Inhibition Of Tyrosine Kinase Activity Research Articles

Structure-Activity Relationship of Substituted Pyrazoline Derivatives as Small Molecule Tyrosine Kinase Inhibitors.

Tyrosine kinase inhibitors (TKIs) target certain cell signalling pathways, and have become a promising class of medications for the treatment of cancer in recent years. Because of their distinct structure and adaptable chemistry, pyrazolines have drawn a lot of interest from organic and medicinal chemists. Their exceptional TKI activity has prompted them to investigate chemotherapy for cancer. We aim to develop agents that inhibit tyrosine kinases highly effective with the least amount of harm possible, perhaps improving the course of cancer treatment. This review compiled current information from recent literature sources, includ-ing in vitro, in vivo, approved medications, active clinical trials, and the structure-activity relationships (SAR) linked to various pyrazoline analogues used as small-molecule Tyro-sine Kinase Inhibitors in cancer treatment. This study focuses on SAR inside the pyrazoline ring and its derivatives as TKIs, and it emphasizes current developments, including patents, authorized medications, and compounds in clinical trials. By enhancing our understanding of these compounds, our goal is to aid in making the roles of pharmacologists, scientists, and researchers who are designing and developing next-generation anticancer drugs with pyrazoline scaffolds easier. The future holds immense potential for the continued evolution of pyrazoline-based therapies, offer-ing renewed hope in the ongoing battle against cancer.

TKI type switching overcomes ROS1 L2086F in ROS1 fusion-positive cancers

ROS1 tyrosine kinase inhibitors (TKIs) are highly effective in ROS1-positive non-small cell lung cancer, but resistance remains a challenge. We investigated the activity of various TKIs against wildtype and mutant ROS1, focusing on the emerging L2086F resistance mutation. Using Ba/F3 and NIH3T3 cell models, CRISPR/Cas9-edited isogenic wildtype and mutant patient-derived cell lines, and in vivo tumor growth studies, we compared type I TKIs (crizotinib, entrectinib, taletrectinib, lorlatinib, and repotrectinib) to type II TKIs (cabozantinib and merestinib) and the type I FLT3 inhibitor gilteritinib. The ROS1 L2086F mutant kinase showed resistance to type I TKIs, while type II TKIs retained activity. Gilteritinib inhibited both wildtype and L2086F mutant ROS1 but was ineffective against the G2032R mutation. Structural analyses revealed distinct binding poses for cabozantinib and gilteritinib, explaining their efficacy against L2086F. Clinical cases demonstrated cabozantinib’s effectiveness in patients with TKI-resistant, ROS1 L2086F mutant NSCLCs. This study provides the first comprehensive report of ROS1 L2086F in the context of later-generation TKIs, including macrocyclic inhibitors. While cabozantinib effectively inhibits ROS1 L2086F, its multi-kinase inhibitor nature highlights the need for more selective and better-tolerated TKIs to overcome kinase-intrinsic resistance. Gilteritinib may offer an alternative for targeting ROS1 L2086F with distinct off-target toxicities, but further studies are required to fully evaluate its potential in this setting.

Abstract LB182: Mutagenesis screens support potential best-in-class profile for selective, brain-penetrant, and TRK-sparing ROS1 inhibitor NVL-520

Abstract Introduction. ROS1 fusions are recurrent oncogenic drivers in non-small cell lung cancer (NSCLC). Tyrosine kinase inhibitors (TKIs) crizotinib, entrectinib, and repotrectinib have been approved for the treatment of ROS1-positive NSCLC. Response to these TKIs can be limited by emergence of drug resistance, with ~40% of patients acquiring on-target ROS1 mutations at progression on crizotinib. NVL-520 is an investigational, brain-penetrant, and TRK-sparing ROS1 TKI designed to maintain activity for ROS1 mutations that confer resistance to approved therapies. To investigate suppression of resistance by NVL-520, we performed two N-ethyl-N-nitrosourea (ENU) mutagenesis screens and determined the frequency and identity of resistance mutations following treatment with NVL-520 versus earlier-generation ROS1 TKIs. Methods. Cells were treated with ENU for 1 day followed by crizotinib, entrectinib, repotrectinib, or NVL-520 for 28 days. Frequency of resistance indicates the fraction of wells that proliferated on day 28 despite continuous TKI treatment. Resistance mutations in the ROS1 kinase domain were identified by Sanger sequencing. Resistant clones harboring ROS1 kinase-domain mutations were expanded and used to profile the inhibitory activity of ROS1 TKIs. Results. The first ENU screen was performed on Ba/F3 CD74-ROS1, simulating a first-line treatment setting. NVL-520 at an unbound drug concentration (Cunbound) of 8.2 nM yielded 1 resistant clone among 480 replicate wells (frequency of resistance = 0.2%). The second screen was performed on Ba/F3 CD74-ROS1 G2032R, which is the most common point mutation after progression on previous-generation therapies, simulating a later-line treatment setting. NVL-520 at Cunbound of 41 nM yielded 6 resistant clones among 480 replicate wells (frequency of resistance = 1.3%). These NVL-520 concentrations (8.2 - 41 nM) were well within its average Cunbound of 166 nM at the recommended phase 2 dose (RP2D) of 100 mg once daily. None of the 7 resistant clones harbored ROS1 kinase-domain mutations, suggesting an off-target resistance mechanism. Crizotinib, entrectinib, and repotrectinib at their respective average human Cunbound yielded frequencies of resistance ranging from >33% to 100% in at least one of the screens, indicating that they were less effective than NVL-520 at suppressing resistance in these screens. Conclusion. NVL-520 was highly effective at suppressing resistance in ENU mutagenesis screens simulating first-line (CD74-ROS1) and later-line (CD74-ROS1 G2032R) treatment. The screens did not reveal any novel ROS1 mutations that could confer resistance to NVL-520 at the RP2D. The broad preclinical activity against ROS1 and diverse ROS1 mutations, together with its brain penetrance and TRK-sparing design, suggests NVL-520 as a potential best-in-class ROS1 therapy. Citation Format: Anupong Tangpeerachaikul, Franklin Gu, Henry E. Pelish. Mutagenesis screens support potential best-in-class profile for selective, brain-penetrant, and TRK-sparing ROS1 inhibitor NVL-520 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB182.

Abstract 5857: Trastuzumab deruxtecan resistance can be mediated by payload resistance or secondary extracellular ERBB2 mutations but sensitivity to HER2 tyrosine kinase inhibitors is maintained

Abstract Among NSCLC patients, the most common HER2 mutation is the exon 20 insertion mutation, Y772dupYVMA, which accounts for over 40% of all HER2 mutations in lung cancer. Trastuzumab deruxtecan (T-DXd), a HER2 antibody-drug conjugate (ADC), received FDA approval for the treatment of HER2 mutant NSCLC yielding a confirmed objective response of 52% and a median duration of response of greater than 9 months. Unfortunately, patients that initially respond to T-DXd will eventually acquire resistance, and the mechanisms of resistance as well as effective targeting strategies to overcome resistance are not yet elucidated. We generated Ba/F3 cells expressing the HER2 YVMA insertion mutation with acquired resistance to T-DXd by culturing cells in T-DXd until resistance occurred. T-DXd resistant cells retained expression of HER2, but were resistant to the payload, deruxtecan, as well as other topoisomerase inhibitors. T-DXd resistant cells exhibited loss of topoisomerase I, a previously reported mechanism of topoisomerase inhibitor resistance. In parallel, we generated a PDX model of acquired T-DXd resistance and observed sustained expression of HER2 but loss of TOP1 in resistant tumors. T-DXd resistant cell lines were sensitive to payloads with alternate mechanisms of action including maytansine and likewise retained sensitivity to the HER2 ADC trastuzumab emtansine (T-DM1) which utilizes DM1 as a payload. Given that T-DXd resistant cells retained HER2 expression, we assessed whether they were sensitive to HER2 tyrosine kinase inhibitors (TKI). T-DXd resistant cells remained highly sensitive to HER2 TKIs including poziotinib, afatinib, BI-4142, and BI-1622. To investigate whether genomic alterations of HER2 could facilitate HER2 ADC resistance, we utilized the LentiMutate approach which employs an error-prone HIV-1 reverse transcriptase to produce a high frequency of mutations to identify resistance-associated mutations in cells treated with a HER2 ADC. Sequencing analysis revealed that resistant cells had an enrichment in point mutations within HER2 domain IV (D582N, F595C/S, E580K, C623Y), which includes the binding site of trastuzumab. To validate the impact of these mutations on HER2 ADC resistance, we generated cells expressing a HER2 exon 20 insertion in combination with the observed domain IV mutations. D582N and F695C/S co-mutations conferred resistance to T-DXd and T-DM1 but not to HER2 TKIs. These data demonstrate that resistance to T-DXd can be mediated by loss of sensitivity to the ADC payload in which case ADCs bearing payloads with alternate mechanisms of action as well as HER2 TKIs retain anti-tumor cell activity. Moreover, resistance to trastuzumab-based ADCs can also be mediated by secondary mutations within domain IV of HER2. However, these resistance mutations do not diminish HER2 TKI activity. Citation Format: Monique B. Nilsson, Xiuning Le, Junqin He, Xiaoxing Yu, Xiaofang Huo, Ashwani Kumar, Alissa Poteete, Qian Huang, Ralf Kittler, John Heymach. Trastuzumab deruxtecan resistance can be mediated by payload resistance or secondary extracellular ERBB2 mutations but sensitivity to HER2 tyrosine kinase inhibitors is maintained [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5857.

Src Tyrosine Kinase Inhibitory and Antioxidant Activity of Black Chokeberry and Bilberry Fruit Extracts Rich in Chlorogenic Acid.

Edible berries such as the fruits of black chokeberry (Aronia melanocarpa (Michx.) Elliott) and bilberry (Vaccinium myrtillus L.) are considered to be rich in phenolic compounds, which are nowadays attracting great interest due to their promising health benefits. The main objective of our study was to investigate, for the first time, their inhibitory properties on Src tyrosine kinase activity, as this enzyme plays an important role in multiple cellular processes and is activated in both cancer and inflammatory cells. In hydroethanolic fruit extracts, 5.0-5.9% of total polyphenols were determined spectrophotometrically, including high amounts of hydroxycinnamic acid derivatives. HPLC analysis revealed that the black chokeberry and bilberry extracts contained 2.05 mg/g and 2.54 mg/g of chlorogenic acid, respectively. Using a time-resolved fluorescence resonance energy transfer (TR-FRET) assay, the extracts studied were found to have comparable inhibitory effects on Src tyrosine kinase, with IC50 values of 366 µg/mL and 369 µg/mL, respectively. The results also indicated that chlorogenic acid contributes significantly to the observed effect. In addition, both fruit extracts exhibited antioxidant activity by scavenging DPPH and NO radicals with SC50 values of 153-352 µg/mL. Our study suggested that black chokeberry and bilberry fruits may be beneficial in cancer and other inflammation-related diseases.

Alk-rearranged lung adenocarcinoma: From molecular genetics to therapeutic targeting.

Anaplastic Lymphoma Kinase (ALK) is a potent oncogenic driver of lung adenocarcinoma (LUAD). ALK is constitutively activated by gene fusion events between the ALK and other gene fusion partners in about 2-3% of LUADs, characterized by few other gene alterations. ALK-fusions are a druggable target through potent pharmacological inhibitors of tyrosine kinase activity. Thus, several ALK-TKIs (Tyrosine Kinase Inhibitors) of first-, second- and third-generation have been developed that improved the outcomes of ALK-rearranged LUADs when used as first- or second-line agents. However, resistance mechanisms greatly limit the durability of the therapeutic effects elicited by these TKIs. The molecular mechanisms responsible for these resistance mechanisms have been in part elucidated, but overcoming acquired resistance to ALK-derived therapy remains a great challenge. Some new therapeutic strategies under investigation aim to induce long-term remission in ALK-fusion positive LUADs.

Potential Biomarkers Associated with Prognosis and Trastuzumab Response in HER2+ Breast Cancer.

Breast cancer (BC) is the most common malignancy among women worldwide. Around 15-25% of BC overexpress the human epidermal growth factor receptor 2 (HER2), which is associated with a worse prognosis and shortened disease-free survival. Therefore, anti-HER2 therapies have been developed, such as monoclonal antibodies (trastuzumab, Tz), antibody-drug conjugates (ado-trastuzumab emtansine, T-DM1), and pharmacological inhibitors of tyrosine kinase activity (lapatinib, Lp). Although Tz, the standard treatment, has significantly improved the prognosis of patients, resistance still affects a significant population of women and is currently a major challenge in clinical oncology. Therefore, this study aims to identify potential biomarkers to predict disease progression (prognostic markers) and the efficacy of Tz treatment (predictive markers) in patients with HER2+ BC. We hypothesize that proteins involved in cell motility are implicated in Tz-resistance. We aim to identify alterations in Tz-resistant cells to guide more efficient oncologic decisions. By bioinformatics, we selected candidate proteins and determined how their expression, localization, and the process they modulate were affected by anti-HER2 treatments. Next, using HER2+ BC patients' data, we assessed these proteins as prognostic and predictive biomarkers. Finally, using Tz-resistant cells, we evaluated their roles in Tz response. We identified deregulated genes associated with cell motility in Tz/T-DM1-resistant vs. -sensitive cells. We showed that Tz, T-DM1, and Lp decrease cell viability, and their effect is enhanced in combinations. We determined synergism between Tz/T-DM1 and Lp, making possible a dose reduction of each drug to achieve the same therapeutic effect. We found that combinations (Tz/T-DM1 + Lp) efficiently inhibit cell adhesion and migration. Furthermore, we demonstrated the induction of FAK nuclear and cortactin peri-nuclear localization after T-DM1, Lp, and Tz/T-DM1 + Lp treatments. In parallel, we observed that combined treatments downregulate proteins essential for metastatic dissemination, such as SRC, FAK, and paxillin. We found that low vinculin (VCL) and cortactin (CTTN) mRNA expression predicts favorable survival rates and has diagnostic value to discriminate between Tz-sensible and Tz-resistant HER2+ BC patients. Finally, we confirmed that vinculin and cortactin are overexpressed in Tz-resistance cells, SKBR3-RTz. Moreover, we found that Tz plus FAK/paxillin/cortactin-silencing reduced cell adhesion/migration capacity in Tz-sensitive and -resistant cells. In conclusion, we demonstrate that combined therapies are encouraging since low doses of Tz/T-DM1 + Lp inhibit metastatic processes by downregulating critical protein expression and affecting its subcellular localization. We propose that vinculin and cortactin might contribute to Tz-sensibility/resistance in BC cells. Finally, we identify potential prognostic and predictive biomarkers that are promising for personalized BC management that would allow efficient patient selection in order to mitigate resistance and maximize the safety and efficacy of anti-HER2 therapies.

Synthesis, Molecular Docking Studies, and In vitro Anticancer Evaluation of Novel Tolfenamic Acid Derivatives

Background: Accumulating experimental evidence has demonstrated that tolfenamic acid (TA) has anticancer activity. TA has been shown to modulate the expression of several cancer-related genes involved in apoptosis and cell cycle arrest, as well as metastasis and angiogenesis. Objective: The current study was carried out to evaluate the anticancer activities of eight newly synthesized TA derivatives by conducting in silico molecular docking studies and in vitro biological evaluations to validate their VEGFR-2 tyrosine kinase inhibitory activities. Methods: The novel TA derivatives (3A–F–5) were obtained by reacting TA hydrazide with substituted aldehydes, phthalic anhydride, and succinic anhydride, respectively. Spectroscopic techniques were used to characterize the target molecules. Docking studies were performed to determine the binding patterns to the potential molecular target VEGFR-2, and these were compared with the results of the in vitro VEGFR-2 tyrosine kinase (TK) inhibition assay. Results: Our findings indicate that the newly synthesized compounds are cytotoxic, with compound 4 being the most potent. Additionally, all compounds inhibited VEGFR-2 TK activity, the EC50 value of compound 4 was nearly identical to that of the conventional VEGFR TK inhibitor sorafenib. SAR studies revealed that the presence of an aryl or a heteroaryl fragment attached to the hydrophilic linker (as found in compound 4) is crucial to the anticancer activity. Conclusion: The outcomes suggest that the isoindoline derivative (compound 4) is a good candidate for further investigation. The docking results provide evidence for a correlation between the experimental and predicted VEGFR-2 TK inhibitory activity. Moreover, ADMET studies indicate that all ligands have good pharmacokinetic properties.

Emerging therapeutics and evolving assessment criteria for intracranial metastases in patients with oncogene-driven non-small-cell lung cancer.

The improved survival outcomes of patients with non-small-cell lung cancer (NSCLC), largely owing to the improved control of systemic disease provided by immune-checkpoint inhibitors and novel targeted therapies, have highlighted the challenges posed by central nervous system (CNS) metastases as a devastating yet common complication, with up to 50% of patients developing such lesions during the course of the disease. Early-generation tyrosine-kinase inhibitors (TKIs) often provide robust systemic disease control in patients with oncogene-driven NSCLCs, although these agents are usually unable to accumulate to therapeutically relevant concentrations in the CNS owing to an inability to cross the blood-brain barrier. However, the past few years have seen a paradigm shift with the emergence of several novel or later-generation TKIs with improved CNS penetrance. Such agents have promising levels of activity against brain metastases, as demonstrated by data from preclinical and clinical studies. In this Review, we describe current preclinical and clinical evidence of the intracranial activity of TKIs targeting various oncogenic drivers in patients with NSCLC, with a focus on newer agents with enhanced CNS penetration, leptomeningeal disease and the need for intrathecal treatment options. We also discuss evolving assessment criteria and regulatory considerations for future clinical investigations.

A Review on Inhibitory Action of Tyrosine Kinase Inhibitors (TKI) by Curbing the ATP-Tyrosine Kinase Interactions

Abstract: According to the latest data, the cancer prevalence fraction has surged to the highest number. This is why cancer has become a prominent disease that must be seen as a serious issue. Inhibitory action and ideas become prominent and necessary because of the rising death incidence daily. The simplifying idea of inhibition of cancer is targeting the complex that forms between the tyrosine kinase and ATP, which ultimately provides a clear way. Tyrosine kinase is a proteinaceous enzyme responsible for various cellular events like cell development, growth, and division. But these functions are performed by the activated tyrosine kinase, and the activation occurs by phosphorylation using ATP. The transfer of the phosphate group from ATP to tyrosine is known as phosphorylation. The basic idea is to enhance the competitive inhibition of the ATP-Tyrosine complex is a promising target for treating cancer. Various molecules have a substantial effect on the above-said target. This review summarizes molecules currently in any drug development or clinical trial with the same effect. This review covers most inhibitory molecules from different categories, which either directly or indirectly inhibit the Tyrosin kinase-ATP complex by incorporating.

Effect of dual properties of regorafenib in osteosarcoma on tumor progression and bone parameters in mouse preclinical models.

e23501 Background: Osteosarcoma (OS) constitutes the most frequent malignant primary bone tumor in children and young adults. Actual treatments in OS are based in a multimodal therapy that combines pre- and post-surgery polychemotherapies including cisplatin, doxorubicin, ifosfamide and/or methotrexate. Regardless the use of this polychemotherapeutic approach, the five years overall survival for patients has not improved during the last four decades and there is a clinical need to identify new active drugs in OS. An interesting strategy implies the use of compounds that showed therapeutic benefits in other solid tumors. Regorafenib, a small molecule with multi-target tyrosine kinase inhibitory activity against FGFR-1 and -2, KIT, PDGFR, RAF, RET, TIE2 and VEGFR-1, -2 and -3, showed an anti-angiogenic/oncogenic properties in various solid tumors that includes metastatic colorectal cancer, hepatocarcinoma and breast cancer. In addition, regorafenib also inhibits in vitro the proliferation of several pediatric solid tumor cell lines such as rhabdomyosarcoma, Ewing sarcoma and OS. Methods: The present work aims to decipher the in vitro and in vivo effects of regorafenib in OS preclinical models. Results: The treatment of MNNG-HOS, KHOS and 143B osteosarcoma cell lines with Regorafenib resulted in the inhibition of cell proliferation in a dose dependent manner. The gene expression profiles of MNNG-HOS and 143B cells treated or not with regorafenib and analyzed by NanoString technology revealed a specific regulation of molecular pathways by regorafenib in both cell lines. The in vivo effect of regorafenib was then analyzed in immunocompetent (mouse MOS-J cells inoculated in C57Bl/6 mice) and immunodeficient (human MNNG-HOS and 143B OS cells injected in Nude mice) murine osteosarcoma models. In all the cases, a significant reduction of primary tumor growth was observed as well as a significant decrease of lung metastases compared to untreated mice. Interestingly, the cortical and trabecular bone mineral density were significantly increased in regorafenib treated mice compared to untreated groups as demonstrated by X ray microtomodensitometry in all models. Conclusions: Our results support the use of regorafenib as an alternative treatment in metastatic OS and demonstrated for the first time the its bone protective effect.

A real-world study of ALK fusion detection, treatment patterns, and survival outcomes of patients with advanced non–small-cell lung cancer (aNSCLC) in Brazil.

e18655 Background: Clinical trials have firmly established that ALK tyrosine kinase inhibitors (TKIs) are safe and effective in aNSCLC, with substantial anti-tumor activity of multiple agents across different treatment lines. Real-world ALK fusion diagnosis and targeted therapy effectiveness have not been described in Brazilian private healthcare setting, with ALK TKIs approved since 2016. Methods: This study retrospectively explored the patterns of use and outcome after initial exposure to ALK TKIs in patients with ALK positive aNSCLC in a real-world clinical cohort from Oncoclínicas Group, Brazil's largest network of community oncology practices, between 2016 and 2022. For data collection we used a platform where structured EHR variables are integrated with unstructured elements from physician notes using technology-based abstraction and expert curation. We also describe molecular epidemiology and ALK fusion variant prevalence for a subset of patients tested with an in-house RNA sequencing assay (GS180, ArcherDx) at Oncoclínicas Precision Medicine Lab from 2020 to 2022. Results: Out of 100 ALK-positive aNSCLC patients treated with ALK TKIs, median age was 60 years and 62% were female. Overall, median number of treatment lines was 2, with 29% receiving 3 or more regimens. In first line, 64% of the patients received an ALK TKI (58% crizotinib, 33% alectinib, and 9% brigatinib, ceritinib or lorlatinib). The remaining 36% were treated with chemo +/- immunotherapy first, and 28% switched to an ALK TKI right after driver diagnosis. When comparing first line treatment practice in 2020-2022 versus 2016-2019 we detected major reduction in crizotinib as first ALK TKI (83% versus 35%). Overall, 14% received 2 or more sequential ALK TKIs. With a median follow-up of 12 months, median Overall Survival (mOS) from start of first ALK TKI was 28 months (CI95% 18-NR). In first line, exposure to alectinib was associated with numerically higher mOS (36 months) when compared to crizotinib (18 months, HR = 0.58, CI95% 0.2-1.6). From 592 cases of non-squamous NSCLC cases with in-house RNA sequencing, ALK fusions were detected in 5.2%. EML4-ALK fusion was found in 84%, with most common variants being E5:A20 (non-canonical) in 41% and E13:E20 (v1) in 38%. We also found novel fusions partners HIP1, KLC1, LTK and STRN in 16%. Conclusions: The largest clinico-genomics cohort in Latin America reveals that the impact of ALK TKIs in ALK fusion aNSCLC may not be as profound in the real world as in clinical trials. Fast access to molecular diagnosis and exposure to ALK TKI in first line are being incentivized. EML4-ALK variant prevalence in Brazilian patients with aNSCLC is different from published cohorts, and the effect that non-canonical variants may have on ALK TKI activity must be studied further.

Tyrosine Kinase Inhibitors Diminish Renal Neoplasms in a Tuberous Sclerosis Model Via Induction of Apoptosis.

Tuberous sclerosis complex (TSC) tumors are presently incurable despite a cytostatic response to mTOR pathway inhibition because recurrence of disease occurs after treatment is discontinued. Here, we explored the hypothesis that inhibiting tyrosine kinase activity in mesenchymal lineage-specific platelet-derived growth factor receptor β (PDGFRβ) signaling in TSC tumors is cytocidal and attenuates tumorigenesis at significantly higher levels than treatment with an mTOR inhibitor. Rapamycin-induced versus tyrosine kinase inhibitor (TKI)-induced renal angiomyolipoma (AML) and pulmonary lymphangioleiomyomatosis (LAM) tumor cells were comparatively analyzed using cell survival assays, RNA sequencing, and bioinformatics to distinguish tumoricidal mechanisms adopted by each drug type. The efficacy of imatinib therapy was validated against spontaneously developing renal cystadenomas in tuberous sclerosis Tsc2+/- mouse models (C57BL/6J mice; N = 6; 400 mg/kg/d; oral gavage) compared with Tsc2+/- mice treated with PBS (C57BL/6J mice; N = 6). Our study revealed that TKIs imatinib and nilotinib were cytocidal to both pulmonary LAM and renal AML cell cultures through the downregulation of the glycoprotein GPVI pathway and resultant disruption in mitochondrial permeability, increased cytosolic cytochrome C, and caspase 3 activation. Importantly, renal tumor growth was significantly attenuated in imatinib-treated Tsc2+/- mice compared with PBS treatment. The preclinical studies reported here provide evidence documenting the effectiveness of TKIs in limiting LAM and AML cell growth and viability with important clinical potential. Furthermore, these drugs elicit their effects by targeting a PDGF pathway-dependent apoptotic mechanism supporting the investigation of these drugs as a novel class of TSC therapeutics.

Synthesis of Some New Phthalazine−piperazine−pyrazole Conjugates; In vitro Anti‐Cancer, ADMET And Molecular Docking Studies

AbstractA new series of phthalazine−piperazine−pyrazole conjugates were synthesised and evaluated for their in vitro anticancer activity against three human cancer cell lines including MCF‐7 (breast), A549 (lung) and DU‐145 (prostate). The result data showed that compound 2‐((3‐(3,5‐dimethoxyphenyl)‐1H‐pyrazol‐5‐yl)methyl)‐4‐(piperazin‐1‐yl methyl)phthalazin‐1(2H)‐one (5 i) showed comparable activity against all the cell lines with the standard drug Etoposide. As well, compounds 2‐((3‐(3‐methoxyphenyl)‐1H‐pyrazol‐5‐yl) methyl)‐4‐(piperazin‐1‐ylmethyl) phthalazin‐1(2H)‐one (5 j) and 2‐((3‐(3,5‐dimethylphenyl)‐1H‐pyrazol‐5‐yl) methyl)‐4‐(piperazin‐1‐ylmethyl) phthalazin‐1(2H)‐one (5 l) exhibited promising activity against three cancer cell lines as compared to Etoposide. Three potent compounds (5 i, 5 j, and 5 l) were examined against the normal breast cell line in a cell viability assay (MCF‐10A), but none of them showed any apparent cytotoxicity with IC50 values above 78 μM. In addition, compounds 5 i, 5 j, and 5 l were screened for the VEGFR‐2 tyrosine kinase inhibitory activity using sorafenib as the standard drug and found that compound 5 i exhibited more potency in inhibiting the VEGFR‐2 tyrosine kinase as compared to sorafenib. Finally, molecular docking studies of compounds 5 i, 5 j and 5 l were also revealed that these compounds showed more binding interactions with VEGFR‐2 than VEGFR‐1. In accumulation to this, in silico pharmacokinetic profile was carried out for the potent compounds 5 i, 5 j and 5 l by SWISS/ADME and pkCSM. Whereas, 5 i, 5 j and 5 l compounds followed Lipinski, Veber, Egan, and Muegge rules without any deviation.

A Receptor Tyrosine Kinase Inhibitor Sensitivity Prediction Model Identifies AXL Dependency in Leukemia.

Despite incredible progress in cancer treatment, therapy resistance remains the leading limiting factor for long-term survival. During drug treatment, several genes are transcriptionally upregulated to mediate drug tolerance. Using highly variable genes and pharmacogenomic data for acute myeloid leukemia (AML), we developed a drug sensitivity prediction model for the receptor tyrosine kinase inhibitor sorafenib and achieved more than 80% prediction accuracy. Furthermore, by using Shapley additive explanations for determining leading features, we identified AXL as an important feature for drug resistance. Drug-resistant patient samples displayed enrichment of protein kinase C (PKC) signaling, which was also identified in sorafenib-treated FLT3-ITD-dependent AML cell lines by a peptide-based kinase profiling assay. Finally, we show that pharmacological inhibition of tyrosine kinase activity enhances AXL expression, phosphorylation of the PKC-substrate cyclic AMP response element binding (CREB) protein, and displays synergy with AXL and PKC inhibitors. Collectively, our data suggest an involvement of AXL in tyrosine kinase inhibitor resistance and link PKC activation as a possible signaling mediator.

One‐Pot Synthesis of Isatin‐Pyrazole Hybrids as VEGFR‐2 Inhibitors and Molecular Docking Studies

AbstractHerein, we described the synthesis of isatin‐pyrazole hybrids (6 a–o) using Acyl‐Sonogashira coupling reaction and evaluated for their in vitro cytotoxicity against three human cancer cell lines such as A549, PC3 and MCF‐7 by using MTT assay and 5‐fluorouracil as the reference drug. Some of the compounds 6 a, 6 j and 6 n were found to be more active than the standard 5‐fluorouracil against three human cancer cell lines. The in vitro VEGFR‐2 tyrosine kinase inhibitory activity reveals that 6 j has exhibited double inhibitory potency with IC50 value of 16.28±1.21 nM compared to reference drug sorafenib and 6 a and 6 n were also shown more inhibition capacity than the standard. Molecular docking studies of the compounds 6 a, 6 j and 6 n towards human VEGFR2 kinase shown that they have good binding interactions with the target protein with binding energies ranging from −10.30 kcal/mol to −10.80 kcal/mol. Finally, the in silico pharmacokinetic profile (ADMET) of potent compounds 6 a, 6 j and 6 n were also studied, where all of them have followed Lipinski rule, Ghose rule, Veber rule, Egan rule and Muegge rule without any deviation and the compounds 6 a and 6 j have shown lead likeness in medicinal chemistry point of view.

Six first-line tyrosine kinase inhibitors reveal novel inhibition potential for the EGFR S768I mutation

Lung cancer, the leading cause of cancer-related mortality, is the most commonly diagnosed cancer. Tyrosine kinase inhibitors (TKIs) are considered a drug-targeted therapy for non-small cell lung cancers (NSCLCs) with epidermal growth factor receptor (EGFR) mutations. However, limited data are available involving the activity of EGFR TKIs against rare EGFR mutations. Here, based on an endogenous EGFR-depleted cell Line H3255 by CRISPR, H3255 cells with rare mutant EGFRS768I and compound mutations EGFRS768I+L858R were tested using cell proliferation assay, cytotoxicity, membrane potential, flow cytometry and Western blot analysis. We conducted cytotoxicity screening of EGFR mutations on six front-line TKIs based on first-, second-, and third-generation TKIs (afatinib, dacomitinib, osimertinib, erlotinib, gefitinib, and icotinib). The results showed that the sensitivity of these mutants containing rare variants EGFRS768I to six front-line TKIs was enriched in the irreversible TKI cytotoxicity assays by determining their change in cytotoxicity, apoptosis, cell proliferation and signal pathway factors. Importantly, the variants harboring EGFRL858R (H3255), EGFRS768I (H3255S768I) and EGFRS768I+L858R (H3255S768I+L858R) were sensitive to six TKIs and induced cytotoxicity through different pathways. Moreover, the compound mutations EGFRS768I+L858R showed more TKI resistance than EGFRS768I mutation and EGFRL858R mutation. We present a comprehensive reference for the sensitivity of EGFRS768I variants to six front-line TKIs. For patients with the EGFR S768I mutation and compound mutations EGFRS768I+L858R, six first-line TKIs appear to be reasonable therapeutic options.

The Impact of Cytogenetic Aberrations in the Clonal Evolution of Chronic Myeloid Leukemia: A Single-Center Experience Among 450 Turkish Patients (Cohort Study)

Chronic myeloid leukemia (CML) is a clonal hematologic disorder characterized by t(9;22) translocation, in which cytogenetic aberrations can occur in Ph(+) and (-) clones. These aberrations develop due to clonal evolution as well as treatment and they have prognostic significance. They are grouped as major and minor route anomalies in terms of their effects on prognostic parameters, such as treatment response, overall survival (OS), disease stage, complete cytogenetic response (CCyR), and major molecular response (MMR). It is stated that major route anomalies have unfavorable prognostic effects compared to minor route anomalies. We aimed to investigate the frequency and prognostic effects of cytogenetic anomalies detected in Ph(+) and (-) clones. In this study, we retrospectively analyzed the cytogenetic results of 450 patients diagnosed with CML between 2005 and 2020. We detected cytogenetic aberrations in Ph-positive and negative clones in 41 of 450 patients. The most common anomalies were trisomy 8 (+8), additional Ph chromosome (+Ph), and loss of chromosome Y. Rarely, aneuploidy of the Y chromosome, dup (22), +11, and +6 were seen in CML patients. We observed that these identified aberrations negatively affected MMR and CCyR, and generally resulted in changing imatinib treatment for second-generation tyrosine kinase activity inhibitors. Our results are compatible with the literature. We suggest that cytogenetic aberrations detected in Ph(+) and (-) clones should be a warning sign in terms of treatment and require close observation. The use of cytogenetic methods for the identification of these anomalies is also important.

EVI1 upregulates PTGS1 (COX1) and decreases the action of tyrosine kinase inhibitors (TKIs) in chronic myeloid leukemia cells.

Tyrosine kinase inhibitors (TKIs) are highly effective in treating chronic myelogenous leukemia (CML). However, primary and acquired drug resistance to TKIs have been reported. In this study, we used RNA sequencing followed by RQ-PCR to show that the proto-oncogene EVI1 targets the drug-metabolizing gene PTGS1 in CML. The PTGS1 promoter element had an EVI1 binding site, and CHIP assay confirmed its presence. Data from a publicly available CML microarray dataset and an independent set of CML samples showed a significant positive correlation between EVI1 and PTGS1 expression in CML. Downregulation of EVI1 in K562 cells and subsequent treatment with TKIs resulted in a lower IC50 in the control cells. Furthermore, combined inhibition of BCR-ABL with imatinib and PTGS1 with FR122047 (PTGS1 inhibitor) synergistically reduced the viability of imatinib-resistant K562 cells. We conclude that elevated EVI1 expression contributes to TKIs resistance and that combined inhibition of PTGS1 and BCR-ABL may represent a novel therapeutic approach.

Discovery of new symmetrical and asymmetrical nitrile-containing 1,4-dihydropyridine derivatives as dual kinases and P-glycoprotein inhibitors: synthesis, in vitro assays, and in silico studies

Two new series of symmetric (1a-h) and asymmetric (2a-l) 1,4-DHP derivatives were designed, synthesised, and evaluated as anticancer agents. In vitro anticancer screening of target compounds via National cancer institute “NCI” revealed that analogues 1g, 2e, and 2l demonstrated antiproliferative action with mean growth inhibition percentage “GI%” = 41, 28, and 64, respectively. The reversal doxorubicin (DOX) effects of compounds 1g, 2e, and 2l were examined and illustrated better cytotoxic activity with IC50 =1.12, 3.64, and 3.57 µM, respectively. The most active anticancer analogues, 1g, 2e, and 2l, were inspected for their putative mechanism of action by estimating their epidermal growth factor receptor (EGFR), human epidermal growth factor receptor 2 (HER-2), and Bruton’s tyrosine kinase (BTK) inhibitory activities. Furthermore, the antimicrobial activity of target compounds was assessed against six different pathogens, followed by determining the minimum inhibitory concentration “MIC” values for the most active analogues. Molecular docking study was achieved to understand mode of interactions between selected inhibitors and different biological targets.