Diving into the dark kinome: lessons learned from LMTK3

With >500 proteins crucially implicated in a large variety of physiological processes and diseases, the human kinome represents an invaluable source of putative targets with great potential for therapeutic intervention. However, exploring the human kinome as a whole is an ambitious goal still far from being accomplished. Nearly two decades ago, Manning et al. [1] defined a set of 518 protein kinases throughout the human genome. To date, the list of human kinases has substantially expanded counting around 634 kinases, which include both conventional active and ‘catalytically dead’ kinases [2]. These so-called pseudokinases have gained increased attention over the last years in light of their essential non-catalytic roles in signalling pathways.

Nevertheless, our knowledge of protein targets is confined to a small, yet highly investigated, portion of the kinome, whereas ~25% of the human kinases are considered to be poorly studied, labelled as ‘dark’ kinases [2]. For quite a long time, academic research has consistently been quite biased towards kinases that have already been intensively studied. This is also mirrored by the lasting dominance of certain ‘favoured’ drug targets, especially in oncology. Consequently, a great fraction of the kinome remains without functional annotation, whereas only ~8% of the kinome has so far been effectively targeted for the treatment of cancer [2].

Such a great knowledge disparity, which could also be translated into an opportunity, was addressed by the US National Institutes of Health (NIH) and led to the establishment of the Illuminating the Druggable Genome programme (IDG) [3]. This project was developed with the purpose of encouraging the study of ‘dark’ proteins that may hold therapeutic relevance and it specifically focused on the understudied members of three large protein families. These included G protein-coupled receptors, ion channels, and kinases, which offer a rather high number of drug targets. As part of the IDG project, the Kinase Data and Resource Generating Center along with its Dark Kinase Knowledgebase (DKK) and the linked DKK expression browser were developed to specifically enhance our understanding of neglected kinases (Table 1) [4]. This highlights how the appreciation towards the ‘dark’ kinome continually increases.

Table 1 The Dark Kinase Knowledgebase (DKK) list of understudied kinases (in alphabetical order) [4].

Over the last years, a great number of largely uncharacterised kinase targets have emerged from screening studies and genome sequencing efforts. Back in 2011, LMTK3 (lemur tyrosine kinase 3), the core protein of interest in our laboratory, was initially identified as a promising therapeutic target in breast cancer through a kinome siRNA screen that aimed to uncover regulators of the estrogen receptor α (Fig. 1) [5]. Determining and studying protein function can certainly be challenging. Not surprisingly, research decisions can frequently be guided by the existence of relevant knowledge and tools. As a consequence, highly studied kinases are regularly given priority over understudied ones, mainly due to the unpredictable nature of research that may sometimes lead towards the search for a balanced compromise between scientific potential and associated risks. In the case of LMTK3, structural and functional studies aiming to delineate the exact role of LMTK3 in cellular signalling represented a stimulating opportunity that opened a new avenue for investigation. However, the limited relevant literature or insufficient publicly accessible information, lack of antibodies as well as readily available experimental tools presented significant obstacles to overcome.

Fig. 1: The location of LMTK3 in the phylogenetic tree of the human kinome [1, 12].

Adapted from the Dark Kinase Knowledgebase (DKK) [4].

Ten years later, LMTK3 is an established cancer driver known to act through diverse mechanisms [5,6,7,8,9,10,11,12,13,14]. Concisely, the crystal structure of the LMTK3 kinase domain has now been solved and its consensus phosphorylation motif has been determined [12]. In addition, interrogating the signalling networks of LMTK3 revealed a highly versatile functional spectrum for this previously underinvestigated molecular target. Most importantly, a potent and selective small-molecule inhibitor against LMTK3, namely ‘C28’, has been identified and characterised. Collectively, our work has cast considerable light on LMTK3 providing the academic community with data as well as research tools for the study of LMTK3, while further proving the potential clinical value of ‘dark’ kinases as relevant cancer targets. Intriguingly, the Clinical Kinase Index [2], a newly developed kinase-prioritisation method aiming to promote the validation and study of understudied kinase targets in cancer, ranked LMTK3 amongst the most clinically relevant kinases across a wide range of cancer types (Table 2). LMTK3, as is the case for other understudied kinases, could serve as a notable example of a druggable target for cancer therapy. Such efforts may potentially lead to the initiation of new drug discovery programmes, while elucidating the role of the numerous, yet unexplored kinases.

Table 2 The highest-scoring understudied protein kinases across different cancer types based on the Clinical Kinase Index (CKI) (in alphabetical order) [2].

Since our present view of cellular signalling is that of an intricate, cooperative and dynamic network, the overriding aim would be to attempt to shed some more light on the ‘dark’ kinome. In fact, only by widening our knowledge, we can broaden the therapeutic horizon and therefore increase the possibility of successfully addressing currently unmet clinical needs. With the development of a growing number of online resources that can provide a comprehensive overview of the up-to-date knowledge surrounding any kinase, highly relevant and rapidly available information can be used to experimentally assess the potential function of a specific kinase of interest. Leveraging these tools and the multiple improved technologies could help define the functional signature of these underprivileged proteins.

In summary, our work on LMTK3 clearly demonstrates the achievability and benefits of functionally characterising a specific ‘dark’ kinase, while narrowing the gap between the intensively studied and less-understood kinases. As in the case of LMTK3, several opportunities could arise from such an endeavour, including the further mapping of biological pathways, the unveiling of the role of ‘dark’ kinases, and the discovery of novel targets. We hope that LMTK3 could serve as an example that may provide other researchers with some of the necessary incentive to take a deep breath and dive into the ‘dark’ kinome.