Endocrine Abstracts

Introduction: LMTK3 is an oncogenic Receptor Tyrosine Kinase (RTK) implicated in resistance to endocrine therapy in breast cancer. Initially, LMTK3 was described as a regulator of Estrogen Receptor alpha (ERα) since it was found able to protect it from ubiquitin-mediated proteasomal degradation. In a cohort of breast cancer (BC) patients (n>600), LMTK3 protein levels and intronic polymorphisms were significantly associated with disease-free and overall survival and predicted response to endocrine therapy. These data were validated in an Asian cohort in which it was shown that LMTK3 was associated with more aggressive tumours. In addition, LMTK3 was demonstrated to contribute in BC invasion and migration. Recently, a new scaffolding function of LMTK3 was described that results in cancer progression through chromatin remodelling.

Aim: This study aims to identify selective LMTK3 inhibitors that can be used to enable pathway investigation and establish onward tractability of these compounds for future translational activities.

Materials and methods: The Bellbrook Laboratories Transcreener® assay kit was employed and 30,000 compounds were screened to detect novel LMTK3 inhibitors. Nearly 100 of them significantly inhibited LMTK3 activity and were therefore chosen for 10-point concentration-response profiling in duplicate and LC-MS analysis. The top 50 test compounds were clustered into unique chemotypes and were further tested using radiolabelled in vitro kinase assays. The top 5 compounds from two chemotypes were selected to be evaluated with hit-to-lead medicinal chemistry. Subsequently, an active site-directed competition binding assay (DiscoveRx KINOMEscan) was used to quantitatively measure the interactions between the top 5 hit compounds and more than 450 purified human kinases and disease relevant mutant variants.

Results and discussion: Two (C28 and C36) out of the 30,000 compounds that were screened inhibited by >95% the activity of only 10 and 8 kinases respectively. Moreover, the S(35) selectivity index of C28 was 0.186 while the selectivity index of C36 was 0.114. Interestingly, quantitative analysis of 38 kinase inhibitors currently used in clinical oncology showed a comparably low S(35) score as C28 and C36. It is expected that the crystallization of the LMTK3 kinase domain that is currently being conducted as well as co-crystallization experiments with these inhibitors and other analogues will guide a rational optimisation strategy of each chemotype.

Conclusion: More work is required; however, these data represent a step towards the development of the first LMTK3 inhibitors that may have potential for broad clinical utility in breast cancer.