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.
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 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.
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.
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