Biology subjects

Bruter, A. V.

Publications and source records attributed to Bruter, A. V..

4 recordsLinked to original sources

bioRxiv2025

The sensitivity of acute myeloid leukemia to CDK8/19 inhibitors is determined by their metabolic profile

BackgroundCyclin-dependent kinases CDK8/19 are serine/threonine kinases that regulate transcription as part of the Mediator complex and phosphorylate several non-transcriptional substrates in both normal and tumor cells. Several studies have demonstrated that inhibition of CDK8/19 leads to selective cell death in acute myeloid leukemia (AML) cells with favorable adverse effect profile. However, the exact mechanism of AML sensitivity to CDK8/19 inhibitors (CDK8/19i) is poorly understood. One of the key goals of the current research was to identify the molecular mechanisms underlying this sensitivity. MethodsAML cell lines were stratified by CDK8/19i and CCNC KO sensitivity using the DepMap database and published cytotoxicity data. Mean KO dependency scores grouped by Hallmark categories were correlated with CDK8/19i sensitivity (Spearman), and metabolic indices were calculated as mean log2(TPM+1) expression of pathway genes. Viability of AML lines MV4;11, KG-1, THP-1, Kasumi-1 was assessed after 120 h CDK8/19i (SenB and SNX631) via resazurin or flow cytometry cell cycle analysis. RNA sequencing of all AML lines after 72 h with 1 M SenB was analyzed with DESeq2 (p_adj < 0.05). Oxygen consumption rate of all AML lines was measured after 72 h with 1 M SNX631 using Seahorse XF Mito Stress Test or Seahorse XF Glyolysis Stress Test (Two-way ANOVA, p < 0.05). Intracellular metabolite profiling of all four AML lines after 72 h treatment with 1 {micro}M SNX631 was performed by GC-MS, with peak areas normalized to total protein content. Metabolites with p_val < 0.05 and |log FC| [≥] 0.6 (Welchs two-sample t-test) were considered significant. ResultsAccording to the DepMap database CDK8/19i sensitivity is associated with sensitivity to knockout of metabolic-associated genes. Resazurin assay ranked sensitivity as KG-1, MV4;11 > THP-1 > Kasumi-1 for both inhibitors. Cell cycle analysis after 120 h showed that inhibition of CDK8/19 caused induction of cell death in MV4;11, while KG-1 cells exhibited reduced metabolic activity. GlycoStress assay showed that the MV4;11 cells which are the most sensitive to CDK8/19i have the highest glycolytic capacity. RNA-seq analysis showed that CDK8/19i after 72 h downregulated glycolysis genes only in sensitive KG-1 and MV4;11. MitoStress parameters are reduced most in MV4;11, then KG-1/THP-1, but not in resistant Kasumi-1. CDK8/19i led to decrease in glutaminolysis metabolites only in sensitive KG-1 and MV4;11 cells. ConclusionCDK8/19i in AML predominantly targets cells with a glycolytic-active metabolic phenotype, leading to changes in metabolic composition, disruption of TCA, reduced expression of glycolysis-related genes and decreased glycolysis. Our data suggest that the metabolic profile of AML cells may serve as a functional marker for identifying tumors most likely to respond to CDK8/19i.

cell biology↗

bioRxiv2025

Eg5 Inhibitor SB743921 Causes p53-Dependent Cell Cycle Arrest, Senescence and Death in Tumor Cells

Mitotic inhibitors, such as Vinca alkaloids and taxanes, are one of the most effective chemotherapeutic agents used in the clinic. Despite their advantages, there are drawbacks to their use - primarily development of resistance and a high rate of side-effects, including damage to non-proliferating tissues. A range of new inhibitors targeting mitosis, whose activity does not depend on the binding to tubulin, are currently tested in clinical trials. Among such agents, inhibitors of Eg5 kinesin are highly promising due to their high activity and specificity. Here we show that compared to other drugs that target mitosis, an Eg5 inhibitor, SB743921, preferentially eliminates TP53-mutated cells and induces irreversible senescence, even after the drug washout, regardless of the p53 status. These effects are not defined by the immediate block of mitosis where SB743921 and a clinically used mitotic inhibitor Ixabepilone induce similar rates of mitotic arrest, apoptosis and induction of p53 and p21, but rather a long-term reaction, with absence of proteins required for replication, such as Cyclin A, E2F1, pRB. While after the washout Ixabepilone-treated cells can exit senescence and resume proliferation, cells treated with SB743921 did not exit the senescent state and did not resume proliferation as based on SA-{beta}-galactosidase staining and EdU incorporation. The remaining senescent cells were effectively eliminated by Bcl2/Bcl-xL/Bcl- w inhibitor ABT-263, showing a potential of the combinational therapy with senolytic drugs. In total, we show the capacity of Eg5-targeting drugs for therapy of high-risk TP53-mutated tumors, which are potentially resistant to clinically approved mitotic inhibitors.

cancer biology↗

bioRxiv2023

Knockout of cyclin dependent kinases 8 and 19 leads to depletion of cyclin C and suppresses spermatogenesis and male fertility in mice

Paralogs CDK8 and CDK19 are regulatory kinases associated with the transcriptional Mediator complex. We have e generated mice with the systemic inducible Cdk8 knockout on the background of Cdk19 constitutive knockout. Cdk8/19 double knockout (iDKO) males, but not single Cdk8 and Cdk19 KO, had an atrophic reproductive system and were infertile. The iDKO males lacked postmeiotic spermatids and spermatocytes after meiosis I pachytene. Testosterone levels were decreased whereas the amounts of the luteinizing hormone were unchanged. Single cell RNA sequencing showed marked differences in the expression of steroidogenic genes (such as Cyp17a1, Star and Fads) in Leydig cells concomitant with alterations in Sertoli cells and spermatocytes likely associated with impaired synthesis of steroids. Star and Fads were also downregulated in cultivated Leydig cells after iDKO. The treatment of primary Leydig cells culture with a CDK8/19 inhibitor did not induce the same changes in gene expression as iDKO, and prolonged treatment of mice with a CDK8/19 inhibitor did not affect the size of testes. iDKO, in contrast to single knockouts or treatment with a CDK8/19 kinase inhibitor, led to depletion of cyclin C (CcnC), the binding partner of CDK8/19 that has been implicated in CDK8/19-independent functions. This suggests that the observed phenotype was likely mediated through kinase-independent activities of CDK8/19, such as CcnC stabilization.

developmental biology↗

bioRxiv2023

CDK8/19 Inhibition Attenuates G1 Arrest Induced by BCR-ABL Antagonists and Accelerates Death of Chronic Myelogenous Leukemia Cells

Imatinib mesylate (IM) and other BCR-ABL tyrosine kinase inhibitors (BCR-ABLi) are the mainstay of chronic myelogenous leukemia (CML) treatment. However, activation of circumventing signaling pathways and quiescence may limit BCR-ABLi efficacy. CDK8/19 Mediator kinases have been implicated in the emergence of non-genetic drug resistance. Dissecting the effects of pharmacological CDK8/19 inhibition on CML survival in response to BCR-ABLi, we found that a selective, non-toxic CDK8/19 inhibitor (CDK8/19i) Senexin B (SenB) and other CDK8/19i sensitized K562 cells to different BCR-ABLi via attenuation of cell cycle arrest. In particular, SenB prevented IM-induced upregulation of genes that negatively regulate cell cycle progression. SenB also antagonized IM-activated p27Kip1 elevation thereby diminishing the population of G1-arrested cells. After transient G1 arrest, cells treated with IM+SenB re-entered the S phase, where they were halted and underwent replicative stress. Consequently, the combination of IM and SenB intensified apoptotic cell death, measured by activation of caspase 9 and 3, subsequent cleavage of poly(ADPriboso)polymerase 1, positive Annexin V staining and increase of subG1 fraction. In contrast, IM-treated BCR-ABL-positive KU812 CML cells, which did not induce p27Kip1, readily died regardless of SenB treatment. Thus, CDK8/19i prevent the quiescence-mediated escape from BCR-ABLi-induced apoptosis, suggesting a strategy for avoiding the CML relapse.

cancer biology↗