In situ single-cell therapeutic response imaging facilitated by the TRIPODD fluorescence imaging platform.

Purpose: Small molecule drugs such as tyrosine kinase inhibitors (TKIs) targeting tumoral molecular dependencies have become standard of care for numerous cancer types. Notably, epidermal growth factor receptor (EGFR) TKIs (e.g., erlotinib, afatinib, osimertinib) are the current first-line treatment for non-small cell lung cancer (NSCLC) due to their improved therapeutic outcomes for EGFR mutated and overexpressing disease over traditional platinum-based chemotherapy. However, many NSCLC tumors develop resistance to EGFR TKI therapy causing disease progression. Currently, the relationship between in situ drug target availability (DTA), local protein expression and therapeutic response cannot be accurately assessed using existing analytical tools despite being crucial to understanding the mechanism of therapeutic efficacy. Procedure: We have previously reported development of our fluorescence imaging platform termed TRIPODD (Therapeutic Response Imaging through Proteomic and Optical Drug Distribution) that is capable of simultaneous quantification of single-cell DTA and protein expression with preserved spatial context within a tumor. TRIPODD combines two complementary fluorescence imaging techniques: intracellular paired agent imaging (iPAI) to measure DTA and cyclic immunofluorescence (cyCIF), which utilizes oligonucleotide conjugated antibodies (Ab-oligos) for spatial proteomic expression profiling on tissue samples. Herein, TRIPODD was modified and optimized to provide a downstream analysis of therapeutic response through single-cell DTA and proteomic response imaging. Results: We successfully performed sequential imaging of iPAI and cyCIF resulting in high dimensional imaging and biomarker assessment to quantify single-cell DTA and local protein expression on erlotinib treated NSCLC models. Pharmacodynamic and pharmacokinetic studies of the erlotinib iPAI probes revealed that administration of 2.5 mg/kg each of the targeted and untargeted probe 4 h prior to tumor collection enabled calculation of DTA values with high Pearson correlation to EGFR, the erlotinib molecular target, expression in the tumors. Analysis of single-cell biomarker expression revealed that a single erlotinib dose was insufficient to enact a measurable decrease in the EGFR signaling cascade protein expression, where only the DTA metric detected the presence of bound erlotinib. Conclusion: We demonstrated the capability of TRIPODD to evaluate therapeutic response imaging to erlotinib treatment as it relates to signaling inhibition, DTA, proliferation, and apoptosis with preserved spatial context.

Synergy of EGFR and AURKA Inhibitors in KRAS-mutated Non-small Cell Lung Cancers.

The most common oncogenic driver mutations for non-small cell lung cancer (NSCLC) activate EGFR or KRAS. Clinical trials exploring treatments for EGFR- or KRAS-mutated (EGFRmut or KRASmut) cancers have focused on small-molecule inhibitors targeting the driver mutations. Typically, these inhibitors perform more effectively based on combination with either chemotherapies, or other targeted therapies. For EGFRmut NSCLC, a combination of inhibitors of EGFR and Aurora-A kinase (AURKA), an oncogene commonly overexpressed in solid tumors, has shown promising activity in clinical trials. Interestingly, a number of recent studies have indicated that EGFR activity supports overall viability of tumors lacking EGFR mutations, and AURKA expression is abundant in KRASmut cell lines. In this study, we have evaluated dual inhibition of EGFR and AURKA in KRASmut NSCLC models. These data demonstrate synergy between the EGFR inhibitor erlotinib and the AURKA inhibitor alisertib in reducing cell viability and clonogenic capacity in vitro, associated with reduced activity of EGFR pathway effectors, accumulation of enhanced aneuploid cell populations, and elevated cell death. Importantly, the erlotinib-alisertib combination also synergistically reduces xenograft growth in vivo. Analysis of signaling pathways demonstrated that the combination of erlotinib and alisertib was more effective than single-agent treatments at reducing activity of EGFR and pathway effectors following either brief or extended administration of the drugs. In sum, this study indicates value of inhibiting EGFR in KRASmut NSCLC, and suggests the specific value of dual inhibition of AURKA and EGFR in these tumors. SIGNIFICANCE: The introduction of specific KRAS G12C inhibitors to the clinical practice in lung cancer has opened up opportunities that did not exist before. However, G12C alterations are only a subtype of all KRAS mutations observed. Given the high expression of AURKA in KRASmut NSCLC, our study could point to a potential therapeutic option for this subgroup of patients.

PDZK1 suppresses TNBC development and sensitizes TNBC cells to erlotinib via the EGFR pathway.

Epidermal growth factor receptor (EGFR)-targeted drugs (erlotinib, etc.) are used to treat multiple types of tumours. EGFR is highly expressed in most triple-negative breast cancer (TNBC) patients. However, only a small proportion of TNBC patients benefit from EGFR-targeted drugs in clinical trials, and the resistance mechanism is unclear. Here, we found that PDZ domain containing 1 (PDZK1) is downregulated in erlotinib-resistant TNBC cells, suggesting that PDZK1 downregulation is related to erlotinib resistance in TNBC. PDZK1 binds to EGFR. Through this interaction, PDZK1 promotes EGFR degradation by enhancing the binding of EGFR to c-Cbl and inhibits EGFR phosphorylation by hindering EGFR dimerisation. We also found that PDZK1 is specifically downregulated in TNBC tissues and correlated with a poor prognosis in TNBC patients. In vitro and in vivo functional assays showed that PDZK1 suppressed TNBC development. Restoration of EGFR expression or kinase inhibitor treatment reversed the degree of cell malignancy induced by PDZK1 overexpression or knockdown, respectively. PDZK1 overexpression sensitised TNBC cells to erlotinib both in vitro and in vivo. In conclusion, PDZK1 is a significant prognostic factor for TNBC and a potential molecular therapeutic target for reversing erlotinib resistance in TNBC cells.

Decoding dynamic interactions between EGFR-TKD and DAC through computational and experimental approaches: A novel breakthrough in lung melanoma treatment.

In the quest for effective lung cancer treatments, the potential of 3,6-diaminoacridine-9-carbonitrile (DAC) has emerged as a game changer. While DAC's efficacy against glioblastoma is well documented, its role in combating lung cancer has remained largely untapped. This study focuses on CTX-1, exploring its interaction with the pivotal EGFR-TKD protein, a crucial target in lung cancer therapeutics. A meticulous molecular docking analysis revealed that CTX-1 exhibits a noteworthy binding affinity of -7.9 kcal/mol, challenging Erlotinib, a conventional lung cancer medication, which displayed a binding affinity of -7.3 kcal/mol. For a deeper understanding of CTX-1's molecular mechanics, this study employed rigorous 100-ns molecular dynamics simulations, demonstrating CTX-1's remarkable stability in comparison with erlotinib. The Molecular Mechanics Poisson-Boltzmann Surface Area (MM-PBSA) method further corroborated these results, with CTX-1 showing a free binding energy of -105.976 ± 1.916 kJ/mol. The true prowess of CTX-1 was tested against diverse lung cancer cell lines, including A549, Hop-62 and H-1299. CTX-1 not only significantly outperformed erlotinib in anticancer activity but also exhibited a spectrum of therapeutic effects. It effectively diminished cancer cell viability, induced DNA damage, halted cell cycle progression, generated reactive oxygen species (ROS), impaired mitochondrial transmembrane potential, instigated apoptosis and successfully inhibited EGFR-TKD. This study not only underscores the potential of CTX-1 a formidable contender in lung cancer treatment but also marks a paradigm shift in oncological therapeutics, offering new horizons in the fight against this formidable disease.

Monolayer culture alters EGFR inhibitor response through abrogation of microRNA-mediated feedback regulation.

Ex vivo drug screening is a potentially powerful tool for the future of cancer care, but the accuracy of results is contingent on the culture model. Both monolayer (2D) and spheroid (3D) culture systems offer advantages, but given the differences in mechanical environment, we hypothesized that that the suitability of one system over another would be critical for screening drugs with mechanical targets in mechanical tissues. HCC827 lung adenocarcinoma cells were challenged with EGFR tyrosine kinase inhibitors in monolayer and spheroid culture. RNA sequencing was performed on cells in both conditions to assess culture-induced transcriptional changes that could account for differences in drug response and differences in EGFR expression detected by immunostain. A microRNA microarray was performed to assess culture-induced differences in regulation of microRNA, and the impact of miR-146a-5p on drug response was verified by inhibition. Results were confirmed in human lung adenocarcinoma tissue. HCC827 spheroids were resistant to erlotinib and gefitinib, but significantly more sensitive in 2D culture. RNA-seq and immunostaining show a discrepancy in EGFR transcript and protein expression between the two conditions, which we attribute to miR-146a-5p. This microRNA targets EGFR and is differentially expressed between 2D and 3D culture. Inhibition of miR-146a-5p significantly increased erlotinib cytotoxicity, but validation in patient-derived spheroids suggests that the effect may be mutation-specific. Analysis of RNA-seq data suggests that cells in 2D culture become highly dependent on EGFR signaling to drive proliferation and cell spreading, resulting in a misleading level of sensitivity to EGFR TKIs, while the same cells in spheroid culture retain microRNA-driven EGFR feedback regulation that leaves them less vulnerable to EGFR inhibition. These findings underscore the need for close scrutiny of culture-induced effects on drug target regulation in model design for ex vivo drug screening.

Activation of the PI3K/AKT signaling pathway by ARNTL2 enhances cellular glycolysis and sensitizes pancreatic adenocarcinoma to erlotinib.

BACKGROUND: Pancreatic adenocarcinoma (PC) is an aggressive malignancy with limited treatment options. The poor prognosis primarily stems from late-stage diagnosis and when the disease has become therapeutically challenging. There is an urgent need to identify specific biomarkers for cancer subtyping and early detection to enhance both morbidity and mortality outcomes. The addition of the EGFR tyrosine kinase inhibitor (TKI), erlotinib, to gemcitabine chemotherapy for the first-line treatment of patients with advanced pancreatic cancer slightly improved outcomes. However, restricted clinical benefits may be linked to the absence of well-characterized criteria for stratification and dependable biomarkers for the prediction of treatment effectiveness. METHODS AND RESULTS: We examined the levels of various cancer hallmarks and identified glycolysis as the primary risk factor for overall survival in PC. Subsequently, we developed a glycolysis-related score (GRS) model to accurately distinguish PC patients with high GRS. Through in silico screening of 4398 compounds, we discovered that erlotinib had the strongest therapeutic benefits for high-GRS PC patients. Furthermore, we identified ARNTL2 as a novel prognostic biomarker and a predictive factor for erlotinib treatment responsiveness in patients with PC. Inhibition of ARNTL2 expression reduced the therapeutic efficacy, whereas increased expression of ARNTL2 improved PC cell sensitivity to erlotinib. Validation in vivo using patient-derived xenografts (PDX-PC) with varying ARNTL2 expression levels demonstrated that erlotinib monotherapy effectively halted tumor progression in PDX-PC models with high ARNTL2 expression. In contrast, PDX-PC models lacking ARNTL2 did not respond favorably to erlotinib treatment. Mechanistically, we demonstrated that the ARNTL2/E2F1 axis-mediated cellular glycolysis sensitizes PC cells to erlotinib treatment by activating the PI3K/AKT signaling pathway. CONCLUSIONS: Our investigations have identified ARNTL2 as a novel prognostic biomarker and predictive indicator of sensitivity. These results will help to identify erlotinib-responsive cases of PC and improve treatment outcomes. These findings contribute to the advancement of precision oncology, enabling more accurate and targeted therapeutic interventions.

Humanization of SLCO2B1 in Rats Increases rCYP3A1 Protein Expression but Not the Metabolism of Erlotinib to OSI-420.

The organic anion transporting polypeptide (OATP)2B1 [(gene: solute carrier organic anion transporter family member 2B1 (SLCO2B1)] is an uptake transporter that facilitates cellular accumulation of its substrates. Comparison of SLCO2B1+/+ knockin and rSlco2b1-/- knockout rats showed a higher expression of rCYP3A1 in the humanized animals. We hypothesize that humanization of OATP2B1 not only affects cellular uptake but also metabolic activity. To further investigate this hypothesis, we used SLCO2B1+/+ and rSlco2b1-/ - rats and the OATP2B1 and rCYP3A1 substrate erlotinib, which is metabolized to OSI-420, for in vivo and ex vivo experiments. One hour after administration of a single dose of erlotinib, the knockin rats exhibited significantly lower erlotinib serum levels, but no change was observed in metabolite concentration or the OSI-420/erlotinib ratio. Similar results were obtained for liver tissue levels comparing SLCO2B1+/+ and rSlco2b1-/- rats. Liver microsomes isolated from the erlotinib-treated animals were characterized ex vivo for rCYP3A activity using testosterone, showing higher activity in the knockin rats. The contrary was observed when microsomes isolated from treatment-naïve animals were assessed for the metabolism of erlotinib to OSI-420. The latter is in contrast to the higher rCYP3A1 protein amount observed by western blot analysis in rat liver lysates and liver microsomes isolated from untreated rats. In summary, rats humanized for OATP2B1 showed higher expression of rCYP3A1 in liver and reduced serum levels of erlotinib but no change in the OSI-420/erlotinib ratio despite a lower OSI-420 formation in isolated liver microsomes. Studies with CYP3A-specific substrates are warranted to evaluate whether humanization affects not only rCYP3A1 expression but also metabolic activity in vivo. SIGNIFICANCE STATEMENT: Humanization of rats for the organic anion transporting polypeptide (OATP)2B1 increases rCYP3A1 expression and activity in liver. Using the OATP2B1/CYP3A-substrate erlotinib to assess the resulting phenotype, we observed lower erlotinib serum and liver concentrations but no impact on the liver/serum ratio. Moreover, there was no difference in the OSI-420/erlotinib ratio comparing humanized and knockout rats, suggesting that OSI-420 is not applicable to monitor differences in rCYP3A1 expression as supported by data from ex vivo experiments with rat liver microsomes.

Design, synthesis, in silico molecular docking, and ADMET studies of quinoxaline-isoxazole-piperazine conjugates as EGFR-targeting agents.

In this paper, we report the synthesis of quinoxaline-isoxazole-piperazine conjugates. The anticancer activity was evaluated against three human cancer cell lines, including MCF-7 (breast), HepG-2 (liver), and HCT-116 (colorectal). The outcomes of the tested compounds 5d, 5e, and 5f have shown more potent activity when compared to the standard drug erlotinib. In a cell survivability test (MCF-10A), three potent compounds (5d, 5e, and 5f) were evaluated against the normal breast cell line, although neither of them displayed any significant cytotoxicity with IC50 values greater than 84 µM. Furthermore, the compounds 5d, 5e, and 5f were tested for tyrosine kinase EGFR inhibitory action using erlotinib as the reference drug and compound 5e was shown to be more potent in inhibiting the tyrosine kinase EGFR than sorafenib. In addition to this, molecular docking studies of compounds 5d, 5e, and 5f demonstrated that these compounds had more EGFR-binding interactions. The potent compounds 5d, 5e, and 5f were subjected to in silico pharmacokinetic assessment by SWISS, ADME, and pkCSM. While the compounds 5d, 5e, and 5f followed Lipinski, Veber, Egan, and Muegge rules without any deviation.

Erlotinib combination with a mitochondria-targeted ubiquinone effectively suppresses pancreatic cancer cell survival.

BACKGROUND: Pancreatic cancer is a leading cause of cancer-related deaths. Increased activity of the epidermal growth factor receptor (EGFR) is often observed in pancreatic cancer, and the small molecule EGFR inhibitor erlotinib has been approved for pancreatic cancer therapy by the food and drug administration. Nevertheless, erlotinib alone is ineffective and should be combined with other drugs to improve therapeutic outcomes. We previously showed that certain receptor tyrosine kinase inhibitors can increase mitochondrial membrane potential (Δψm), facilitate tumor cell uptake of Δψm-sensitive agents, disrupt mitochondrial homeostasis, and subsequently trigger tumor cell death. Erlotinib has not been tested for this effect. AIM: To determine whether erlotinib can elevate Δψm and increase tumor cell uptake of Δψm-sensitive agents, subsequently triggering tumor cell death. METHODS: Δψm-sensitive fluorescent dye was used to determine how erlotinib affects Δψm in pancreatic adenocarcinoma (PDAC) cell lines. The viability of conventional and patient-derived primary PDAC cell lines in 2D- and 3D cultures was measured after treating cells sequentially with erlotinib and mitochondria-targeted ubiquinone (MitoQ), a Δψm-sensitive MitoQ. The synergy between erlotinib and MitoQ was then analyzed using SynergyFinder 2.0. The preclinical efficacy of the two-drug combination was determined using immune-compromised nude mice bearing PDAC cell line xenografts. RESULTS: Erlotinib elevated Δψm in PDAC cells, facilitating tumor cell uptake and mitochondrial enrichment of Δψm-sensitive agents. MitoQ triggered caspase-dependent apoptosis in PDAC cells in culture if used at high doses, while erlotinib pretreatment potentiated low doses of MitoQ. SynergyFinder suggested that these drugs synergistically induced tumor cell lethality. Consistent with in vitro data, erlotinib and MitoQ combination suppressed human PDAC cell line xenografts in mice more effectively than single treatments of each agent. CONCLUSION: Our findings suggest that a combination of erlotinib and MitoQ has the potential to suppress pancreatic tumor cell viability effectively.

Hexane Fraction of Adenophora triphylla var. japonica Root Extract Inhibits Angiogenesis and Endothelial Cell-Induced Erlotinib Resistance in Lung Cancer Cells.

The aim of this study was to investigate the anti-angiogenic effects of the hexane fraction of Adenophora triphylla var. japonica root extract (HAT) and its influence on the development of erlotinib resistance in human lung cancer cells. HAT significantly reduced the migration, invasion, and tube formation of human umbilical vein endothelial cells (HUVECs). The phosphorylation levels of vascular endothelial growth factor receptor 2 (VEGFR2) and its downstream molecules were decreased via HAT, indicating its anti-angiogenic potential in endothelial cells (ECs). A docking analysis demonstrated that ß-sitosterol and lupeol, representative components of HAT, exhibit a high affinity for binding to VEGFR2. In addition, conditioned media from HAT-pretreated H1299 human lung cancer cells attenuated cancer-cell-induced chemotaxis of HUVECs, which was attributed to the decreased expression of angiogenic and chemotactic factors in H1299 cells. Interestingly, co-culture of erlotinib-sensitive PC9 human lung cancer cells with HUVECs induced erlotinib resistance in PC9 cells. However, co-culture with HAT-pretreated HUVECs partially restored the sensitivity of PC9 cells to erlotinib. HAT inhibited the development of erlotinib resistance by attenuating hepatocyte growth factor (HGF) production by ECs. Taken together, our results demonstrate that HAT exerts its anticancer effects by regulating the crosstalk between ECs and lung cancer cells.

Design, synthesis, and apoptotic antiproliferative action of new 1,2,3-triazole/1,2,4-oxadiazole hybrids as dual EGFR/VEGFR-2 inhibitors.

A novel series of 1,2,3-triazole/1,2,4-oxadiazole hybrids (7a-o) was developed as dual inhibitors of EGFR/VEGFR-2. Compounds 7a-o were evaluated as antiproliferative agents with Erlotinib as the reference drug. Results demonstrated that most of the tested compounds showed significant antiproliferative action with GI50 values ranging from 28 to 104 nM, compared to Erlotinib (GI50 = 33 nM), and compounds 7i-m were the most potent. Compounds 7h, 7i, 7j, 7k, and 7l were evaluated as dual EGFR/VEGFR-2 inhibitors. These in vitro experiments demonstrated that compounds 7j, 7k, and 7l are potent antiproliferative agents that may operate as dual EGFR/VEGFR-2 inhibitors. Compounds 7j, 7k, and 7l were evaluated for their apoptotic potential activity, where findings indicated that compounds 7j, 7k, and 7l promote apoptosis by activating caspase-3, 8, and Bax and down-regulating the anti-apoptotic Bcl-2. Molecular docking simulations show the binding mode of the most active antiproliferative compounds within EGFR and VEGFR-2 active sites.

Self-Assembly of Erlotinib-Platinum(II) Complexes for Epidermal Growth Factor Receptor-Targeted Photodynamic Therapy.

Due to cell mutation and self-adaptation, the application of clinical drugs with early epidermal growth factor receptor (EGFR)-targeted inhibitors is severely limited. To overcome this limitation, herein, the synthesis and in-depth biological evaluation of an erlotinib-platinum(II) complex as an EGFR-targeted anticancer agent is reported. The metal complex is able to self-assemble inside an aqueous solution and readily form nanostructures with strong photophysical properties. While being poorly toxic toward healthy cells and upon treatment in the dark, the compound was able to induce a cytotoxic effect in the very low micromolar range upon irradiation against EGFR overexpressing (drug resistant) human lung cancer cells as well as multicellular tumor spheroids. Mechanistic insights revealed that the compound was able to selectively degrade the EGFR using the lysosomal degradation pathway upon generation of singlet oxygen at the EGFR. We are confident that this work will open new avenues for the treatment of EGFR-overexpressing tumors.

Design, synthesis and bioactivity evaluation of the combination of evodiamine and erlotinib linked by indolequinone.

Compared with single-targeted therapy, the design and synthesis of heterozygous molecules is still a significant challenge for the discovery of antitumor drugs. Quinone oxidoreductase-1 (NQO1) is a potential target for selective cancer therapy due to its overexpression in many cancer cells and its unique bioredox properties. Based on the principle of combinatorial drug design, we successfully synthesized a new hybrid molecules 13 with an indolequinone structure. We found that the synthesized compounds exhibited much higher cytotoxicity against the tested cancer cells than free drugs. Further mechanism studies confirmed that compound 13 induced cell apoptosis was achieved by regulating p53-dependent mitochondrial pathway and cell cycle arrest at the G0/G1 phase.

In vitro biodistribution studies on clinically approved FGFR inhibitors ponatinib, nintedanib, erlotinib and the investigational inhibitor KP2692.

Binding towards human serum albumin (HSA) and α1-acid glycoprotein (AGP) of three approved fibroblast growth factor receptor (FGFR) inhibitors ponatinib (PON), nintedanib (NIN) and erdafitinib (ERD), as well as the experimental drug KP2692 was studied by means of spectrofluorometric and UV-visible spectrophotometric methods. Additionally, proton dissociation processes, lipophilicity, and fluorescence properties of these four molecules were investigated in detail. The FGFR inhibitors were predominantly presented in their single protonated form (HL+) at pH 7.4 (at blood pH). At gastric pH (pH 1-2) the protonated forms (+1 - +3) are present, which provide relatively good aqueous solubility of the drugs. All of the four inhibitors are highly or extremely lipophilic at pH 7.4 (logD7.4 ≥ 2.7). At acidic pH 2.0 PON and ERD are rather lipophilic, NIN is amphiphilic, while KP2692 is highly hydrophilic. All four compounds bind to HSA and AGP. Moderate binding of PON, KP2692 and NIN was found towards albumin (logK' = 4.5-4.7), while their affinity for AGP was about one order of magnitude higher (logK' = 5.2-5.7). ERD shows a larger affinity for both proteins (logK'HSA ≈ 5.2, logK'AGP ≈ 7.0). The computed constants were used to model the distribution of the FGFR inhibitors in blood plasma under physiological and pathological (acute phase) conditions. The changing levels of the two proteins under pathological conditions compensate each other for PON and NIN, so that the free drug fractions do not change considerably. In the case of ERD the higher AGP levels distinctly reduce the free available fraction of the drug. Comparison with clinical pharmacokinetic data indicates that the here presented solution distribution studies can very well predict the conditions in cancer patients.

Radiation-Activated Cobalamin-Kinase Inhibitors for Treatment of Pancreatic Ductal Adenocarcinoma.

Pancreatic ductal adenocarcinoma (PDAC) remains one of the most dismal diagnoses that a patient can receive. PDAC is extremely difficult to treat, as drug delivery is challenging in part due to the lack of vascularization, high stromal content, and high collagen content of these tumors. We have previously demonstrated that attaching drugs to the cobalamin scaffold provides selectivity for tumors over benign cells due to a high vitamin demand in these rapidly growing cells and an overexpression of transcobalamin receptors in a variety of cancer types. Importantly, we have shown the ability to deliver cobalamin derivatives to orthotopic pancreas tumors. Tyrosine kinase inhibitors have shown promise in treating PDAC as well as other cancer types. However, some of these inhibitors suffer from drug resistance, and as such, their success has been diminished. With this in mind, we synthesized the tyrosine kinase inhibitors erlotinib (EGFR) and dasatinib (Src) that are attached to this cobalamin platform. Both of these cobalamin-drug conjugates cause visible light-induced apoptosis, and the cobalamin-erlotinib conjugate (2) causes X-ray-induced apoptosis in MIA PaCa-2 cells. Both visible light and X-rays provide spatial control of drug release; however, utilizing X-ray irradiation offers the advantage of deeper tissue penetration. Therefore, we explored the utilization of 2 as a synergistic therapy with radiation in athymic nude mice implanted with MIA PaCa-2 tumors. We discovered that the addition of 2 caused an enhanced reduction in tumor margins in comparison with radiation therapy alone. In addition, treatment with 2 in the absence of radiation caused no significant reduction in tumor size in comparison with the controls. The cobalamin technology presented here allows for the spatial release of drugs in conjunction with external beam radiation therapy, potentially allowing for more effective treatment of deep-seated tumors with less systemic side effects.

Zebrafish drug screening identifies Erlotinib as an inhibitor of Wnt/ß-catenin signaling and self-renewal in T-cell acute lymphoblastic leukemia.

The Wnt/ß-catenin pathway's significance in cancer initiation, progression, and stem cell biology underscores its therapeutic potential. However, the clinical application of Wnt inhibitors remains limited due to challenges posed by off-target effects and complex cross-talk of Wnt signaling with other pathways. In this study, we leveraged a zebrafish model to perform a robust and rapid drug screening of 773 FDA-approved compounds to identify Wnt/ß-catenin inhibitors with minimal toxicity. Utilizing zebrafish expressing a Wnt reporter, we identified several drugs that suppressed Wnt signaling without compromising zebrafish development. The efficacy of the top hit, Erlotinib, extended to human cells, where it blocked Wnt/ß-catenin signaling downstream of the destruction complex. Notably, Erlotinib treatment reduced self-renewal in human T-cell Acute Lymphoblastic Leukemia cells, which rely on active ß-catenin signaling for maintenance of leukemia-initiating cells. Erlotinib also reduced leukemia-initiating cell frequency and delayed disease formation in zebrafish models. This study underscores zebrafish's translational potential in drug discovery and repurposing and highlights a new use for Erlotinib as a Wnt inhibitor for cancers driven by aberrant Wnt/ß-catenin signaling.

Different EGF-induced receptor dimer conformations for signaling and internalization.

The structural basis of the activation and internalization of EGF receptors (EGFR) is still a matter of debate despite the importance of this target in cancer treatment. Whether agonists induce dimer formation or act on preformed dimers remains discussed. Here, we provide direct evidence that EGF-induced EGFR dimer formation as best illustrated by the very large increase in FRET between snap-tagged EGFR subunits induced by agonists. We confirm that Erlotinib-related TK (tyrosine kinase) inhibitors also induce dimer formation despite the inactive state of the binding domain. Surprisingly, TK inhibitors do not inhibit EGF-induced EGFR internalization despite their ability to fully block EGFR signaling. Only Erlotinib-related TK inhibitors promoting asymmetric dimers could slow down this process while the lapatinib-related ones have almost no effect. These results reveal that the conformation of the intracellular TK dimer, rather than the known EGFR signaling, is critical for EGFR internalization. These results also illustrate clear differences in the mode of action of TK inhibitors on the EGFR and open novel possibilities to control EGFR signaling for cancer treatment.

Binding Thermodynamics of Fourth-Generation EGFR Inhibitors Revealed by Absolute Binding Free Energy Calculations.

The overexpression or mutation of the kinase domain of the epidermal growth factor receptor (EGFR) is strongly associated with non-small-cell lung cancer (NSCLC). EGFR tyrosine kinase inhibitors (TKIs) have proven to be effective in treating NSCLC patients. However, EGFR mutations can result in drug resistance. To elucidate the mechanisms underlying this resistance and inform future drug development, we examined the binding affinities of BLU-945, a recently reported fourth-generation TKI, to wild-type EGFR (EGFRWT) and its double-mutant (L858R/T790M; EGFRDM) and triple-mutant (L858R/T790M/C797S; EGFRTM) forms. We compared the binding affinities of BLU-945, BLU-945 analogues, CH7233163 (another fourth-generation TKI), and erlotinib (a first-generation TKI) using absolute binding free energy calculations. Our findings reveal that BLU-945 and CH7233163 exhibit binding affinities to both EGFRDM and EGFRTM stronger than those of erlotinib, corroborating experimental data. We identified K745 and T854 as the key residues in the binding of fourth-generation EGFR TKIs. Electrostatic forces were the predominant driving force for the binding of fourth-generation TKIs to EGFR mutants. Furthermore, we discovered that the incorporation of piperidinol and sulfone groups in BLU-945 substantially enhanced its binding capacity to EGFR mutants. Our study offers valuable theoretical insights for optimizing fourth-generation EGFR TKIs.

Engineering of Amphiphilic Erlotinib Analogue as Novel Nanomedicine for Non-Small Cell Lung Cancer Therapy.

Purpose: Molecular targeted therapy is one of the most pivotal strategies in the treatment of non-small cell lung cancer, yet its curative effect is severely compromised by the poor aqueous solubility, low bioavailability and inadequate tumor accumulation of targeted agents. To enhance the efficacy of targeted agents, we demonstrate a novel self-assemble amphiphilic molecule based on erlotinib as an effective nanodrug for anti-cancer treatment. Methods: An amphiphilic molecule composed of hydrophobic erlotinib and hydrophilic biotin block was synthesized and characterized by nuclear magnetic resonance (NMR) as well as high-resolution mass spectrometry (HRMS). Then, nanoassemblies of the amphiphilic molecules are formulated by using nanoprecipitation method. Subsequently, the size, morphology, cell uptake, the anticancer activity and in vivo distribution of the newly constructed erlotinib nanodrug were systematically assessed by some methods, including transmission electron microscopy (TEM), dynamic light-scattering (DLS), flow cytometry, in vivo imaging system etc. Results: We developed a novel nanoformulation of erlotinib, which possesses a high drug loading of 45%. With the features of well-defined structure and small size, the obtained nanodrug could be effectively accumulated in tumor sites and rapidly internalized by cancer cells. Finally, the erlotinib-based nanoformulation showed considerably better anticancer activity compared to free erlotinib both in vitro and in vivo. Moreover, the nanodrug displayed great tolerability. Conclusion: Combining the advantageous features of both nanotechnology and self-assemble, this novel erlotinib nanomedicine constitutes a promising therapeutic candidate for cancer treatment. This study also underlines the potential use of amphiphilic molecule for improving drug efficacy as well as reducing drug toxicity, which could become a general strategy for the preparation of nanodrugs of active agents.

New 1,2,3-Triazole/1,2,4-triazole Hybrids as Aromatase Inhibitors: Design, Synthesis, and Apoptotic Antiproliferative Activity.

A novel series of 1,2,3-triazole/1,2,4-triazole hybrids 5a, 5b, and 6a-i was designed and synthesized as antiproliferative agents targeting aromatase enzymes. The antiproliferative activity of the new hybrids against four cancer cells was studied using Erlotinib as a control. Compounds 6a and 6b demonstrated the highest antiproliferative activity among these hybrids, with GI50 values of 40 nM and 35 nM, respectively. Compound 6b was the most potent derivative, with a GI50 of 35 nM, comparable to Erlotinib's GI50 of 33 nM. Compound 6b inhibited all cancer cell lines with comparable efficacy to Erlotinib. Compounds 5a, 5b, and 6a-i were tested for inhibitory action against aromatase as a potential target for their antiproliferative activity. Results revealed that compounds 6a and 6b were the most potent aromatase inhibitors, with IC50 values of 0.12 ± 0.01 µM and 0.09 ± 0.01 µM, respectively, being more potent than the reference Ketoconazole (IC50 = 2.6 ± 0.20 µM) but less potent than Letrozole (IC50 = 0.002 ± 0.0002). These findings indicated that compounds 6a and 6b had significant aromatase inhibitory action and are potential antiproliferative candidates. The findings were further linked to molecular docking investigations, which gave models of strong interactions with the aromatase domain for inhibitors with high binding scores.