Biology subjects

Romanienko, P.

Publications and source records attributed to Romanienko, P..

5 recordsLinked to original sources

bioRxiv2026

Cardiomyocyte-derived Wnt5a drives doxorubicin-induced cardiomyopathy by amplifying cellular senescence

Doxorubicin (DOX) is an effective anthracycline chemotherapeutic agent, but its use is limited by cardiotoxicity that can progress to cardiomyopathy and heart failure. Cellular senescence contributes to DOX-induced cardiac injury, yet the upstream signals that initiate and propagate senescence in the injured heart remain unclear. Here, we identify Wnt5a, a non-canonical Wnt ligand, as a mediator of anthracycline cardiomyopathy. WNT5A was increased in serum from cancer patients receiving anthracycline therapy and in a pathologic human cardiomyocyte population in the context of DOX-induced cardiomyopathy. In mouse hearts, DOX induced early cardiomyocyte-enriched Wnt5a expression before overt cardiac dysfunction. Cardiomyocyte-specific Wnt5a deletion attenuated DOX-induced cardiac dysfunction, fibrosis and senescence marker induction, whereas recombinant Wnt5a and cardiomyocyte-targeted Wnt5a overexpression were sufficient to promote cardiomyocyte senescence and cardiac dysfunction. Mechanistically, DOX activated a Wnt5a-Fzd2 feed-forward axis that amplified Wnt5a expression in cardiomyocytes and propagated senescence to neighboring fibroblasts. Genetic disruption of this pathway in cardiomyocytes, fibroblasts or senescent cells reduced DOX-induced cardiomyopathy. Pharmacological inhibition of Wnt5a signaling with secreted frizzled-related protein 5 suppressed DOX-induced cardiac injury without compromising the anticancer efficacy of DOX. These findings identify Wnt5a-Fzd2 signaling as a senescence-amplifying mechanism in anthracycline cardiomyopathy and suggest a therapeutic strategy to mitigate DOX cardiotoxicity.

cell biology↗

bioRxiv2026

dCas allele sequestration (das-CRISPR): A Versatile New Method to Achieve Monoallelic Gene Editing in Mouse Embryos and in cell culture.

CRISPR-Cas9 technology is a powerful tool extensively used for genome editing in mouse and many other species. Streptococcus pyogenes Cas9 efficiently cuts both alleles in mouse zygotes leaving many edited embryos without a functional protein that might be needed to sustain development, to survive postnatally or to reproduce, thus complicating its overwhelmingly advantageous use in making gene modifications. About 25% of mouse genes are essential for embryonic development and another 7% are necessary for fertility, thus for these genes it is desirable to maintain a functional allele to establish viable lines from CRISPR-Cas9 edited mouse embryos. However, exclusive monoallelic editing is challenging to achieve with current CRISPR methods. Controlling the activity of Cas9 in genome editing is an ongoing research field focused on developing new methods to curtail its damage caused by excess of on-target and off-target editing. In this study we describe a novel and a simple method, we termed das-CRISPR, for dCas allele sequestration in combination with CRISPR system, that allows monoallelic editing of targeted allele in mouse and in cultured cell lines. This method incorporates the use of a nuclease deficient deadCas9 (dCas9) present at higher levels than an active Cas9, both complexed with the same single guide RNA (sgRNA) sequence. We showed the delivery of the two proteins as ribonucleoprotein complexes (RNP) into mouse zygotes leads to the generation of viable and fertile mice carrying lethal mutations in an essential gene. We found that greater amounts of dCas9 RNPs bind and protect a target site while the lower amount of functional Cas9 RNPs accessed the unoccupied target site resulting in higher frequency of monoallelic gene editing, compared to using just Cas9 alone. We also showed this method can mitigate and control the activity of Cas9 in mouse NIH3T3 cells in culture to achieve monoallelic editing. This method is a versatile approach to controlling excessive Cas9 activity on-target and off-target both in vitro and in vivo.

molecular biology↗

bioRxiv2024

The Mammalian KU70 C-terminus SAP Domain Is Required to Repair Exogenous DNA Damage

The mammalian non-homologous end joining (NHEJ) is required for V(D)J recombination as well as coping with exogenously induced DNA double strand breaks (DSBs). Initiated by the binding of KU70/KU80 (KU) dimer to DNA ends and the subsequent recruitment of the DNA- dependent protein kinase catalytic subunit (DNA-PKcs), NHEJ plays a key role in DNA repair. While there has been significant structural understandings of how KU70 participates in NHEJ, the specific function of its highly conserved C-terminal SAP domain remains elusive. In this study, we developed a novel mouse model by deleting the SAP domain but preserving the KU70 nuclear localization and its dimerization ability with KU80. We found that the KU70 SAP deletion did not affect the V(D)J recombination or animal development but significantly impaired the animals and cells in repairing exogenously induced DSBs. We further showed an inability of KU70-{Delta}SAP cells to retain the DNA Ligase IV (LIG4) and other NHEJ co-factors on chromatin, and a spreading pattern of DSB marker {gamma}H2AX in KU70-{Delta}SAP cells after DNA damage. Our findings suggest that a specific inhibition of the SAP function may offer an opportunity to modulate cell sensitivity to therapeutic DSB-inducing agents without interfering with the developmental function of KU70. KeyPointsO_LIGeneration of a novel transgenic mouse line lacking the C-terminal conserved KU70-SAP domain C_LIO_LIKU70-SAP defends against exogenous DSBs, but unessential for development and V(D)J recombination C_LIO_LIKU70-SAP aids in recruiting and retaining NHEJ components, such as LIG4, to DSB sites C_LI

cancer biology↗

bioRxiv2023

Distinct functions of EHMT1 and EHMT2 in cancer chemotherapy and immunotherapy

EHTM1 (GLP) and EHMT2 (G9a) are closely related protein lysine methyltransferases often thought to function together as a heterodimer to methylate histone H3 and non-histone substrates in diverse cellular processes including transcriptional regulation, genome methylation, and DNA repair. Here we show that EHMT1/2 inhibitors cause ATM-mediated slowdown of replication fork progression, accumulation of single-stranded replication gaps, emergence of cytosolic DNA, and increased expression of STING. EHMT1/2 inhibition strongly potentiates the efficacy of alkylating chemotherapy and anti-PD-1 immunotherapy in mouse models of tripe negative breast cancer. The effects on DNA replication and alkylating agent sensitivity are largely caused by the loss of EHMT1-mediated methylation of LIG1, whereas the elevated STING expression and remarkable response to immunotherapy appear mainly elicited by the loss of EHMT2 activity. Depletion of UHRF1, a protein known to be associated with EHMT1/2 and LIG1, also induces STING expression, and depletion of either EHMT2 or UHRF1 leads to demethylation of specific CpG sites in the STING1 promoter, suggestive of a distinct EHMT2-UHRF1 axis that regulates DNA methylation and gene transcription. These results highlight distinct functions of the two EHMT paralogs and provide enlightening paradigms and corresponding molecular basis for combination therapies involving alkylating agents and immune checkpoint inhibitors.

cancer biology↗

bioRxiv2017

MEF2C phosphorylation is required for chemotherapy resistance in acute myeloid leukemia

HIGHLIGHTSO_LIMEF2C S222 phosphorylation is a specific marker of chemotherapy resistance in diagnostic AML patient specimens.\nC_LIO_LIMEF2C S222 phosphorylation is dispensable for normal hematopoiesis in mice, as established using genome editing in vivo, but is required for MLL-AF9 induced leukemogenesis.\nC_LIO_LIMARK kinases specifically phosphorylate MEF2C S222, potentiating its transcriptional activity.\nC_LIO_LIChemical inhibition of MARK-induced MEF2C phosphorylation overcomes chemotherapy resistance of and exhibits selectivity toxicity against MEF2C-activated human AML cells.\nC_LI\n\nSUMMARYIn acute myeloid leukemia, chemotherapy resistance remains prevalent and poorly understood. Using functional proteomics of patient AML specimens, we identified MEF2C S222 phosphorylation as a specific marker of primary chemoresistance. We found that Mef2cS222A/S222A knock-in mutant mice engineered to block MEF2C phosphorylation exhibited normal hematopoiesis, but were resistant to leukemogenesis induced by MLL-AF9. MEF2C phosphorylation was required for leukemia stem cell maintenance, and induced by MARK kinases in cells. Treatment with the selective MARK inhibitor MRT199665 caused apoptosis of MEF2C-activated human AML cell lines and primary patient specimens, but not those lacking MEF2C phosphorylation. These findings identify kinase-dependent dysregulation of transcription factor control as a determinant of therapy response in AML, with immediate potential for improved diagnosis and therapy for this disease.

cancer biology↗