Functional measurement of mitogen-activated protein kinase pathway activation predicts responsiveness of RAS-mutant cancers to MEK inhibitors

Highlights

• Various RAS mutations and impact of the mitogen-activated protein kinase (MAPK)/ERK kinase (MEK) inhibitor is not well established.

• RAS mutants have variable levels of MAPK activity as assessed by a functional assay.

• High MAPK activity demonstrated longer progression-free survival with MEK inhibitors.

Abstract

Background

RAS variant–related functional impact on the mitogen-activated protein kinase (MAPK) pathway, and correlation between MAPK activation and MAPK/ERK kinase (MEK) inhibitor responsiveness, is not established.

Patients and methods

Of 1,693 tumours sequenced, 576 harboured a RAS alteration; 62 patients received an MEK inhibitor (MEKi) and had RAS mutations that were functionally characterised. We report that RAS mutants have variable levels of MAPK activity, as measured by a functional cell-based assay that quantified MAPK pathway activation after transfection with a variety of RAS mutations.

Results

Patients with tumours harbouring RAS alterations with high versus low MAPK activity who were treated with an MEKi showed significantly longer median progression-free survival (PFS) (5.0 vs. 2.3 months; p = 0.0034) and overall survival (20.0 vs. 5.0 months; p = 0.0146) and a trend towards higher rates of clinical benefit (stable disease ≥6 months or partial/complete remission) (38% versus 15%; p = 0.095) (p-values as per univariate analysis). PFS remained statistically significant after the multivariate analysis (p = 0.003).

Conclusions

These results support a correlation between RAS-mutant cancers with greater MAPK signalling and PFS after MEKi treatment.

Introduction

The mitogen-activated protein kinase (MAPK) signal transduction pathway is a chain of proteins responsible for the integration of extracellular signals and coordination of responses for control of multiple cellular functions including cellular growth, survival and differentiation [1]. Aberrant activation of this pathway is a major oncogenic event in many human cancer types. Mutations in three related ‘rat sarcoma’ (RAS) genes—KRAS, NRAS and HRAS—are among the most frequently altered oncogenes in the MAPK pathway, occurring in approximately 30% of all tumour types [2,3]. These RAS genes encode highly homologous proteins that function as molecular regulators or switches in the MAPK pathway that cycle between on and off conformations, depending on the binding of GTP and GDP, respectively, and hence control a broad spectrum of cellular activities such as proliferation and cell survival [3]. When mutated, there is a higher likelihood that RAS is GTP bound, often related to impairment of intrinsic or GTPase-activating protein–stimulated hydrolysis of GTP [4,5], which in turn activates effector proteins such as RAF or PI3K, subsequently activating the RAF/MAPK/ERK kinase (MEK)/ERK and PI3K/AKT/MTOR cascades, found to drive many cancers types [6]. Of note, RAS mutations are highly prevalent in some of the deadliest cancers, including lung, colon, rectal and pancreatic cancers [2,7], and associated with poor survival outcomes [8].

Despite more than three decades of research, RAS has often been considered an undruggable target, in part owing to the relative cellular abundance and the high affinity of RAS for binding GTP as well as the lack of a suitable binding location in critical regions on the smooth surface of the RAS oncoprotein [2,6,9]. Only recently has clinical activity been observed with selective KRAS G12C inhibitors, with responses being noted especially in patients with non-small cell lung cancer [[10], [11], [12]].

Owing to the frequency and poor prognosis of patients whose tumours harbour RAS mutations, multiple other approaches apart from direct inhibition have been undertaken to develop drugs to treat malignancies with RAS mutations, including interfering with trafficking and localisation of RAS to the plasma membrane and suppression of downstream MEK and ERK signals [2,13,14]. Although many of these approaches have shown promise in the preclinical setting, clinical application has largely failed to demonstrate significant clinical effectiveness. Overall, the treatment of patients with cancers harbouring RAS mutations continues to represent an ongoing clinical challenge and unmet medical need [15].

Some of the challenges in targeting RAS may derive from the fact that the functional consequences of distinct RAS alterations may differ [3,4]. We hypothesised that certain RAS mutations may have higher impact on MAPK activity and thus may be more sensitive to intervention with MEK inhibition. Herein, we evaluated 62 patients with diverse RAS-mutated solid tumours whose RAS activity was interrogated and treatment outcomes after treatment with MEK inhibitor–based compounds were available. MAPK functional activity of different RAS mutations was evaluated and scored by examining the degree of localisation of ERK from the cytoplasm to nucleus in vitro. We demonstrate that intervention with MEK inhibitors in patients with high versus low MAPK activity was associated with prolonged progression-free survival (PFS) and overall survival (OS).