A simple and rapid pre-clinical in vivo model reveals comparative cardiotoxicity profiles of kinase inhibitors.

Despite significant success, targeted therapeutics such as kinase inhibitors (KIs) still pose adverse events such as the cardiotoxicity. There is a lot of variation in the type and intensity of cardiotoxicity caused by different KIs and current pre-clinical models are inadequate to predict it. Thus, there is a need to develop more simple and rapid models for screening of novel KIs at the pre-clinical step itself. We thus aimed to establish a rapid and robust pre-clinical animal model for predicting cardiotoxicity of KIs and identify comparative cardiotoxicity profiles of a panel of FDA-approved KIs. Heart rate measurement and survival analysis of Daphnia was performed at regular intervals following treatment with ten KIs that were approved for the treatment of various cancers. The heart rates of Daphnia as well as the survival varied between KIs in a dose and time dependent manner suggesting differential cardiotoxicity profiles of various KIs. Further, the correlation between the cardiotoxicity and survival also varied among the ten KIs. Importantly, sorafenib and vemurafenib displayed maximum and least cardiotoxicity, respectively. The comparative cardiotoxicity profiles also are in conformity with the previous studies indicating the utility of Daphnia as a valuable and relevant animal model to rapidly predict the cardiotoxicity of novel KIs at a pre-clinical stage.

AMPK-induced novel phosphorylation of RUNX1 inhibits STAT3 activation and overcome imatinib resistance in chronic myelogenous leukemia (CML) subjects.

Imatinib resistance remains an unresolved problem in CML disease. Activation of JAK2/STAT3 pathway and increased expression of RUNX1 have become one reason for development of imatinib resistance in CML subjects. Metformin has gained attention as an antileukemic drug in recent times. However, the molecular mechanism remains elusive. The present study shows that RUNX1 is a novel substrate of AMP-activated kinase (AMPK), where AMPK phosphorylates RUNX1 at Ser 94 position. Activation of AMPK by metformin could lead to increased cytoplasmic retention of RUNX1 due to Ser 94 phosphorylation. RUNX1 Ser 94 phosphorylation resulted in increased interaction with STAT3, which was reflected in reduced transcriptional activity of both RUNX1 and STAT3 due to their cytoplasmic retention. The reduced transcriptional activity of STAT3 and RUNX1 resulted in the down-regulation of their signaling targets involved in proliferation and anti-apoptosis. Our cell proliferation assays using in vitro resistant cell line models and PBMCs isolated from CML clinical patients and normal subjects demonstrate that metformin treatment resulted in reduced growth and improved imatinib sensitivity of resistant subjects.

Large-scale pathogenicity prediction analysis of cancer-associated kinase mutations reveals variability in sensitivity and specificity of computational methods.

BACKGROUND: Mutations in kinases are the most frequent genetic alterations in cancer; however, experimental evidence establishing their cancerous nature is available only for a small fraction of these mutants. AIMS: Predicition analysis of kinome mutations is the primary aim of this study. Further objective is to compare the performance of various softwares in pathogenicity prediction of kinase mutations. MATERIALS AND METHODS: We employed a set of computational tools to predict the pathogenicity of over forty-two thousand mutations and deposited the kinase-wise data in Mendeley database (Estimated Pathogenicity of Kinase Mutants [EPKiMu]). RESULTS: Mutations are more likely to be drivers when being present in the kinase domain (vs. non-kinase domain) and belonging to hotspot residues (vs. non-hotspot residues). We identified that, while predictive tools have low specificity in general, PolyPhen-2 had the best accuracy. Further efforts to combine all four tools by consensus, voting, or other simple methods did not significantly improve accuracy. DISCUSSION: The study provides a large dataset of kinase mutations along with their predicted pathogenicity that can be used as a training set for future studies. Furthermore, a comparative sensitivity and selectivity of commonly used computational tools is presented. CONCLUSION: Primary-structure-based in silico tools identified more cancerous/deleterious mutations in the kinase domains and at the hot spot residues while having higher sensitivity than specificity in detecting deleterious mutations.

Metformin exerts antileukemic effects by modulating lactate metabolism and overcomes imatinib resistance in chronic myelogenous leukemia.

Imatinib is the frontline treatment option in treating chronic myelogenous leukemia (CML). Hitherto, some patients relapse following treatment. Biochemical analysis of a panel of clonally derived imatinib-resistant cells revealed enhanced glucose uptake and ATP production, suggesting increased rates of glycolysis. Interestingly, increased lactate export was also observed in imatinib-resistant cell lines. Here, we show that metformin inhibits the growth of imatinib-resistant cell lines as well as peripheral blood mononuclear cells isolated from patients who relapsed following imatinib treatment. Metformin exerted these antiproliferative effects by inhibiting MCT1 and MCT4, leading to the inhibition of lactate export. Furthermore, glucose uptake and ATP production were also inhibited following metformin treatment due to the inhibition of GLUT1 and HK-II in an AMPK-dependent manner. Our results also confirmed that metformin-mediated inhibition of lactate export and glucose uptake occurs through the regulation of mTORC1 and HIF-1α. These results delineate the molecular mechanisms underlying metabolic reprogramming leading to secondary imatinib resistance and the potential of metformin as a therapeutic option in CML.

Applications of promiscuity of FDA-approved kinase inhibitors in drug repositioning and toxicity.

Promiscuity of therapeutics has important implications in treatment and toxicity. So far, a comprehensive understanding of promiscuity related to kinase inhibitors is lacking and such an analysis may offer potential opportunities for drug repurposing. In the present study, profiling of inhibitor-specific kinases based on the available biochemical IC50s was performed, fold-change of IC50 values for additional targets were calculated by taking the primary target as the reference kinase, and finally the promiscuity degree (PD) for FDA-approved kinase inhibitors was calculated. Surprisingly, class II inhibitors showed more PD than that of the class I inhibitors. We further identified cancer types and sub-types in which additional kinase targets or off-targets of inhibitors were overexpressed for potential drug repurposing. In addition, the expression of these kinases in normal human tissues were also profiled to predict toxicity following drug repositioning. Taken together, the study offers opportunities for cancer treatment in a kinase-specific manner.

Molecular docking analysis reveals differential binding affinities of multiple classes of selective inhibitors towards cancer-associated KRAS mutants.

KRAS is the most frequently mutated oncogene in solid cancers, and inhibitors that specifically target the KRAS-G12C mutant were recently approved for clinical use. The limited availability of experimental data pertaining to the sensitivity of KRAS-non-G12C mutants towards RAS inhibitors made it difficult to predict the response of KRAS-mutated cancers towards RAS-targeted therapies. The current study aims at evaluating sensitivity profiles of KRAS-non-G12C mutations towards clinically approved sotorasib and adagrasib, and experimental RAS inhibitors based on binding energies derived through molecular docking analysis. Computationally predicted sensitivities of KRAS mutants conformed with the available but limited experimental data, thus validating the usefulness of molecular docking approach in predicting clinical response towards RAS inhibitor treatment. Our results indicate differential sensitivity of KRAS mutants towards both clinical and experimental therapeutics; while certain mutants exhibited broad cross-resistance to most inhibitors, some mutants showed resistance towards specific inhibitors. These results thus suggest the potential of emergence of more resistance mutations in future towards RAS-targeted therapy and points to an urgent need to develop novel classes of inhibitors that are able to overcome both primary and secondary drug resistance. Supplementary Information: The online version contains supplementary material available at 10.1007/s13205-022-03407-9.

Estimated sensitivity profiles of lung cancer specific uncommon BRAF mutants towards experimental and clinically approved kinase inhibitors.

Current experimental and clinical data are inadequate to conclusively predict the oncogenicity of uncommon BRAF mutants and their sensitivity towards kinase inhibitors. Therefore, the present study aims at estimating sensitivity profiles of uncommon lung cancer specific BRAF mutations towards clinically approved as well as experimental therapeutics based on computationally derived direct binding energies. Based on the data derived from cBioportal, BRAF mutants displayed significant mutual exclusivity with KRAS and EGFR mutants indicating them as potential drivers in lung cancer. Predicted sensitivity of BRAF-V600E conformed to published experimental and clinical data thus validating the usefulness of computational approach. The BRAF-V600K displayed higher sensitivity to most inhibitors as compared to that of the BRAF-V600E. All the uncommon mutants displayed higher sensitivity than both the wild type and BRAF-V600E towards PLX 8394 and LSN3074753. While V600K, G469R and N581S displayed favorable sensitivity profiles to most inhibitors, V600L/M, G466A/E/V and G469A/V displayed resistance profiles to a variable degree. Notably, molecular dynamic (MD) simulation revealed that increased number of interactions caused enhanced sensitivity of G469R and N581S towards sorafenib. RAF kinase inhibitors were further classified into two groups as per their selectivity (Group I: BRAF-V600E-selective and Group II: CRAF-selective) based on which potential mutation-wise combinations of RAF kinase inhibitors were proposed to overcome resistance. Based on computational inhibitor sensitivity profiles, appropriate treatment strategies may be devised to prevent or overcome secondary drug resistance in lung cancer patients with uncommon mutations.

Squamous cell carcinoma of the lung: improving the detection and management of immune-related adverse events.

INTRODUCTION: Immune checkpoint inhibitors (ICIs) have revolutionized treatment for patients with non-small lung cancer (NSCLC). Currently approved ICIs are monoclonal antibodies that target programmed death receptor 1 (PD-1), its ligand PD-L1, or CTLA-4. With ICIs comes a novel collection of toxicities: immune-related adverse events (IRAEs). Management of IRAEs requires multidisciplinary expertise. We review the biology of IRAEs and their management in patients with squamous NSCLC. AREAS COVERED: We review the pathophysiology of ICIs and IRAEs. For IRAEs related to squamous NSCLC, Cochrane Central, EMBASE, and PubMed were queried for trials with patients with squamous cell carcinoma or adenocarcinoma histology, who were assessed for incidence rates of IRAEs. Thirteen trials met inclusion criteria. National guidelines are reviewed to outline management strategies for IRAEs. EXPERT OPINION: IRAEs are unique compared to standard chemotherapy. As the role of ICIs expand across all stages of squamous cell NSCLC and with different combinations of antineoplastics, management of IRAEs will become crucial. Optimal management of IRAEs requires multidisciplinary teamwork. Further investigation into the pathophysiology of IRAEs can enhance current management strategies.

Synthesis and cytotoxicity of novel 14α-O-(1,4-disubstituted-1,2,3-triazolyl) ester derivatives of andrographolide.

A series of novel 14α-O-(1,4-disubstituted-1,2,3-triazolyl) ester derivatives of andrographolide (5a-n) were synthesized from andrographolide (1). For this endeavour, selective esterification at C-14 hydroxyl group of andrographolide (1) with propiolic acid via protection, deprotection strategy followed by 1,4-regioselective [1,3]dipolar cycloaddition of alkyne, azide using Cu(I) catalyzed Click chemistry. All the synthesized derivatives were screened for their cytotoxicity on HCT-15, HeLa and K562 cell lines. Compounds 5c and 5j showed highest activity against HCT-15 and K562 cell lines whereas compound 5a displayed activity in all the three cell lines. Loss of cell viability was not observed with the non-transformed cell line MRC-5 with compounds 5j, 5k, 5h and 2 indicating cytotoxic activity of these compounds towards cancer cell lines. Further, molecular docking analysis and SAR studies of highly active compounds 5c and 5j revealed enhanced binding affinity to the target NF-κB protein.

Analysis of cellular models of clonal evolution reveals co-evolution of imatinib and HSP90 inhibitor resistances.

Treatment relapse due to clonal evolution was shown to be an independent factor for poor prognosis in advanced stages of chronic myeloid leukemia. Overcoming secondary resistance arising due to clonal evolution is still an unmet need and lack of adequate pre-clinical models hampers the identification of underlying mechanisms and testing of alternate treatment strategies. The current study thus aimed to create cellular models to study molecular mechanisms underlying clonal evolution and identify strategies to overcome the secondary drug resistance. Analysis of cell lines derived from three independent cell-based screens revealed the co-evolution specifically of imatinib and HSP90 inhibitor (HSP90i) resistances despite their exposure to a single inhibitor alone. Molecular and biochemical characterization of these cell lines revealed additional cytogenetic abnormalities, differential activation of pro-survival signaling molecules and over expression of ABL kinase and HSP90 genes. Importantly, all the imatinib-HSP90i dual resistant cell lines remained sensitive to sorafenib and vorinostat suggesting their utility in treating patients who relapse upon imatinib treatment due to clonal evolution. In addition, we cite similar examples of dual resistance towards various kinase inhibitors and HSP90i in some cell lines that represent solid cancers suggesting co-evolution leading to secondary drug resistance as a pan-cancer phenomenon. Taken together, our results suggest the efficacy of HSP90i in overcoming drug resistance caused by point mutations in the target kinase but not in cases of clonal evolution.

Epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors in non-small cell lung cancer harboring uncommon EGFR mutations: Focus on afatinib.

The development of first-, second-, and third-generation epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) has revolutionized the treatment of patients with non-small cell lung cancer (NSCLC) harboring mutations in the EGFR. However, limited data are available regarding the activity of available EGFR TKIs against uncommon EGFR mutations. This is an important question because improvements in screening techniques are facilitating the identification of patients with uncommon mutations for whom optimal treatment has not yet been clarified. This uncertainty reflects the fact that most prospective clinical trials of EGFR TKIs have been restricted to patients with tumor harboring common (Del19 or L858R) mutations. In this article, we discuss the nature of EGFR mutation heterogeneity in NSCLC and review recent preclinical and clinical data that have assessed the sensitivity of different mutations to different EGFR TKIs. Recent preclinical data indicate that second-generation ErbB family blockers, such as afatinib, have a broad activity profile across uncommon EGFR mutations. Emerging evidence indicates that the preclinical data for afatinib are reflected in the clinic. Subanalysis of clinical trials, and real-world data, demonstrate that EGFRs with defined, but uncommon mutations such as G719X, S768I, and L861Q are sensitive to afatinib, which is now approved for tumors harboring these mutations. A recent clinical trial has demonstrated that EGFRs harboring some of these less common mutations also appear to be sensitive to the third-generation EGFR TKI, osimertinib. Treatment options for tumors with other uncommon mutations, notably exon 20 insertion, remain an area of unmet need, although osimertinib has shown preclinical activity in this setting, and early clinical activity has been seen with the dual EGFR/HER2 TKIs, poziotinib and TAK-788. Further data are required to help drive appropriate treatment decisions in patients whose tumors harbor these uncommon EGFR mutations. To see an abstract video summarising the content of the paper, please visit http://usscicomms.com/oncology/masood/seminars-in-oncology/.

Emergence of ERBB2 Mutation as a Biomarker and an Actionable Target in Solid Cancers.

The oncogenic role ERBB2 amplification is well established in breast and gastric cancers. This has led to the development of a well-known portfolio of monoclonal antibodies and kinase inhibitors targeting the ERBB2 kinase. More recently, activating mutations in the ERBB2 gene have been increasingly reported in multiple solid cancers and were shown to play an oncogenic role similar to that of ERBB2 amplification. Thus, ERBB2 mutations define a distinct molecular subtype of solid tumors and serve as actionable targets. However, efforts to target ERBB2 mutation has met with limited clinical success, possibly because of their low frequency, inadequate understanding of the biological activity of these mutations, and difficulty in separating the drivers from the passenger mutations. Given the current impetus to deliver molecularly targeted treatments for cancer, there is an important need to understand the therapeutic potential of ERBB2 mutations. Here we review the distribution of ERBB2 mutations in different tumor types, their potential as a novel biomarker that defines new subsets in many cancers, and current data on preclinical and clinical efforts to target these mutations. IMPLICATIONS FOR PRACTICE: A current trend in oncology is to identify novel genomic drivers of solid tumors and developing precision treatments that target them. ERBB2 amplification is an established therapeutic target in breast and gastric cancers, but efforts to translate this finding to other solid tumors with ERBB2 amplification have not been effective. Recently the focus has turned to targeting activating ERBB2 mutations. The year 2018 marked an important milestone in establishing ERBB2 mutation as an important actionable target in multiple cancer types. There have been several recent preclinical and clinical studies evaluating ERBB2 mutation as a therapeutic target with varying success. With increasing access to next-generation sequencing technologies in the clinic, oncologists are frequently identifying activating ERBB2 mutations in patients with cancer. There is a significant need both from the clinician and bench scientist perspectives to understand the current state of affairs for ERBB2 mutations.

Evolvement of nutraceutical onion plants engineered for resveratrol biosynthetic pathway.

KEY MESSAGE: Genetically engineered onion expressing codon-optimized VvSTS1 gene accumulated stilbenes and extended life span in yeast and can serve as potential nutraceutical. Resveratrol (RV) is a natural polyphenolic compound found in certain plant species including grapes. RV is well known for its nutraceutical properties and to assuage several disease conditions. Onion is the second most consumed vegetable worldwide and contains large quantities of precursor molecules, malonyl-CoA and para-coumaroyl-CoA that are needed for RV biosynthesis. The present study reports the development of nutraceutical onion by engineering RV biosynthetic pathway. A codon-optimized grapevine synthetic stilbene synthase gene (VvSTS1) was synthesized using native grapevine sequence. Six-week-old healthy yellowish compact nodular calli were co-cultivated with Agrobacterium tumefaciens harbouring pCAMBIA1300-hpt II-CaMV35S-VvSTS1-nos. PCR analysis revealed the presence of VvSTS1 and hpt II genes in putative transgenics. Southern blot analysis confirmed the integration of VvSTS1 gene and independent nature of transformants. LC-ESI-HRMS analysis revealed the accumulation of variable quantities of RV (24.98-50.18 µg/g FW) and its glycosylated form polydatin (33.6-67.15 µg/g FW) in both leaves and bulbs, respectively, indicating the successful engineering of RV biosynthetic pathway into onion. The transgenic onion bulb extracts extended the life span in haploid yeast. The transgenic onion accumulating RV and polydatin, developed for the first of its kind, may serve as a potential nutraceutical resource.

Molecular docking analysis of imine stilbene analogs and evaluation of their anti-aging activity using yeast and mammalian cell models.

The NAD+-dependent histone deacetylase SIRT1 was shown to be associated with aging and longevity. A stilbene, resveratrol (RV) was shown to exert anti-aging activity by stimulating the SIRT1 activity. However, the utility of RV is limited by its low bioavailability and structural instability. It is thus envisaged to test imine stilbene (IMS) analogs of RV for their potential anti-aging activity. In the present study, molecular docking analysis of five IMS analogs (3a, 3b, 3c, 3d and 3e) against the SIRT1 protein has been carried out. All the five IMS analogs displayed enhanced binding affinity towards SIRT1; three out of five IMS analogs (3a, 3 b, 3e) showed significantly higher affinity with lower binding energies (-9.58, -9.54, and -9.82 kcal mol-1) than RV (-8.11 kcal mol-1). Further, experimental validation of anti-aging activity was performed by measuring the chronological life span in vitro using yeast and cellular replicative senescence (CRS) in mammalian cell line models. All IMS analogs extended the chronological life span in yeast as compared to untreated cells as well as RV treated cells. Enhanced anti-aging activity was also observed in an analogous mammalian cell line model upon treatment with either RV or IMS analogs. The results thus suggest that most of the IMS analogs tested may serve as potent drug lead molecules with anti-aging activity.

Elucidation of conformational diversity of druggable enzymes and classification of chemical modulators based on inhibitor-bound structures.

Communicated by Ramaswamy H. Sarma.

Computational Analysis of Epidermal Growth Factor Receptor Mutations Predicts Differential Drug Sensitivity Profiles toward Kinase Inhibitors.

INTRODUCTION: A significant proportion of patients with lung cancer carry mutations in the EGFR kinase domain. The presence of a deletion mutation in exon 19 or L858R point mutation in the EGFR kinase domain has been shown to cause enhanced efficacy of inhibitor treatment in patients with NSCLC. Several less frequent (uncommon) mutations in the EGFR kinase domain with potential implications in treatment response have also been reported. The role of a limited number of uncommon mutations in drug sensitivity was experimentally verified. However, a huge number of these mutations remain uncharacterized for inhibitor sensitivity or resistance. METHODS: A large-scale computational analysis of clinically reported 298 point mutants of EGFR kinase domain has been performed, and drug sensitivity profiles for each mutant toward seven kinase inhibitors has been determined by molecular docking. In addition, the relative inhibitor binding affinity toward each drug as compared with that of adenosine triphosphate was calculated for each mutant. RESULTS: The inhibitor sensitivity profiles predicted in this study for a set of previously characterized mutants correlated well with the published clinical, experimental, and computational data. Both the single and compound mutations displayed differential inhibitor sensitivity toward first- and next-generation kinase inhibitors. CONCLUSIONS: The present study provides predicted drug sensitivity profiles for a large panel of uncommon EGFR mutations toward multiple inhibitors, which may help clinicians in deciding mutant-specific treatment strategies.

Synthesis of novel forskolin isoxazole derivatives with potent anti-cancer activity against breast cancer cell lines.

Forskolin C1-isoxazole derivatives (3,5-regioisomers) (11a-e, 14, 15a-h and 15, 16a-g) were synthesized regioselectively by adopting 1,3-dipolar cycloadditions. These derivatives were tested using estrogen receptor positive breast cancer cell lines MCF-7 and BT-474. Majority of the compounds exhibited activity against the p53-positive MCF-7 breast cancer cells but not against the p53-negative BT-474 breast cancer cells. Among forskolin derivatives, compounds 11a, 11c, 14a, 14f, 14g, 14h, 15b, 16g and 17b exhibited higher anti-cancer activity against MCF-7 cell line with an IC50≤1µM. The derivative 14f exhibited highest activity in both p53-positive (MCF-7) and p53-negative (BT-474) breast cancer cell lines with an IC50 of 0.5µM.

Computational analysis of ABL kinase mutations allows predicting drug sensitivity against selective kinase inhibitors.

The ABL kinase inhibitor imatinib has been used as front-line therapy for Philadelphia-positive chronic myeloid leukemia. However, a significant proportion of imatinib-treated patients relapse due to occurrence of mutations in the ABL kinase domain. Although inhibitor sensitivity for a set of mutations was reported, the role of less frequent ABL kinase mutations in drug sensitivity/resistance is not known. Moreover, recent reports indicate distinct resistance profiles for second-generation ABL inhibitors. We thus employed a computational approach to predict drug sensitivity of 234 point mutations that were reported in chronic myeloid leukemia patients. Initial validation analysis of our approach using a panel of previously studied frequent mutations indicated that the computational data generated in this study correlated well with the published experimental/clinical data. In addition, we present drug sensitivity profiles for remaining point mutations by computational docking analysis using imatinib as well as next generation ABL inhibitors nilotinib, dasatinib, bosutinib, axitinib, and ponatinib. Our results indicate distinct drug sensitivity profiles for ABL mutants toward kinase inhibitors. In addition, drug sensitivity profiles of a set of compound mutations in ABL kinase were also presented in this study. Thus, our large scale computational study provides comprehensive sensitivity/resistance profiles of ABL mutations toward specific kinase inhibitors.

FeCl3 catalysed 7-membered ring formation in a single pot: a new route to indole-fused oxepines/azepines and their cytotoxic activity.

Various oxepine and azepine fused N-heterocyclic derivatives were synthesized using a new and one-pot reaction of 2,3-dichloro quinoxaline/pyrazine with 2-(1H-indol-2-yl)phenol/aniline in the presence of 25 mol% FeCl3. The reaction proceeded via C-C bond followed by C-X (X = O or N) bond formation to construct the central 7-membered ring, affording the desired products in good yields. The structure assignment was confirmed by the single crystal X-ray analysis of a synthesized oxepine fused N-heterocycle derivative. Most of the synthesized compounds were found to be promising when tested for their anti-proliferative properties against cervical and breast cancer cell lines.

Targeting oncoprotein stability overcomes drug resistance caused by FLT3 kinase domain mutations.

FLT3 is the most frequently mutated kinase in acute myeloid leukemia (AML). Internal tandem duplications (ITDs) in the juxta-membrane region constitute the majority of activating FLT3 mutations. Several FLT3 kinase inhibitors were developed and tested in the clinic with significant success. However, recent studies have reported the development of secondary drug resistance in patients treated with FLT3 inhibitors. Since FLT3-ITD is an HSP90 client kinase, we here explored if targeting the stability of drug-resistant FLT3 mutant protein could be a potential therapeutic option. We observed that HSP90 inhibitor treatment resulted in the degradation of inhibitor-resistant FLT3-ITD mutants and selectively induced toxicity in cells expressing FLT3-ITD mutants. Thus, HSP90 inhibitors provide a potential therapeutic choice to overcome secondary drug resistance following TKI treatment in FLT3-ITD positive AML.