Breast cancer (BC) is a complex and heterogenous disease, and various approaches have been used to classify BC into several subtypes to improve diagnostic and therapeutic outcomes. One of the features of BC is deregulated transcription, which allows for classification of the disease based on gene expression signature into four basic types: Luminal A, Luminal B, HER2-enriched, and triple negative (TNBC)/basal-like. Novel therapeutic approaches targeting oncogenic transcriptional programs may represent a promising strategy, in particular for TNBC, where the lack of common genetic alterations has so far limited the development of targeted therapies. The CDK8 module of the mediator complex represents an effective therapeutic target across multiple hematologic malignancies and solid tumors. CDK8 module of mediator functions as a master coordinator of transcription, bridging enhancers and core promoters. Meta-analysis of transcriptomic data revealed that higher CDK8 expression and its paralog CDK19 is associated with shorter relapse-free survival (RFS) in all molecular subtypes of BC. Our analysis of curated TCGA data revealed that >15% of all BC have alterations in either CDK8 or CDK19. Increased expression of CDK8 in BC can be partially attributed to copy number gains and amplifications. High CDK8 expression was inversely correlated with the expression of estrogen receptor (ER) and positively correlated with occurrence of TP53 mutations. In order to identify whether increased expression of CDK8/19 in BC could be associated with increased sensitivity to pharmacological inhibition of both kinases, we have interrogated a panel of BC cell lines representing various subtypes with RVU120 - a specific, selective inhibitor of CDK8/CDK19, currently being tested in a first-in-human Phase Ib clinical trial. The highest sensitivity to RVU120 in clonogenic assays has been observed for TNBC and ER-/PR-/HER2+ cells with high STAT3 phosphorylation levels. In contrast, neither RVU120 nor other CDK8 inhibitors were able to inhibit mitogenic effect of estrogen, confirming differential efficacy in hormone - independent BC. Detailed transcriptional profiling of responder cells revealed high enrichment of TNF/NFKB and STAT target genes (signatures associated with inflammatory phenotypes) and SOX4 target genes (signatures associated with invasiveness and stemness). Non-responder cells were characterized by enrichment of transcriptional signatures of ER activity. Efficacy of RVU120 in TNBC cells has been corroborated in a three-dimensional (3D) spheroid viability assay that could predict in vivo efficacy of RVU120 in tested xenograft models and recapitulated using other chemically non-related CDK8 inhibitors, indicating a class effect. Single agent efficacy of RVU120 has been confirmed in subcutaneous TNBC xenograft models in vivo at well tolerated doses. These studies provide rationale for further development of RVU120 in TNBC patients. Citation Format: Tomasz Rzymski, Aniela Gołas, Milena Mazan, Urszula Pakulska, Magdalena Masiejczyk, Agata Stachowicz, Justyna Martyka, Michał Combik, Katarzyna Wiklik, Kristina Goller, Marta Obacz, Elżbieta Adamczyk, Krzysztof Brzózka. Selective CDK8/CDK19 inhibitor RVU120 demonstrates efficacy against hormone-independent breast cancer cells in vitro and in vivo [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P5-17-13.
Background: CDK8 and its paralog CDK19 are part of the kinase module of the mediator complex, which functions as a bridge between enhancers and core promoters. The CDK8 module functions as a master regulator of transcription and lineage development, including regulation of various oncogenic programs and importantly also hematopoiesis and differentiation. The CDK8/CDK19 inhibitor RVU120 (SEL120) is being investigated in a Phase Ib clinical study (NCT04021368) in AML and HR-MDS patients. Preclinical data indicate the high efficacy of RVU120 in AML models, particularly in cells with stem cell-like characteristics, where the treatment leads to lineage commitment and eventually cell death. Results from the patient cohorts of the dose-escalation phase indicate signs of clinical efficacy, including a complete response (CR) in a relapsed/refractory (R/R) AML patient. Aim: It is now critical to establish the relationship between preclinical and clinical efficacy results and molecular characteristics to identify actionable biomarkers predicting response to CDK8/CDK19 inhibitors. Methods: Association between gene mutations and gene expression patterns with responses to RVU120 has been analyzed on 27 genetically annotated AML patient-derived cells (PDCs). The activity and efficacy of RVU120 were assessed in both non-differentiating and differentiating media followed by flow cytometry and bioinformatics analysis. DNMT3A mutant PDCs were implanted intravenously into NSG-SGM3 mice and after disease onset, animals were treated orally with RVU120. Profiling of transcriptional response to RVU120 in DMNT3A mutant cells has been performed by RNA-seq. The first-in-human Phase1b study CLI120-001 (NCT04021368) of RVU120 is currently enrolling R/R AML and HR-MDS patients, in a dose-escalation design aimed at exploring safety/tolerability and identifying the randomized Phase II dose (RPD2). RVU120 is administered orally, every other day (EOD), 7 total doses per cycle, in 21-day treatment cycles until disease progression/unacceptable toxicity. Local assessment of Bone Marrow (BM) and Peripheral Blood (PB) are performed at different time points according to investigator guidelines to define response to study drug according to Dohner 2017 response criteria. Results: Screening of 27 AML PDCs against RVU120 and other non-related CDK8/CDK19 inhibitors indicated high anti-cancer efficacy in >40% of tested samples (12 out of 27). Correlation of efficacy with the genetic profile of samples showed specific enrichment of DNMT3A mutants (8 out of 12) in the responder group (p=0.015). Notably, efficacy results were further corroborated in vivo in a disseminated PDX AML model, showing complete clearance of blasts positive for DNMT3A mutation and recovery of normal murine BM in animals treated with RVU120. Molecular profiling of responder cells indicated transcriptional reprogramming and lineage commitment. At the date of this abstract submission, 7 patients have been enrolled into the Phase 1b CLI120-001 trial: 5 AML and 2 HR-MDS patients, median age 73 years, failing 2 median previous lines of therapy. Notably, a 62 YO R/R AML patient that has achieved a CR was positive for DNMT3A R882C mutation. At study entry, this patient had progressed after venetoclax/decitabine, with pancytopenia and >50% BM monocytic blasts and skin leukemia. At the end of the first cycle of the study drug, BM showed complete clearance of blasts with hematological recovery and strong monocytic differentiation starting in cycle 2. Skin leukemia lesions improved gradually during treatment with a complete resolution in cycle 7, resulting in CR. After 1 month from CR patient progressed with 65% BM blasts with the same phenotype as at the study entry. Conclusion: AML PDCs with DNMT3 mutations show increased sensitivity to RVU120 treatment both in vitro and in vivo. The anti-cancer efficacy of RVU120 was strongly associated with transcriptomic reprogramming and lineage commitment. Preliminary evidence of response to RVU120 has also been shown in a R/R AML patient positive for DNMT3A mutation that has achieved a CR. Further molecular studies in more patients treated with RVU120 are ongoing and could provide evidence for predictive biomarkers of response to RVU120 in AML. Figure 1 Figure 1. Disclosures Rzymski: Ryvu Therapeutics: Current Employment, Current equity holder in publicly-traded company. Pakulska: Ryvu Therapeutics: Current Employment, Current equity holder in publicly-traded company. Burris: Boehringer Ingelheim: Consultancy, Other: research grant ; AstraZeneca: Consultancy, Other: research grant ; Bayer: Consultancy, Other: research grant ; Daiichi Sankyo: Consultancy; Grail: Consultancy; Incyte: Consultancy, Other: research support; Novartis: Consultancy, Other: research grant, Expert Testimony; Pfizer: Consultancy, Other: research grant ; Vincerix Pharma: Consultancy; Abbvie: Other: research grant ; Agios: Other: research grant ; ARMO Biosciences: Other: research grant ; Array BioPharma: Other: research grant ; BioAtla: Other: research grant ; BioMed Valley Discoveries: Other: research grant ; Boehringer Ingelheim: Other: research grant ; Bristol Myers Squibb: Other: research grant ; CALGB: Other: research grant ; CicloMed: Other: research grant ; eFFECTOR Therapeutics: Other: research grant ; Lilly: Other: research grant ; EMD Serono: Other: research grant ; Roche/Genetech: Other: research grant ; GlaxoSmithKline: Other: research grant ; Harpoon: Other: research grant ; Hengrui Therapeutics: Other: research grant ; Infinity Pharmaceuticals: Other: research grant ; Janssen: Other: research grant ; Jounce: Other: research grant ; Kymab: Other: research grant ; MacroGenics: Other: research grant ; MedImmune: Other: research grant ; Merck: Other: research grant ; Millennium Pharmaceuticals: Other: research grant ; Moderna: Other: research grant ; Foundation Medicine: Other: research grant ; Revolution Medicine: Other: research grant ; Seattle Genetics: Other: research grant ; Tesaro: Other: research grant ; TG Therapeutics: Other: research grant ; Verastem: Other: research grant ; Vertex Pharmaceuticals: Other: research grant ; XBiotech: Other: research grant ; Zymeworks: Other: research grant ; Arch Oncology: Other: research grant ; Arvinas: Other: research grant ; Coordination Pharmaceuticals: Other: research grant ; NGM Biopharmaceuticals: Other: research grant ; Gossamer Bio: Other: research grant ; Ryvu Therapeutics: Other: research grant ; BioTheryX: Other: research grant ; HCA Healthcare: Other: stock ownership. Obacz: Ryvu Therapeutics: Current Employment, Current equity holder in publicly-traded company. Goller: Ryvu Therapeutics: Current Employment, Current equity holder in publicly-traded company. Combik: Ryvu Therapeutics: Current Employment, Current equity holder in publicly-traded company. Mazan: Ryvu Therapeutics: Current Employment, Current equity holder in publicly-traded company. Juszczynski: Ryvu Therapeutics: Current equity holder in publicly-traded company. Zawadzka: Ryvu Therapeutics: Current Employment, Current equity holder in publicly-traded company. Brzozka: Selvita SA: Current Employment, Current equity holder in publicly-traded company, Membership on an entity's Board of Directors or advisory committees; Ardigen: Current Employment, Membership on an entity's Board of Directors or advisory committees; Ryvu Therapeutics: Current Employment, Current equity holder in publicly-traded company, Membership on an entity's Board of Directors or advisory committees. Shamsili: Ryvu Therapeutics: Current Employment, Current equity holder in publicly-traded company. Angelosanto: Ryvu Therapeutics: Current Employment.
Breast cancer (BC) is a complex and heterogenous disease, and various approaches have been used to classify BC into several subtypes to improve diagnostic and therapeutic outcomes. One of the features of BC is deregulated transcription, which allows for classification of the disease based on gene expression signature into four basic types: Luminal A, Luminal B, HER2-enriched, and triple negative (TNBC)/basal-like. Novel therapeutic approaches targeting oncogenic transcriptional programs may represent a promising strategy, in particular for TNBC, where the lack of common genetic alterations has so far limited the development of targeted therapies. The CDK8 module functions as a master coordinator of transcription, bridging enhancers and core promoters. Meta-analysis of curated TCGA data reveals that >15% of all BC have increased expression of CDK8 and/or CDK19, and that elevated expression of both genes is associated with decreased overall survival. High CDK8 expression is inversely correlated with the expression of estrogen receptor (ER) and positively correlated with occurrence of TP53 mutations. RVU120 is a specific, low nM, selective inhibitor of CDK8/CDK19, currently being tested in a first-in-human Phase Ib clinical trial in patients with metastatic or advanced solid tumors progressing from previous lines of therapy (ClinicalTrials.gov: NCT05052255). In order to establish a rationale for treatment of BC with RVU120 we have interrogated a panel of cell lines representing various subtypes of BC. These studies revealed that TNBC and ER-/PR-/HER2+ cells were highly sensitive to RVU120 and also to two other non-related CDK8/19 inhibitors, indicating a class effect. In contrast, CDK8/19 inhibitors were not able to inhibit mitogenic effect of estrogen, confirming differential efficacy in hormone - independent BC. Detailed transcriptional profiling of RVU120-responder cells revealed high enrichment of STAT3 target genes, SOX4 target genes and gene hallmarks of epithelial to mesenchymal transition (EMT), signatures associated with invasiveness and stemness. Non-responder cells were characterized by enrichment of transcriptional signatures of ER activity. Further proteomic profiling show that the top responder cells were the Mesenchymal Stem-Like (MSL) molecular subtype of TNBC and were positive for transactivated pSTAT3. Transcriptomic RNAseq profiling of MSL TNBC cell lines indicated that among genes inhibited by RVU120 there was high enrichment of STAT1 (interferon signature), STAT3 (IL6- signaling) and STAT5 (IL2 signaling) dependent genes. Single agent efficacy of RVU120 has been confirmed in subcutaneous TNBC xenograft models in vivo at well tolerated doses. Overall, these studies provide rationale for further development of RVU120 in TNBC patients. Citation Format: Urszula Pakulska, Marta Obacz, Aniela Gołas, Milena Mazan, Magdalena Masiejczyk, Agata Stachowicz, Justyna Martyka, Michał Combik, Kinga Kęska, Katarzyna Wiklik, Kristina Goller, Elżbieta Adamczyk, Krzysztof Brzózka, Tomasz Rzymski. RVU120, a selective CDK8/CDK19 inhibitor, demonstrates efficacy against hormone-independent breast cancer cells in vitro and in vivo [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2647.
Background Acute myeloid leukemia (AML) is characterized by rapid proliferation of myeloid blood cells. Due to its heterogeneity and the high rate of acquired drug resistance, new treatment modalities are needed. SEL120 is a specific type I selective inhibitor of Cyclin-dependent kinase 8 (CDK8) and Cyclin-dependent kinase 19 (CDK19). A first- in- human phase Ib clinical trial with SEL120 in patients with AML or HR-MDS was initiated in June 2019. Preclinical studies demonstrated high efficacy of SEL120 in experimental AML models via mechanisms involving differentiation and induction of apoptosis. Transcriptomic analysis of hematological cell lines demonstrated that SEL120 treatment upregulated expression of an apoptotic activator BIM from BCL-2 family of proteins. Methods Efficacy of the compound alone or in combination was tested in viability assays in a broad panel of cancer cell lines. Activity and mechanism of action of CDK8 inhibitor - SEL120 alone and in combination was investigated by flow cytometry, western blotting, co-immunoprecipitation, differential gene expression and ChIPseq analysis. In vivo efficacy was tested in mice injected with MV4-11 cell line both subcutaneously and intravenously. Results Here we provide a strong rationale for combination of SEL120 and BCL-2-selective inhibitor Venetoclax (ABT-199). We found that SEL120 synergistically induced apoptosis with Venetoclax in AML cells. Combination of both compounds significantly reduced levels of prosurvival protein MCL-1 and induced hallmarks of apoptosis including Caspase-3 activation and PARP cleavage. Previous studies associated Venetoclax resistance with increased sequestration of proapoptotic BIM by high levels of MCL-1. While a SEL120 treatment alone had no effects on MCL-1 levels, combination of both compounds resulted in sensitization of Venetoclax-resistant AML cells. We demonstrated that mechanism of Venetoclax resistance can be abrogated by the cotreatment with SEL120 leading to changes in proportions of BIM and MCL-1 levels. Synergistic interaction between SEL120 and Venetoclax resulted in apoptotic cell death in established cell lines and patient derived AML cells. Finally, using murine models of subcutaneous or disseminated AML, we found complete remissions of AML and associated recovery of normal cells in bone marrow of animals treated with both SEL120 and Venetoclax. Conclusion Taken together, these data provided rationale for a novel clinical strategy that may lead to durable responses in AML patients. Citation Format: Tomasz Rzymski, Marta Obacz, Milena Mazan, Marion Chappelier, Marcus Järås, Michal Mikula, Elżbieta Adamczyk, Katarzyna Wiklik, Michal Combik, Aniela Golas, Magdalena Masiejczyk, Przemyslaw Juszczynski, Jerzy Ostrowski, Krzysztof Brzózka. Synergistic effect of CDK8 and BCL-2 inhibition in AML through regulation of MCL-1 and BIM balance [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 6217.
Background: Myelodysplastic syndromes (MDS) are a group of malignant blood disorders characterized by ineffective hematopoiesis and cytopenias and frequent evolution to acute myeloid leukemia (AML). MDS results from the expansion of genetically and epigenetically changed clones with impaired differentiation and maturation. Primary clinical goals in MDS are to achieve remissions, alleviate symptoms associated with cytopenias, and minimize the transfusion burden. While supportive red blood cell transfusions, erythropoiesis-stimulating agents and novel targeted agents may lead to clinical improvement, an allogeneic bone marrow transplant (BMT) remains the only potential curative option for patients with MDS. RVU120 (SEL120) is a specific, selective inhibitor of CDK8 and its paralog CDK19. A first-in-human Phase Ib clinical trial with RVU120 in patients with AML or high risk (HR)-MDS is currently ongoing. Preclinical studies indicated the strong antileukemic potential of RVU120 that was often associated with the multilineage commitment of CD34+ AML cells. Moreover, RVU120 could improve proliferation and induce erythroid differentiation of CD34+ cells derived from Diamond-Blackfan anemia (DBA) patients. Aims: Primary aim was to evaluate the erythroid differentiation potential of RVU120 in primary MDS and transformed cord blood CD34+ blood cells characterized with an early block in erythroid differentiation. Methods: Efficacy and mechanism of action of RVU120 and other CDK8 inhibitors were investigated in cord blood (CB) cells transduced with TLS-ERG, a fusion gene generated from t(16;21)(p11;q22). Transformed cells displayed an increased capacity for self-renewal, proliferation, and altered erythroid differentiation. Efficacy was further tested in CD34+ cells isolated from BM of MDS patients and established MDS cell lines. Cells were treated with RVU120 and global transcriptional changes and chromatin status were analyzed by RNA-seq, ATAC-seq, and ChIP-seq. Cell cycle, proliferation, and lineage-specific markers were studied by flow cytometry in liquid and semi-solid methylcellulose-based media. Results: RVU120 treatment leads to transcriptional reprogramming of transformed CD34+ CB cells. The most profound changes included decreased CDK8 occupancy followed by increased STAT5 and RNA Polymerase II loading at transcription start site and gene bodies. RVU120 treatment transcriptionally represses multiple genes associated with hematopoietic and leukemia stem cells such as CD34, FLI1, ENG and RGS18 and importantly induce the expression of genes involved in erythroid commitment, including regulators of erythroid/megakaryocytic fate, such as RGS18, KLF1, FLI1, INHBA, GATA1/2 and hemoglobin genes. Detailed analysis of transformed CB and MDS CD34+ cells by flow cytometry at early and late time points reflected sequential changes in the expression of lineage-specific surface markers, leading to erythroid differentiation. Conclusions: Presented results indicate strong erythroid differentiation potential of RVU120 in CD34+ cells that acquired genetic abnormalities leading to arrested erythroid commitment, characteristics of many MDS and AML subtypes. Observed differentiation phenotype strikingly resembles effects of RVU120 in DBA cells caused by disruption of genes encoding ribosomal proteins. Detailed transcriptomic profiling strongly links the differentiation with enrichment of genes representing regulators of erythroid commitment and hemoglobin metabolism. Further studies are warranted to investigate the efficacy of RVU120 in chronic anemias associated with bone marrow failures in AML and MDS patients. Figure 1 Figure 1. Disclosures Pakulska: Ryvu Therapeutics: Current Employment, Current equity holder in publicly-traded company. Obacz: Ryvu Therapeutics: Current Employment, Current equity holder in publicly-traded company. Goller: Ryvu Therapeutics: Current Employment, Current equity holder in publicly-traded company. Combik: Ryvu Therapeutics: Current Employment, Current equity holder in publicly-traded company. Keska: Ryvu Therapeutics: Current Employment, Current equity holder in publicly-traded company. Mazan: Ryvu Therapeutics: Current Employment, Current equity holder in publicly-traded company. Juszczynski: Ryvu Therapeutics: Current equity holder in publicly-traded company. Brzozka: Ardigen: Current Employment, Membership on an entity's Board of Directors or advisory committees; Selvita SA: Current Employment, Current equity holder in publicly-traded company, Membership on an entity's Board of Directors or advisory committees; Ryvu Therapeutics: Current Employment, Current equity holder in publicly-traded company, Membership on an entity's Board of Directors or advisory committees. Dziedzic: Ryvu Therapeutics: Current Employment, Current equity holder in publicly-traded company. Angelosanto: Ryvu Therapeutics: Current Employment. Shamsili: Ryvu Therapeutics: Current Employment, Current equity holder in publicly-traded company. Rzymski: Ryvu Therapeutics: Current Employment, Current equity holder in publicly-traded company.
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.
Background Acute myeloid leukemia (AML) is a heterogeneous disease characterized by rapid proliferation of leukemic blasts and high rate of acquired resistance to drugs. Differentiating agent ATRA has been established as a backbone of APL treatment, however its activity in other leukemias is limited. Knowing that Cyclin Dependent Kinase 8 (CDK8) can maintain tumor dedifferentiation and embryonic stem cell pluripotency, we investigated whether CDK8 inhibitor SEL120 could effectively target leukemia by induction of lineage commitment. SEL120 is a specific, selective inhibitor of CDK8 and its paralog CDK19. A first-in-human phase Ib clinical trial with SEL120 in AML or HR-MDS patients is currently ongoing. To better understand the mechanism of action of SEL120 in AML, we studied effects of SEL120 on differentiation as one of the important anti-leukemic activities of the compound. Methods Global transcriptional changes were analyzed by RNAseq at different time points to capture early and long-term effects of SEL120. Genome-wide profiling of DNA-binding was performed by CHIPseq. Cell cycle, proliferation and lineage specific markers were studied by flow cytometry. Differentiation potential of AML cells was studied in semi-solid methylcellulose-based media to asses colony formation of SEL120 treated blasts. Results SEL120 treatment leads to decreased CDK8 occupancy and increased RNA Pol II occupancy as well as changes in the peak distribution among promoter and enhancer regions. SEL120 could repress many “stemness” genes and induce the expression of genes involved in lineage commitment, including regulators of erythroid/megakaryocytic fate, such as RGS18, KLF1, FLI1 and GATA1/2. Moreover, for the first time, we showed that prolonged exposure of AML CD34+ cells to SEL120 could lead to colony formation in semi-solid media. Detailed analysis by flow cytometry at early and late time points reflected sequential changes in the expression of lineage-specific surface markers, characterizing differentiation and the presence of cells of myeloid or erythroid/megakaryocytic origin. Conclusion In addition to established role of CDK8 in regulation of tumor suppressor genes we present evidence for an essential role of CDK8 in lineage controlling-functions. Previously we reported specific cytotoxicity of SEL120 on cells positive for stemness markers such as CD34+. We further expanded on these studies, showing profound morphological changes in AML blasts during prolonged exposure to SEL120, correlating with increased expression of myeloid and erythroid/megakaryocytic markers. Based on these findings further studies are warranted to investigate the efficacy of SEL120 in anemia associated with bone marrow failures in AML and MDS. Combination of direct effects of SEL120 on viability of cells, with a strong differentiation potential, represents a promising profile for a drug in successful leukemia treatment. Citation Format: Urszula Pakulska, Elzbieta Adamczyk, Katarzyna Dziedzic, Katarzyna Wiklik, Michal Combik, Michal Mikula, Aniela Golas, Marta Obacz, Magdalena Masiejczyk, Przemyslaw Juszczynski, Magdalena Cybulska, Milena Mazan, Krzysztof Brzozka, Tomasz Rzymski. SEL120, a CDK8/CDK19 inhibitor, possesses strong multilineage differentiation potential in AML [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 1018.
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