Oncogenic protein condensates suppress growth factor perception and modulate drug tolerance

Abstract

Drug resistance remains a central challenge towards durable cancer therapy, including for cancers driven by the EML4-ALK oncogene. EML4-ALK and related fusion oncogenes form cytoplasmic protein condensates that transmit oncogenic signals through the Ras/Erk pathway. However, whether such condensates play a role in drug response or resistance development is unclear. Here, we applied optogenetic functional profiling to examine how EML4-ALK condensates impact signal transmission through transmembrane receptor tyrosine kinases (RTKs), a common route of resistance signaling. We found that condensates dramatically suppress signaling through activated RTKs including EGFR. Conversely, ALK inhibition restored and hypersensitized RTK signals. Modulation of RTK sensitivity occurred because EML4-ALK condensates sequestered downstream adapters that are required to transduce signals from both EML4-ALK and ligand-stimulated RTKs. Strikingly, EGFR hypersensitization resulted in rapid and pulsatile Erk signal reactivation within 10s of minutes of drug addition. EGFR reactivation originated from paracrine signals from neighboring apoptotic cells, and reactivation could be blocked by inhibition of either EGFR or matrix metalloproteases. Paracrine signals promoted survival during ALK inhibition, and blockade of paracrine signals accelerated cell killing and suppressed drug tolerance. Our results uncover a regulatory role for protein condensates in cancer signaling and drug response and demonstrate the potential of optogenetic profiling for drug discovery based on functional biomarkers in cancer cells.

Simple visualization of submicroscopic protein clusters with a phase-separation-based fluorescent reporter

“…We validated this fast decay through a fixed-cell time course experiment and obtained similar fast decay of the EML4-ALK/CluMPS condensates (t 1/2 = 3.5 minutes) ( Supplementary Figure 10 ). This faster measured rate may reflect the differences in measuring endogenous aggregates versus aggregates of overexpressed EML4-ALK, as performed previously 79 . We also observed that, despite extended drug treatment, ALKi did not induce full dissociation of CluMPS condensates, in line with previous reports showing that EML4-ALK (V1) condensates are relatively stable 10, 80 .…”

“…The ability to visualize endogenous aggregates allowed us to track their dynamics in response to the ALK inhibitor crizotinib ( Figure 5F, Supplementary Figure 11, Supplementary Movie 4 ). Although it is known that crizotinib blocks EML4-ALK activity and suppresses its condensation, in part due to dissociation of Grb2 10, 79, 80 , the kinetics and extent of condensate dissolution has not been observed in living cells. We found that, upon drug addition, CluMPS condensate area rapidly decreased within 10 minutes, followed by a slow but measurable decrease thereafter ( Figure 5G, Supplementary Figure 11 ).…”

Principles and functions of condensate modifying drugs

“…Resistance to cancer therapies like cirzotinib against fusion oncogenic proteins, such as EML4-ALK in non-small cell lung cancer (NSCLC), can be explained by a localizer c-mod mechanism of action ( Gonzalez-Martinez et al, 2022 ). EML4-ALK is a chimeric receptor tyrosine kinase, hyperactivation of which drives cancer.…”

“…Drug resistance to EML4-ALK-targeted therapies results in a post-therapy rescue of EGFR signaling. Activators of EGFR signaling are sequestered and inactivated by EML4-ALK condensates ( Gonzalez-Martinez et al, 2022 ). Upon treatment with EML4-ALK inhibitor and localizer c-mod cirzotinib ( Table 1 and shown in Figure 2A ), the EGFR activators are re-localized away from the EML4-ALK condensate community resulting in an active EGFR signaling driven drug resistance.…”

“…A clinical trial for the combined inhibition for EML4-ALK and EGFR by cirzotininb and erlotinib, respectively failed ( Ou et al, 2017 ). Current understanding of the condensate-based mechanisms of EGFR signaling might allow us to discover additional specific inhibitors to target activators of the signaling pathway downstream of the current target of erlotinib, the kinase active site of EGFR ( Gonzalez-Martinez et al, 2022 ). For example, a novel localizer c-mod may restrict the migration of EGFR activators from the EML4-ALK condensate.…”