Ekaterina A. Varlamova

Background/Objectives: Duchenne muscular dystrophy (DMD) is caused by frameshift mutations in the DMD gene encoding dystrophin. Beyond skeletal muscle, dystrophin’s full and shortened isoforms are expressed in non-muscle tissues, including the male reproductive system, where it is reported to engage in spermatogenesis, sperm motility and overall fertility in patients and mouse models. We have previously generated two genetically modified mouse strains carrying insertions (insT or insG) in exon 51 of the Dmd gene to model DMD. Here, we characterize male fertility and related reproductive phenotypes in both strains. Methods: Fertility was assessed by mating, recording copulatory plugs, litter production and pregnancy rates over an extended breeding period. Sperm concentration and motility were quantified in vas deferens samples. Testicular dystrophin expression was evaluated by qPCR and immunofluorescence. Germ cell populations were examined by flow cytometry of testicular cell suspension; H&E histology with morphometry of tubular and peritubular compartments was carried out, along with immunofluorescence for cytoskeletal markers (dystrophin, actin, vimentin). Results: Both strains retained immunoreactivity for an N-terminal/rod-domain dystrophin epitope in testes, with modest nonsense-mediated decay of Dp427m transcripts and unaltered Dp71 levels. In a small breeding trial, mutant males, especially insG, produced fewer offspring despite comparable litter sizes, with fewer pregnancies after a copulatory plug. Sperm concentration in the vas deferens was approximately halved in both strains, with occasional azoospermia. Flow cytometry showed a selective decrease in the mature haploid (elongating) population in insG animals. Conclusions: In exon-51 frameshift models, the phenotype is lower sperm concentration in the vas deferens, to roughly half of control levels, with occasional azoospermia. InsG also fails to maintain a normal elongating-to-round spermatid ratio. Whether this fully accounts for the poorer breeding performance in the cohorts studied remains open. Overall, these findings underscore the functional role of dystrophin in spermatogenesis.

Background: Cyclin-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. Methods: AML 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 uM SenB was analyzed with DESeq2 (p_adj < 0.05). Oxygen consumption rate of all AML lines was measured after 72 h with 1 uM 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 uM SNX631 was performed by GC-MS, with peak areas normalized to total protein content. Metabolites with p_val < 0.05 and |log2FC| < 0.6 (Welch's two-sample t-test) were considered significant. Results: According 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. Conclusion: CDK8/19i 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.

Age-related changes are associated with mitochondrial dysfunction, which is often caused by the accumulation of mutations in mitochondrial DNA (mtDNA). One common model of aging and age-related diseases involves mice with a mutant DNA polymerase γ (PolG^mut) whose proofreading function is impaired, which leads to the accumulation of mutations in mtDNA. The main limitation of such a model is that introducing a mutation into the mouse’s own gene leads to the accumulation of mutations in mtDNA over several generations, making it impossible to rule out whether mtDNA mutations or compensatory effects are the cause of functional impairments such as accelerated aging. This paper describes two lines of transgenic animals with inducible expression of PolG^mut. This inducible system prevents mutation accumulation in the germline, promoting stable reproduction and reproducibility of mice, increasing experimental flexibility for various studies of mitochondrial diseases. PolG^mut activation at different stages of life and different tissues allows us to study the progression of pathological changes during mitochondrial aging over time and detect the onset of mutation accumulation. The simplicity, reproducibility, and temporal control of this system represent a significant methodological improvement for studying mitochondrial mutagenesis and the pathophysiology of aging. Using this model, we demonstrated that the most pronounced pathology in these animals is accelerated thymus involution and the accumulation of cytotoxic effector CD8+ T cells in the peripheral immune organs, while no significant abnormalities were observed in other organs and systems. These data probably indicate that mtDNA mutations primarily impair T-cell immune function.

Transcriptional cyclin-dependent kinases 8 and 19 (CDK8 and CDK19) are enzymatic components of the Mediator complex. CDK19 is presumably a minor paralog of CDK8 and is thought to compensate for lack of CDK8 in certain cases, but evidence for unique functions of the two kinases remains sporadic. Experiments with tumor cell lines have yielded the vast majority of information on the role and mechanisms of action of the kinases, and the results might be irrelevant because of the changes accumulated by tumor cells. Mice with an inducible Cdk8 knockout and a constitutive Cdk19 knockout ( Cdk 8 ^fl/fl / Cdk19 ^–/– / Rosa26/Cre/ER ^T2 ) were therefore engineered for the first time to study the joint role of CDK8 and CDK19 in the while organism and primary cell cultures. The mice were used to obtain Cdk19 ^–/– mouse embryonic fibroblasts (MEFs) in which a Cdk8 knockout could be induced with 4-hydroxytamoxifen (4-OHT). Unlike most tumor cells, MEFs were sensitive to CDK8/19 inhibition and knockout: the inhibition led to significant cell death within 5 days, while the knockout decreased the cell proliferation rate. RNA sequencing revealed alterations in the Wnt signaling pathway, cytokine response, and osteoclast differentiation in Cdk8/Cdk19 knockout MEFs. Expression of genes associated with steroidogenesis was lower, consistent with earlier data. Changes associated with the cytoskeleton, adipogenic differentiation, osteogenic differentiation, cell adhesion, extracellular matrix formation, and mitochondrial biogenesis were described for the first time. Studies of the stress responses in MEFs showed that responses to DNA damage from X-ray irradiation and to serum stimulation after starvation were also mediated by CDK8/19 and were significantly reduced in Cdk8/Cdk19 knockout cells.

CDK8 and CDK19 paralogs are regulatory kinases associated with the transcriptional Mediator complex. We have 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 or 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, and were likely associated with an impaired synthesis of steroids. Star and Fads were also downregulated in cultured Leydig cells after iDKO. The treatment of primary Leydig cell culture with a CDK8/19 inhibitor did not induce the same changes in gene expression as iDKO, and a prolonged treatment of mice with a CDK8/19 inhibitor did not affect the size of testes. iDKO, in contrast to the 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.

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.

Duchenne muscular dystrophy (DMD) is a severe X-linked genetic disorder caused by an array of mutations in the dystrophin gene, with the most commonly mutated regions being exons 48–55. One of the several existing approaches to treat DMD is gene therapy, based on alternative splicing and mutant exon skipping. Testing of such therapy requires animal models that carry mutations homologous to those found in human patients. Here, we report the generation of two genetically modified mouse lines, named “insT” and “insG”, with distinct mutations at the same position in exon 51 that lead to a frameshift, presumably causing protein truncation. Hemizygous males of both lines exhibit classical signs of muscular dystrophy in all muscle tissues except for the cardiac tissue. However, pathological changes are more pronounced in one of the lines. Membrane localization of the protein is reduced to the point of absence in one of the lines. Moreover, an increase in full-length isoform mRNA was detected in diaphragms of insG line mice. Although further work is needed to qualify these mutations as sole origins of dissimilarity, both genetically modified mouse lines are suitable models of DMD and can be used to test gene therapy based on alternative splicing.

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.

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 p27 ^Kip1 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 p27 ^Kip1 , 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.