ASTX295 is a potent MDM2 antagonist designed to have a shorter half-life (t1/2 4-6 hours in plasma), which leads to an improved safety profile (bone-marrow sparing), making it more amenable to combinations compared to other MDM2 antagonists. The recent success of targeting TP53:Y220C mutant cancers with a p53 corrector molecule holds great promise for treatment of all Y220C TP53 mutant cancers. However, for effective therapy, a high level of p53 signalling will be required to overcome the elevated apoptotic thresholds seen in some cancer cells with co-mutations in pro-survival signalling pathways. We rationalised that this could be achieved by removing the MDM2-driven negative feedback loop that is triggered following Y220C correction. Corrected p53 signalling was determined in two TP53:Y220C mutant pancreatic cancer cell lines with additional MAPK mutations: BxPC3 (BRAF:L485-P490del) and T3M-4 (KRAS:Q61H). Two small-molecule TP53:Y220C correctors were tested alone and in combination with ASTX295 using real-time microscopy, western blotting, and qPCR. Markers of p53 signalling (at the protein and mRNA level), senescence (β-galactosidase), apoptosis (Annexin V), and DNA damage (phospho-H2A.X) were measured after treatment with compounds. A mouse xenograft model was established in male CB17 SCID mice inoculated subcutaneously with BxPC3 cells, allowing PK/PD and efficacy evaluation. The combination of ASTX295 and the Y220C corrector led to increased cell death in both BxPC3 and T3M-4 cell lines in vitro, with elevated p53 signalling (p21), apoptosis (PUMA), and DNA damage (phospho-H2A.X) when MDM2 is antagonised. The KRAS mutant line, T3M-4, was less sensitive to the combination, but could be driven to apoptosis at higher concentrations of the corrector. PK/PD evaluation in BxPC3-xenograft-bearing mice demonstrated prolonged p53 signalling following combination treatment up to 16 h post dose, with a subsequent reduction at 24 h consistent with the ASTX295 exposure profile. We have shown that MDM2 antagonism leads to sustained corrected p53 signalling over time, which drives cell death, even in tumor cells harbouring co-mutations conferring higher apoptotic resistance. Indeed, our study provides proof of concept that efficacy could be achieved in multiple tumor types in which the p53 pathway can be targeted therapeutically through combination with ASTX295, which would drive and maintain p53 signalling levels above the apoptotic threshold. George Ward, Judit Espana-Agusti, Keisha Hearn, Mark Wade, Lynsey Fazal, Hugh Walton, Andrea Biondo, John Lyons, Martin Sims, Simone Jueliger, Jessica Brothwood, Andrew Woodhead, Louise Walsh, Tomoko Smyth, Nicola Wilsher, Maria Ahn. Switching cell fate from senescence to apoptosis by the combination of a p53 corrector with the MDM2 antagonist ASTX295 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 1627.

Abstract Purpose: Deregulated phosphatidylinositol 3-kinase pathway signaling through AGC kinases including AKT, p70S6 kinase, PKA, SGK and Rho kinase is a key driver of multiple cancers. The simultaneous inhibition of multiple AGC kinases may increase antitumor activity and minimize clinical resistance compared with a single pathway component. Experimental Design: We investigated the detailed pharmacology and antitumor activity of the novel clinical drug candidate AT13148, an oral ATP-competitive multi-AGC kinase inhibitor. Gene expression microarray studies were undertaken to characterize the molecular mechanisms of action of AT13148. Results: AT13148 caused substantial blockade of AKT, p70S6K, PKA, ROCK, and SGK substrate phosphorylation and induced apoptosis in a concentration and time-dependent manner in cancer cells with clinically relevant genetic defects in vitro and in vivo. Antitumor efficacy in HER2-positive, PIK3CA-mutant BT474 breast, PTEN-deficient PC3 human prostate cancer, and PTEN-deficient MES-SA uterine tumor xenografts was shown. We show for the first time that induction of AKT phosphorylation at serine 473 by AT13148, as reported for other ATP-competitive inhibitors of AKT, is not a therapeutically relevant reactivation step. Gene expression studies showed that AT13148 has a predominant effect on apoptosis genes, whereas the selective AKT inhibitor CCT128930 modulates cell-cycle genes. Induction of upstream regulators including IRS2 and PIK3IP1 as a result of compensatory feedback loops was observed. Conclusions: The clinical candidate AT13148 is a novel oral multi-AGC kinase inhibitor with potent pharmacodynamic and antitumor activity, which shows a distinct mechanism of action from other AKT inhibitors. AT13148 will now be assessed in a first-in-human phase I trial. Clin Cancer Res; 18(14); 3912–23. ©2012 AACR.

Abstract Aim We aimed to design an MDM2-p53 antagonist with a differentiated tolerability profile that could be used to treat patients with wild-type TP53 malignancies. As part of an alliance between Newcastle University, Astex Pharmaceuticals, and Cancer Research Horizons, we discovered ASTX295, a potent inhibitor of the MDM2-p53 interaction that is currently under clinical investigation in patients with solid tumors (NCT03975387). We selected ASTX295 as a compound with a predicted short plasma half-life, which we hypothesised would help to mitigate the dose-limiting neutropenia and thrombocytopenia observed with earlier MDM2-p53 antagonists in clinical studies. To examine this hypothesis in vitro, we determined time- and concentration-dependent responses to ASTX295 treatment in healthy volunteer-derived human bone marrow cells, megakaryocytes, and in a panel of human tumor cell lines. Methods Samples containing bone marrow cells from healthy patients undergoing hip surgery were obtained under the ethical approval of the Newcastle Biobank (REC 12/NE/0395). Following Lymphoprep™ separation, cells were treated ex vivo with ASTX295 and seeded for Granulocyte-macrophage (GM) colony-forming assays in methylcellulose. Megakaryocytes were obtained from in vitro differentiation of CD34+ stem/progenitor cells. Human tumor cell lines (including MDM2-amplified SJSA-1) were treated with ASTX295 in vitro and seeded at low density for colony-forming assays. Exposures of 6, 12, or 24h were examined, and the data plotted to calculate LC50 values. Results The clonogenic survival of tumor cells was time-dependent, with LC50 values (mean ± SEM) in SJSA1 cells being 238 ± 46nM and 75 ± 7nM respectively (n = 3-4), following a 12h or 24h exposure to ASTX295. Time-dependent effects were also evident in five human bone marrow samples but with LC50 values of 1.9, >3, >10, >10, and >10uM being achieved at 12h, and 860 ± 268nM at 24h. Megakaryocytes showed similar time-dependent sensitivities in which daily treatment of 2 or 6h over three days did not induce apoptosis while significant cell death was observed when the treatment time was extended to 16-24h daily. In contrast, short, daily pulse treatment of 2-6h in cell lines (MV4-11, MOLM-13, SJSA-1) over three days was sufficient to induce cell death. Conclusions ASTX295 is a potent antagonist of the MDM2-p53 interaction. Collectively, our in vitro data suggest that a shorter exposure to ASTX295 (up to 12h), may help to spare healthy bone marrow cells whilst killing tumor cells. Hence, intermittent exposure to an MDM2-p53 antagonist could favourably modulate its therapeutic index. The predicted short plasma half-life of ASTX295 should provide flexibility in controlling the duration of exposure in vivo, potentially enabling a more bone-marrow sparing approach to MDM2-p53 antagonism to be utilised. Citation Format: Elaine Willmore, Maria Ahn, Suzanne Kyle, Yan Zhao, Huw Thomas, Kenneth S. Rankin, Luke Bevan, Lynsey Fazal, Keisha Hearn, Nicola Wilsher, Justyna Kucia-Tran, Nicola Ferrari, Nicola Wallis, Neil Thompson, John Lyons, Duncan Miller, Celine Cano, Martin E. Noble, Ian R. Hardcastle, Steven Howard, Gianni Chessari, John Lunec, David R. Newell, Steve R. Wedge. Targeting the MDM2-p53 interaction: Time- and concentration-dependent studies in tumor and normal human bone marrow cells reveal strategies for an enhanced therapeutic index [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 3333.

Abstract In response to cellular stress, the tumor suppressor p53 is activated to modulate cell cycle progression, DNA repair, and apoptosis. Inhibition of the MDM2-p53 interaction in tumors carrying wild-type p53 prevents its degradation and can reactivate p53 to elicit an anti-cancer effect. Targeting the p53-MDM2 interaction therefore remains a promising strategy for cancer therapy. However, development of first generation MDM2 antagonists has been challenged by dose-limiting, on-target bone marrow toxicities. Understanding of differential effects of p53 pathway activation in normal hematopoietic versus cancer cells (to be presented in a separate abstract) together with our expertise in structure-based drug design have led to the discovery of ASTX295, a potent MDM2 antagonist with differentiated pharmacokinetic profile aimed at sparing bone marrow toxicities and increasing the therapeutic index. Here, we present the first disclosure of the structure and pre-clinical characterisation of ASTX295. ASTX295 exhibits potent activity (IC50<1 nM) against MDM2 in an ELISA-based in vitro assay and induces significant growth reduction in p53 wild-type, MDM2-amplified SJSA-1 cells (GI50=27 nM). Antiproliferative activity of ASTX295 was further demonstrated in a panel of 219 p53 wild-type cell lines, with 143 cell lines showing GI50 values less than 1 μM and 50 showing values less than 0.1 μM. Effects of ASTX295 are shown in cell lines carrying functional p53 as confirmed in three p53 wild-type and mutant cell line pairs (SJSA1 and SN40R2, A2780 and A2780CP, HCT116 and HCT116 p53−/−). In addition to inhibiting cell cycle progression and cell proliferation, ASTX295 also potently induces apoptosis following 24-48 hour treatment. Further in vitro analyses of ASTX295 demonstrated an increase in the levels of p53 (EC50=10 nM after 2 hours) and its transcriptional targets such as p21 and MDM2. In vivo, ASTX295 shows robust induction of p53 and its target genes at 3 and 6 hours after oral administration together with dose-dependent inhibition of tumour growth in the SJSA-1 xenograft model. Importantly, ASTX295 exhibits optimised pharmacokinetic and pharmacodynamic profiles with relatively short duration of pathway modulation and a desired predicted human half-life of 2-8 hours. Based on our pre-clinical hypothesis on differential time-dependent sensitivities of normal versus cancer cells to p53 activation, achieving such a profile while maintaining potency may increase the therapeutic index. These data highlight the therapeutic potential of ASTX295, which is currently being tested in a Phase 1/2 clinical trial in advanced solid tumors with wild-type p53 (NCT03975387). We plan to present preliminary clinical data in a separate abstract at this meeting. Citation Format: Maria Ahn, Luke Bevan, Ildiko Buck, Celine Cano, Juan Castro, Ben Cons, Jane Endicott, Lynsey Fazal, Nicola Ferrari, Ian Hardcastle, Keisha Hearn, Rhian Holvey, Steven Howard, Chris Johnson, Claire Jennings, Justyna Kucia-Tran, Suzanne Kyle, John Lunec, John Lyons, Duncan Miller, David Rees, Martin Noble, David R. Newell, Judith Reeks, Harpreet Saini, Jeffrey St. Denis, Emiliano Tamanini, Huw Thomas, Neil Thompson, Mladen Vinkovic, George Ward, Nicola Wallis, Hugh Walton, Stephen Wedge, Pamela Williams, Elaine Willmore, Nicola Wilshire, Yan Zhao, Gianni Chessari. Discovery of ASTX295, a potent, next-generation small molecule antagonist of MDM2 with differentiated pharmacokinetic profile [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 6588.

Abstract Programmed cell death mechanisms are important for the regulation of tumor development and progression. Evasion of and resistance to apoptosis are significant factors in tumorigenesis and drug resistance. Bypassing apoptotic pathways and eliciting another form of regulated cell death, namely necroptosis, an immunogenic cell death (ICD), may override apoptotic resistance. Here, we present the mechanistic rationale for combining tolinapant, an antagonist of the inhibitor of apoptosis proteins (IAP), with decitabine, a hypomethylating agent (HMA), in T-cell lymphoma (TCL). Tolinapant treatment alone of TCL cells in vitro and in syngeneic in vivo models demonstrated that ICD markers can be upregulated, and we have shown that epigenetic priming with decitabine further enhances this effect. The clinical relevance of ICD markers was confirmed by the direct measurement of plasma proteins from patients with peripheral TCL treated with tolinapant. We showed increased levels of necroptosis in TCL lines, along with the expression of cancer-specific antigens (such as cancer testis antigens) and increases in genes involved in IFN signaling induced by HMA treatment, together deliver a strong adaptive immune response to the tumor. These results highlight the potential of a decitabine and tolinapant combination for TCL and could lead to clinical evaluation. Significance: The IAP antagonist tolinapant can induce necroptosis, a key immune-activating event, in TCL. Combination with DNA hypomethylation enhances tolinapant sensitivity and primes resistant cells by re-expressing necrosome proteins. In addition, this combination leads to increases in genes involved in IFN signaling and neoantigen expression, providing further molecular rationale for this novel therapeutic option.

Figure S1. Additional Western blots showing different CRISPR clones generated from each construct. (Refers to Figure 1)

Abstract Background High-throughput drug screening and computational methods to associate genomic features of cell lines to drug sensitivity are valuable tools for predicting biomarkers of sensitivity. In addition, integrating genomic features to expression-based patterns could improve biomarker prediction and patient stratification. Particularly weighted gene co-expression networks represent an effective approach to identify key modules and possible biological mechanisms of sensitivity. Using a combination of cell panel screening and gene co-expression networks, we predicted markers of sensitivity to ASTX295 (MDM2i) in cancer cell lines and confirmed expression-based signatures in publicly available clinical datasets. Method ASTX295 was screened in a panel of 210 p53 wild-type cancer cell lines derived from a range of tumor tissues. ANOVA was used to identify significant associations of genomics features of cell lines to drug response. Transcriptomics profiling of apoptotic and non-apoptotic patient-derived cell lines was performed and significant differentially expressed genes were identified. Further, pathway enrichment and expression-based signatures identified in cell lines were further confirmed in TCGA patient data using weighted gene co-expression network analysis (WGCNA). Expression modules identified by WGCNA were correlated to genomic features and clinical parameters to identify potentially clinically relevant biomarkers. Results Analysis of the cell panel data identified CDKN2A loss as a statistically significant biomarker predictive of sensitivity to ASTX295. As mesothelioma is one of the indications with high prevalence of loss of CDKN2A, the anti-proliferative activity of ASTX295 was further confirmed in an independent panel of p53 wild-type, patient-derived mesothelioma cell lines. Further, pathway and transcriptional regulator analysis identified the Interferon signalling as significantly enriched in apoptotic cell lines and was confirmed in a TCGA mesothelioma patient data set and the module was found to be significantly correlated with a subgroup of P53 wild-type patients with CDKN2A loss. In conclusion, combining cell panel drug screening with co-expression networks helped to identify biomarkers associated with ASTX295 sensitivity, and provided new insights into the underlying mechanism of ASTX295 response. Citation Format: Harpreet Kaur Saini, Maria Ahn, George Ward, Justyna Kucia-Tran, Christina Gewinner, Nicola Ferrari, Jessica Brothwood, Luke Bevan, Matthew Davis, Lynsey Fazal, Martin Sims, Marc O'Reilly, Gianni Chessari, Roberta Ferraldeschi, John Lyons, Nicola Wallis, Neil Thompson. Identification of biomarkers of response to MDM2 inhibition in solid tumours using computational, multi-omics approaches [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 667.

Cell death proteins play a central role in host immune signaling during sepsis. These interconnected mechanisms trigger cell demise via apoptosis, necroptosis, and pyroptosis while also driving inflammatory signaling. Targeting cell death mediators with novel therapies may correct the dysregulated inflammation seen during sepsis and improve outcomes for septic patients.

Effect of necrosome protein expression in TCL cell lines on tolinapant-induced cell death. A, Western blots of parental BW5147, RIPK3−/− BW5147 and MLKL−/− BW5147 cell lysates (untreated). B, Cytotox-NIR signal captured by real-time microscopy (IncuCyte), detecting membrane permeabilization during lytic cell death after treatment of parental BW5147, RIPK3−/− BW5147 and MLKL−/− BW5147 cells with tolinapant in the absence or presence of zVAD. C, Western blots of Karpas-299 cells transduced with control gRNA (KARPAS-299-CTR gRNA) or with RIPK3 gRNA (KARPAS-299-RIPK3 gRNA) constructs treated with tolinapant ± zVAD for 0, 2, or 6 hours. D, Real-time microscopy (IncuCyte) measurement of membrane permeabilization by measuring Cytotox-NIR signal after treatment of KARPAS-299-CTR gRNA or KARPAS-299-RIPK3 gRNA cells with tolinapant. E, Western blots of EL4-PAR and CASP8 KO EL4 (EL4-C8KO) cell lysates after 24-hour treatment with tolinapant. F, Real-time microscopy (IncuCyte) measurement of membrane permeabilization by measuring Cytotox-NIR signal after treatment of EL4-PAR or EL4-C8KO cells with tolinapant.

Abstract MAPK pathway activation is a feature of multiple tumor types. Drugging KRAS and BRAF, the two main oncogenic drivers in the MAPK pathway, has proven successful in the clinic. Inhibition of the downstream effectors, MEK and ERK, can also induce tumor regression. Despite this, many tumors are intrinsically resistant to MAPK pathway inhibitors, or acquire resistance under selective pressure to drug treatment. This creates a need for combination treatments to improve clinical responses. A synthetic lethal (SL) interaction between inhibition of the MAPK pathway and blockade of JNK-JUN signaling has recently been described1. Specifically, data from yeast genetics and CRISPR knockout experiments in human cells have identified MAP2K4 as a potential therapeutic target that could be combined with MAPK inhibitors. To date, however, no potent MAP2K4 inhibitors with in vitro and cellular selectivity against key anti-targets have been reported. Here, we describe the development of potent covalent inhibitors of MAP2K4 kinase activity. Biochemical and cell-based assays show that the compound(s) are selective for MAP2K4 versus anti-targets including MAP2K7 and ERK kinases. The combination of MAP2K4 and MEK/ERK inhibitors was effective in cell lines driven by MAPK signaling. These data provide the rationale for further development of MAP2K4 inhibitors to advance our understanding of this novel drug combination. References: 1. Xue Z, Vis DJ, Bruna A, Sustic T, van Wageningen S, Batra AS, et al. MAP3K1 and MAP2K4 mutations are associated with sensitivity to MEK inhibitors in multiple cancer models. Cell Res. 2018; 28:719-29. Citation Format: Mark Wade, Emiliano Tamanini, Mathieu Unbekandt, Nicola Wallis, John Lyons, Joanne Munck, Andrew Woodhead, Patrick Schopf, Jessie Stow, Charlotte East, Mellissa Clark, Jeffrey St. Denis, Puja Pathuri. Targeting MAPK-driven tumors via inhibition of MAP2K4 [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 5902.