Multitargeted Tyrosine Kinase Inhibitor Research Articles

Antineoplastic Activity of Pazopanib in Anaplastic Thyroid Cancer in Primary Culture.

Anaplastic thyroid cancer (ATC) is a rare and rapidly fatal human cancer. Its usual treatment includes the combination of surgery, external hyperfractionated radiation therapy, and chemotherapy. These treatments permit achieving about 6-10 months of median survival. For this reason, it is challenging to predict the ATC patient clinical therapy responsiveness. Pazopanib is a multitarget tyrosine kinase inhibitor of VEGF receptors, PDGF, and c-Kit. Until now, the effect of pazopanib in primary human ATC cells (pATC) has not been reported in the literature. The aim of our study was to evaluate in vitro the antineoplastic effect of pazopanib in pATC. Surgical thyroidal tissues were collected from five patients with ATC, from thyroid biopsy at the moment of first surgical operation. An inhibition of proliferation, migration, and invasion, and an increase in apoptosis were demonstrated upon treating pATC cells with pazopanib (p < 0.05). Moreover, pazopanib was able to significantly decrease the VEGF expression in pATC cells (p < 0.05). To conclude, in this study, we demonstrate the antineoplastic activity of the antiangiogenic inhibitor, pazopanib, in human pATC in vitro.

Retrospective review of safety and efficacy of anlotinib in advanced osteosarcoma with metastases after failure of standard multimodal therapy.

To study the safety and efficacy of anlotinib, a multitargeted tyrosine kinase inhibitor, in the treatment of advanced osteosarcoma (OSS) with metastases. We retrospectively studied patients with advanced OSS and metastases who received anlotinib treatment in our hospital from June 2018 to April 2020. All patients had received standard multimodal therapies, before taking anlotinib. Therapeutic doses of anlotinib were 12mg for adults and 10mg for children and adolescents once a day for 2 consecutive weeks, followed by a week of withdrawal. This 3-week cycle of treatment was continued until the tumor progressed rapidly or the patients failed to tolerate the side effects. Adverse drug reactions were recorded, and therapeutic efficacy was evaluated based on progression-free survival (PFS), disease control rate (DCR), overall survival (OS), and objective response rate (ORR). The median PFS was 9.8±.9 months, and the 6- and 10-month PFS rates were 73% and 33%, respectively. The median OS was 11.4±.6 months. No patients achieved complete response. After 6 months of treatment, the DCR and ORR were 80% and 13%, respectively. No drug-related deaths or Grade 4 adverse events occurred in the patients. Five patients (33%) had Grade 3 adverse events. The most common drug-related adverse events were hand-food syndrome, fatigue, high blood pressure, anorexia, and pneumothorax. Anlotinib had a modest therapeutic effect in patients with advanced OSS after the failure of standard treatment. The adverse events were mostly tolerable or relieved after treatment.

Advanced development of biomarkers for immunotherapy in hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is the most common liver cancer and one of the leading causes of cancer-related deaths in the world. Mono-immunotherapy and combination therapy with immune checkpoint inhibitors (ICIs) and multitargeted tyrosine kinase inhibitors (TKIs) or anti-vascular endothelial growth factor (anti-VEGF) inhibitors have become new standard therapies in advanced HCC (aHCC). However, the clinical benefit of these treatments is still limited. Thus, proper biomarkers which can predict treatment response to immunotherapy to maximize clinical benefit while sparing unnecessary toxicity are urgently needed. Contrary to other malignancies, up until now, no acknowledged biomarkers are available to predict resistance or response to immunotherapy for HCC patients. Furthermore, biomarkers, which are established in other cancer types, such as programmed death ligand 1 (PD-L1) expression and tumor mutational burden (TMB), have no stable predictive effect in HCC. Thus, plenty of research focusing on biomarkers for HCC is under exploration. In this review, we summarize the predictive and prognostic biomarkers as well as the potential predictive mechanism in order to guide future research direction for biomarker exploration and clinical treatment options in HCC.

PS-BPR02-1: A CASE OF ADVANCED ENDOMETRIAL CANCER AFTER NEPHRECTOMY FOR RENAL CELL CANCER WITH HYPERTENSION AND PROTEINURIA INDUCED BY LENVATINIB

Background: The combination of pembrolizumab plus lenvatinib is approved for advanced endometrial cancer that is not MSI-H or dMMR, is progressive on prior systemic therapy, and is not amenable to curative surgery or radiation. Lenvatinib is an oral, multi-targeted tyrosine kinase inhibitor (TKI) that exerts anti-malignant effects by inhibiting many tyrosine kinase receptors, such as VEGF receptors 1, 2, and 3, fibroblast growth factor receptors 1 through 4, platelet-derived growth factor receptor α, rearranged during transfection (RET), and stem cell factor receptor (KIT) signaling networks. Although lenvatinib's usefulness has been demonstrated, adverse effects such as hypertension and proteinuria often necessitate the discontinuation of administration. We encountered a patient with advanced endometrial cancer after nephrectomy for renal cell cancer who developed hypertension and proteinuria due to lenvatinib. We discussed TKI induced hypertension and proteinuria in patients on pre-existing eGFR decline. Case presentation: A 65-year-old Japanese female was diagnosed in 2011 with concurrent endometrial cancer and right renal cell cancer. She underwent total hysterectomy, bilateral salpingo oophorectomy, and right nephrectomy. She received 6 courses of CAP (cyclophosphamide, epirubicin and cisplatin) therapy as adjuvant chemotherapy for endometrial cancer. More than 7 years later, the patient presented with a vaginal cuff recurrence that was histologically confirmed and radiotherapy was indicated. In 2019, there was a recurrence in pelvic lymph nodes. Between 2019 and 2021, she underwent 9 cycles of chemotherapy with carboplatin plus paclitaxel, however the cancer progression was observed. In February 2021, the patient was started on pembrolizumab and Lenvatinib (20 mg/day) combination therapy. Her creatinine was 0.85 mg/dL, with an eGFR of 52 mL/min/1.73m2 before the administration of lenvatinib. The patient experienced continuous grade 3 hypertension (systolic blood pressure Hiragino Sans 160 mmHg or diastolic blood pressure ≧ 100 mmHg), and 2 antihypertensive agents(amlodipine and ormesartan) were required to control hypertension. Despite lenvatinib dose reduction and the aggressive use of antihypertensive agents, grade 3 hypertension had recurred several times, and then proteinuria gradually increased to 3.0 g/day. Lenvatinib was decreased to 10 mg/day (2 steps of dose reduction) within 2 months of treatment initiation, and finally, hypertention and proteinuria were ameliorated. Conclusion: A history of nephrectomy, pre-existing eGFR decline and hypertension are associated with developing TKI induced proteinuria. These patients should be monitored carefully, and appropriate measures for the early control of hypertension and proteinuria may result in higher-dose treatment with lenvatinib, thereby maximizing its therapeutic effect.

Anti-Anaplastic Thyroid Cancer (ATC) Effects and Mechanisms of PLX3397 (Pexidartinib), a Multi-Targeted Tyrosine Kinase Inhibitor (TKI)

Background Anaplastic thyroid cancer (ATC) is the greatest lethal thyroid neoplasm with a low incidence and lacks an effective treatment strategy and standardized treatment protocol. PLX3397 (Pexidartinib) is an FDA-approved multitarget tyrosine kinase inhibitor. The research is designed to explore the possible anti-proliferative activity of pexidartinib on ATC, as well as its related molecular mechanisms. Methods The cell viability was assessed by CCK-8, LDH release, colony formation, and EdU detection assays. Apoptosis and the alteration on cell cycle arrest were characterized by flow cytometry (FCM). ER stress was evaluated by immunofluorescence (IF). ROS levels were determined by flow cytometry. Western blot assays were conducted to evaluate changes in key molecules related to apoptosis and ER stress. The ATC xenografts model was established, and immunohistochemistry was performed to validate the anti-ATC effects of pexidartinib in vivo. Results Pexidartinib significantly inhibited ATC cell proliferation and induced apoptosis and cell cycle arrest. Moreover, pexidartinib potently induced ER stress and elevated ROS in ATC cells, and the apoptotic cells and ER stress in ATC after administration of pexidartinib could be reversed by an ER stress inhibitor and ROS scavenger, respectively. Furthermore, pexidartinib treatment induced Nrf2 accumulation in nuclei and reduced the interaction of Nrf2 with Keap-1, and knockdown of Nrf2 enhanced the anti-ATC effects of pexidartinib in vitro. In addition, pexidartinib significantly inhibited ATC xenograft growth and proliferation in vivo, and the combination of ML385, an Nrf2 inhibitor, potently enhanced the anti-ATC effects of pexidartinib in vivo. Conclusion Our findings suggest pexidartinib is a potential agent for treating ATC. Co-administration with an Nrf2 inhibitor is an effective synergistic strategy.

Anlotinib Suppressed Ovarian Cancer Progression via Inducing G2/M Phase Arrest and Apoptosis

Ovarian cancer remains the most common gynecologic malignancy, because of its chemotherapy resistance and relapse. Anlotinib, a new oral multi-targeted tyrosine kinase inhibitor, has shown encouraging antitumor activity in several preclinical and clinical trials, while its effect on ovarian cancer has not been reported. In this study, we investigated the antitumor activity and underlying mechanism of anlotinib in ovarian cancer. Cell viability was analyzed by Cell Counting Kit-8 assay. Migration was measured by wound-healing assay. The cell cycle distribution and cell apoptosis rate were detected by flow cytometry. In vivo antitumor effect was analyzed in mouse ovarian carcinoma peritoneal metastasis model. We found that anlotinib inhibited the proliferation of ovarian cancer cells in a dose- and time- dependent manner by inducing G2/M phase arrest and apoptosis. Moreover, anlotinib upregulated the the phosphorylation of Histone H3, and expression of p21 protein in vitro. In addition, anlotinib inhibited the migration of ovarian cancer cells in vitro. Furthermore, anlotinib inhibited tumor growth by inhibiting cell proliferation and suppressing ovarian cancer angiogenesis in vivo. This study demonstrated the extraordinary anti-ovarian cancer effect of anlotinib, which may provide a promising therapeutic strategy for ovarian cancer.

Whole-exome sequencing identifies a set of genes as markers of hepatocellular carcinomaearly recurrence.

Hepatocellular carcinoma (HCC) is characterized by a high recurrence rate and poor prognosis. In recent years, the therapeutic regimen of programmed cell death protein 1 (PD-1) antibody combined with multi-targeted tyrosine kinase inhibitors (mTKIs) has achieved better results in the clinical application of hepatocellular carcinoma. Whole-exome sequencing can reflect the mutational characteristics of patients' exons and guide the clinical selection of molecular targeting drugs more accurately, which is in line with the concept of precision medicine. We performed exome sequencing on 63 patients with HCC treated with radical surgery at our hospital and collected their clinical indexes and postoperative follow-up data. Using machine learning, a prediction model for recurrence within 1year was constructed and the model was presented in a nomogram. Patients treated with PD-1 antibodies in combination with mTKIs after relapse were grouped by prognosis, and the valuable mutated genes were screened according to whole-exome sequencing data. The tumor tissue immune cells were analyzed using the UCSC Xena database. The expressions of target proteins were verified by Polymerase Chain Reaction (PCR) and Immunohistochemistry (IHC), respectively, on commercial HCC cell lines and pathological specimens of hepatocellular carcinoma collected clinically. The proportion of patients who relapsed within a year was 41% and the prognosis of those patients was poor. The characteristic exon mutation profile with a high frequency of variants in multiple mucin genes was present in Chinese HCC patients. Multiple nidi and 30 exon variants were brought into the prediction model with an area under the curve (AUC) = 0.94. MUC6 gene mutation was obvious in patients with an early recurrence, and MUC3A and MUC4 gene mutations were evident in patients with poorer responses to PD-1 antibodies combined with mTKIs. Those three mucins were negatively correlated with immune infiltrating cells. We depicted the exon characteristics of hepatocellular carcinoma in the Chinese population and established a predictive model for recurrence within 1year after radical surgical treatment. Moreover, we found that mucins were worthy targets of hepatocellular carcinoma.

TGF-β Signaling Activation Confers Anlotinib Resistance in Gastric Cancer.

Gastric cancer (GC) has always been a great threat to human health due to its aggressiveness and lethality. Anlotinib, a novel multi-target tyrosine kinase inhibitor (TKI), has been certified its anti-tumor effects on various tumors. Nonetheless, there are few studies on applying anlotinib as a treatment for GC. The underlying mechanism of acquired resistance during anlotinib administration remains unclear. We investigated the toxicologic effects of anlotinib on GC cells through CCK8, colony-forming, and flow cytometry assays in vitro and xenograft models in vivo. Anlotinib-resistant GC cells, AGS-R and MGC803-R, were generated and characterized by cell proliferation and apoptosis assays. The signaling pathways involved in anlotinib resistance were probed using Cignal™ Finder 10-Pathway Reporter Array. Western blot and dual-luciferase reporter assays were performed to confirm the relationships. The TGF-β inhibitor LY364947 was introduced to demonstrate the importance of TGF-β signaling in anlotinib resistance via a series of functional assays. Anlotinib suppressed cell growth and induced apoptosis in vitro and inhibited tumorigenesis and metastasis in vivo, while its anti-tumor effects were impaired in anlotinib-resistant cells. The results of dual-luciferase reporter assays and western blot indicated TGF-β signaling was activated in anlotinib-resistant GC cells. LY364947 combined with Anlotinib exerted a better antineoplastic effect than monotherapy and considerably reversed the anlotinib resistance in GC. Our findings suggested that TGF-β signaling may take a significant part in anlotinib resistance in GC. The suppression of TGF-β signaling may be a possible and promising approach for the GC oncotherapy when combined with anlotinib.

The multitargeted tyrosine kinase inhibitor lenvatinib shows promising efficiency and overcomes resistance in FGFR2-driven cholangiocarcinoma

The multitargeted tyrosine kinase inhibitor lenvatinib shows promising efficiency and overcomes resistance in FGFR2-driven cholangiocarcinoma

Anlotinib Exerts Inhibitory Effects against Cisplatin-Resistant Ovarian Cancer In Vitro and In Vivo.

Background: Anlotinib is a highly potent multi-target tyrosine kinase inhibitor. Accumulating evidence suggests that anlotinib exhibits effective anti-tumor activity against various cancer subtypes. However, the effects of anlotinib against cisplatin-resistant (CIS) ovarian cancer (OC) are yet to be elucidated. The objective of this study was to investigate the inhibitory effect of anlotinib on the pathogenesis of cisplatin-resistant OC. Materials and Methods: Human OC cell lines (A2780 and A2780 CIS) were cultured and treated with or without anlotinib. The effects of anlotinib on cell proliferation were determined using cell-counting kit-8 and colony-formation assays. To evaluate the invasion and metastasis of OC cells, we performed wound-healing and transwell assays. The cell cycle was analyzed via flow cytometry. A xenograft mouse model was used to conduct in vivo studies to verify the effects of anlotinib. The expression of Ki-67 in the tumor tissue was detected via immunohistochemistry. Quantitative real-time polymerase chain reaction and Western blotting were used to measure the mRNA and protein levels. Results: Our study revealed that anlotinib significantly inhibited the proliferation, migration, and invasion of A2780 and A2780 CIS in a dose-dependent way in vitro (p < 0.05). Through R software ‘limma’ package analysis of GSE15372, it was found that, in comparison with A2780, PLK2 was expressed in significantly low levels in the corresponding cisplatin-resistant strains. The ERK1/2/Plk2 signaling axis mediates the inhibitory effect of anlotinib on the proliferation and migration of ovarian cancer cell lines. Moreover, our research found that anlotinib effectively inhibited the growth of tumor cells in an OC xenograft mouse model. Conclusions: In this study, anlotinib showed excellent inhibitory effects against cisplatin-resistant OC both in vitro and in vivo. These results add to the growing body of evidence supporting anlotinib as a potential anticancer agent against OC.

DCZ19931, a novel multi-targeting kinase inhibitor, inhibits ocular neovascularization

Neovascularization is a prominent cause of irreversible blindness in a variety of ocular diseases. Current therapies for pathological neovascularization are concentrated on the suppression of vascular endothelial growth factors (VEGF). Despite the remarkable efficacy of anti-VEGF drugs, several problems still exist, including ocular complications and drug resistance. Thus, it is still required to design novel drugs for anti-angiogenic treatment. This study aimed to investigate the anti-angiogenic effects of a small molecule multi-target tyrosine kinase inhibitor, DCZ19931, on ocular neovascularization. The results showed that administration of DCZ19931 at the tested concentrations did not cause obvious cytotoxicity and tissue toxicity. DCZ19931 could reduce the size of choroidal neovascularization (CNV) lesions in laser-induced CNV model and suppress ocular neovascularization in oxygen-induced retinopathy (OIR) model. DCZ19931 could suppress VEGF-induced proliferation, migration, and tube formation ability of endothelial cells, exhibiting similar anti-angiogenic effects as Ranibizumab. DCZ19931 could reduce the levels of intercellular cell adhesion molecule-1 (ICAM-1) expression in vivo and in vitro. Network pharmacology prediction and western blots revealed that DCZ19931 exerted its anti-angiogenic effects through the inactivation of ERK1/2-MAPK signaling and p38-MAPK signaling. In conclusion, this study indicates that DCZ19931 is a promising drug for anti-angiogenic therapy for ocular diseases.

High-Dose Intermittent Treatment with the Multikinase Inhibitor Sunitinib Leads to High Intra-Tumor Drug Exposure in Patients with Advanced Solid Tumors

Patients with advanced cancer refractory to standard treatment were treated with sunitinib at a dose of 300 mg once every week (Q1W) or 700 mg once every two weeks (Q2W). Tumor, skin and plasma concentrations were measured and immunohistochemical staining for tumor cell proliferation (TCP), microvessel density (MVD) and T-cell infiltration was performed on tumor biopsies before and after 17 days of treatment. Oral administration of 300 mg sunitinib Q1W or 700 mg Q2W resulted in 19-fold (range 5-35×) and 37-fold higher (range 10-88×) tumor drug concentrations compared to parallel maximum plasma drug concentrations, respectively. Patients with higher tumor sunitinib concentrations had favorable progression-free and overall survival than those with lower concentrations (p = 0.046 and 0.024, respectively). In addition, immunohistochemistry of tumor biopsies revealed an induction of T-cell infiltration upon treatment. These findings provide pharmacological and biological insights in the clinical benefit from high-dose intermittent sunitinib treatment. It emphasizes the potential benefit from reaching higher tumor drug concentrations and the value of measuring TKI tumor- over plasma-concentrations. The finding that reaching higher tumor drug concentrations provides most clinical benefit in patients with treatment refractory malignancies indicates that the inhibitory potency of sunitinib may be enforced by a high-dose intermittent treatment schedule. These results provide proof of concept for testing other clinically available multitargeted tyrosine kinase inhibitors in a high-dose intermittent treatment schedule.

Berbamine dihydrochloride suppresses the progression of colorectal cancer via RTKs/Akt axis

Ethnopharmacological relevanceBerberis amurensis Rupr. is used to treat cancer as a traditional herbal medicine. Berbamine (BBM) is a natural bisbenzylisoquinoline alkaloid extracted from Berberis amurensis which possesses multiple pharmacological activity including anticancer. Aim of the studyTo investigate the influence of BBM on the progression of colorectal cancer (CRC) and further explore the underlying mechanism of BBM based on the RTKs/Akt signaling pathway. Materials and methodsIn vitro, cell viability and colony formation were conducted to detect BBM inhibitory of CRC cell lines. Transwell was detected the ability of migration and invasion by BBM. Apoptosis detection assay, cell cycle assay and the measurement of ROS were detected to confirm the inductive effect of cell apoptosis. RT-qPCR and Western blot to clarify the specific mechanism of anticancer. Finally, we conducted HE staining, Ki67, Tunnel and immunochemistry were confirmed the anti-colorectal cancer activity of BBM from vivo study. ResultsWe found that BBM could inhibit CRC cell lines growth. Moreover, BBM presented an inhibitory effect the ability of migration and invasion in CRC cells. Furthermore, the occurrence of apoptosis was involved in the anti-colorectal cancer role of BBM. BBM also triggered ROS accumulation in CRC cells that might be a key factor for the inductive effect of BBM in cell apoptosis. Cell cycle assay revealed that BBM induced the arrest of G1-S phase and increased the p21 levels but decreased CyclinE1, CyclinE2, CDK6, CyclinD1. RT-qPCR manifested that the down-regulation effect of BBM on AKT1, EGFR, PDGFRα and FGFR4 genes. The results also showed that BBM could decreased the expression levels of phosphor-AKT, PDGFRα, PDGFRβ, EGFR, FGFR3 and FGFR4 which belong to RTKs family. Consistently, BBM remarkably suppressed tumor xenograft growth in nude mice. ConclusionTaken together, all the results as presented above suggest that BBM as a novel multitargeted receptor tyrosine kinase inhibitor plays a crucial role in the inhibitory effect of CRC and may be a promising therapeutic agent for the CRC in clinic.

80P Penpulimab (Anti-PD-1) combined with anlotinib as first-line therapy for unresectable hepatocellular carcinoma (uHCC): Updated overall survival results from a phase Ib/II study

The combination of immune checkpoint inhibitors and tyrosine kinase inhibitors (TKIs) manifested high efficacy in uHCC. Anlotinib was a novel oral multi-targeted TKI selective for VEGF receptors 1/2/3, FGF receptors 1-4, PDGF receptors α and β, and c-kit. Penpulimab, a humanized anti-PD-1 IgG1 monoclonal antibody, was engineered to eliminate FcγR binding, consequently eliminating ADCC, ADCP and ADCR effects. This AK105-203 trial (NCT04172571) aimed to explore the efficacy and safety of penpulimab plus anlotinib in patients (pts) with histologically or cytologically confirmed uHCC.

MNK/eIF4E inhibition overcomes anlotinib resistance in non-small cell lung cancer.

Anlotinib is approved for refractory cases in advanced non-small-cell lung cancer (NSCLC). This is a novel oral multitarget tyrosine kinase inhibitor, but patients inevitably face prospects of drug resistance during the treatment process. Using anlotinib-resistant NSCLC models, this work investigated the underlying molecular mechanism and systematically addressed the issue of anlotinib resistance. We demonstrated that expression and activity of eukaryotic translation initiation factor 4E (eIF4E) were upregulated in NSCLC cells due to prolonged exposure to anlotinib. eIF4E depletion resulted in significant effects to anlotinib-resistant cells, showing proliferation inhibition and apoptosis inducement. We further showed that MAP kinase interacting serine/threonine kinase (MNK)-dependent eIF4E inhibition by cercosporamide was active against anlotinib-resistant cells and significantly augmented anlotinib's efficacy in parental NSCLC cells. Importantly, observations from in-vitro experiments are consistent in in vivo anlotinib-resistant and anlotinib-sensitive NSCLC cancer xenograft mouse models. Our work is the first to reveal that eIF4E is involved intimately in anlotinib resistance development in NSCLC, and this eIF4E activation can be reversed by cercosporamide or other MNK inhibitors.

Efficacy and Safety of PD-1/PD-L1 Inhibitors in Advanced Hepatocellular Carcinoma: A Systematic Review and Meta-analysis.

Programmed cell death1 (PD-1) and programmed cell death ligand1 (PD-L1) inhibitors have been increasingly employed for the treatment of various cancers in clinical practice. This study aimed to systematically evaluate the efficacy and safety of PD-1/PD-L1 inhibitors for advanced hepatocellular carcinoma (HCC). PubMed, EMBASE, Cochrane library, Web of Science, and Abstracts of American Society of Clinical Oncology proceedings databases were searched. Objective response rate (ORR), disease control rate (DCR), median progression-free survival (PFS), median overall survival (OS), and incidence of adverse events (AEs) and drug withdrawal were pooled. Odds ratio (OR) and hazard ratio (HR) were calculated to analyze the difference in the ORR, DCR, PFS, and OS between groups. Among the 14,902 initially identified papers, 98 studies regarding use of PD-1/PD-L1 inhibitors in advanced HCC were included. Based on different criteria of response in solid tumors, the pooled ORR, DCR, and median PFS was 16-36%, 54-74%, and 4.5-6.8months, respectively. The pooled median OS was 11.9months. Compared to multitarget tyrosine kinase inhibitors (TKIs), PD-1/PD-L1 inhibitors monotherapy significantly increased ORR (OR 2.73, P < 0.00001) and OS (HR 0.97, P = 0.05), and PD-1/PD-L1 inhibitors combined with TKIs significantly increased ORR (OR 3.17, P < 0.00001), DCR (OR 2.44, P < 0.00001), PFS (HR 0.58, P < 0.00001), and OS (HR 0.58, P < 0.00001). The pooled incidence of all-grade AEs, grade ≥ 3 AEs, and drug withdrawal was 71%, 25%, and 7%, respectively. On the basis of the present systematic review and meta-analysis, PD-1/PD-L1 inhibitors should be the preferred treatment choice for advanced HCC owing to their higher antitumor effect and improved outcomes.

Development of optimized pyrimido-thiazole scaffold derivatives as anticancer and multitargeting tyrosine kinase inhibitors using computational studies

Cancer is one of the frontier causes of leading death ratesglobally and one of the most researched domains. Despite many drugs available in the market currently, their effectiveness towards resistance, randomly mutating cancer targets, and side effects initiate the necessity of developing target selective and effective anti-cancer drugs.This study aimed to develop a congeneric series of fused pyrimidine and thiazole-containing hybrid series and evaluate its anticancer potential using in-silico tools. Molecular modeling strategies were used to develop various derivatives of fused thiazole-pyrimidines as anticancer agents for target-based drug discovery. Strategically, based on Structural Activity Relationship (SAR), of the synthesized or studied compounds, a combinatorial library was generated using the lead grow tool to develop a possible combination of various derivatives. Using Swiss ADME and Osiris; the ADMETproperties were predicted and screened molecules were docked using Auto-dock vina and Swiss Dockto obtain possible interactions with 5 listed targets of Tyrosine Kinase Inhibitors (TKI's) namely BCR-ABL kinase (PDB- 2GQG), c-ABL Kinase (PDB-3PYY), EGFR(PDB-1M17), VEGFR(PDB- 4ASD), and BRAF inhibitors (PDB- 2FB8). SAR of the pyrimido-thiazole molecules was derived from the literature survey and substitutions that enhance the anticancer activity were incorporated into the Combilib to generate a library of 221 compounds. This library of compounds was subjected to a Lipinski Filter. About 45 compounds were screened and tested for their drug-likeliness parameters. The 24 compounds that qualified with good absorption, drug-likeliness, and ADME were tested for their toxicity for developing relatively safe drugs with low side effects. These molecules were docked intofivedifferent PDB's and their binding affinity scores along with prominent interactions were noted. Compound 33 has shown comparable binding affinity and key interactions with all the five PDB's and thus can be used as a lead for further investigations. These studies have helped to optimize the relationship between fused pyrimidine and thiazoles as anticancer Tyrosine Kinase Inhibitors.

Ponatinib modulates the metabolic profile of obese mice by inhibiting adipose tissue macrophage inflammation.

Obesity-induced metabolic syndrome is a rapidly growing conundrum, reaching epidemic proportions globally. Chronic inflammation in obese adipose tissue plays a key role in metabolic syndrome with a series of local and systemic effects such as inflammatory cell infiltration and inflammatory cytokine secretion. Adipose tissue macrophages (ATM), as one of the main regulators in this process, are particularly crucial for pharmacological studies on obesity-related metabolic syndrome. Ponatinib, a multi-targeted tyrosine kinase inhibitor originally used to treat leukemia, has recently been found to improve dyslipidemia and atherosclerosis, suggesting that it may have profound effect on metabolic syndrome, although the mechanisms underlying have not yet been revealed. Here we discovered that ponatinib significantly improved insulin sensitivity in leptin deficient obese mice. In addition to that, ponatinib treatment remarkably ameliorated high fat diet-induced hyperlipidemia and inhibited ectopic lipid deposition in the liver. Interestingly, although ponatinib did not reduce but increase the weight of white adipose tissue (WAT), it remarkably suppressed the inflammatory response in WAT and preserved its function. Mechanistically, we showed that ponatinib had no direct effect on hepatocyte or adipocyte but attenuated free fatty acid (FFA) induced macrophage transformation from pro-inflammatory to anti-inflammatory phenotype. Moreover, adipocytes co-cultured with FFA-treated macrophages exhibited insulin resistance, while pre-treat these macrophages with ponatinib can ameliorate this process. These results suggested that the beneficial effects of ponatinib on metabolic disorders are achieved by inhibiting the inflammatory phenotypic transformation of ATMs, thereby maintaining the physiological function of adipose tissue under excessive obesity. The data here not only revealed the novel therapeutic function of ponatinib, but also provided a theoretical basis for the application of multi-target tyrosine kinase inhibitors in metabolic diseases.

Implementing RNAi Therapy Targeting FLT3 in Acute Myeloid Leukemia (AML)

Approximately 25% of newly diagnosed AML patients display an internal tandem duplication (ITD) in the fms-like tyrosine kinase 3 (FLT3) gene. Although both multi-targeted and FLT3 specific tyrosine kinase inhibitors (TKIs) are being utilized for clinical therapy, drug resistance, short remission periods, and high relapse rates are challenges that still need to be tackled (Gebru et. al. J. Hematol. Oncol. 2020). RNA interference (RNAi), mediated by short interfering RNA (siRNA) presents a novel therapeutic platform with the potential of personalization due to its gene sequence-driven mechanism of action. The success of RNAi therapy to a great extent is dictated by the siRNA delivery vehicle employed. This study explored the use of a lipid-substituted low molecular weight polyethyleneimine (lipopolymer) for delivering FLT3 siRNA in FLT3-ITD positive AML models and assess the efficacy of combining this treatment with chemotherapeutic drugs currently used in clinic. Low molecular weight polyethyleneimine (PEI) polymers were modified with aliphatic lipids to yield lipopolymers that self-assembled into nanocomplexes with siRNA. Treatment of MV4-11 cells with lipopolymer/FLT3 siRNA nanocomplexes resulted in ~50% reduction in cell proliferation rates (p<0.001), as determined by taking direct cell counts after the siRNA treatment. Quantitative analysis of relative mRNA transcript levels conducted by qPCR revealed downregulation of FLT3 gene by 30% (p<0.01). Furthermore, a similar decline (~35%, p<0.005) in FLT3 protein levels was observed when measured by flow cytometry through immunostaining with an anti-FLT3 antibody. Evaluation of apoptosis by FITC-Annexin V/propidium iodide (PI) staining showed a 3-fold increase in percentage of cells in the late apoptosis stage after FLT3 specific siRNA treatment (p<0.005). The effect of combining lipopolymer/FLT3 siRNA treatment with daunorubucin manifested as a decline in cell proliferation rates as well as 3-fold increase in apoptosis (p<0.05). To assess the impact of combination treatment on stem cells, colony forming ability was measured by seeding treated cells in methylcellulose and the colonies generated subsequently were counted after two weeks. A notable drop in colony counts was observed in the combination relative to individual treatments. However, similar analyses with the FLT3 targeting TKI midostaurin led to only a modest augmentation of anti-leukemic activity. Subsequently, the more recently approved FLT3 targeting TKI gilteritinib was explored in combination with lipopolymer/FLT3 siRNA nanocomplexes both in vitro and in vivo. For in vivo studies, lipopolymer/FLT3 siRNA nanocomplexes were administered via tail-vein injection at a siRNA dose of 25 µg per mouse (~1 mg/kg) once the tumor volume was ≥50mm3 in AML MV4-11 subcutaneous xenograft models created in male NCG (ABL/VAF) mice. Four injections of the nanocomplexes were administered, each 72 hours apart with simultaneous measurements of the tumor volume. Gilteritinib was administered four times intraperitonially at a dose of 5 mg/kg, with a spacing of 72 hours between doses. A significant reduction in tumor volume was observed in the combination treated tumors compared to gilteritinib alone treated tumors. Refractory/relapsed tumors underwent a second round of treatment (with both nanocomplexes and gilteritinib being administered four times each). The combination therapy significantly increased median survival to 65 days as compared to 45 days with gilteritinib treatment alone (p<0.05). These findings demonstrate the promising potential of RNAi implemented with lipopolymer nanocomplexes for AML molecular therapy. The study prospectively supports the addition of RNAi therapy to current treatment modalities available to target the heterogeneity prevalent in AML.

Strategies for Alleviating Tyrosine Kinase Resistance in Acute Myeloid Leukemia By Modulating p53 Signaling Pathway with Small Molecules

Background:FLT3-ITD can be found in up to 30% of acute myeloid leukemia (AML) patients and confers a poor prognosis. While midostaurin and gilteritinib are FDA-approved for these high-risk patients, they only result in a temporary response without transplantation, suggesting the importance of identifying the acquired resistance. Previously, we demonstrated that cabozantinib, an oral multi-target tyrosine kinase inhibitor, was selectively cytotoxic to AML cells with FLT3-ITD (MV4-11 and Molm13). Subsequently, we established cabozantinib-resistant human leukemic MV4-11 derivative (MV4-11-XR) and Molm13 derivative (Molm13-XR) after gradual escalating concentration of cabozantinib incubation from parental MV4-11 (MV4-11-P) and Molm13 (Molm13-P) cells, respectively. Subsequently, cell cytotoxicity assay showed these two cabozantinib-resistant cells were also resistant to quizartinib, sorafenib and gilteritinib. RNA-seq analysis revealed that MV4-11-XR and Molm13-XR cells shared 87 identical differentially down-regulated genes compared to their respective parental cells. Further Metascape for gene enriched pathway analysis demonstrated the most affected was p53 signaling pathway. Aim: To investigate whether modulation with p53 activator RITA or MDM2 inhibitor RG7388 could alleviate the drug resistance and their underlying molecular mechanism. Materials and Methods: Human leukemia MV4-11 and Molm13 cells were used in this study. The differential expression genes (DEGs) were examined using RNA-seq (Illumina NextSeq-500). Metascape enrichment pathway analyses were preformed to predict the biological functions of DEGs. Cabozantinib, RITA, and RG7388 (Idasanutlin) were used. MTS assay was performed to the effects of various drug on cell growth. Western blotting, flow cytometry and quantitative real-time PCR were applied to elucidate potential molecular mechanism. Fluorescent leukemic cell xenografts in zebrafish and high content imaging system were used for investigating the efficacy of various drugs in vivo. Results: The gene expression profile in MV4-11-P and Molm13-P cells was used as a baseline to establish the up- and down-regulated genes in MV4-11-XR and Molm13-XR cells. The FPKM were estimated with the selection criteria of p value <0.05 and [log2 (fold change)] <1 for significantly differential expression for downregulation. We identified a total of 747 DEGs between the MV4-11-P and -XR cells and 407 DEGs between the Molm13-P and -XR cells. Among them, 87 DEGs were shared by these two cohorts. Metascape enrichment analysis revealed that those down-regulated DEGs were enriched in p53-signaling pathway, in line with decreased expression of P53-associated genes, BBC3, CCNG2, CKDN1A, ZMAT3, confirmed by using q-RT-PCR. IGV (Integrative Genomics Viewer) analysis showed that the variant allele frequency of R248W progressed from 57% in MV4-11-P to 100% in MV4-11-XR cells, a similar phenomenon also appeared in P72R (65% to 98%). Microsatellite analysis confirmed the loss of heterozygosity in the TP53 gene of MV4-11-XR. On the other hand, there were not TP53 variants in both Molm13-P and Molm13-XR cells, but increased level of MDM2 in Molm13-XR cells was noted. Further study revealed that p53 activator RITA with 77.6nM could reduce IC50 of cabozantinib from 1026nM to 310.7nM, coupled with increased gene expression and protein levels of BBC3 (PUMA), CCNG2 (cyclin G2), CDKN1A (p21) and ZMAT3. In addition, RITA had synergistic effect with cabozantinib on cell cycle arrest on G2/M phase, increased apoptosis and decreased colony formation and proliferation. On the other hand, MDM2 inhibitor RG7388 reduced the IC50 of cabozantinib from 517nM to 101nM with only 50.6 nM in Molm13-XR cells. Similarly, RG7388 could increase those P53-associated gene expression and protein levels, and had synergistic effect with cabozantinib on cell cycle arrest on G0/G1 phase, increased apoptosis and decreased colony formation and proliferation. In addition, in vivo zebrafish experiment demonstrated that the combination of RG7388 and cabozantinib had a synergistic and significant inhibitory effect compared with single use. Conclusion: These findings suggested that reactivation of p53 is a potential therapeutic option to alleviate drug resistance in FLT3-mutated AML, provided that AML patients are tested for p53 dysfunction, including TP53 mutational status and downregulation.