Antisense Blockade of a THRAP3-Binding RNA Site Restrains Prostate Cancer Organoid Growth

Following Citron Kinase from Phosphorylation to RNA Binding

Selective inhibitors of citron kinase, or CIT, are not available, although earlier work implicated the enzyme in prostate cancer progression. A study led by Cleveland Clinic and collaborating academic teams therefore investigated whether a downstream RNA-processing event could offer another way to interfere with its activity. The work in Science Advances combines kinase assays, transcriptomics, engineered cells and organoid models to trace a CIT-controlled splicing program.

The investigators focused on THRAP3, a protein associated with the spliceosome. In vitro kinase assays supported direct phosphorylation at serines 243 and 379, but not 320. Functional experiments singled out serine 243: a mutation that prevents phosphorylation at this site eliminated the growth-promoting effect of wild-type THRAP3, while mutation at 379 did not. The distinction persisted across cell lines representing treatment-naive, castration-resistant and neuroendocrine prostate cancer states.

CIT loss and the serine 243 mutation also altered THRAP3 localization and RNA recruitment. Cross-linking and immunoprecipitation sequencing detected 3396 THRAP3-bound genes; binding at 311 changed after CIT silencing, with reduced binding for 300. These CIT-dependent targets were enriched for cell-proliferation functions, providing a defined subset for further functional tests.

Transcript Variants Behave Differently in Patient-Derived Models

The study examined two transcripts of CPT1A, a representative THRAP3 target. Variant 2 depended on CIT and THRAP3, whereas variant 1 did not. Expressing the variants separately showed that their effects could differ despite originating from the same gene. In prostate cancer cell lines, the CIT-dependent variants of CPT1A and ESYT2 preferentially increased viability and Ki67-positive-cell measurements.

Organoids extended this comparison beyond established cell lines. Patient-derived organoids were generated from treatment-naive prostatectomy tissue, while a second system came from the castration-resistant LuCaP 86.2 patient-derived xenograft (PDX). Over 15 days, control patient-derived organoids increased in size, organoids expressing CPT1A variant 1 became smaller, and those expressing variant 2 grew more. The PDX-derived organoids showed similar directional differences.

The growth analysis followed the surface area of 100 individual patient-derived organoids per transduction group. The two origins also remain distinct: one model starts from fresh patient tissue and the other from a tumor propagated as a xenograft. Mouse experiments separately supported growth-promoting effects of CPT1A variant 2; they did not test the final antisense treatment.

Blocking a THRAP3 Recruitment Site

The researchers next designed an antisense oligonucleotide, or ASO, complementary to a THRAP3-binding region in CPT1A pre-mRNA. It blocked recruitment at this RNA site without reducing THRAP3 abundance. A scrambled oligonucleotide served as a control.

At 5–10 nM, the specific ASO reduced proliferation across cell lines representing several disease stages but did not reduce proliferation of the benign RWPE-1 prostate epithelial comparator. It lowered CPT1A variant 2 production and reduced THRAP3 binding to the target pre-mRNA. Trypan-blue measurements also showed fewer live cells and more dead cells. In the LuCaP 86.2 PDX-derived organoids, the ASO reduced growth, adding a three-dimensional treatment-resistant model to the response evidence.

Low-dose combinations with enzalutamide or docetaxel produced additive effects in castration-resistant cells. These combination experiments belong to the cell-line evidence; the paper does not establish the same combinations in the organoid system. Similarly, preferential effects on the tested malignant cells do not define clinical tolerability.

The work identifies a route from kinase-dependent phosphorylation to a specific RNA-binding and splicing event that can be perturbed experimentally. The authors emphasize that THRAP3 is only one of several relevant CIT substrates. Their ASO evidence remains confined to in vitro cell and organoid models, and delivery, formulation, chemistry and in vivo applicability still require development. The organoid experiments connect transcript-selective perturbations with growth responses in fresh-patient and PDX-derived tumor cultures.