HDAC6 promotes osimertinib resistance evolution in non-small cell lung cancer by activating EGFR degradation through the ubiquitin-proteasome pathway

Abstract

Osimertinib is the standard first-line treatment for patients with non-small cell lung cancer (NSCLC) harboring EGFR-sensitive mutations. However, drug resistance inevitably develops, highlighting the critical need for strategies to overcome this resistance and prolong therapeutic efficacy. Understanding the mechanisms underlying drug resistance is essential, and drug-resistant cell models serve as valuable tools for investigating acquired resistance. In this study, we establish an osimertinib resistance evolution model in vitro by continuous high-dose drug induction and identify cell lines exhibiting “permanent” resistance to osimertinib (osimertinib resistant, OR). Transcriptome sequencing (RNA-seq), gain- and loss-of-function assay, including lentiviral-mediated overexpression and RNAi knockdown, pharmacological inhibition, and protein degradation analysis reveal significant alterations in genes associated with epigenetic regulation, notably a marked upregulation of histone deacetylase 6 (HDAC6) in OR cells. Knockdown of HDAC6 or pharmacological inhibition of HDAC6 restores the sensitivity of OR cells to osimertinib, whereas overexpression of HDAC6 in sensitive cells reduces drug efficacy and accelerates the onset of resistance. Furthermore, we find that HDAC6 upregulation promotes EGFR degradation, thereby contributing to resistance. Collectively, our findings demonstrate the utility of drug resistance evolution models in identifying key resistance factors. HDAC6 plays a pivotal role in osimertinib resistance, and targeting HDAC6 may represent a novel therapeutic strategy to overcome resistance and enhance treatment efficacy.

1. Introduction

In 2022, lung cancer accounted for approximately 2.5 million new cases and 1.8 million deaths worldwide [1], underscoring its persistent global health burden [2]. The identification of driver genes and the development of targeted therapies have substantially improved treatment outcomes for lung cancer patients with specific genetic mutations [3]. However, the inevitable emergence of drug resistance remains a major clinical challenge, adversely affecting patient survival and limiting the durability of treatment response. Therefore, extending the effective use of targeted therapies and delaying or overcoming resistance represent urgent and unresolved priorities in clinical oncology.

EGFR was the first driver gene identified in lung cancer [4]. Osimertinib, a third-generation EGFR tyrosine kinase inhibitor (TKI), is now used as a first-line treatment in clinical practice [5]. It extends progression-free survival (PFS) in lung cancer patients to 10–18.9 months [6,7], with a median overall survival (OS) of 26.8–38.6 months [7]. Several studies have characterized key mechanisms of osimertinib resistance, including target mutations, bypass signaling activation and histological transformation [8,9]. However, these studies have largely focused on patients after resistance has already developed. Drug resistance is a dynamic process, and understanding its evolution along with developing effective interventions is critical to overcoming clinical resistance.

Traditional resistance models typically involve gradually increasing drug concentrations to induce resistance, yielding a homogeneous population of resistant cells [10]. In contrast, high-dose “hard-hit” approaches more closely recapitulate the clinical onset of resistance and provide a superior platform for mechanistic investigation [11,12]. In this approach, a high concentration of osimertinib eliminate most sensitive cells, while the surviving cells gradually evolve drug-resistant traits under continuous drug pressure and ultimately establish stable drug resistance through adaptive mutagenesis [13].