New study finds classical RAS proteins are not essential for RAF inhibitor-induced paradoxical ERK activation

The classical RAS family of proteins functions as molecular switches at the cell’s plasma membrane where it cycles between an active and inactive state. The active state initiates a cascade of events that promote downstream signaling to regulate cellular functions, such as cell proliferation. 

Mutations and dysregulation of the RAS-RAF-MEK-ERK signaling pathway are among the most common causes of cancer. The National Cancer Institute’s RAS Initiative aims to discover more about  RAS proteins and ways to modulate their signaling involved in cancer. 

Potential roadblocks in disrupting the pathway 

To block cancer-associated signaling in the RAS-RAF-MEK-ERK pathway, previous research developed drugs to inhibit RAF, the second step of the signaling cascade. Frederick National Laboratory RAS Initiative scientist Nicole Fer calls RAF and RAS “partners in crime” since RAF binds to RAS. “RAF is an effector,” she said. “It is one of the major players in helping RAS to start the signaling cascade.” 

Three generations of RAF inhibitors have been developed to target this pathway and to block the signaling response downstream of RAS. However, targeting RAF has not provided therapeutic benefits to patients with RAS-driven cancers. RAF inhibitors were found to cause “paradoxical ERK activation.” Rather than turning off the signal, RAF inhibitors instead can activate it through mechanisms that are not yet fully understood, however, the current consensus is that it is RAS-dependent.  

In the recent study published in the Proceedings of the National Academy of Science (PNAS) by Fer and her FNL colleagues, they discovered that other RAS proteins may be involved in the paradoxical ERK activation. The activation occurred even in the absence of KRAS, NRAS and HRAS in certain cellular contexts. 

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Scientific graphic in two panels
Panel A: Active RAS is present. It binds to the RBD (RAS binding domain) of RAF and recruits it to the membrane where several events, including phosphorylation, dephosphorylation and conformational changes occur to activate RAF. Once activated, RAF phosphorylates and activates MEK, MEK then activates ERK by phosphorylation. Phosphorylated ERK subsequently leads to the activation of several transcription factors that lead to proliferation and development of cancer. Rather than turning off the ERK signal, the RAF inhibitors activate it instead. 
Panel B: According to PNAS paper, when classical RAS is absent or inhibited (such as with G12C inhibitors), MRAS likely binds to RAF directly through the RBD, but weakly. This interaction is further enhanced  when RAF inhibitors are present due to the open and active conformation of the inhibitor-bound RAF which allows for easier MRAS access. Once MRAS interacts with RAF, the activation cascade begins: MEK then ERK and finally the transcription factors that increase proliferation and cancer development, all in the absence of classical RAS. 

Using engineered cell lines that lack all three classical variants of RAS, Fer and her colleagues treated with a pan-RAF inhibitor and surprisingly found paradoxical ERK activation. They observed that MRAS – a non-classical variant of RAS – was compensating for the absence of the classical protein and activating the signal.  

“We always thought it (paradoxical activation of ERK signal) was RAS-dependent,” Fer said. “But even when you remove the classical RAS proteins, you have other RAS-related proteins that can come in and do the job. MRAS compensates for the lack of classical RAS proteins. It is the key player here.” 

Fer said the study highlights the need to better understand some of the other poorly characterized potential players that emerge in what she calls the “whack- a-mole” game scientists play when attempting to inhibit KRAS. “Compensatory mechanisms of resistance will be a concern when inhibiting something as critical as KRAS in cancer growth and this time the target to follow is MRAS,” she said. 

Implications for clinical care 

Fer and her colleagues’ findings could be relevant for patients being treated with the first KRAS-blocking drug, approved by the U.S. Food and Drug Administration  in May 2021. Sotorasib is used to treat patients with advanced non-small cell lung cancer carrying KRAS-G12C mutations. This study suggests that a treatment regimen that includes a combination of a KRAS inhibitor and a RAF inhibitor is may not be optimal. It could result in paradoxical activation of the ERK pathway, thereby encouraging the cancer growth rather than inhibiting it.  

“We have found that in most cellular contexts, when KRAS is inhibited, MRAS protein expression significantly increases in an effort to compensate and there is the potential over time for this to contribute to the resistance to KRAS inhibition.” Fer said.