Paul Andrew Clarke

Human DHX8 is a spliceosomal DEAH-box RNA helicase involved in releasing mRNA from the spliceosome and crucial in ensuring splicing fidelity. DHX8 was identified as a promising therapeutic oncology target due to its role in regulating stress-adaptive gene expression, including HSF1-dependent transcription, while having broader transcriptional effects in cells under oncogenic stress. We report the discovery of novel RNA-competitive DHX8 inhibitors based on a 2-(phenethylthio)nicotinic acid scaffold, which were optimized using a structure-guided design approach, following a biophysical fragment screen. This yielded compound 53 with nanomolar biochemical potency, good in vitro PK, and activity in a cellular target engagement assay. Optimizing inhibitor binding between Arg647 and the nonconserved His693, coupled with extending into a pocket in the DHX8 Winged-Helix domain, was crucial for potency improvement. By binding in the Winged-Helix domain, these inhibitors restrict the helicase domain’s conformational plasticity, stabilizing a closed, inactive conformation while sterically blocking ssRNA translocation.

Oestrogen receptor (ER) activation leads to the formation of DNA double strand breaks (DSB), promoting genomic instability and tumour heterogeneity. The single-stranded DNA cytosine deaminase APOBEC3B (A3B) serves as a co-activator of ER and is implicated in inducing DSBs at transcriptional enhancers regulated by ER. Using whole-genome sequencing in an engineered cell model lacking base excision repair (BER) function, we demonstrate that A3B preferentially targets transcriptionally active regulatory regions in an R-loop-dependent manner. Strand-specific DNA:RNA immunoprecipitation sequencing (ssDRIP-seq) and ssDNA-associated protein immunoprecipitation sequencing (SPI-seq) confirm that A3B binds to and deaminates ssDNA within R-loops, a process facilitated by ER transactivation. Furthermore, BER-mediated processing of A3B-induced uracil bases contributes to the formation of R-loop-associated DSBs, which are essential for ER-regulated gene activation. These findings establish a role for A3B in R-loop homeostasis and transcriptional regulation, with implications for understanding ER-driven genomic instability and potential therapeutic targeting of A3B.

Transcription factor heat shock factor 1 (HSF1) orchestrates the cellular stress response, promoting malignant transformation, unchecked proliferation, and stress-resilient survival of tumour cells. We set out to discover potentially druggable regulators of HSF1 activation and identified DEAH-box RNA helicase 8 (DHX8). We investigated the role of DHX8 in regulating HSF1 within the broader context of DHX8 function in cancer cells. DHX8 silencing induces intron retention in HSF1 transcripts, reducing HSF1 protein. Importantly, DHX8 loss significantly alters RNA processing of an HSF1-regulated cancer-associated gene signature linked to poor clinical outcomes, as well as additional oncogenic and stress-response pathways. DHX8 binds between the pre-messenger RNA (mRNA) lariat branch point and the 3′ splice site, consistent with the predominance of intron-retained transcripts following DHX8 loss. We show that both the ATPase and RNA-binding activities of DHX8 are essential for its role in splicing, including processing of HSF1 mRNA. We also find that DHX8 silencing triggers apoptosis more effectively in human cancer cells than in non-tumorigenic cells. Our findings identify DHX8 as a critical regulator of stress-adaptive gene expression, highlighting its promise as a therapeutic target not only to disrupt HSF1-dependent transcriptional programs but also having broader effects in cancer cells under oncogenic stress.

Eukaryotic translation initiation factor 4E (eIF4E) has long been recognised as a pivotal regulator of cap-dependent protein synthesis initiation. More recently, eIF4E has emerged as a multifunctional factor proposed to influence various aspects of RNA metabolism, including nuclear export of mRNA to the cytoplasm. Its versatile roles are largely attributed to its ability to bind the methyl-7-guanosine cap (m7G-cap) of mRNAs and participate in critical protein–protein interactions. Deregulated eIF4E expression or activity has been implicated in several diseases, but it is most prominently studied as an oncogene where its activity can drive cancer onset, progression and drug resistance. Consequently, eIF4E is a highly attractive target for the development of novel anti-tumour therapeutics. Recent advancements have provided new insights into the mechanism of action of eIF4E, leveraging fragment-based compound screening and genetically modified cell models to identify and characterise binding sites on this challenging-to-drug protein target. In this review, we summarise the multiple roles of eIF4E and features that underpin its activity in both the cytoplasm and nucleus, and the key findings related to the modulation of its activity and therapeutic potential.

The Mediator complex is a regulator of gene expression, influencing chromatin structure and RNA polymerase II-mediated transcription. Its activity is controlled by a protein kinase module, which includes cyclin-dependent kinases 8 and 19, that phosphorylates RNA polymerase II and transcription factors to regulate gene expression. Using orthogonal approaches combining chemical and genetic tools, we demonstrated the selectivity of our small-molecule inhibitors derived from 3,4,5-trisubstituted pyridine and 3-methyl-1H-pyrazolo[3,4-b]pyridine chemical series in human colorectal cell culture and tumor xenograft models. The lack of activity of our inhibitors in CDK8/19 double knockout models, with respect to molecular, proliferative, and antitumor end points, revealed their specificity and dependence on these kinases. Using our chemical probes and knockout models, we explored Mediator kinase function in human colorectal cancer cells. Phospho-proteome profiling revealed substrates enriched with transcription and chromatin regulators, while promoter reporter experiments identified transcription factor binding sites, including TCF/LEF and AP1, regulated by Mediator kinases. Additionally, altered phosphorylation of several Mediator subunits suggests a mechanism for the rapid regulation of the Mediator complex. Overall, our results demonstrate that CDK8 and CDK19 play pivotal roles in regulating gene expression associated with oncogene activation and signaling pathways. Further studies are warranted to elucidate their broader cellular roles and regulatory mechanisms. The selective inhibitors validated in this study will provide valuable tools for such mechanistic investigations into Mediator kinase functions and their potential therapeutic exploitation.

NXP800 activates the UPR and inhibits E2F-mediated transcription to drive antitumor activity against the castration-resistant VCaP prostate cancer cell line–derived mouse xenograft. A and B, Castration-resistant emergent VCaP prostate cancer cell line–derived mouse xenografts were treated with vehicle (n = 7, gray) or 35 mg/kg NXP800 (active, n = 8, red) after tumors had established castration-resistant growth with a defined dosing schedule for 38 days. Mean tumor volume (normalized to start; defined as 100%) with SEM is shown. P values were calculated for NXP800 compared with vehicle using the unpaired Student t test at 38 days. P values ≤ 0.05 are shown (*; A). Time to reach 200% starting tumor volume was used as a surrogate endpoint for survival. HR with 95% CI and P value for univariate Cox survival model are shown (B). C–G, Castration-resistant VCaP prostate cancer cell line–derived mouse xenografts were treated with vehicle (n = 4) or 35 mg/kg NXP800 (active, n = 4) daily after tumors had established castration-resistant growth for 5 days with tumor collection 6 hours after final dose for pharmacodynamic studies. RNA-seq was performed on tumor samples. Analysis of RNA-seq with gene set enrichment analysis shows the enrichment and de-enrichment of Hallmark pathways comparing NXP800 (active) and vehicle. NES and FDR are shown as volcano plots. Colored dots denote significantly (FDR 0.05) enriched (red dots) and de-enriched (blue dots) pathways (C). Log2 fold expression level changes of “activating” E2F (E2F1–3) family members treated with NXP800 (active) were compared with vehicle. P values were calculated by DESeq2 using the Wald test. P values ≤ 0.05 are shown (*; D). PERK, phospho-eIF2α, total-eIF2α, and ATF4 (PERK arm); ATF6 (ATF6 arm); IRE1 (IRE1 arm); and GAPDH (housekeeping) protein expression was determined by Western blot (E). Cleaved caspase 3 (F) and Ki-67 (G) protein expression was determined by IHC on formalin-fixed, paraffin-embedded tumors. Representative micrographs are shown. Scale bar, 50 μm. Mean percentage positive cells with SD are shown. P values were calculated for NXP800 (red) compared with vehicle (white) using the unpaired Student t test. P values ≤ 0.05 are shown (*).

Supplementary figure 5: Enzalutamide inhibits AR signaling in enzalutamide responsive VCaP PCa cells but not enzalutamide resistant LNCaP95 and 22Rv1 PCa cells.

Supplementary figure 2: AR and AR-V7 bind members of the 70KDa heat shock protein family and heat shock mediated cellular stress increases HSP72 and AR-V7 protein expression, and associates with GO Cellular Response to Heat gene expression signature, in PCa cells

NXP800 inhibits AR transactivation and AR signaling to inhibit the growth of AR aberrant prostate cancer models. A–C, VCaP (A), LNCaP95 (B), and 22Rv1 (C) prostate cancer cells were treated with vehicle (DMSO 0.1%) or various concentrations (5, 10, 50, 100, and 250 nmol/L) of NXP800 (active, red line) or CCT365248 (inactive, blue line), and growth was determined after 5 days by CellTiter-Glo Luminescent Cell Viability Assay. Mean growth (compared with vehicle; defined as 1) with SD from a single experiment with six replicates is shown. P values were calculated for active compound compared with inactive compound for each concentration using the unpaired Student t test. P values ≤ 0.05 are shown (*). VCaP (A), LNCaP95 (B), and 22Rv1 (C) prostate cancer cells were treated with vehicle (DMSO 0.1%) or various concentrations (50, 100, and 250 nmol/L) of NXP800 (active) or CCT365248 (inactive) for 48 hours, and AR-FL, AR-V7, PSA, and GAPDH protein expression was determined from one experiment performed in triplicate. Single Western blot representative of three is shown. VCaP (A), LNCaP95 (B), and 22Rv1 (C) prostate cancer cells were treated with vehicle (DMSO 0.1%) or various concentrations (50, 100, and 250 nmol/L) of NXP800 (active, red line), or CCT365248 (inactive, blue line) for 48 hours, and KLK2, KLK3, TMPRSS2, and FKBP5 RNA expression was determined. Mean RNA expression (normalized to average of GAPDH/B2M/HRPT1/RPLP0 and vehicle; defined as 1), with SD from a single experiment with three replicates is shown. P values were calculated for active compared with inactive compound for each concentration using the unpaired Student t test. P values ≤ 0.05 are shown (*). D, PC3 cells were transfected with CONTROL-FLAG, AR-FL-FLAG, or AR-V7-FLAG and PSA-ARE3-luciferase, prior to treatment with vehicle (DMSO 0.1%), 250 nmol/L CCT365248 (inactive), 250 nmol/L NXP800 (active), or 3 μmol/L enzalutamide for 1 hour prior to stimulation with or without 10 nmol/L DHT for 16 hours. Mean luciferase activity (compared with CONTROL-FLAG/vehicle/without DHT) with SD from two experiments with five replicates is shown. P values were calculated for each plasmid (with and without DHT stimulation) with vehicle compared with other treatments using the unpaired Student t test. P values ≤ 0.05 are shown (*). E, ChIP with an AR antibody was carried out in 22Rv1 cells that were plated in starved (charcoal-striped serum/phenol red–free) media for 72 hours prior to treatment with 250 nmol/L CCT365248 (inactive) or 250 nmol/L NXP800 (active) for 1 hour prior to stimulation with or without 10 nmol/L DHT for 5 hours. AR recruitment to AR-responsive genes (KLK2, KLK3, FKBP5, TMPRSS2, CHRNA2, and ANKRD30B) was determined. Mean binding as percentage of input with SD from two experiments with three replicates is shown. P values were calculated for 250 nmol/L CCT365248 (inactive) compared with 250 nmol/L NXP800 (active) with and without DHT stimulation using the unpaired Student t test. P values ≤ 0.05 are shown (*).