Abstract The translation initiation factor eIF4E is a rate-limiting factor for protein synthesis that binds the mRNA m7G-cap to initiate the recruitment and binding of eIF4F components such as eIF4G. Targeting eIF4E has long been considered a promising anticancer strategy but it has remained undruggable using conventional screening approaches. Fragment-based crystallographic screening is a powerful technique for probing the surface of proteins to identify potentially druggable binding sites. Here, we describe fragment-based screening using a combination of Xray crystallography and NMR to identify low affinity fragment hits binding to the mRNA cap-binding site or at an additional site of unknown functional relevance (site 2). Subsequent rounds of iterative structure-based design yielded a lead molecule, with 100nM binding potency for site 2, that could disrupt the eIF4E:eIF4G interaction and inhibit cap-dependent translation in cell lysates. To explore the functional relevance of site 2 we established a cell model expressing an eIF4E-FKBP12F36V fusion protein that was sensitive to degradation following treatment with the heterobifunctional dTAGV-1 molecule. Degradation of eIF4E resulted in inhibition of cell growth and reduced expression of MCL1 protein, that were rescued by re-expression of a non-degradable wild-type eIF4E. A mutation reported to block canonical eIF4G binding (W73F) disrupted binding to eIF4G but to our surprise retained the ability to rescue the eIF4E degradation phenotype. Mutation of site 2 (L85R and L134R) also disrupted the eIF4E:eIF4G interaction. The L85R mutant remained able to partially rescue the cellular responses, in contrast to the L134R mutant that could not rescue eIF4E loss. The W73F/L85R double mutant resulted in a combinatorial loss of eIF4E function. Despite disrupting eIF4E function in lysates, there was a significant drop-off of compound activity in cells. However, treatment of the W73F mutant with compound recapitulated the combination of the W73F canonical eIF4G binding mutation with the L85R site 2 mutation. This approach demonstrates the power of coupling fragment screening with target-degradation and genetic rescue approaches to find and explore novel functional pockets. Our data suggest it may be necessary to disrupt the extended protein-protein interaction made by both the canonical and non-canonical regions of eIF4E to drive a strong functional effect in cells. However, the discovery of a lead compound, the associated structural understanding, and the knowledge that one site 2 mutation inactivates eIF4E provides hope that a significantly more potent small molecule inhibitor or degrader could drive a more profound cellular effect which may lead to improved cellular activity and a deeper understanding of eIF4E biology. Citation Format: Paul A. Clarke, Swee Y. Sharp, Marianna Martella, Christopher I. Milton, Charlotte East, Mladen Vinkovic, Nicola Wallis, George Ward, Caroline Richardson, Andrew Woodhead. Integrating fragment-based crystallographic screening with dTAG-PROTAC degradation and genetic rescue to explore the function of eIF4E [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7073.

Abstract Eukaryotic initiation factor 4E (eIF4E) serves as a regulatory hub for oncogene-driven protein synthesis and is considered a promising anticancer target. Here we screen a fragment library against eIF4E and identify a ligand-binding site with previously unknown function. Follow-up structure-based design yields a low nM tool compound (4, Kd = 0.09 µM; LE 0.38), which disrupts the eIF4E:eIF4G interaction, inhibits translation in cell lysates, and demonstrates target engagement with eIF4E in intact cells (EC50 = 2 µM). By coupling targeted protein degradation with genetic rescue using eIF4E mutants, we show that disruption of both the canonical eIF4G and non-canonical binding sites is likely required to drive a strong cellular effect. This work highlights the power of fragment-based drug discovery to identify pockets in difficult-to-drug proteins and how this approach can be combined with genetic characterization and degrader technology to probe protein function in complex biological systems.

Abstract Background: NXP800 is a potent, oral activator of the Integrated Stress response (ISR), inhibitor of heat shock factor 1 (HSF1) activation and tumor cell proliferation, which is in early clinical studies in ARID1A-mutated, platinum resistant, clear cell ovarian cancer (NCT05226507). We discovered NXP800 using multiparameter medicinal chemistry optimization of a hit identified from a cell-based phenotypic screen. Owing to the unbiased nature of phenotypic screening, target identification is crucial to understand the biological and therapeutic activity of hit compounds. Methods and Results:: By RNAseq profiling human cancer cells treated with NXP800 we identified changes in expression of genes regulated by HSF1 or ATF4 - effects accompanied by eIF2alpha (eIF2a) phosphorylation and resulting activation of the ISR. To further understand this response, we explored whether NXP800 resistance models could inform on its mechanism of action (MoA). We used ARID1A mutant SK-OV-3 human ovarian carcinoma cells that are 1) highly sensitive to NXP800, 2) model the target patient population and 3) MSI-high so likely to have an elevated mutation rate contributing to acquisition of resistance. We generated two independent NXP800-resistant SK-OV-3 cell lines in which NXP800-mediated ATF4 induction and concomitant inhibition of global translation were abolished. By whole exome sequencing, we identified a heterozygous L99P mutation in the alpha subunit of eIF2B (eIF2Ba), the nucleotide exchange factor for eIF2a. Expression of eIF2BaL99P, but not wild-type, in parental SK-OV-3 cells reduced sensitivity to NXP800 to the same level as cells with NXP800-induced resistance. Using fluorescence recovery after photobleaching to monitor the dynamic association of the eIF2B:eIF2a complex, we elucidated that the eIF2BaL99P mutation reduces NXP800-mediated inhibition of eIF2a-GFP recycling through eIF2B bodies. Phosphorylation of eIF2a is regulated by four stress-controlled kinases GCN2, HRI, PKR and PERK. Using systematic siRNA knockdown or small-molecule inhibitors, we showed that GCN2 alone is required for ISR activation by NXP800 and that ISR induction inhibited HSF1 activation. Furthermore, inactivation of GCN2 reduced the antiproliferative activity of NXP800 to the same extent observed in NXP800-resistant SK-OV-3 cells. In contrast, exposure of resistant or parental SK-OV-3 cells expressing eIF2BaL99P to GCN2 inhibitors did not cause further reduction in NXP800 sensitivity - confirming the importance of the eIF2BaL99P mutation in the resistance mechanism. Conclusions: We have used acquired resistance to understand the MoA of NXP800 as a potent activator of GCN2 and the ISR pathway. Further studies are underway to determine the exact proximal molecular target of NXP800 and the mechanism of GCN2/ISR activation. Citation Format: Marissa V. Powers, Rachel Hodgson, Swee Y. Sharp, Toby Roe, K. Elizabeth Allen, Susan Campbell, Robert te Poele, Matthew Cheeseman, Keith Jones, Paul A. Clarke, Paul Workman. Using acquired resistance to explore the mechanism of action of the integrated stress response/GCN2 activator NXP800 - A new developmental agent for platinum-resistant ARID1A mutant ovarian cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 524.

Abstract Background. HSF1 helps cancer cells cope with multiple stresses caused by oncogene activation. Methods. See details below. Results. We discovered the bisamide NXP800 as an inhibitor of HSF1-mediated transcription through phenotypic screening and med chem optimization. Gene expression microarray analysis of human cancer cell lines and tumor xenografts treated with bisamide inhibitors indicated activation of the ATF4 axis of the ISR as a key mechanism of action. In SK-OV-3 ovarian cancer xenografts, NXP800 showed clear PK/PD relationships with increased expression of ATF4 transcriptional target genes alongside decreased expression of HSF1 transcriptional targets. NXP800 also caused tumor regressions in this model. Expanding in additional human ovarian cancer xenograft models, we observed efficacy in five and a complete lack of response in three others. The sensitive models all had homozygous deleterious mutations in the ARID1A gene, whereas the non-responding xenografts were all wild type (WT). ARID1A is a component of the SWI/SNF chromatin remodelling complex involved in repression and activation of target genes. Subsequent screening of the large Sanger human cancer cell line panel confirmed ARID1A as the most significant common disease-related alteration predicting sensitivity to NXP800 in ovarian cancer cell lines. This predictive relationship was confirmed in an ARID1A isogenic HCT-116 cell line pair; sensitivity was greater in the homozygous ARID1A mutant cells compared to WT, resulting in PARP cleavage in the mutant cells only. In vivo there was no effect in WT HCT-116 xenografts whereas growth inhibition was observed in the homozygous mutant cells, resulting in significantly smaller tumors. In addition, the induction of ATF4 target genes was stronger and more prolonged in the mutant cells. It is known that whereas short term ATF4 activation is adaptive, persistent activation can promote the induction of apoptosis. CHIP-seq analysis confirmed clear relationships between ARID1A status, ATF4 and HSF1 promoter occupancy, and the distribution of BRG1 and RNA pol II at target sites, although these were often gene-specific and complex. A relatively simple example is the regulation of the INHBE gene (expression of which is a PD biomarker). In untreated samples there is no binding of ATF4, BRG1 and RNA pol II at the INHBE promoter. NXP800 treatment results in the recruitment of BRG1, ATF4 and RNA pol II in ARID1A mutant TOV-21G cells but not in the RMGI WT cells, leading to increased expression in the mutant cells only. Conclusions. We propose that ARID1A loss alters the binding and recruitment of ATF4 and HSF1 leading to the altered and prolonged expression of ATF4 target genes and increased sensitivity to NXP800. NXP800 is currently in phase Ib for the treatment of ARID1A mutant platinum resistant ovarian cancer (NCT05226507). Citation Format: Robert H. te Poele, Marissa Powers, Swee Sharp, Emmanuel de Billy, Maria Taskinen, Loredana Pellegrino, Sharon Gowan, Asadh Miah, Angela Hayes, Matthew Cheeseman, Keith Jones, Suzanne Eccles, Florence Raynaud, Paul Clarke, Paul Workman. Discovery of ARID1A loss as a patient biomarker for NXP800 - A developmental activator of the integrated stress response (ISR) and inhibitor of the HSF1 pathway in ovarian cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6441.

Adobe PDF - MCT-07-0149--Suppl_Data.pdf from Inhibition of the heat shock protein 90 molecular chaperone in vitro and in vivo by novel, synthetic, potent resorcinylic pyrazole/isoxazole amide analogues

HSF1 is a stress-inducible transcription factor that regulates the eukaryotic heat shock response (HSR). HSF1 activation induces the expression of multiple proteins needed for cellular recovery from stress. HSF1 also plays a key role in tumorigenesis and regulates the expression of a cancer-specific gene signature which is unique to malignant cells and distinct from that activated during the HSR. We discovered NXP800, the first-in-class orally active HSF1 pathway inhibitor which is now undergoing Phase 1 clinical trial, based on a phenotypic pathway screen. Here we employed various techniques to investigate the mechanism of action of NXP800. Using RNAseq in a panel of human carcinoma cell lines, we identified overlapping gene expression changes in response to NXP800. These included genes regulated by HSF1 and interestingly also genes associated with activation of the integrated stress response (ISR). In contrast, we found no evidence for activation of the unfolded protein response. Consistent with the observed NXP800-induced phosphorylation of eIF2α which is a critical regulator of the ISR, NXP800 increased the protein expression of downstream ISR markers ATF4, CHOP and CHAC1, both in human tumor cells in vitro and in human tumor xenograft models in vivo. Induction of the ISR is controlled by four stress-activated protein kinases (PKs) that phosphorylate eIF2α. To further explore these kinases in the mechanism of action of NXP800 we used genetic knockdown by siRNA and inhibition by small-molecule tool compounds. Silencing each of the ISR-regulatory PKs revealed that GCN2 was required for ISR activation by NXP800. This was confirmed using two GCN2 inhibitors from different chemical series. Global phospho-proteome analysis showed that altered protein phosphorylation following NXP800 exposure was reversed upon co-treatment with a GCN2 inhibitor. We also demonstrated that activation of the ISR caused inhibition of HSF1 activation when stimulated with an HSF1 activator, thus confirming the link between ISR induction and inhibition of HSF1 activation. Activation of GCN2 and the ISR can occur in response to a variety of stimuli including amino acid deprivation. However, we did not detect a difference in the uptake of amino acids following exposure to NXP800, indicating that NXP800 does not directly impair amino acid uptake. Using an siRNA approach to determine if activation of the ISR components was contributing to growth inhibition following NXP800 exposure, we found that blocking the induction of ATF4 reduced the response of NXP800-sensitive SK-OV-3 human ovarian carcinoma cells to NXP800 treatment. In summary, NXP800 acts on cancer cells to induce activation of the ISR pathway via GCN2, which then leads to inhibition of HSF1 activation. Further studies are underway to determine the precise molecular target of NXP800 and the mechanism of HSF1 pathway inhibition. Citation Format: Marissa V. Powers, Swee Y. Sharp, Eirini-Maria Lampraki, Toby Roe, Loredana Pellegrino, Maria Taskinen, Robert te Poele, Florence Raynaud, Suzanne Eccles, Matthew Cheeseman, Keith Jones, Paul A. Clarke, Paul Workman. Activation of the integrated stress response by the developmental HSF1 pathway inhibitor NXP800 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr LB234.

Supplementary Table 1, Figure Legends 1-3 from Acquired Resistance to 17-Allylamino-17-Demethoxygeldanamycin (17-AAG, Tanespimycin) in Glioblastoma Cells

Supplementary Figure 1C from NVP-AUY922: A Novel Heat Shock Protein 90 Inhibitor Active against Xenograft Tumor Growth, Angiogenesis, and Metastasis

CCT251236 1, a potent chemical probe, was previously developed from a cell-based phenotypic high-throughput screen (HTS) to discover inhibitors of transcription mediated by HSF1, a transcription factor that supports malignancy. Owing to its activity against models of refractory human ovarian cancer, 1 was progressed into lead optimization. The reduction of P-glycoprotein efflux became a focus of early compound optimization; central ring halogen substitution was demonstrated by matched molecular pair analysis to be an effective strategy to mitigate this liability. Further multiparameter optimization led to the design of the clinical candidate, CCT361814/NXP800 22, a potent and orally bioavailable fluorobisamide, which caused tumor regression in a human ovarian adenocarcinoma xenograft model with on-pathway biomarker modulation and a clean in vitro safety profile. Following its favorable dose prediction to human, 22 has now progressed to phase 1 clinical trial as a potential future treatment for refractory ovarian cancer and other malignancies.

Supplementary Materials, Figure Legends 1-6, Tables 1-5 from NVP-AUY922: A Novel Heat Shock Protein 90 Inhibitor Active against Xenograft Tumor Growth, Angiogenesis, and Metastasis

Supplementary Figure 6 from NVP-AUY922: A Novel Heat Shock Protein 90 Inhibitor Active against Xenograft Tumor Growth, Angiogenesis, and Metastasis

Supplementary Figure 3A-B from NVP-AUY922: A Novel Heat Shock Protein 90 Inhibitor Active against Xenograft Tumor Growth, Angiogenesis, and Metastasis

Supplementary Figure 1 from Acquired Resistance to 17-Allylamino-17-Demethoxygeldanamycin (17-AAG, Tanespimycin) in Glioblastoma Cells

Supplementary Data from Mechanistic Evaluation of the Novel HSP90 Inhibitor NVP-AUY922 in Adult and Pediatric Glioblastoma

Supplementary Methods, Figures 1-3 from Silencing of HSP90 Cochaperone AHA1 Expression Decreases Client Protein Activation and Increases Cellular Sensitivity to the HSP90 Inhibitor 17-Allylamino-17-Demethoxygeldanamycin

Supplementary Data from EGFRvIII Deletion Mutations in Pediatric High-Grade Glioma and Response to Targeted Therapy in Pediatric Glioma Cell Lines

Supplementary Materials, Table 1 and Figures 1-3 from In vitro Biological Characterization of a Novel, Synthetic Diaryl Pyrazole Resorcinol Class of Heat Shock Protein 90 Inhibitors

Initiation of translation is considered the main rate-limiting step of protein synthesis and requires the recognition of 5’ m7G-cap on mature mRNAs and the formation of eukaryotic translation initiation factor 4F (eIF4F) multi-protein mRNA cap-binding complex. Formation of this complex requires the interaction of eIF4E and the scaffold protein eIF4G, and RNA helicase eIF4A. This eIF4F complex along with eIF3 mediate the recruitment of the 40S ribosomal particle to the 5′ cap of mRNA. Activation of eIF4E is a regulatory hub of many major oncogenic pathways, thus, targeting eIF4E has emerged as a potential therapeutic strategy in cancer. Here we have used a targeted protein degradation approach coupled with genetic rescue to explore the molecular and cellular dependency of cancer cells on eIF4E. Stable H1299 human NSCLC clones expressing FKBP12F36V-tagged eIF4E but lacking endogenous eIF4E were established. Treatment of multiple N- or C-tagged-eIF4E clones with dTAGv-1, an FKBP12F36V selective heterobifunctional molecule that recruits VHL, induced rapid degradation of eIF4E to undetectable levels by 6hr exposure. This also resulted in reduced expression of MCL1, a previously reported biomarker of eIF4E activity. Longer exposures to dTAGv-1 resulted in a cytostasis that was not associated with cell death. A diastereomer negative control of dTAGv-1 that cannot recruit VHL did not elicit loss of eIF4E or the downstream events associated with its loss. Global analysis of protein synthesis initiation by RIBOseq and proteome profiling following dTAGv-1 treatment out to 32hr exposure demonstrated surprisingly few alterations in protein expression despite the significant effect on cancer cell growth. We also expressed wild-type or eIF4E mutants predicted to disrupt key functions and determined their ability to rescue molecular or cellular phenotype associate with eIF4E-loss following dTAGv-1 treatment. Expression of wild-type eIF4E completely rescued cell growth and MCL1 expression. A W56A mutant predicted to disrupt mRNA-cap binding was unable to rescue eIF4E loss. In contrast, expression of W73F or S290A mutants (predicted to disrupt eIF4G binding or exhibit reduced eIF4E activity, respectively) were able to rescue the loss of eIF4E. In summary, our rescue experiments show that mRNA-cap binding by eIF4E is required and that eIF4E:eIF4G interaction in cells may be more complex than predicted. This may also explain the challenges associated with developing selective and cellularly potent inhibitors of the eIF4E:eIF4G interaction and that targeting mRNA-cap binding may be a more effective strategy. We predicted that removing eIF4E would impact on the global synthesis of many proteins. However, our data demonstrate that targeting eIF4E leads to limited effects on protein synthesis that remain sufficient to inhibit cancer cell growth. Further experiments are underway to understand which proteins drive this dependency on eIF4E. Citation Format: Swee Y. Sharp, Marianna Martella, Christopher I. Milton, George Ward, Caroline Richardson, Andrew Woodhead, Paul A. Clarke. Exploring the role of eIF4E in cancer cells with targeted protein degradation. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3722.