How ARNT “protein binding competition” can influence cancer cell resistance to targeted therapies.
BRAFV600E mutations drive aggressive tumor growth in multiple cancer types by activating the MAPK proliferation pathway. While BRAFV600E-targeted therapies show initial promise, most patients develop resistance through escape mechanisms. Our study investigated these resistance pathways in thyroid cancer (TC) and melanoma to develop new treatment strategies. The first remarkable finding of our genome-wide CRISPR genetic knockout screening in TC is that the different strategies used by BRAFV600E TC cells to acquire resistance against targeted therapies are often also found in other BRAFV600E+ cancer types, including lung and skin cancer1, suggesting that resistance transcends cell-specific contexts.
Loss of AhR promotes resistance to targeted therapies.
We also got intrigued by the presence of Aryl hydrocarbon receptor (AhR) as a top promoter of targeted therapy resistance. AhR is an environmental sensor integrating extracellular, endogenous, and metabolic signals to equilibrate cell activity. In cancer, the role of AhR is complex and context-dependent; both pro-oncogenic and tumor-suppressive effects of the receptor have been reported2. AhR was among the top hits on our screen: Knocking out AhR offered a selective advantage to TC and melanoma cells against targeted therapies. We validated the screening results and presented evidence that indeed AhR knockout TC and melanoma cells are more resistant to the combined treatment of dabrafenib and trametinib than cells expressing AhR.
Transcriptomic investigations gave us the first mechanistic clue, revealing elevated expression of genes linked to SMAD3. However, we found no evidence of changes in phosphorylation levels of the TGF-β/SMAD pathway in AhR knockout cells, Akt pathways, or any of the 37 phosphoproteins tested, meaning that a different resistance mechanism was at play.
Coupling competition for ARNT
Transcriptomics also revealed higher expression levels of genes regulated by HIF-1α, which is known to heterodimerize with ARNT (HIF-1β) proteins3. We hypothesized that suppressing AhR could liberate ARNT availability and allow it to seek concurrent binding partners. We confirmed by co-immunoprecipitation of ARNT binding partners that in the absence of AhR (and consequently AhRR), SMAD3 and SMAD2 have higher interactions with ARNT. Molecular docking modelization supported the co-immunoprecipitation data and predicted that the binding priority for ARNT is AhRR > AhR > SAMD3 > SMAD2 > HIF-1α.
Thus, the loss of AhR permits a higher level of interaction between ARNT and SMAD2/3 binding, promoting increased expression of TGF-β-dependent genes. This suggests a remarkably intricate regulatory complex of gene transcription, with ARNT at the center, in which AhR, AhRR, HIF-1α, and SMAD2/3, among others, compete to heterodimerize. The capacity to bind to AhR seems to be defined by affinity, presence of ligands, activation, and protein levels in the nucleus. Another similar transcription factor binding competition occurs for RUNX1, between FOXP3 and RORγt, and direct Th17 and Treg polarisation4.
Revisiting resistance
We were surprised to find a resistance mechanism in which no mutation or epigenetic changes are directly involved. Our findings suggest that cancer cells can develop drug resistance through protein binding shifts rather than genetic mutations. The lack of AhR activation due to the cell metabolic profile can also reduce AhR levels in the nucleus, facilitating ARNT coupling with SMADs or other co-transcription factors and promoting a more resistant phenotype. Determining how often this mechanism influences drug resistance could open new therapeutic avenues.
Acknowledgement:
Cover and figure 1 illustrations by Michael Konomos, MS, CMI. Emory University.
Atlanta, GA, USA.