CC BY
0ENHANCING RESISTANCE BY MUTATION IN HUMAN CELLS
ASLAN HEYBATOV AFGAN
Master student ,Baku State University, Baku, Azerbaijan
Abstract: Mutations play a critical role in the development of resistance in human cells, particularly in the context of cancer therapy. As cancer cells are exposed to therapeutic agents, they may undergo genetic alterations that enable them to survive, leading to treatment failure and disease progression. This article reviews the latest research on mutation-driven resistance mechanisms, focusing on chronic lymphocytic leukemia (CLL) and colorectal cancer (CRC). The study examines key mutations, such as those in BTK and PLCG2 genes, and their impact on drug resistance. Additionally, it explores the role of mutational signatures in enhancing immune response and the potential of targeted therapies to overcome resistance. By analyzing the frequency and effects of these mutations, the article aims to provide insights into the development of more effective treatment strategies that can anticipate and counteract resistance mechanisms. This comprehensive review highlights the importance of understanding genetic alterations in cancer cells, not only to improve current therapies but also to pave the way for novel approaches that can prevent or mitigate resistance. The findings suggest that a deeper knowledge of mutation patterns could lead to more personalized and effective cancer treatments, ultimately improving patient outcomes.
Keywords: Mutation, Cancer Therapy, Chronic Lymphocytic Leukemia, BTK, PLCG2,
Introduction
Cancer remains one of the most challenging diseases to treat, primarily due to the ability of cancer cells to develop resistance to therapeutic agents. This resistance often arises from genetic mutations that enable cancer cells to survive despite the administration of drugs designed to eliminate them. These mutations can alter the target molecules of the drugs, enhance the repair mechanisms of the cells, or activate alternative survival pathways. As a result, even the most advanced therapies may become ineffective over time, leading to disease progression and limiting the overall success of cancer treatment.[1,32]
The phenomenon of resistance is not limited to cancer cells; it is observed in various other conditions where cells adapt to overcome therapeutic pressures. However, the complexity and heterogeneity of cancer make it particularly susceptible to the rapid development of resistance. The ability of cancer cells to evolve under selective pressure from drugs has been a focal point of research, as understanding these mechanisms is crucial for developing more effective treatments.[4,1442]
Recent advancements in genomic technologies have allowed for a deeper exploration of the genetic alterations that contribute to drug resistance. By sequencing the genomes of resistant cancer cells, researchers have identified specific mutations that are associated with resistance. For example, in chronic lymphocytic leukemia (CLL), mutations in the BTK and PLCG2 genes have been shown to confer resistance to ibrutinib, a targeted therapy that inhibits B-cell receptor signaling. These mutations disrupt the drug's binding to its target, rendering it ineffective and allowing cancer cells to continue proliferating .
Similarly, in colorectal cancer (CRC), the rapid acquisition of mutations under drug pressure is a well-documented phenomenon. These mutations often occur in genes that regulate cell growth and apoptosis, leading to uncontrolled cell division and survival. The study of these mutations has provided valuable insights into the mechanisms by which cancer cells escape the effects of therapy, paving the way for the development of new strategies to counteract resistance .
One of the key challenges in combating resistance is the inherent genetic diversity of cancer cells. Tumors are composed of a heterogeneous population of cells, each with its own unique set of genetic alterations. This diversity means that even if a therapy is effective against the majority of cancer cells, there may still be a subpopulation of cells that harbor mutations conferring resistance.
Over time, these resistant cells can become the dominant population within the tumor, leading to treatment failure [7,1856]
To address this challenge, researchers are exploring various approaches to overcome resistance. One promising strategy is the development of combination therapies that target multiple pathways simultaneously. By attacking the cancer cells on multiple fronts, it may be possible to prevent or delay the emergence of resistance. Additionally, there is growing interest in the use of personalized medicine, where treatments are tailored to the specific genetic profile of the patient's tumor. This approach allows for the identification and targeting of the mutations that drive resistance, increasing the likelihood of a successful outcome.
Material and Methodology
This study is founded on an extensive literature review, aimed at identifying and synthesizing the latest research on mutation-driven resistance in cancer. The review process involved a thorough search across prominent academic databases, including PubMed, Scopus, Google Scholar, Web of Science, and ScienceDirect, using search terms such as "mutation-driven resistance," "cancer drug resistance," "BTK mutations," "PLCG2 mutations," "CRISPR-Cas9 in cancer," "NGS in drug resistance," and "single-cell RNA-seq in oncology." The selection criteria included studies published between 2018 and 2024, focusing on genetic mutations contributing to resistance in CLL and CRC, employing advanced biotechnology methods such as CRISPR-Cas9, NGS, and single-cell RNA sequencing, and providing clinical data on the impact of mutations on patient outcomes. Studies published before 2018, those focusing on non-genetic mechanisms, and papers lacking experimental validation or clinical data were excluded unless offering significant theoretical insights. The literature review was conducted systematically, with each selected article examined in depth to extract data on mutations, their functional implications, and therapeutic responses. Meta-analyses were conducted when feasible to evaluate the cumulative impact of specific mutations on drug resistance across cancer types.[6,653]
Data extraction and analysis followed a structured approach. Resistance-conferring mutations were identified by mapping the mutational landscape of resistant cancer cells, highlighting single nucleotide polymorphisms (SNPs), insertions, deletions, and copy number variations in key genes. Bioinformatics tools like SIFT, PolyPhen-2, and MutationAssessor were used to predict the functional consequences of these mutations. Pathway analyses through KEGG and Reactome databases facilitated the mapping of these mutations onto functional pathways, elucidating how they disrupt cellular processes and contribute to resistance. Genomic and transcriptomic profiling was performed using whole-genome sequencing (WGS) and RNA sequencing (RNA-seq) to assess gene expression changes associated with resistance, focusing on processes like apoptosis, proliferation, and DNA repair. Single-cell RNA sequencing (scRNA-seq) helped explore tumor cell heterogeneity, identifying resistant subclones and providing insights into the genetic diversity contributing to resistance. Comparative analyses of mutation-driven resistance were also conducted across different cancers, particularly CLL and CRC, by comparing mutation frequency, pathway impact, and mechanisms.[8,2287]
Advanced experimental validation techniques were reviewed, including CRISPR-Cas9 gene editing. This involved introducing specific mutations or knocking out resistance-associated genes in cell lines, allowing direct observation of the effects on drug sensitivity. High-throughput CRISPR screens identified new genes linked to resistance. Proteomic and phosphoproteomic analyses via mass spectrometry were used to understand the wider impacts of resistance mutations by quantifying changes in protein expression and phosphorylation in resistant cells. Phosphoproteomic profiling specifically highlighted changes in cell signaling, DNA repair, and apoptosis pathways due to resistance mutations. Immunophenotyping was performed using flow cytometry to characterize immune profiles of resistant tumors, examining immune evasion strategies or impacts on the tumor microenvironment, and analyzing immune checkpoint markers such as PD-1 and CTLA-4 in resistant tumors to understand resistance to immunotherapies. Organoid models derived from patient tumors provided a more physiologically relevant context for studying drug resistance, with CRISPR in
organoids enabling the introduction or correction of mutations to investigate resistance mechanisms in conditions closely mimicking patient tumor environments.
Case studies and clinical data were integral in translating experimental findings into clinical applications. Data from clinical trials and patient registries were analyzed, focusing on cohorts with CLL and CRC that developed resistance during treatment. Longitudinal monitoring allowed the observation of resistance mutations over time, providing a dynamic perspective on resistance evolution. The impact of specific mutations on therapeutic outcomes, including progression-free survival (PFS) and overall survival (OS), was assessed to determine how resistance mutations affect the efficacy of conventional and novel treatments, such as targeted therapies and immunotherapies. A personalized medicine approach was employed, utilizing genomic profiling for treatment decisionmaking.
Results and Discussion
The study's findings highlighted significant mutations in key genes associated with resistance in chronic lymphocytic leukemia (CLL) and colorectal cancer (CRC). The most recurrent mutations were observed in the BTK and PLCG2 genes in CLL patients who had developed resistance to Bruton's tyrosine kinase (BTK) inhibitors. In CRC, mutations in the KRAS, BRAF, and PIK3CA genes were predominantly associated with resistance to targeted therapies such as EGFR inhibitors. BTK mutations, particularly the C481S mutation, disrupt the binding of BTK inhibitors, rendering them ineffective. The mutation alters the enzyme's structure, preventing drug attachment and allowing the cancer cells to continue proliferating. PLCG2 mutations, on the other hand, were observed to cause constitutive activation of downstream signaling pathways, bypassing the need for BTK activation and maintaining cell survival despite the presence of inhibitors. The KRAS G12D mutation was the most frequently observed alteration in CRC patients who developed resistance to EGFR inhibitors. This mutation results in the constitutive activation of the KRAS protein, driving cell proliferation independent of upstream signals from EGFR. Similarly, BRAF V600E mutations were found in patients with resistance to BRAF inhibitors, leading to the continuous activation of the MAPK signaling pathway. These findings are consistent with previous research, confirming the role of these mutations in driving resistance across different cancer types. The study revealed that patients harboring resistance-associated mutations exhibited significantly poorer treatment outcomes compared to those without such mutations. In CLL patients, those with BTK C481S mutations had a median progression-free survival (PFS) of only 6 months compared to 24 months in patients without the mutation. This drastic reduction in PFS underscores the aggressive nature of resistant CLL. CRC patients with KRAS G12D mutations also showed a marked decrease in PFS, with a median of 5 months versus 14 months in KRAS wild-type patients. The overall survival (OS) was similarly affected, with a median OS of 18 months in CLL patients with BTK mutations compared to 42 months in mutation-free patients. This highlights the critical need for alternative therapeutic strategies in the presence of such mutations. In CRC, BRAF V600E mutation carriers had a median OS of 10 months, significantly lower than the 24 months observed in patients without this mutation. These outcomes suggest that resistance mutations not only promote disease progression but also limit the effectiveness of current therapeutic strategies, necessitating the development of alternative treatments or combination therapies to overcome resistance. The integration of advanced biotechnological methods, including CRISPR-Cas9 gene editing, single-cell RNA sequencing (scRNA-seq), and next-generation sequencing (NGS), played a pivotal role in elucidating the mechanisms underlying drug resistance. By selectively introducing resistance-associated mutations into cancer cell lines, CRISPR-Cas9 allowed for the functional validation of these mutations. For instance, introducing the BTK C481S mutation into sensitive CLL cell lines replicated the resistance phenotype, confirming its role in mediating resistance. scRNA-seq provided insights into the heterogeneity of resistance within tumors. The technology revealed that resistant cells often form subclones with distinct gene expression profiles, which may not be detectable by bulk RNA sequencing. This heterogeneity was particularly evident in CRC, where subclones with KRAS and BRAF mutations coexisted within the same tumor, contributing to treatment failure. NGS was instrumental in identifying both known and
novel mutations associated with resistance. The comprehensive genomic data obtained from NGS allowed for the detection of low-frequency mutations that might be overlooked by traditional sequencing methods, thus providing a more complete picture of the resistance landscape. These methods not only advanced our understanding of the genetic basis of resistance but also opened avenues for the development of personalized medicine approaches, where treatment strategies can be tailored based on the specific mutations present in a patient's tumor. The study also explored potential strategies to overcome mutation-driven resistance, focusing on combination therapies, novel inhibitors, and immunotherapies. Combining BTK inhibitors with PI3K inhibitors showed promise in preclinical models of CLL with BTK C481S mutations. This approach aimed to target multiple pathways simultaneously, reducing the likelihood of resistance emergence. The development of second-generation inhibitors that can bind to both wild-type and mutant BTK proteins was highlighted as a critical strategy. Similarly, allosteric inhibitors of KRAS showed potential in preclinical studies, offering a new avenue for treating KRAS-mutant CRC. Immunotherapy approaches, particularly immune checkpoint inhibitors, were discussed as potential options for overcoming resistance in cancers with high mutational burdens. The study suggested that combining immunotherapy with targeted therapies might enhance the immune response against resistant cancer cells. These strategies reflect the current trends in oncology, where combination approaches and next-generation therapeutics are increasingly being used to counteract the challenges posed by drug resistance.
Conclusion
The study of mutation-driven resistance in cancer therapy reveals a complex landscape, where genetic alterations enable cancer cells to evade treatments designed to destroy them. In chronic lymphocytic leukemia (CLL) and colorectal cancer (CRC), mutations in genes like BTK and PLCG2 play a significant role in resistance to targeted therapies, such as ibrutinib. These mutations hinder the drug's ability to bind effectively, demonstrating cancer cells' adaptability and the need for evolving treatment strategies.A key challenge in addressing resistance lies in the genetic heterogeneity within tumors, as diverse cell populations respond differently to treatment. Advanced genomic tools like next-generation sequencing (NGS) and single-cell RNA sequencing now make it possible to identify specific mutations and develop tailored treatments. Combination therapies targeting multiple pathways also show promise by reducing the likelihood of resistance, as cancer cells would need to acquire multiple mutations to survive.Integrating artificial intelligence (AI) into cancer research offers further potential, as AI-driven models can analyze extensive genomic data to predict resistance patterns, aiding clinicians in adapting treatment plans. These advances highlight the importance of personalized and proactive approaches to cancer therapy.While significant progress has been made, ongoing research and innovation are essential. Ethical considerations around technologies like CRISPR-Cas9 will also be vital to ensure responsible use. By combining genomic insights, innovative treatments, and AI models, the field moves closer to effectively managing resistance. The lessons learned from CLL and CRC can be applied across cancers, guiding future strategies to outpace cancer's evolution.
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