Biology subjects

Itkonen, H. M.

Publications and source records attributed to Itkonen, H. M..

5 recordsLinked to original sources

bioRxiv2026

Castration-resistant prostate cancer cells are addicted to the high activity of cyclin-dependent kinase 2

BackgroundCyclin-dependent kinases drive the progression through the cell cycle and thereby form classical targets for cancer therapy. In prostate cancer (PC), the first line of therapy typically targets androgen receptor (AR), but it frequently leads to development of incurable form of the disease, castration-resistant PC (CRPC). Here, we sought to understand if CRPC cells are selectively addicted to a specific cell cycle kinase. MethodsWe used PC and CRPC patient data to evaluate transcriptional changes and modeled the responses in vitro using multiple models of PC, CRPC and normal cells. Development of a CDK2 inhibitor-resistant CRPC cell line, and a compound screen were used to identify chronic and acute vulnerabilities to augment the efficacy of our candidate therapy in multiple PC, CRPC and also normal cells, to assure selectivity. ResultsWe show that the emergence of CRPC is associated with significant upregulation of cyclins that positively regulate cyclin-dependent kinase 2 (CDK2) and downregulation of CDK4 cyclins. Accordingly, CDK2-specific inhibitors and its knock down efficiently reduce proliferation of PC and CRPC cells. CDK2 inhibitor-resistant CRPC model displayed transcriptional rewiring of cell cycle regulators, characterized by a shift towards CDK4/6-dependency and increased AR-signaling. Combinatorial drug screen discovered both antagonistic and additive combinations, and we show that AR inhibitors selectively augment the efficacy of CDK2 inhibitors against PC and CRPC cells, but the combination is not toxic to normal cells. ConclusionWe discovered that CRPC cells are addicted to high CDK2 activity and show that combination of CDK2 inhibitors with the currently used anti-CRPC therapies selectively augment their efficacy.

cancer biology↗

bioRxiv2026

Chromatin compaction upon CDK12 inhibition drives long gene silencing and a combinatorial lethal dependency on PAF1

Loss-of-function mutations in cyclin-dependent kinase 12 (CDK12) define an aggressive subtype of metastatic castration-resistant prostate cancer (mCRPC) characterized by genomic instability, and depletion of CDK12 activity downregulates the expression of long DNA repair genes. While these transcriptional defects are well-documented, potential alterations in chromatin that may topologically affect this silencing and the subsequent adaptive survival mechanisms of tumor cells remain poorly understood. Here, we employ a dynamic multi-omics strategy to map the remodeling of the RNA Polymerase II (RNA Pol II) complex and chromatin landscape. We utilized 5-ethynyl uridine (5-EU) metabolic labeling combined with mass spectrometry (MS) to specifically capture the transcriptionally active RNA Pol II complex, distinguishing active elongation complexes from static background, and map how its interactome changes when the activity of the major transcriptional kinases CDK7, CDK9 or CDK12/13 is inhibited. These data are then integrated with ATAC-seq, transcriptomics, and targeted synthetic lethality screen to validate findings from the RNA Pol II capture. We show that, unlike CDK9 inhibition, CDK12/13 inhibition triggers a progressive chromatin compaction. This is characterized by the decrease in the binding activity of CTCF to chromosomes closing, which acts as a physical blockade specifically silencing long genes when CDK12 activity is lost. In response to this topological remodeling, cells launch an adaptive survival program by recruiting the transcription elongation factor PAF1 and the DNA damage sensor DDB1 to the stalled RNA Pol II. We identify PAF1 as a critical vulnerability in this context. Collectively, we identify CDK12 as a regulator of chromatin topology rather than merely an elongation kinase. The chromatin compaction upon CDK12/13 inhibition creates a unique dependency on the PAF1-mediated adaptive remodeling. Specifically, targeting PAF1 converts this stress response into a lethal trap, establishing a potent, kinase-selective combination lethal strategy for CDK12-deficient prostate cancer.

cancer biology↗

bioRxiv2025

RNA polymerase II O-GlcNAcylation promotes nuclear entry to drive transcription

O-GlcNAcylation of RNA Polymerase II (RNA Pol II) was described in 1993 and here we wanted to probe the functional significance of this modification. O-GlcNAc transferase (OGT) is the sole enzyme performing nucleo-cytoplasmic O-GlcNAcylation. The role of RNA Pol II O-GlcNAcylation has remained enigmatic partly due to the lack of reagents selectively recognizing the O-GlcNAcylated polymerase. We developed a novel approach, wheat germ agglutinin (WGA) lectin-based enrichment for O-GlcNAcylated proteins followed by traditional ChIP for RNA Pol II, to identify chromatin-binding sites of the O-GlcNAcylated RNA Pol II. We termed this technique as wChIP and used it to demonstrate that the O-GlcNAcylated RNA Pol II is enriched to the promoters of highly expressed genes. RNA Pol II has a repetitive c-terminal domain (CTD) of YSPTSPS, which is heavily post-translationally modified. Using structural modeling and WGA pulldown-experiments, we identify Ser-5 as a CTD site that is dynamically modified by OGT. Mass spectrometry-based detection of proteins that recognize CTD that is O-GlcNAcylated on Ser-5 identified nuclear transport protein, karyopherin subunit beta 1 (KPNB1), as a reader protein for this modification. Functionally, high activity of both OGT and KPNB1 is required for the efficient entry of RNA Pol II to nucleus and chromatin. OGT and KPNB1 are significantly co-expressed in prostate cancer tissue, and their combined inhibition is more toxic to prostate cancer cells than normal prostate cells. We propose that RNA Pol II O-GlcNAcylation promotes entry to nucleus, and this could integrate metabolic information to regulate global transcription.

cancer biology↗

bioRxiv2024

FINCA disease mouse model exhibits altered behaviour and immune response

Fibrosis, neurodegeneration and cerebral angiomatosis (FINCA) is a childhood-onset multi-organ neurodevelopmental disorder associated with multi-organ manifestations and recurrent infections. The disease is caused by variants in NHLRC2 initiating a cascade of unknown pathological events. Previously, we have demonstrated that despite the significant decrease at the molecular level, the compound heterozygosity of knock out and p.Asp148Tyr alleles in NHLRC2 does not lead to a severe phenotype in mice. Here, we analysed the behavioural and immunological phenotype of the FINCA mice and studied the molecular pathways affected by p.Asp148Tyr in NHLRC2 using mouse and human-derived cell culture models. The FINCA mice displayed a mild hyperactivity and deficient early immune response when challenged with LPS leading to altered cytokine responses, including IFN{gamma}, IL-12, and TNF. By comparing gene expression and putative interaction partners affected by p.Asp148Tyr, we identified Rho GTPase signalling as the common pathway. Altogether, these results establish a multi-dimensional impact of the p.Asp148Tyr variant in NHLRC2. Knowledge of the molecular pathways affected by NHLRC2 and the natural course of FINCA disease progression are instrumental for the development of effective therapeutics. Summary statementFINCA is a paediatric neurodevelopmental and multi-organ disorder caused by variants in NHLRC2. Here, mild hyperactivity in connection with altered early immune response is described in the FINCA mouse model.

neuroscience↗

bioRxiv2021

Compromised CDK12 activity causes dependency on the non-essential spliceosome components

Prostate cancer (PC) is the most common cancer in men and after development of the castration-resistant PC (CRPC), there are no curative treatment options. Inactivating mutations in cyclin-dependent kinase 12 (CDK12) define an aggressive sub-type of CRPC. We hypothesized that compromised CDK12 activity leads to significant rewiring of the CRPC cells, and that this rewiring results in actionable synthetic lethal interactions. MethodsWe used combinatorial lethal screening, ChIP-seq data, RNA-seq data, global alternative splicing analysis, and comprehensive mass spectrometry (MS) profiling to understand how the compromised CDK12 activity rewires the CRPC cells. In addition, we used DepMap-, PC- and CRPC-datasets as a strategy to identify factors that are selectively required by the CDK12-mutant cells. ResultsWe show that inhibition of O-GlcNAc transferase (OGT) and CDK12 induces cancer cell-selective growth-defect. OGT catalyzes all nucleocytoplasmic O-GlcNAcylation, and we use unbiased MS-profiling to show that the short-term CDK12 inhibition induces hyper-O-GlcNAcylation of the spliceosome-machinery in PC and CRPC cells. Integration of DepMap- and a small scale-drug screen data reveled that depletion of CDK12 activity causes addiction to non-essential spliceosome components (CLK1/4 and SRPK1). CDK12-mutant tumors overexpress CLK1/4 and SRPK1. Finally, we show that the genomes of the CDK12-mutant tumors have lower DNA methylation, and that CDK12 inhibition induces the expression of the genes marked by DNA methylation. ConclusionsCompromised CDK12 activity rewires DNA methylation, transcription and splicing, and this rewiring renders the affected cells addicted on the non-essential spliceosome components. We propose that inactivation of CDK12 is a biomarker for sensitivity against inhibitors of the non-essential spliceosome components just entering the clinical trials. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=85 SRC="FIGDIR/small/470703v1_ufig1.gif" ALT="Figure 1"> View larger version (24K): org.highwire.dtl.DTLVardef@a128e8org.highwire.dtl.DTLVardef@84a70dorg.highwire.dtl.DTLVardef@f37b10org.highwire.dtl.DTLVardef@1145f1b_HPS_FORMAT_FIGEXP M_FIG C_FIG

cancer biology↗