Background: Heat Shock Factor (HSF1) is a stress-inducible transcription factor that plays a key role in the activation of the eukaryotic heat shock response. HSF1 is hijacked by cancer cells to activate a set of genes that overlap with, but are not identical to, the classical heat shock response. Whereas the HSF1 pathway has been shown to play a key role in oncogenesis and multiple hallmark features of malignancy in experimental cancers, and its amplification/expression/activity correlates with poor clinical outcome, knockout of HSF1 does not impair viability in model organisms – suggesting a potential therapeutic window. Drug discovery has been limited as HSF1 is difficult to drug directly. Therefore, we sought to discover inhibitors of the HSF1 pathway using a phenotypic screening approach, from which we discovered the bisamide clinical development candidate NXP800. Here we describe the discovery and validation of biomarkers to support the clinical development of NXP800, forming a Pharmacological Audit Trail. Material and methods: We searched for biomarkers that correlated with sensitivity in human tumour xenografts and human cancer cell line panels and used isogenic models to validate the key predictive biomarker. To identify pharmacodynamic markers we used a combination of gene expression and protein profiling, combining both screening and hypothesis-driven approaches. Results: NXP800 (CCT361814) has good PK properties in mice, including oral bioavailability. Moreover, NXP800 exhibits impressive therapeutic activity in xenografts of human ovarian clear cell ovarian cancer (OCCC) and endometrioid ovarian cancer – two serious conditions of high unmet medical need with limited treatment options – including sustained tumour growth inhibition and regression at well-tolerated doses. The efficacy and tolerability data indicate a clear therapeutic window. We identified genetic loss of ARID1A, a component of the SWI/SNF chromatin remodelling complex, as predictive of greater therapeutic responsiveness. This correlation was confirmed in isogenic models. We have also observed promising therapeutic potential in additional cell lines including, gastric and small cell lung cancer, as well as haematological cancer. Gene expression profiling showed that NXP800 caused activation of the integrated stress response and inhibition of the heat shock response pathways. As PD biomarkers, we selected and validated CHAC1, ATF3, ATF4 and HSP27. We are currently investigating links between CHAC1, glutathione depletion and ferroptosis. Conclusions: A Phase 1 trial (NCT05226507) of NXP800 is underway, incorporating the validated predictive, PK and PD biomarkers that constitute a Pharmacological Audit Trail. Future expansion cohorts will include patients with ARID1A mutation, including ovarian clear cell carcinoma and ovarian endometrioid carcinoma. Conflict of interest: Ownership: Alterome Therapeutics, Black Diamond Therapeutics, Chroma Therapeutics, NextechInvest, Nuvectis Pharma, Storm Therapeutics Advisory Board: Alterome Therapeutics, Astex Pharmaceuticals, Black Diamond Therapeutics, CV6 Therapeutics, NextechInvest, Nuvectis Pharma, Storm Therapeutics, Vividion Therapeutics Board of Directors: Storm Therapeutics Corporate-sponsored Research: Astex Pharmaceuticals, AstraZeneca, BACIT, Cyclacel Pharmaceuticals, Merck KGaA, Nuvectis Pharma, Sixth Element Other Substantive Relationships: Chemical Probes Portal (non profit, Paul Workman, Executive Director)

Background: Acquired resistance to molecularly targeted therapeutics is a key challenge in personalised cancer medicine, highlighting the need for identifying the underlying mechanisms and early biomarkers of relapse, in order to guide subsequent patient management.Methods: Here we use human head and neck squamous cell carcinoma (HNSCC) models and nuclear magnetic resonance (NMR) spectroscopy to assess the metabolic changes that follow acquired resistance to EGFR tyrosine kinase inhibitors (TKIs), and which could serve as potential metabolic biomarkers of drug resistance.Results: Comparison of NMR metabolite profiles obtained from control (CAL(S)) and EGFR TKI-resistant (CAL(R)) cells grown as 2D monolayers, 3D spheroids or xenograft tumours in athymic mice revealed a number of differences between the sensitive and drug-resistant models. In particular, we observed elevated levels of glycerophosphocholine (GPC) in CALR relative to CALS monolayers, spheroids and tumours, independent of the growth rate or environment. In addition, there was an increase in alanine, aspartate and creatine+phosphocreatine in resistant spheroids and xenografts, and increased levels of lactate, branched-chain amino acids and a fall in phosphoethanolamine only in xenografts. The xenograft lactate build-up was associated with an increased expression of the glucose transporter GLUT-1, whereas the rise in GPC was attributed to inhibition of GPC phosphodiesterase. Reduced glycerophosphocholine (GPC) and phosphocholine were observed in a second HNSCC model probably indicative of a different drug resistance mechanism.Conclusions: Our studies reveal metabolic signatures associated not only with acquired EGFR TKI resistance but also growth pattern, microenvironment and contributing mechanisms in HNSCC models. These findings warrant further investigation as metabolic biomarkers of disease relapse in the clinic.

Acquired resistance to selective FLT3 inhibitors is an emerging clinical problem in the treatment of FLT3-ITD+ acute myeloid leukaemia (AML). The paucity of valid pre-clinical models has restricted investigations to determine the mechanism of acquired therapeutic resistance, thereby limiting the development of effective treatments. We generated selective FLT3 inhibitor-resistant cells by treating the FLT3-ITD+ human AML cell line MOLM-13 in vitro with the FLT3-selective inhibitor MLN518, and validated the resistant phenotype in vivo and in vitro. The resistant cells, MOLM-13-RES, harboured a new D835Y tyrosine kinase domain (TKD) mutation on the FLT3-ITD+ allele. Acquired TKD mutations, including D835Y, have recently been identified in FLT3-ITD+ patients relapsing after treatment with the novel FLT3 inhibitor, AC220. Consistent with this clinical pattern of resistance, MOLM-13-RES cells displayed high relative resistance to AC220 and Sorafenib. Furthermore, treatment of MOLM-13-RES cells with AC220 lead to loss of the FLT3 wild-type allele and the duplication of the FLT3-ITD-D835Y allele. Our FLT3-Aurora kinase inhibitor, CCT137690, successfully inhibited growth of FLT3-ITD-D835Y cells in vitro and in vivo, suggesting that dual FLT3-Aurora inhibition may overcome selective FLT3 inhibitor resistance, in part due to inhibition of Aurora kinase, and may benefit patients with FLT3-mutated AML.

PDB ID : 2YM7 Title : Crystal structure of checkpoint kinase 1 (Chk1) in complex with inhibitors Authors : Reader, J.C.; Matthews, T.P.; Klair, S.; Cheung, K.M.J.; Scanlon, J.; Proisy, N.; Addison, G.; Ellard, J.; Piton, N.; Taylor, S.; Cherry, M.; Fisher, M.; Boxall, K.; Burns, S.; Walton, M.I.; Westwood, I.M.; Hayes, A.; Eve, P.; Valenti, M.; Brandon, A.H.; Box, G.; vanMontfort, R.L.M.; Williams, D.H.; Aherne, G.W.; Raynaud, F.I.; Eccles, S.A.; Garrett, M.D.; Collins, I. Deposited on : 2011-06-06 Resolution : 1.81 Å(reported)

J. K. Boult, L. Perryman, G. Box, C. Jones, S. A. Eccles, and S. P. Robinson Cancer Research UK and EPSRC Cancer Imaging Centre, The Institute of Cancer Research and Royal Marsden NHS Trust, Sutton, Surrey, United Kingdom, Paediatric Oncology, The Institute of Cancer Research, Sutton, Surrey, United Kingdom, Cancer Research UK Cancer Therapeutics Unit, The Institute of Cancer Research, Sutton, Surrey, United Kingdom

Previous studies in sarcoma found that a composite gene signature, including high expression of nucleotide excision repair (NER) genes (XPG and/or ERCC1) and low expression of homologous recombination repair (HR) genes (BRCA1), identifies a highly sensitive population of patients with significantly improved outcome to trabectedin. This exploratory phase II trial evaluated a customized trabectedin treatment according to this gene signature in patients with non-small cell lung cancer (NSCLC) after the failure of standard platinum-based treatment.Patients were selected according to their mRNA expression (elevated XPG and/or ERCC1, with low BRCA1) using the following values as cutoff: XPG = 0.99, ERCC1 = 3.47 and BRCA1 = 12.00. Trabectedin was administered as a 1.3 mg/m2 3-hour intravenous infusion every 3 weeks (q3wk). The primary efficacy endpoint was the progression-free survival rate at 3 months. Objective response according to the Response Evaluation Criteria in Solid Tumors (RECIST) was a secondary efficacy endpoint.Two of 18 evaluable patients (11.1%; 95% CI, 1.38–34.7%) achieved progression-free survival rate at 3 months. The primary efficacy objective (at least 3 of 18 patients being progression-free at 3 months) was not met, and therefore the trial was early finalized. No objective responses per RECIST were achieved. Four patients had stable disease. Median PFS was 1.3 months, and median overall survival was 5.9 months. Trabectedin was usually well tolerated, with a safety profile similar to that described in patients with other tumor types.Customized treatment with trabectedin 1.3 mg/m2 3-h q3wk according to composite gene signature (XPG and/or ERCC1 overexpression, and BRCA1 underexpression) was well tolerated, but had modest activity in NSCLC patients pretreated with platinum. Therefore, further clinical trials with trabectedin as single agent in this indication are not warranted.

To investigate critical factors influencing the localization and antitumor effects of monoclonal antibodies (MAb) or toxic conjugates, we have adapted a single rat sarcoma, HSN, for preferential growth in the lungs, liver, and lymph nodes (the major sites of metastasis in humans) and have raised a panel of syngeneic rat MAbs to a stably-expressed cell surface antigen. Using this model we have shown that localization in tumors is significantly influenced by their anatomical location and vascularization, and the degree of MAb interaction with host cells. Uptake in small hepatic tumors was excellent, but access to lung tumors was limited by the poor permeability of pulmonary vessels. HSN cells transfected with the human IL-2 gene and coinjected in low numbers with parental tumors secreted sufficient cytokine to enhance the local permeability of vessels and doubled MAb localization in tumors without any systemic toxicity, suggesting that regional delivery of IL-2 may be used to enhance MAb localization in this situation. In order to extent the applicability of the model to studies of MAbs raised against human tumor targets, we have transfected the human c-erb B-2 gene (homolog of the ratneu) into the highly metastatic HSN.LV subline. MAbs raised against the external domain of the p185 product can now be screened for their ability to localize in metastases, and for various conjugates to inhibit tumor growth either independently of, or in association with, a fully functional immune system.

The formation of new blood vessels (angiogenesis) is required for the growth of most tumors. The tumor microenvironment also induces lymphangiogenic factors that promote metastatic spread. Anti-angiogenic therapy targets the mechanisms behind the growth of the tumor vasculature. During the past two decades, several strategies targeting blood and lymphatic vessels in tumors have been developed. The blocking of vascular endothelial growth factor (VEGF)/VEGF receptor-2 (VEGFR-2) signaling has proven effective for inhibition of tumor angiogenesis and growth, and inhibitors of VEGF-C/VEGFR-3 involved in lymphangiogenesis have recently entered clinical trials. However, thus far anti-angiogenic treatments have been less effective in humans than predicted on the basis of pre-clinical tests in mice. Intrinsic and induced resistance against anti-angiogenesis occurs in patients, and thus far the clinical benefit of the treatments has been limited to modest improvements in overall survival in selected tumor types. Our current knowledge of tumor angiogenesis is based mainly on experiments performed in tumor-transplanted mice, and it has become evident that these models are not representative of human cancer. For an improved understanding, angiogenesis research needs models that better recapitulate the multistep tumorigenesis of human cancers, from the initial genetic insults in single cells to malignant progression in a proper tissue environment. To improve anti-angiogenic therapies in cancer patients, it is necessary to identify additional molecular targets important for tumor angiogenesis, and to get mechanistic insight into their interactions for eventual combinatorial targeting. The recent development of techniques for manipulating the mammalian genome in a precise and predictable manner has opened up new possibilities for the generation of more reliable models of human cancer that are essential for the testing of new therapeutic strategies. In addition, new imaging modalities that permit visualization of the entire mouse tumor vasculature down to the resolution of single capillaries have been developed in pre-clinical models and will likely benefit clinical imaging.