BCR-ABL residues interacting with ponatinib are critical to preserve the tumorigenic potential of the oncoprotein

Patients with chronic myeloid leukemia in whom tyrosine kinase inhibitors (TKIs) fail often present mutations in the BCR-ABL catalytic domain. We noticed a lack of substitutions involving 4 amino acids (E286, M318, I360, and D381) that form hydrogen bonds with ponatinib. We therefore introduced mutations in each of these residues, either preserving or altering their physicochemical properties. We found that E286, M318, I360, and D381 are dispensable for ABL and BCR-ABL protein stability but are critical for preserving catalytic activity. Indeed, only a "conservative" I360T substitution retained kinase proficiency and transforming potential. Molecular dynamics simulations of BCR-ABLI360T revealed differences in both helix αC dynamics and protein-correlated motions, consistent with a modified ATP-binding pocket. Nevertheless, this mutant remained sensitive to ponatinib, imatinib, and dasatinib. These results suggest that changes in the 4 BCR-ABL residues described here would be selected against by a lack of kinase activity or by maintained responsiveness to TKIs. Notably, amino acids equivalent to those identified in BCR-ABL are conserved in 51% of human tyrosine kinases. Hence, these residues may represent an appealing target for the design of pharmacological compounds that would inhibit additional oncogenic tyrosine kinases while avoiding the emergence of resistance due to point mutations.This work was supported by an investigator grant to P.V. from Associazione Italiana per la Ricerca sul Cancro (AIRC) and by funding from the Biotechnology and Biological Sciences Research Council (BB/I023291/1 and BB/H018409/1 to AP and FF). P.B. is the recipient of an AIRC - Marie Curie fellowship

STRUCTURAL AND FUNCTIONAL IMPACT OF G2032R MUTATION IN ROS1 – A THEORETICAL PERSPECTIVE

Objective: Drug resistance is an imperative issue in the treatment of patients with lung cancer. In this work, investigation of the drug resistance mechanism of G2032R mutation in ROS1 is carried out using computational simulation techniques.Methods: Molecular docking and molecular dynamics (MD) simulation approach have been utilized to uncover the mechanism behind crizotinib resistance in ROS1 at a molecular level. Normal mode analysis was carried out using ElNemo server which examines the movements and conformational changes in the protein structure. ArgusLab, PEARLS, and Autodock were employed for the docking analysis, whereas GROMACS package 4.5.3 was used for MD simulation approach.Results: The results from our analysis indicates that wild-type ROS1 (Protein Data Bank Code 3ZBF) could be more crucial for the crizotinib binding as it indicates largest binding affinity, minimum number of H-bonds, and higher flexibility than mutant-type ROS1. Moreover, the theoretical basis for the cause of drug insensitivity is the differences in the electrostatic properties of binding site residues between the wild and mutant ROS1 structures. Our analysis theoretically suggests that E-2027 is a key residue responsible for the ROS1 drug selectivity.Conclusion: Molecular docking and MD simulation results provide an explanation of the resistance caused by G2032R and may give a key clue for the drug design to encounter drug resistance.Â

Making NSCLC Crystal Clear:How Kinase Structures Revolutionized Lung Cancer Treatment

The parallel advances of different scientific fields provide a contemporary scenario where collaboration is not a differential, but actually a requirement. In this context, crystallography has had a major contribution on the medical sciences, providing a “face” for targets of diseases that previously were known solely by name or sequence. Worldwide, cancer still leads the number of annual deaths, with 9.6 million associated deaths, with a major contribution from lung cancer and its 1.7 million deaths. Since the relationship between cancer and kinases was unraveled, these proteins have been extensively explored and became associated with drugs that later attained blockbuster status. Crystallographic structures of kinases related to lung cancer and their developed and marketed drugs provided insight on their conformation in the absence or presence of small molecules. Notwithstanding, these structures were also of service once the initially highly successful drugs started to lose their effectiveness in the emergence of mutations. This review focuses on a subclassification of lung cancer, non-small cell lung cancer (NSCLC), and major oncogenic driver mutations in kinases, and how crystallographic structures can be used, not only to provide awareness of the function and inhibition of these mutations, but also how these structures can be used in further computational studies aiming at addressing these novel mutations in the field of personalized medicine

Exploring the Role of Molecular Dynamics Simulations in Most Recent Cancer Research: Insights into Treatment Strategies

Cancer is a complex disease that is characterized by uncontrolled growth and division of cells. It involves a complex interplay between genetic and environmental factors that lead to the initiation and progression of tumors. Recent advances in molecular dynamics simulations have revolutionized our understanding of the molecular mechanisms underlying cancer initiation and progression. Molecular dynamics simulations enable researchers to study the behavior of biomolecules at an atomic level, providing insights into the dynamics and interactions of proteins, nucleic acids, and other molecules involved in cancer development. In this review paper, we provide an overview of the latest advances in molecular dynamics simulations of cancer cells. We will discuss the principles of molecular dynamics simulations and their applications in cancer research. We also explore the role of molecular dynamics simulations in understanding the interactions between cancer cells and their microenvironment, including signaling pathways, proteinprotein interactions, and other molecular processes involved in tumor initiation and progression. In addition, we highlight the current challenges and opportunities in this field and discuss the potential for developing more accurate and personalized simulations. Overall, this review paper aims to provide a comprehensive overview of the current state of molecular dynamics simulations in cancer research, with a focus on the molecular mechanisms underlying cancer initiation and progression.Comment: 49 pages, 2 figure

FAM129B, an antioxidative protein, reduces chemosensitivity by competing with Nrf2 for Keap1 binding.

BackgroundThe transcription factor Nrf2 is a master regulator of antioxidant response. While Nrf2 activation may counter increasing oxidative stress in aging, its activation in cancer can promote cancer progression and metastasis, and confer resistance to chemotherapy and radiotherapy. Thus, Nrf2 has been considered as a key pharmacological target. Unfortunately, there are no specific Nrf2 inhibitors for therapeutic application. Moreover, high Nrf2 activity in many tumors without Keap1 or Nrf2 mutations suggests that alternative mechanisms of Nrf2 regulation exist.MethodsInteraction of FAM129B with Keap1 is demonstrated by immunofluorescence, colocalization, co-immunoprecipitation and mammalian two-hybrid assay. Antioxidative function of FAM129B is analyzed by measuring ROS levels with DCF/flow cytometry, Nrf2 activation using luciferase reporter assay and determination of downstream gene expression by qPCR and wester blotting. Impact of FAM129B on in vivo chemosensitivity is examined in mice bearing breast and colon cancer xenografts. The clinical relevance of FAM129B is assessed by qPCR in breast cancer samples and data mining of publicly available databases.FindingsWe have demonstrated that FAM129B in cancer promotes Nrf2 activity by reducing its ubiquitination through competition with Nrf2 for Keap1 binding via its DLG and ETGE motifs. In addition, FAM129B reduces chemosensitivity by augmenting Nrf2 antioxidative signaling and confers poor prognosis in breast and lung cancer.InterpretationThese findings demonstrate the important role of FAM129B in Nrf2 activation and antioxidative response, and identify FMA129B as a potential therapeutic target. FUND: The Chang Gung Medical Foundation (Taiwan) and the Ministry of Science and Technology (Taiwan)

Belizatinib is a potent inhibitor for non-small cell lung cancers driven by different variants of EML4-ALK fusion proteins carrying L1196M-mutations

Structural fusions of EML4 with ALK kinase might lead to signaling abnormalities that drive NSCLC. Although approved specific ALK inhibitors have led to excellent initial responses in ALK positive NSCLC patients, acquired resistance to these inhibitors due to the occurrence of mutations is a major clinical challenge. Studies have shown that different mutations are related with unique and specific responses to certain inhibitors, and that the sensitivity of EML4-ALK fusion variants to ALK specific inhibitors varies. To investigate the response spectrum of combinations of each resistance mutation and EML4-ALK fusion gene variant (V) to different ALK specific inhibitors, individual cellular Ba/F3 models were constructed and used for ALK-TKIs screening in this thesis. Based on cell viability assays, the results gained here suggested that brigatinib might be priority recommended for G1269A and L1152R mutations, lorlatinib for C1156Y and I1171T mutations and belizatinib for G1269A and L1196M mutation. Moreover, a tendency was observed that V2 was most sensitive to ALK-TKIs, V1 and V3b had intermediate sensitivity and V3a was the least sensitive. But there were situations where drug sensitivity was not in line with the expected additive effects of mutation- and variant- dependent sensitivities, which indicated the necessity of taking both variants and mutations into consideration. Notably, belizatinib, a drug in development, was approximately twenty times as potent as lorlatinib for ALK-L1196M in all types of fusion variants. The promising efficacy of belizatinib against L1196M mutation of EML4-ALK were also proven by molecular dynamics simulations. In conclusion, the results of this study provided sensitivity spectra with clinical confirmed EML4-ALK mutation-fusion combinations to nine ALK-TKIs, offered a possible optimal sequence of ALK-TKIs for treating ALK-positive patients developing resistance mutations during therapy and suggested belizatinib as a promising possible targeted inhibitor for the L1196M mutation of EML4-ALK in NSCLC

TEPOTINIB REVERSES ABCB1- AND ABCG2-MEDIATED MULTIDRUG RESISTANCE IN CANCER

Overexpression of ATP-binding cassette (ABC) transporters ABCB1 and ABCG2 in cancer cells have been linked to the development of multidrug resistance (MDR), an obstacle to cancer therapy. Therefore, it is important to inhibit ABCB1/ABCG2 activity in order to maintain an effective intracellular level of chemotherapeutic drugs in drug-resistant cancer cells. Tepotinib is an ATP-competitive MET kinase inhibitor approved for the treatment of adult patients with metastatic non–small cell lung cancer harboring MET exon 14 skipping alterations. In the present study, we identified that the MET inhibitor tepotinib can reverse ABCB1- and ABCG2-mediated MDR by directly binding to the drug-binding site of the transporters and reversibly inhibiting drug efflux activity, therefore enhancing the cytotoxicity of substrate drugs in drug-resistant cancer cells. Furthermore, the ABCB1/ABCG2 double-transfected cell model and ABCG2 gene knockout cell model demonstrated that tepotinib specifically inhibits these two MDR-related ABC transporters. The ATPase assay showed that tepotinib concentration-dependently inhibited the ATPase activity of ABCB1 but stimulated the ATPase activity of ABCG2. Furthermore, treatment with tepotinib did not alter protein expression or subcellular localization of ABCB1/ABCG2. The docking simulation suggested a high binding affinity of tepotinib with ABCB1/ABCG2 drug-binding site. In mouse bearing drug-resistant tumors, tepotinib increased the intratumoral accumulation of ABCG2 substrate drug topotecan and enhanced its antitumor effect. Taken together, our study provides a new potential of repositioning tepotinib as a dual ABCB1/ABCG2 inhibitor and combining tepotinib with substrate drugs to antagonize MDR

Design, synthesis, characterization and anti-cancer effects evaluation of interchain cysteine linked antibody drug conjugates

Antibody drug conjugates (ADCs) are emerging therapeutic products specially designed for the treatment of cancers. Potent cytotoxic agents are linked to antibodies via linkers. Antibody drug conjugates, which take advantage of both antibodies and cytotoxic agents, showed high selectivity towards cancer cells. Cytotoxic agents are delivered to the cancer cells selectively and kill them from inside while leaving low or non-toxicity towards normal cells. Antibody drug conjugates are actually heterogeneous mixtures. The major heterogeneities mainly lie in the drug to antibody ratio (DAR) and drug linking positions, both of which potentially affect the therapeutic index of ADCs. In this research, we focused on the interchain cysteine linked ADCs, which are the most popular class. We first developed methods and characterized the drug linking position heterogeneity. Positional isomers of interchain cysteine linked ADCs were separated and their relative abundance was determined. In addition, novel LC/HRMS methods were developed for the accurate determination of the DAR with cleavable and non-cleavable linkers. Meanwhile, we also designed and synthesized new ADCs, namely Cetuximab-Staurosporine for EGFR over expressed Non-Small Cell Lung Cancer. The anticancer effects were evaluated on A549 human lung cancer cells --Abstract, page iv

A secondary RET mutation in the activation loop conferring resistance to vandetanib

Resistance to vandetanib, a type I RET kinase inhibitor, developed in a patient with metastatic lung adenocarcinoma harboring a CCDC6-RET fusion that initially exhibited a response to treatment. The resistant tumor acquired a secondary mutation resulting in a serine-to-phenylalanine substitution at codon 904 in the activation loop of the RET kinase domain. The S904F mutation confers resistance to vandetanib by increasing the ATP affinity and autophosphorylation activity of RET kinase. A reduced interaction with the drug is also observed in vitro for the S904F mutant by thermal shift assay. A crystal structure of the S904F mutant reveals a small hydrophobic core around F904 likely to enhance basal kinase activity by stabilizing an active conformer. Our findings indicate that missense mutations in the activation loop of the kinase domain are able to increase kinase activity and confer drug resistance through allosteric effects