Tracking and blocking interdependencies of cellular BRAF-MEK oncokinase activities

Abstract

The selective targeting of mutated kinases in cancer therapies has the potential to improve the therapeutic success and thereby the survival of patients. In the case of melanoma, the constitutively active MAP kinase pathway is targeted by combinatorial inhibition of BRAF and MEK activities. These MAPK pathway players may display patient-specific differences in the onco-kinase mutation spectrum, which needs to be considered for the design of more efficient personalized therapies. Here, we extend a bioluminescence-based kinase conformation biosensor (KinCon) to allow for live-cell tracking of interconnected kinase activity states. First, we show that common MEK1 patient mutations promote a structural rearrangement of the kinase to an opened and active conformation. This effect was reversible by binding of MEK inhibitors to mutated MEK1, as shown in biosensor assays and molecular dynamics simulations. Second, we implement a novel application of the KinCon technology for tracking the simultaneous, vertical targeting of the two functionally linked kinases BRAF and MEK1. Thus, we demonstrate that, in the presence of constitutively active BRAF-V600E, specific inhibitors of both kinases are efficient in driving MEK1 into a closed, inactive conformation state. We compare current melanoma treatments and show that combinations of BRAFi and MEKi displayed a more pronounced structural change of the drug sensor than the respective single agents, thereby identifying synergistic effects among these drug combinations. In summary, we depict the extension of the KinCon biosensor technology to systematically validate, anticipate, and personalize tailored drug arrangements using a multiplexed setup.

Impact of protein and small molecule interactions on kinase conformations

“…Overall these findings underline that the KinCon technology can be extended to track the impact of binary protein complexes and related cancer mutations on kinase activity dynamics. Moreover, this data demonstrated that in contrast to the previously published MEK1 and BRAF KinCon reporter ( Fleischmann et al, 2021 , Fleischmann et al, 2023 , Röck et al, 2019 , Mayrhofer et al, 2020 ), the more closed STRADα and LKB1 KinCon reporter conformations represent the more active full-length kinase states.…”

“…We have previously applied the technology to gain insights into the functioning of two kinases that belong to the MAPK pathway. In these proof-of-concept studies, we showed that BRAF and MEK1 KinCon reporters are direct real-time readouts for kinase activities in intact cell settings caused or altered by mutation and drug treatments ( Röck et al, 2019 , Mayrhofer et al, 2020 , Fleischmann et al, 2021 , Fleischmann et al, 2023 ).…”

“…One cell-based technology for studying cellular kinase activity states is the KinCon reporter technology ( Röck et al, 2019 , Mayrhofer et al, 2020 , Fleischmann et al, 2023 ). It is a highly sensitive assay for tracking kinase activities which is mirrored by the alterations of full-length kinase structures in cells.…”

“…We provide a summary of the changes in KinCon activity conformations. We have previously shown that for both kinases of the MAPK pathway, MEK1 and BRAF, the respective inhibitors affect primarily the active kinase conformation ( Röck et al, 2019 , Mayrhofer et al, 2020 , Fleischmann et al, 2021 , Fleischmann et al, 2023 ). Exemplarily we illustrate that both kinases which are activated through cancer patient mutations (BRAF-V600E and MEK1-K57E) bind the respective kinase inhibitor, resulting in a change in the activity conformations ( Figure 5A and B ).…”

“…Here, we show that genetically encoded KinCon reporters are extendable to many more kinases, to enable systematic monitoring of cellular kinase activity states in living cells. In addition to its predictive capabilities, the KinCon technology serves as a valuable tool for uncovering cellular factors influencing drug efficacy ( Mayrhofer et al, 2020 , Fleischmann et al, 2023 , Röck et al, 2019 ). Such insights into the molecular structure dynamics of kinases in intact cells upon interactions with small molecule inhibitors or regulatory proteins is necessary for the design of more effective therapeutic strategies.…”