The conformational dynamics of HER2-HER3 heterodimerisation are an important consideration for evaluating existing and future targeted therapy intervention strategies against HER2+ breast cancer and other HER family driven cancers. Here, we show that the HER2 inhibitor lapatinib is paradoxically able to promote proliferative behaviour in HER2+ breast cancer cells when administered in the presence of the HER3 ligand NRG. The synergy between growth factor and inhibitor requires an intricate, multi-step cascade of conformational events.

Lapatinib itself is able to promote heterodimerisation between the kinase domains of HER2 and HER3, stabilising an orientation consistent with a symmetric, head-to-head kinase domain heterodimer that is distinct from the canonical, asymmetric, head-to-tail active kinase domain orientation that occurs throughout the EGFR family. An analogous interface has previously been observed in the HER3 kinase domain crystal lattice (Jura et al., 2009b); here, we have provided modelling and cellular evidence of a heterodimer with an interface consistent to the one observed in the HER3 kinase domain crystal lattice. Sequestering HER2 and HER3 in these inactive, lapatinib-bound heterodimers was of benefit to NRG-mediated proliferative signalling. Our results, in which inhibitor binding drives dimer formation that boosts signalling and proliferation, shows some parallels with the inhibitor-induced signalling phenotypes in the RAF-family (Eyers et al., 1998; Hatzivassiliou et al., 2010; McKay et al., 2011; Poulikakos et al., 2010; Thevakumaran et al., 2015)

While the FRET-FLIM analysis of the lapatinib-induced dimerisation was not able to differentiate between heterodimers or higher order oligomers, our clustering data shows that lapatinib is likely to induce higher order oligomers. Because of the modelled symmetrical nature of these lapatinib-induced dimers, in which both lapatinib-bound HER2 and HER3 would be conformationally available as ‘activator’ receptors for additional oligomerization partners, it is not inconceivable they may act as nucleation points for larger oligomeric signalling platforms. Such signalling arrays, in which mutual cooperativity increases signaling output, have been proposed for EGFR oligomers (Huang et al., 2016).

The addition of ligand potentially causes rearrangements within these platforms through the ligand-induced conformational ballet of multi-level interactions between the various extracellular and intracellular domains of EGFR family receptors (reviewed in (Lemmon et al., 2014)). The formation of lapatinib-induced oligomeric platforms may facilitate a transition into active signalling heterodimers upon ligand binding, due to the availability of dimerisation partners in immediate proximity within these drug-induced oligomer platforms.

Both the lapatinib-induced HER2-HER3 heterodimerisation and the downstream lapatinib-NRG synergistic effects on proliferation depended on the ability of HER3 to bind ATP. Although usually classified as a pseudokinase, HER3 has been shown to retain a measure of autophosphorylation activity (not transphosphorylation) under specific circumstances (Shi et al., 2010). We show HER2-HER3 heterodimerisation and downstream proliferative effects can be elicited by the addition of the HER3-binding inhibitor bosutinib, indicating that nucleotide binding pocket occupation performs a structural role that is critical to HER3 function, and apparently independent of any retained catalytic activity. Observing increased heterointeractions and cellular proliferation due to inhibition of an activity-deficient kinase is a strong indication of the importance of ATP-binding in certain pseudokinases, and the necessity of pocket-occupied structural conformers in sustaining protein–protein interactions and subsequent downstream signalling output.

Because of the importance of HER3 in HER2-targeted therapy resistance, its conserved ATP binding raised the possibility of targeting HER3 with ATP-competitive kinase inhibitors. Our data show, however, that stabilisation of the HER3 kinase domain with an ATP-competitive kinase inhibitor can have a stimulating effect on HER2+ breast cancer cell proliferation. This indicates that the development of small molecule targeted therapy against HER3 for use in HER2+ breast cancer needs to be directed away from stabilising the HER3 ATP binding pocket occupied conformer and rather towards stabilising the apo, inactive conformer. An exception to this might be the development of irreversible, adamantane-linked inhibitors of HER3 that target the receptor for proteosomal degradation (Xie et al., 2014).

The substantial effect that lower doses of lapatinib have on proliferation in the presence of growth factor may have an impact on the establishment of lapatinib-resistance in vivo. This is in accordance with the observation from xenograft models that resistance occurs much more readily if lapatinib is administered continuously at low doses than if it’s used intermittently at high dose (Amin et al., 2010). Increased production of growth factors (including NRG) is a well-described resistance mechanism against HER2-targeted therapy (reviewed in [Claus et al., 2014]). NRG production by the microenvironment has also been shown to play a role in metastatic spread of ovarian cancer cells that express high levels of HER3 (Pradeep et al., 2014). High expression levels of NRG in HER2+ breast cancer patients showed a strong correlation with disease recurrence (Xia et al., 2013). Several somatic mutations in HER3 observed in cancer fall within the extracellular domain and have a potential effect on ligand-binding affinity (Jaiswal et al., 2013). These mutations may exacerbate the inhibitor-growth factor synergistic behaviour reported here.

Our results provide a potential molecular mechanism for the disappointing results observed in a recent Phase III study of lapatinib used in an adjuvant setting (ALTTO trial) (Piccart-Gebhart et al., 2016). The lapatinib-only arm of this study was terminated prematurely, and the effects observed in the adjuvant setting for both lapatinib-trastuzumab co-treatment and trastuzumab treatment followed by lapatinib were not significant. These clinical results indicate there are complicating factors in hindering lapatinib efficacy in patients, which may involve the expression levels of HER3 and NRG stimulation by a complex tumour microenvironment. The complex relationships between distinct protein conformation dynamics, formation of oligomeric assemblies, the availability of ligand, and the various effects on downstream signalling all need to be considered when applying targeted therapy to avoid potentially unexpected enhanced cancer cell proliferation after inhibitor treatment.