Small Molecule Screens Identify Cdk8-Inhibitors As Candidate Diamond-Blackfan Anemia Drugs

Abstract A safe and efficient therapy for patients with the bone marrow failure syndrome Diamond-Blackfan Anemia (DBA) is urgently needed. To identify novel drug candidates for DBA, a small molecule screen was performed using c-Kit positive E14.5 fetal liver cells from a DBA mouse model in which the DBA phenotype is induced upon doxycycline (DOX)-inducible silencing of Rps19, the most frequently mutated gene in DBA (Jaako et al. PMID: 21989989). Test compounds were added 24 hours after cell seeding and DOX addition. After four days the number of live metabolically active cells in each well was estimated based on quantitation of intracellular ATP. Fifteen commercial annotated small-molecule libraries (3 800 molecules) and 10 500 selected compounds from a diverse compound library were screened. Between the screens we identified 20 molecules that reproducibly increased proliferation of rps19-deficient erythroid progenitors 4-8 fold in a concentration-dependent manner. Hits from annotated libraries included inhibitors of TGFb receptor, DYRK and Casein kinases. The most potent hits however were compounds in a series of thienopyridines, with an unknown target profile, but with a core structure suggesting kinase inhibition activity. Database searches revealed that the structure-activity relationships (SAR) of 12 active and 10 inactive analogues were similar to that of a series of thienopyridines previously reported as bone anabolic agents by unknown mechanism (Saito et al. PMID: 23453217). The most potent analogues described by Saito et al. as bone anabolic agents were synthesized, and the compounds rescue also RPS19-deficient erythroid cell proliferation in a potent manner (EC50= 20-50 nM). In an attempt to identify the molecular target of these compounds, six actives including thienopyridines 15k and 15w were subjected to kinase profiling against 468 kinases (DiscoverX). Three of the compounds targeted cyclin- dependent kinases CDK8 and its paralog CDK19, and in particular the most potent molecule (15w) is a highly selective CDK8 inhibitor. To confirm whether CDK8 inhibition underlies the rescue of the DBA phenotype, structurally unrelated and potent CDK8 inhibitors including CCT-251545, Senexin A, Senexin B, and Sel120-34A were also evaluated. All tested CDK8-inhibitors rescue proliferation and erythroid maturation of c-kit+ cells from the DBA mouse in a concentration-dependent manner. To further investigate the potential of CDK8 as a therapeutic target in DBA several CDK8 inhibitors were evaluated in erythroid cultures of primary DBA patient cells. CDK8 inhibitors 15w, Senexin B and Sel120-34A increase erythroid progenitor proliferation 5-10 fold of CD34+ peripheral blood cells from three DBA patients (RPS19, RPS26, RPL35a mutations), and increase the fraction of transferrin receptor (CD71) and glycophorin A positive cells in culture. Healthy CD34+ peripheral blood treated with CDK8 inhibitors show no increase in proliferation. Finally, we show that bone marrow failure and anemia in the DBA mouse model is partially rescued (RBC and Hemoglobin levels, p<0.001) after 8 daily oral doses (30mg/kg) of CDK8 inhibitor Sel120-34A. Since CDK8 is the regulatory subunit of the Mediator complex, acting as a regulator of RNA pol II transcription the therapeutic effect of CDK8 inhibitors likely involves direct changes in gene expression. RNA-Seq and gene set enrichment analysis on DBA mouse model cells treated with CDK8 inhibitor Sel120-34A shows p53 target genes are induced in RPS19-deficient cells and that CDK8 inhibitor treatment reverses this change, agreeing with previous reports that CDK8 can regulate transcription of p53 target genes. We confirm CDK8-inhibitor treatment reverses nuclear localization of p21 in RPS19-deficient cell lines. The most significant effect in gene set analysis however is the rescue of MYC-target gene expression, including increased expression of several ribosomal protein genes. In summary, selective and potent CDK8 inhibitors dramatically improve proliferation and maturation of erythroid progenitors in an animal model for DBA and in primary DBA patient cells. The therapeutic effect involves reduced expression of p53-target genes and increased expression of MYC-target genes. Given lack of adverse effects at effective doses in vivo, further development of SEL120-34A as a treatment for DBA patients is warranted. Disclosures Rzymski: Selvita S.A.: Employment, Equity Ownership. Johansson:LU Holding: Patents & Royalties. Lundbäck:LU Holding: Patents & Royalties. Mazan:Selvita S.A.: Employment. Majewska:Selvita S.A.: Employment. Brzózka:Selvita S.A.: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees. Flygare:LU Holding: Patents & Royalties: Patent.