The Complex Mechanisms of Tolerance and Resistance in Lung Adenocarcinoma: Insights into KRAS Mutations and Targeted Therapies


Lung adenocarcinoma (LAUD) is a formidable adversary, contributing to approximately 40% of all non-small cell lung cancer (NSCLC) cases. Within this subset, KRAS mutations emerge as the most common gain-of-function alterations, accounting for roughly 30% of lung adenocarcinomas.

These mutations predominantly affect codon 12, with a smaller fraction impacting codons 13 and 61. The functional consequence of these mutations is the impairment of K-ras p21 protein (KRAS) guanosine triphosphatase (GTPase) activity, rendering the oncoprotein constitutively active. Remarkably, different amino acid substitutions within KRAS induce distinct biological behaviors, affecting patient prognosis and responses to targeted therapies and chemotherapy.

Recent advancements in cancer research have seen the development of covalent inhibitors targeting KRAS G12C, such as sotorasib and adagrasib. These inhibitors work by interacting with the mutant cysteine residue and locking the molecule in the guanosine diphosphate (GDP)-bound inactive state.

However, challenges persist in the quest for effective therapies, as progression-free survival with sotorasib was limited to a mere 6.3 months, and only 45% of patients exhibited a partial response to adagrasib. Additionally, many of these partial responders ultimately develop resistance.

Understanding Drug Resistance

Drug resistance in the context of lung adenocarcinoma is typically thought to occur through genetic alterations that are often irreversible. This notion of “resistance” encompasses both inherent resistance and acquired resistance. Inherent resistance relates to genetic changes that preclude a patient from responding to therapy entirely.

On the other hand, acquired resistance is mediated through a reversible tolerant state. Tolerance arises when a tumor initially responds to a drug but subsequently becomes unresponsive due to nongenetic mechanisms. This reversible phenotype allows the cells to revert to their original state and repopulate, leading to tumor proliferation in the absence of the drug. Over time, the prolonged exposure of tolerant cells to drugs can result in the acquisition of mutations, leading to the development of irreversible resistance.

While the genetic basis of drug resistance is well-established, the nongenetic mechanisms that drive the development of tolerance and ultimately acquired resistance remain relatively poorly understood.

The Role of Intrinsically Disordered Proteins

Emerging research has begun to shed light on the role of intrinsically disordered proteins (IDPs) in driving nongenetic mechanisms that lead to irreversible drug-resistant phenotypes. IDPs are proteins characterized by significant disordered regions within their structures. Several studies have highlighted how IDPs play a critical role in the development of drug resistance in cancer.

For instance, integrin β4 (ITGB4) and paxillin (PXN), both key components of the focal adhesion complex, have been identified as IDPs that can induce cisplatin resistance in KRAS-mutant NSCLC through nongenetic mechanisms. Coexpression of these proteins has been correlated with poor patient survival, and perturbing their signaling using the FDA-approved proteasome inhibitor, carfilzomib (CFZ), led to cell growth inhibition and sensitization to cisplatin.

However, the precise contribution of ITGB4 and PXN in acquiring tolerance or resistance against sotorasib remains elusive. Furthermore, the potential of CFZ in reverting sotorasib resistance is yet to be explored.

The Complexity of KRAS Mutations

KRAS is a complex, hybrid protein, with several intrinsically disordered regions interspersed among highly ordered regions. Thus, any amino acid substitutions occurring within the disordered regions can induce conformational changes, altering the interaction of KRAS with downstream signaling transducers and resulting in variable responses to therapy.

Furthermore, it remains unclear whether the failure to respond to sotorasib also results in a loss of sensitivity to adagrasib, as both molecules bind and inhibit KRAS G12C in its GDP-bound state.

Research Objectives

In this study, our aim is to address these crucial questions and shed light on the mechanisms underlying the development of tolerance and resistance in lung adenocarcinoma with KRAS mutations, particularly when treated with sotorasib. Specifically, we will investigate the significance of ITGB4 and Wnt/β-catenin signaling in the acquisition of tolerance to sotorasib. Additionally, we will explore the potential role of the small-molecule inhibitor CFZ in reverting drug-tolerant phenotypes.

This research will deepen our understanding of the intricacies of lung adenocarcinoma with KRAS mutations and targeted therapies, offering new insights into the development of drug resistance and potential avenues for improved patient outcomes. It is a vital step toward the advancement of precision medicine in the fight against this deadly disease.


Sotorasib and adagrasib, two promising mutant-selective KRAS G12C inhibitors, have offered a glimmer of hope in the treatment of lung adenocarcinoma with KRAS mutations. However, the emergence of resistance to these inhibitors, whether inherent or acquired, has raised significant concerns, highlighting the critical need for a comprehensive understanding of the underlying resistance mechanisms. Traditionally, resistance has been attributed primarily to the accumulation of random genetic mutations and the subsequent selection of mutant clones, aligning with the principles of Darwinian selection. This reductionist, gene-centric perspective has dominated the field. However, it is increasingly clear that therapy resistance can also arise from heterogeneous drug-tolerant persister cells or minimal residual disease, mediated by both genetic and nongenetic mechanisms.

In this study, we delved into the complex landscape of resistance by utilizing three different KRAS-G12C NSCLC cell lines with distinct genetic backgrounds and responses to sotorasib. These cell lines, H358, H23, and SW1573, all shared KRAS G12C and p53 mutations, but they also carried additional genetic alterations. H23 had STK11 and ATM mutations, while SW1573 carried mutations in CDKN2A, SMAD4, CTNNB1, PIK3CA, and SMARCB1. Notably, STK11 mutations frequently co-occurred with KRAS mutations in patients, with poor survival outcomes reported for such cases. Conversely, the PIK3CA and CTNNB1 mutations found in SW1573 were less common in KRAS-mutant NSCLC.

Strikingly, the H358 cells, with two mutations (KRAS G12C and p53), exhibited high sensitivity to sotorasib. In contrast, the H23 cells, harboring four mutations, displayed tolerance to the drug, and the SW1573 cells, with five mutations, were inherently resistant. Tolerant cells often comprise a heterogeneous mix of sensitive and persister cells. Over time, under sustained selection pressure, these persister cells can contribute to the development of acquired resistance. Consistent with this hypothesis, we successfully generated isogenic sotorasib-resistant H23 cells that could tolerate high concentrations of sotorasib without gaining any function-altering mutations in genes previously associated with resistance. However, these resistant cells exhibited significant changes in the expression, interactions, and signaling of ITGB4 and β-catenin.

Notably, increased ITGB4 expression in the resistant cells promoted AKT activation, subsequently inhibiting GSK-3β function and activating β-catenin. This complex signaling cascade acted as a bypass mechanism to overcome sotorasib toxicity. These findings indicate that the transition from a tolerant to overtly resistant state can be driven by nongenetic mechanisms. This implies that patients with KRAS G12C and STK11 co-mutations may initially respond to sotorasib but eventually develop resistance and become unresponsive to the treatment. Moreover, our results suggest that patients with mutations in genes contributing to bypass pathways, such as PIK3CA and CTNNB1, may be inherently resistant to sotorasib, similar to the SW1573 cells. In summary, this study highlights that not all tumors evolve to a resistant state akin to H358 cells. Some may progress from a sensitive to a tolerant state via nongenetic mechanisms, while others may evolve into a highly resistant state, resembling SW1573 cells by acquiring mutations in the stressed network. These scenarios underscore the genetic/nongenetic duality of drug resistance in cancer.

Moreover, the study demonstrates that acquired resistance to sotorasib can be reversed using a combination of CFZ and sotorasib. This combination effectively suppressed the expression of ITGB4 and β-catenin, along with their downstream signaling pathways, ultimately enhancing sensitivity to the treatment. Our interest in exploring the potential of CFZ in mitigating sotorasib resistance was rooted in our previous success in alleviating cisplatin resistance in a mutant KRAS LAUD cell line. Despite its classification as a proteasome inhibitor, we speculate that CFZ disrupts expression and signaling downstream of ITGB4/PXN and WNT/β-catenin independently of proteasomal inhibition. Thus, it is apparent that the nuanced roles of components within the focal adhesion complex and WNT/β-catenin signaling can be suppressed by the combination of sotorasib and CFZ.

This study highlights the pivotal role of protein interaction networks in the nongenetic mechanisms of resistance. These networks have the remarkable ability to “rewire” cellular circuitry. In particular, the conformational dynamics of proteins, especially intrinsically disordered proteins (IDPs) that occupy central positions within these circuits, influence various cellular signaling pathways. While KRAS may not be a pure IDP, it can be regarded as a “hybrid” protein, with both ordered and disordered regions. The disordered regions, such as the P loop and switch regions, contribute to conformational flexibility.

The conformational ensemble of the KRAS molecule varies significantly when comparing the apo form to the GDP/guanosine 5′-triphosphate-bound state. Various point mutations in KRAS alter the conformational preferences of the protein, affecting downstream signaling pathways, thereby influencing clinical outcomes. Notably, the most common mutations occurring in the P loop and switch II regions underscore the critical role of conformational changes and downstream signaling. These differences in conformation may account for the varied efficacy and mechanisms of action of sotorasib and adagrasib. This is further supported by the observed disparities in molecular weights of drug-free KRAS, sotorasib-bound KRAS, and adagrasib-bound KRAS using SDS gel electrophoresis. These differences can be attributed to variations in conformation or post-translational modifications.

In summary, this study has unraveled the role of ITGB4 and β-catenin signaling as a bypass mechanism for acquiring resistance to sotorasib. However, this resistance developed against sotorasib may not necessarily extend to adagrasib. These findings underscore the significance of nongenetic resistance mechanisms in cancer and emphasize the need to consider the distinct interactions of different drug-bound ensembles of mutant KRAS. Overall, our research unveils previously unrecognized nongenetic mechanisms underlying resistance to sotorasib and proposes a promising treatment strategy to overcome this resistance, offering new hope for patients with lung adenocarcinoma and KRAS mutations.

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