An essay I wrote for a contest. I didn’t win, but this serves as (I hope) a nice summary of the questions we’re trying to answer particularly for any TAM-interested folks.
Multicellular organisms require communication and coordination both within and between cells. Cellular information sharing occurs through a variety of components including cytokines, growth factors, and hormones, which diffuse across the cell membrane or use transmembrane carriers. These signals operate in concert, and so a relaxation of reductionism in the form of systems biology can more completely capture their effects. Receptor tyrosine kinases (RTKs) are a class of these receptors which transduce extracellular information to modulate and coordinate intracellular processes. Cancer is in part a breakdown of intercellular regulation, and cancer cells frequently utilize RTKs to drive many hallmarks of the disease.
Accordingly, targeting RTKs has been therapeutically effective in a subset of tumors. The ultimate benefit of these therapies, however, is limited by resistance. Resistance occurs through a panoply of mechanisms, including mutation of the drug target to block the effect of therapy, amplification of the drug target to overcome inhibition, and “bypass” switching to alternative pathways not targeted by therapy. In the case of RTK-targeted therapies, often non-targeted RTKs may become activated to provide bypass resistance. One RTK in particular, AXL, while not accompanied by oncogenic mutations, has frequently been identified as a resistance mechanism to targeted therapies. AXL potently drives metastatic dissemination of cancer cells at the same time, and so its activation is especially dire. Despite its importance as identified through genetic studies, little was known about the signaling function of AXL. Therefore, as a graduate student in Douglas Lauffenburger and Frank Gertler’s laboratories at MIT, I became interested in defining how AXL signals, to identify when and where targeting the receptor might be effective.
Information transmission through AXL. Left) Triple-negative breast carcinoma cells frequently co-express AXL and EGFR. Transactivation of AXL by EGFR serves to amplify a subset of pathways downstream of both RTKs. This amplification results in qualitatively distinct EGFR signaling and drives cell invasion. Right) The activity of TAM (Tyro3, AXL, MerTK) receptors depends upon interaction of their ligands with phosphatidylserine. Signal is transduced from lipid to ligand and receptor by constraining the diffusion of ligand-receptor complexes, leading to dimerization.
RTKs frequently act in concert to drive specific phenotypic outcomes. When cells develop resistance to RTK-targeted agents, it can involve co-activation of receptors, and inhibiting combinations of these receptors is then required to overcome resistance. However, the role of these combinations—where multiple receptors are simultaneously important to cancer cell survival—perplexed me. The combination must provide something not available through either receptor alone. We wondered whether RTK co-activation may play a role in redirecting cell response to extracellular growth factor cues. Examining the response of breast carcinoma cells to EGF, I found that EGFR transactivates AXL. Varying the amount of EGFR activation with and without AXL present showed that this serves to quantitatively amplify the activation of certain pathways, producing a qualitatively distinct signaling response. This pattern of activation potently promoted the migration response to EGF over direct EGFR signaling itself. Using a new experimental approach of chemically cross-linking receptors to one another and then quantifying the pairs of cross-linked receptors in parallel, I identified that diffusional proximity of receptor pairs was predictive of their cross-talk capacity. Thus, RTK co-activation not only can lead to therapeutic resistance but endows cells with novel phenotypic traits, and cross-talking receptors are closely localized. Through their communication, the sum of RTK activation was greater than the individual receptor parts.
But how was the activity of AXL itself regulated? RTKs are often, on a most basic level, growth factor concentration sensors. Taking advantage of this observation, RTK signaling is most commonly studied by removing growth factors from cell culture to reduce a receptor’s activity, reintroducing the growth factor after a period of time, then measuring the resulting dynamic responses. This basic experiment frequently does not work for AXL however; adding the AXL ligand Gas6 leads to no measurable phosphorylation response on its own. To explain this perplexing lack of response, I built a mathematical model of the receptor’s binding processes and fit it to our experiments lacking the expected activation. From there, it was clear: ligand was bound to the receptor, but the receptor-ligand complexes had to be brought together more tightly somehow for activation. AXL’s ligand, Gas6, simultaneously binds to a lipid, phosphatidylserine (PS), which is normally found only inside cells but is exposed during processes such as apoptosis, T cell activation, and photoreceptor turnover. The importance of PS for activating AXL has been recognized since 1997, almost as long as its ligand has been known, yet how the lipid functions to promote activation has remained elusive. In contrast to PS activating AXL through some conformational change transmitted through the ligand-receptor complex, our model pointed toward its role being to shepherd AXL-Gas6 complexes together. Indeed, by varying the amount of PS added to culture, we could see a biphasic response where very high concentrations of PS in fact inhibited AXL activation. As expected by the model, PS moieties served as a molecular corral for diffusing receptor-ligand complexes thus promoting activation of the receptor within limited spots on the cell surface. In a sense, the AXL-Gas6 complex itself is the receptor—for spots of PS presentation. To sense this important lipid complex, our cells have constructed this elegant, diffusion-driven sensor of ligand localization that is robust to changes in ligand concentration.
These studies provided a first quantitative analysis of how AXL functions within cells. Later studies have continued to elucidate the role of AXL in cancer including how and when it mediates resistance to targeted therapies in other tumor types. AXL and the TAM receptor family to which it belongs have accrued considerable interest due to their widespread roles in the immune system. The molecular features that drive their activation will help to understand where, how, and when these receptors are activated. In cancer, the microenvironmental changes that lead to activation of AXL will allow us to identify the patients who will benefit from targeted therapies against the receptor.
More generally, this work shows the essential role systems-level studies will play uncovering the etiology of and therapeutic opportunities in complex diseases. In each case, focusing on a single manipulation or measurement would have prevented us from learning how these systems of molecular processes function. Strikingly, even a single receptor-ligand pair can pass information from the extracellular environment to a cell in a subtle and complex manner. We will need the systems biology toolbox on even these relatively focused scales to decipher how molecular and cellular components function together to produce cells, tissues, and ourselves.