated with CT26 murine colon carcinoma or EMT6 murine breast carcinoma cells and the compound was administered, followed by the regular tumor growth and body weight monitoring. Results Ryvu’s small-molecule agonists demonstrate strong binding affinity to recombinant STING proteins across all tested species. The compounds bind to all human STING protein variants and trigger pro-inflammatory cytokine release from human immune cells regardless of the STING haplotype. Moreover, new generation of developed agonists show significantly improved binding to human protein as well as in vitro activity on human cells. Systemic, intravenous in vivo administration leads to a dose-dependent upregulation of STING-dependent pro-inflammatory cytokines, which results in a dose-dependent antitumor efficacy observed in CT26 and EMT6 mouse cancer models, leading to complete tumor remissions in all treated animals. Furthermore, observed efficacy is accompanied by development of a lasting immunological response demonstrated by lack of tumor engraftment or a delayed tumor growth in cured animals challenged with repeated inoculation of cancer cells. Conclusions New generation Ryvu’s STING agonists are strong and selective activators of STING-dependent signaling in both mouse and human immune cells promoting anti-tumor immunity. Treatment with Ryvu’s small-molecule STING agonists leads to engagement of the immune system which results in a complete tumor remission and development of immunological memory of the cancer antigens. The compounds show good selectivity and ADME properties enabling development for systemic administration. In addition developed compounds maintain small functional handles amenable to linker attachment making the series suitable for versatile development as single agents, for combinations with immunotherapies or as targeted agents.

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.

Background. Stimulator of interferon genes, known as STING, is an intracellular sensor of nucleic acids and one of key regulators in activating the innate immune response. Employing synthetic STING agonists has been shown to promote immune-mediated antitumor response in preclinical animal models. Ryvu is developing small-molecule STING agonists suitable for systemic administration. Herein we present unpublished results from characterization of the new generation of our agonist series with significantly improved potency on human immune cells. Methods. Binding to recombinant STING protein was examined using Fluorescence Thermal Shift and Fluorescence Polarisation and was confirmed by X-ray crystallography. Primary screen was performed in THP-1 Dual reporter cells and selectivity was confirmed in THP-1 reporter cells with knocked out STING or expressing varying STING variants. T cell viability and proliferation was assessed by flow cytometry using activated, human T cells exposed to STING agonists. STING pathway activation pattern in cells treated with Ryvu's molecules was confirmed using Western blot analysis. BALB/c mice were injected with compounds and the levels of cytokine release were measured in the plasma. Mice were inoculated with CT26 or EMT6 tumor cells and the compound was administered intravenously followed by the regular monitoring of tumor growth. Results. New generation Ryvu STING agonists are strong binders of human STING protein. Ryvu's compounds show high cellular potency inducing cytokine production in human immune cells at low nM range. Moreover, high activity of developed agonists is maintained irrespective of the natural human STING variant as seen in THP-1 reporter cells as well as in human primary immune cells. High cellular potency of developed compounds also translates into efficacy observed in vivo, where systemic intravenous administration leads to significant tumor growth inhibition and complete tumor regressions in mouse syngeneic models. Conclusion. Ryvu has developed a new generation of potent, direct and selective small-molecule STING agonists. The compounds are characterized by drug-like properties and high in vitro potency on par or outperforming known references. Ryvu agonists are suitable for systemic administration and allow to achieve excellent antitumor efficacy. Taken together, the promising results suggest that the developed series holds high potential for improving immunotherapy in cancer patients. Citation Format: Maciej Krzysztof Rogacki, Stefan Chmielewski, Jolanta Mazurek, Magdalena Zawadzka, Katarzyna Wnuk-Lipińska, Kamil Kuś, Katarzyna Wójcik-Jaszczyńska, Aleksandra Poczkaj, Łukasz Dudek, Wojciech Schonemann, Urszula Głowniak-Kwitek, Marcin Leś, Marek Wronowski, Tushar Mahajan, Urszula Kulesza, Magdalena Zastawna, David Synak, Karol Zuchowicz, Karolina Gluza, Katarzyna Banaszak, Karolina Wiatrowska, Izabela Strojny, Mirosława Gładysz, Justyna Jabłońska, Ewelina Gabor-Worwa, Monika Dobrzańska, Raghuram Tangirala, Peter Littlewood, Krzysztof Brzózka. New generation of STING agonists: Development and characterization of a novel series of systemic immunomodulators with improved potency [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1280.

Background: Stimulator of Interferon Genes (STING) is a major player in the activation of robust innate immune response leading to initiation and enhancement of tumor-specific adaptive immunity. Several clinical and pre-clinical programs are developing cyclic dinucleotides - analogues of endogenous STING ligands. However their chemical nature and stability limit their use as systemic immuno-therapeutics. Herein, we present potent and selective non-nucleotide, non-macrocyclic, small molecule direct STING agonists, structurally unrelated to known chemotypes and suitable for systemic administration. Methods: Binding to recombinant STING protein was examined using FTS, MST, FP and crystallography studies. Phenotypic screen was performed in THP-1 Dual reporter cells. Human macrophages (HMDM) and dendritic cells (HMDC) were differentiated from monocytes (obtained from PBMC) in the presence of M-CSF and GM-CSF/IL-4 for HMDM and HMDC, respectively. Mouse bone marrow-derived dendritic cells (BMDC) were obtained from C57BL/6 or STING KO mice and differentiated with mIL-4 and mGM-CSF. STING agonists were administered into BALB/c mice and cytokine release was measured in plasma. Additionally, mice were inoculated with CT26 murine colon carcinoma cells and the compound was administered, followed by the regular tumor growth monitoring. Finally, the compound was administered to C57BL/6 WT and STING KO mice in several escalating doses. Results: Ryvu's agonists demonstrate a strong binding affinity to recombinant STING proteins across tested species. They trigger pro-inflammatory cytokine release from human PBMC and HMDC and induce dendritic cell maturation regardless of the STING haplotype. Systemic in vivo administration leads to dose-dependent upregulation of STING-dependent pro-inflammatory cytokines, suggesting immune activation which translates into efficacy in vivo in CT26 mouse colorectal cancer model and complete tumor remissions. Furthermore, cured animals develop lasting immunological response demonstrated by diminished tumor growth or lack of palpable tumors in re-challenged mice. Conclusion: Ryvu's STING agonists selectively activate STING-dependent signaling in both mouse and human immune cells promoting anti-tumor immunity. Treatment with Ryvu's STING agonists leads to engagement of the immune system which results in complete tumor remission and development of immunological memory against cancer cells. The compounds show good selectivity and ADME properties enabling development for systemic administration as a single agent or in combinations with immunotherapies or targeted agents. Citation Format: Stefan Chmielewski, Magdalena Zawadzka, Jolanta Mazurek, Maciej K. Rogacki, Karolina Gluza, Katarzyna Wójcik-Jaszczyńska, Aleksandra Poczkaj, Grzegorz Ćwiertnia, Grzegorz Topolnicki, Maciej Kujawa, Eliza Zimoląg, Urszula Głowniak-Kwitek, Magdalena Mroczkowska, Agnieszka Gibas, Marcin Leś, Sylwia Sudoł, Marek Wronowski, Kinga Michalik, Katarzyna Banaszak, Katarzyna Wiklik, Federico Malusa, Michał Combik, Karolina Wiatrowska, Łukasz Dudek, Jose Alvarez, Anna Rajda, Faustyna Gajdosz, Aniela Gołas, Katarzyna Wnuk-Lipińska, Kamil Kuś, Ewelina Gabor-Worwa, Charles Fabritius, Luigi Stasi, Peter Littlewood, Krzysztof Brzózka, Monika Dobrzańska. Development of selective small molecule STING agonists suitable for systemic administration [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4532A.

MJ and JF provided equal contribution as senior authors Acute myeloid leukemia (AML) is associated with poor survival and characterized by an accumulation of immature myeloid blasts in the bone marrow. To efficiently target AML, new therapies directed to leukemia stem cells (LSCs), a self-renewing population that constitutes a chemo-resistant reservoir responsible for disease relapse, are warranted. Cyclin-dependent kinase 8 inhibitors' (CDK8i) anti-cancer activity has been demonstrated in human acute myeloid leukemia (AML) cell lines. Efficacy has been associated with activation of super enhancer regions and reduction of STAT5 S726 phosphorylation in sensitive cells (Pelish, Nature, 2015). SEL120-34A (Selvita, Poland), a selective low nanomolar CDK8/CDK19 inhibitor, has been shown to have antileukemic effect in a panel of AML cell lines (Rzymski, Oncotarget 2017). To evaluate whether primitive AML cells, enriched for LSCs, are sensitive to CDK8 inhibition, we tested the CDK8i SEL120-34A and Senexin B on TEX cells, an AML cell line that exhibits a hierarchical organization and can be used as a LSC model (Warner, Leukemia, 2005). Treatment of TEX cells with SEL120-34A or Senexin B resulted in an inhibition of cell growth (IC50 of 8 and 31 nM respectively at 10 days of culture), associated with reduced activation of STAT5 and STAT1. Both STAT proteins were previously identified as biomarkers for SEL120-34A activity (Rzymski, Oncotarget 2017). In addition, RNA sequencing followed by gene-set enrichment analysis (GSEA) revealed a loss of a LSC signature. To functionally address the effects of CDK8i on LSCs, we used a murine dsRed+ AML model driven by retroviral MLL-AF9 expression. This model has a well-defined LSC population and initiates AML with a short latency, enabling rapid follow-up experiments in syngeneic hosts. Treatment of c-Kit+ AML cells with SEL120-34A or Senexin B in vitro resulted in strong inhibition of leukemia cell growth (IC50 of 119 and 143 nM, respectively, at 7 days of culture), associated with increased apoptosis and reduced cycling of the cells. To address the in vivo therapeutic efficacy of SEL120-34A, mice injected with AML cells 10 days earlier were treated orally for 12 consecutive days using doses of 20 and 40 mg/kg before sacrificed. No tolerability issues were observed with mice maintaining a stable weight through the treatment. Whereas the control group had 87% leukemia cells in the peripheral blood at the end-point analysis, SEL120-34A treated animals showed a dose-dependent selective anti-leukemic activity with a corresponding 78% (20 mg/kg) and 67% (40 mg/kg, p=0.011) of leukemia cells. Similarly, a significant selective dose-dependent anti-leukemic activity of SEL120-34A was observed also in the bone marrow. In addition, a dose-dependent reduced white blood cell count and smaller spleen size upon SEL120-34A treatment was observed, demonstrating that CDK8 inhibition has selective anti-leukemic activity in vivo. Notably, SEL120-34A treatment resulted in granulocytic (Gr1+CD11b-) differentiation of the AML cells (5.8% of the AML cells in the control group; 21.9% at 20 mg/kg, p=0.03; and 22.3%, p=0.0037 at 40 mg/kg). Moreover, SEL120-34A treatment resulted in strong inhibition of Stat5 S726 and Stat1 S727 phosphorylation in AML bone marrow cells harvested from the mice. To test the efficacy of CDK8 inhibition on AML patient cells, four AML patient derived xenografts were treated with SEL120-34A ex vivo. In all four samples, two of which contained activating mutations in FLT3, SEL120-34A treatment resulted in a significant antileukemic activity with decreased number of AML cells and an increase in apoptotic cells. Taken together, our data from murine and human AML models indicate that CDK8 inhibition has therapeutic efficacy in primitive AML cells. SEL120-34A treatment in vivo resulted in reduced leukemia cell burden in both blood and bone marrow accompanied by granulocytic differentiation. Treatment of AML cells in cultures consistently resulted in a reduction in AML cell number and increased apoptosis. Ongoing and future experiments will address whether SEL120-34A treatment also extends survival of mice with AML in syngeneic and patient-derived xenograft models. These data highlight CDK8 as a promising therapeutic target in AML and provides preclinical proof of concept for anti-leukemic efficacy of the clinical candidate SEL120-34A in relevant AML models. Disclosures Mazan: Selvita S.A.: Employment. Majewska:Selvita S.A.: Employment. Wiklik:Selvita S.A.: Employment. Combik:Selvita S.A.: Employment. Masiejczyk:Selvita S.A.: Employment. Fiedor:Selvita S.A.: Employment. Obacz:Selvita S.A.: Employment. Bialas:Selvita S.A.: Employment. Chesy:Selvita S.A.: Employment. Gabor-Worwa:Selvita S.A.: Employment. Brzózka:Selvita S.A.: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees. Rzymski:Selvita S.A.: Employment, Equity Ownership. Fioretos:Cantargia: Equity Ownership, Membership on an entity's Board of Directors or advisory committees.

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

Despite huge effort spent on understanding acute myeloid leukemia (AML), current standards of care are still based on the same chemotherapy agents as two decades ago - typically based on cytarabine chemotherapy with an anthracycline. Although patients aged PIM kinases are expressed in various cancers including AML but also other liquid tumors as well as in some solid tumors. Inhibition of PIM kinases which are downstream in the FLT3 signaling cascade have already shown influence on cancer cell survival. In addition there are great hopes related to the fact that PIM kinases were shown to contribute in resistance to FLT3 inhibitors. This leads to the conclusion that combined inhibition of PIM and FLT3 may be a rational strategy. Selvita has developed series of inhibitors combining activity against PIM and FLT3 kinases. Selected clinical candidate SEL24-B489 - has shown excellent anticancer efficacy. It is active against broad panel of AML cell lines and primary blasts, but also against other hematological cancers: DLBCL, CLL, HL - both in vitro on cell lines, patient samples and in vivo. Head to head comparison of SEL24-B489 with PIM (AZD1208) and FLT3 (AC220) inhibitors currently in clinical development will be presented. Comparison shows strong activity of SEL24-B489 against broader panel of cell lines in vitro and in vivo (in xenograft models) than AZD1208 or AC220. SEL24-B489 is currently in preclinical development and details of toxicology profile will also be discussed. Citation Format: Krzysztof Brzózka, Wojciech Czardybon, Aniela Gołas, Renata Windak, Michał Gałęzowski, Ewelina Gabor-Worwa, Bożena Winnik, Agnieszka Przybyłowicz, Maciej Szydłowski, Emilia Białopiotrowicz, Tomasz Sewastianik, Elżbieta Mądro, Ewa Lech-Marańda, Krzysztof Warzocha, Przemysław Juszczyński. First-in-class dual PIM/FLT3 kinase inhibitor SEL24-B489 for the treatment of hematological malignancies. [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 5394. doi:10.1158/1538-7445.AM2015-5394