Taletrectinib promotes pyroptosis in colorectal carcinoma via SRC/AKT/mTOR axis inhibition

Abstract

The persistent and significant challenge of drug resistance following surgical intervention for colorectal carcinoma (CRC) underscores an urgent and pressing need for the development of innovative and more effective therapeutic strategies. Despite the initial success achieved through surgical resection, the recurrence and subsequent progression of this aggressive malignancy, which are frequently driven by complex mechanisms of acquired drug resistance, severely compromise the long-term prognosis and overall survival rates for patients. This formidable clinical problem highlights the imperative to meticulously explore novel therapeutic agents and pathways that possess the capacity to circumvent these sophisticated adaptive cellular responses, ultimately offering more durable and sustained therapeutic benefits to patients battling CRC.

Within this critical therapeutic landscape, Taletrectinib, known by its chemical identifiers DS-6051b and AB-106, emerges as a particularly promising synthetic small molecule inhibitor. Its primary known mechanism of action involves potent and selective inhibition of ROS1 and NTRK kinases, which are recognized oncogenic drivers in various specific cancer types. This compound has already demonstrated remarkable and clinically meaningful antitumor activity in preliminary investigations, especially in malignancies characterized by activating ROS1 or NTRK gene fusions. Currently, taletrectinib is progressing through advanced clinical trials, with a specific strategic aim to address and overcome the targeted resistance observed with earlier generation therapies in other cancer contexts. This positions taletrectinib as a potentially pivotal addition to the arsenal of precision oncology. However, despite its established efficacy against other molecular targets, the specific anti-cancer effects and the precise underlying mechanisms through which taletrectinib might exert its therapeutic influence within the unique biological milieu of colorectal carcinoma have, until the present study, remained largely uncharacterized and obscure, representing a substantial gap in the understanding of its broader therapeutic applicability.

The overarching purpose of the present study was, therefore, meticulously designed to comprehensively evaluate the inherent cytotoxicity of taletrectinib in various colorectal carcinoma models. Our thorough investigation encompassed both rigorously controlled in vitro cellular assays, utilizing two well-characterized colorectal carcinoma cell lines, and a physiologically relevant in vivo mouse tumor model, thereby providing a robust and translationally pertinent assessment of its direct anti-tumor efficacy. Beyond merely quantifying its growth-inhibitory effects, a paramount focus of our research was dedicated to the intricate elucidation of the precise molecular and cellular mechanisms through which taletrectinib orchestrates its profound cytotoxicity.

To achieve this in-depth mechanistic understanding, a multifaceted array of sophisticated experimental techniques was judiciously employed. The fundamental assessment of cellular viability and any accompanying morphological changes was initially conducted using conventional light microscopy, providing a macroscopic view. This was then complemented by high-resolution scanning electron microscopy, which offered an intimate and detailed examination of cellular ultrastructural alterations, shedding light on the early stages of cell damage. To probe the distinct hallmarks of specific programmed cell death pathways, immunofluorescence assays were strategically utilized, enabling the direct visualization of critical intracellular components and their reorganization during cell demise. Concurrently, quantitative measurements of apoptosis were performed using annexin V-FITC and propidium iodide detection, a gold standard for distinguishing different cell death modalities. Furthermore, the integrity of cellular membranes and the extent of lytic cell death were precisely quantified through lactate dehydrogenase (LDH) release assays. The dynamic changes in protein expression levels and phosphorylation patterns, indicative of specific intracellular signaling pathway modulation, were extensively analyzed via western blotting, providing crucial molecular insights. Most importantly, to comprehensively characterize the genetic landscape of our selected cellular models and to ascertain the presence or absence of specific gene fusions, unbiased and comprehensive transcriptome sequencing was meticulously performed.

Our initial experimental findings provided compelling evidence of a clear and highly encouraging dose-dependent relationship: the viability of the colorectal carcinoma cells exhibited a significant and progressive decrease as concentrations of taletrectinib were incrementally elevated. This fundamental observation established a strong foundational basis for the compound’s intrinsic cytotoxic potential against CRC. A truly critical and unexpected insight emerged from the comprehensive transcriptome sequencing data, which unequivocally demonstrated that neither the HCT116 nor the LOVO colorectal carcinoma cell lines utilized in our rigorous in vitro investigations harbored any detectable ROS1- or NTRK-related gene fusions. This striking absence of the primary molecular targets, for which taletrectinib was originally developed and clinically advanced, profoundly indicated a previously unrecognized and novel, ROS1/NTRK-independent mechanism of action within these specific colorectal cancer contexts.

Further exhaustive in-depth mechanistic evaluations definitively established that the observed potent cytotoxic effect of taletrectinib was exerted via a distinctive and increasingly recognized form of programmed cell death known as pyroptosis. This unique mode of cell death, characterized by its inherently inflammatory nature and distinct from the more widely known apoptosis, was found to be critically dependent upon the precise activation of caspase-3, an initiating protease, and the subsequent proteolytic cleavage of gasdermin E (GSDME), which functions as a key executioner protein. The definitive involvement of gasdermin E (GSDME) in this process unequivocally signifies a lytic and profoundly inflammatory form of cellular demise, a characteristic that holds significant promise as it can potentially elicit robust and beneficial anti-tumor immune responses within the tumor microenvironment.

Moreover, our investigations extended beyond simply identifying pyroptosis to unraveling its upstream regulatory elements. We made a significant discovery that the potent effect of taletrectinib in promoting GSDME-mediated pyroptosis could be substantially attenuated or, in some cases, entirely reversed through strategic treatment with tolimidone, a well-established and potent agonist of the SRC kinase. This crucial observation was consistently and reproducibly demonstrated across both our sophisticated in vitro cellular models and, critically, was robustly validated in the in vivo mouse tumor model. This dual validation established a strong and undeniable mechanistic link between SRC kinase activity and the efficacy of taletrectinib-induced pyroptosis in CRC cells. These findings collectively indicated that taletrectinib exerts its profound anti-tumor activity by intricately modulating key intracellular signaling cascades, specifically involving the SRC pathway.

In conclusion, the comprehensive findings emanating from this study collectively and robustly suggest that taletrectinib suppresses the relentless growth and unchecked proliferation of colorectal carcinoma tumors through a previously unappreciated and novel mechanism: by directly inducing GSDME-mediated pyroptosis. This critical and inflammatory cell death process is intricately linked to the precise modulation of the SRC/AKT/mTOR signaling pathway, highlighting a previously unrecognized and exploitable vulnerability within colorectal cancer cells. This newly elucidated mechanism of action significantly elevates taletrectinib’s profile, underscoring its substantial potential as a promising and innovative therapeutic agent against colorectal carcinoma. Furthermore, it directly addresses the critical unmet clinical need for novel strategies to overcome prevalent drug resistance in this challenging and often refractory malignancy. The profound implications of these findings are poised to pave the way for accelerated and targeted preclinical and subsequent clinical development of taletrectinib for patients afflicted with colorectal cancer, offering a new avenue for improved patient outcomes.

Declarations

Conflict of Interest Statement

The authors explicitly declare that they possess no competing financial interests, whether direct or indirect, nor any non-financial competing interests that could potentially be perceived as influencing or biasing the outcomes, results, or interpretation of the research presented herein.

Ethical Approval

This comprehensive study, including all experimental protocols involving both cellular and animal models, received meticulous and comprehensive approval from the Ethics Committee of the Chengdu Medical College. The officially assigned approval number for this study is 2020-15. Furthermore, it is affirmed that all experimental methods and procedures related to animal care and use are rigorously reported in strict adherence and full compliance with the ARRIVE guidelines (Animal Research: Reporting of In Vivo Experiments), thereby ensuring the highest standards of transparency, reproducibility, and ethical conduct throughout the entire research process.