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Development of tyrosine kinase inhibitors (TKIs) against oncogenic drivers has significantly improved survival of patients with oncogene-mutated non-small cell lung cancer (NSCLC). However, acquired resistance to TKIs emerges over time in essentially all patients who initially respond. Recent evidence suggests that drug-tolerant persister (DTP) cells, which survive and adapt to targeted therapies during an early phase of treatment, play an important role in the emergence of drug resistance. A previous study reported that cluster of differentiation 74 (CD74) expression is upregulated in epidermal growth factor receptor (EGFR)-mutated lung cancer after treatment with EGFR-TKIs and that CD74 can be one of the DTP cell markers. However, both the mechanism underlying CD74 expression and the role of CD74 in DTP cells remain unclear. In the current study, an attempt was made to identify the mechanism using cell culture systems and transgenic mouse models. The results confirmed CD74 upregulation at the messenger RNA (mRNA) level after treatments with TKIs in various oncogene-mutated cell lines, including those with EGFR mutations, ROS1 fusions, and ALK fusions. The class II transactivator (CIITA), upstream of CD74, and tumor necrosis factor (TNF)-α expression were induced by treatments with TKIs in tumor cells, leading to an increase in CD74 expression. In addition, the results showed that treatments with TKIs enhance the autocrine secretion of macrophage migration inhibitory factor (MIF), a ligand of CD74, from tumor cells. This implied that autocrine stimulation of CD74 signaling blocks apoptosis and causes emergence of DTP cells. To examine whether CD74 plays an important role in the emergence of resistance to TKIs in vivo, experiments were completed in which lung-specific EGFR-L858R-T790M transgenic mice were crossed with Cd74 knockout mice. The results showed that complete deletion of CD74 overcomes or delays resistance to TKIs. Taken together, the results of this study suggest that the MIF-CD74 axis can be a novel target to overcome resistance in driver-mutated NSCLC. / 2026-02-28T00:00:00Z

AND Hypoxia in tumours is known to cause resistance to conventional chemotherapeutic drugs. In contrast, little is known about the effects of hypoxia on targeted anti-cancer drugs. This study evaluated the effect of hypoxia on a series of clinically approved tyrosine kinase inhibitors (TKIs). EXPERIMENTAL APPROACH: The effect of hypoxia (0.1% oxygen) on the activity of conventional cytotoxic drugs (5-fluorouracil, doxorubicin and vinblastine), the hypoxia-activated prodrug tirapazamine and 9 TKIs was determined in a panel of cell lines. Where hypoxia had a marked effect on chemosensitivity, Western blot analysis was conducted to determine the effect of hypoxia on target expression and the effect of TKIs on cell signalling response under aerobic and hypoxic conditions. KEY RESULTS: Three patterns of chemosensitivity were observed: resistance under hypoxia, equitoxic activity against hypoxic and aerobic cells, and preferential cytotoxicity to hypoxic cells. Significant hypoxia selectivity (independent of HIF1) was observed in the case of dasatinib and this correlated with the ability of dasatinib to inhibit phosphorylation of Src at tyrosine 530. Sorafenib was significantly less effective under hypoxic conditions but resistance did not correlate with hypoxia-induced changes in Raf/MEK/ERK signalling. CONCLUSIONS AND IMPLICATIONS: Hypoxia influences the activity of TKIs but in contrast to conventional cytotoxic drugs, preferential activity against hypoxic cells can occur. The search for hypoxia-targeted therapies has been long and fruitless and this study suggests that some clinically approved TKIs could preferentially target the hypoxic fraction of some tumour types.

Tyrosine kinase inhibitors (TKI) are small organic molecules designed for the targeted cancer therapy. They perform the inhibition of activated receptor tyrosine kinases in tumor cells, that defeats tumor growth, proliferation, metastasis and angiogenesis in tumor tissue. Two TKI, lenvatinib and vandetanib, are used in thyroid cancer treatment. This thesis investigates the ways leading to enhancement of efficiency of these anticancer drugs for therapy. One of the studied anticancer drug - lenvatinib - was investigated to be prepared in a nanoform. Nanoparticles were based on protein apoferritin as well as on lipids. Theoretical model of lenvatinib interaction with an apoferritin cavity, as well as the model of its encapsulation obtained by computer modeling indicated that lenvatinib seems not to be suitable for preparation of apoferritin nanoparticles. Since lenvatinib occurs in its neutral form during preparation of nanoparticles, it does not interact with nanoparticle. The unsuccessful experimental preparation of lenvatinib-loaded apoferritin nanoparticles confirmed that lenvatinib is not suitable for its preparation. However, the theoretical model can serve for screening of other potentially suitable drugs before the experimental nanoparticle preparation. Since the experimental preparation of...

In recent years, the inhibiition of tyrosine kinases,which may incorrectly regulate some singaling pathway has been used to treat cancer as so-called biological therapy. An example of such inhibitors are vandetanib and lenvatinib. These two substances are used to treat thyroid gland tumors because they affect vascular growth factor receptor or endothelial growth factor receptor that can regulate tumor growth and metastasis. Ellipticine, which has anti-tumor effects on lots of tumor disease, has been investigated in this study together with vandetanib and lenvatinib. In this diploma thesis, the effect of mentioned tyrosine kinase inhibitors, ellipticine and their combinations on gene and protein expression of CYP1A1, 1A2, 3A1 and 3A2 in rat liver in vivo was determined. Protein expression was studied using Western blot method with imunodetection. Gene expression was assessed by quantitative PCR. Moreover, the effect of tested substances and their combinations on CYP1A activity (measured as 7-ethoxyresorufin O-deethylation), CYP1A2 activity (measured as 7-methoxyresorufin O-demethylation), CYP1A1 activity (measured as Sudan I oxidation), CYP3A specific activity (measured as testosteron 6β-hydroxylation) and ellipticine, vandetanib, lenvatinib metabolism was determined. It has been confirmed that...

Medullary thyroid carcinoma (MTC) is a rare malignant tumor originating from thyroid parafollicular C cells. This tumor accounts for 3-4% of thyroid gland neoplasias. MTC may occur sporadically or inherited. The hereditary MTC is part of syndromes of multiple endocrine neoplasia (MEN) 2A and 2B, familial medullary thyroid carcinoma (FMTC). Germline mutations of the RET (REarranged during Transfection) protooncogene cause hereditary form of cancer, whereas somatic mutations can be present in sporadic form of the disease. The RET gene encodes a receptor tyrosine kinase involved in the activation of intracellular signaling pathways leading to proliferation, growth, differentiation, migration and survival. Nowadays, the only possibility of cure for MTC patients consists of total thyroidectomy associated with lymph node dissection. Based on the knowledge of the pathogenic mechanisms of MTC, new drugs have been developed in attempt to control metastatic disease. Of these, the small-molecule tyrosine kinase inhibitors (TKIs) represent one of the most promising agents for MTC treatment and clinical trials have shown encouraging results. Hopefully, the cumulative knowledge about the targets of action of these drugs as well as TKI-associated side effects will help on choosing the best therapeutic approach in order to enhance its benefits.

Whilst progress has been made in reducing mortality in some cancers, mortality rates remain high in many cancers and there is a need to develop novel therapeutic strategies. In this thesis, various pharmacological strategies designed to enhance the activity of existing therapeutic drugs were evaluated. Cancer cells are dependent upon aerobic glycolysis (the Warburg effect) and glutamine uptake. Using clinically approved tyrosine kinase inhibitors and Bortezomib, significant enhancement of chemosensitivity was observed when used in combination with inhibitors of lactate dehydrogenase (Gossypol) and pyruvate kinase dehydrogenase (Dichloroacetate). In contrast, depletion of glutamine from media had to be extensive in order to induce cell death and cell death only occurred after prolonged exposure to glutamine-deprived conditions. This suggests that glutamine depletion strategies alone are unlikely to be successful but may be useful in combination with other agents targeting glutamine addiction in cancer cells. Finally, Temozolomide (TMZ) is an important drug in the treatment of glioblastomas but its activity is reduced by resistance mechanisms including O6 methyl guanine methyltransferase (MGMT) and mismatch repair (MMR). This thesis has identified analogues of TMZ (EA02-45, EA02-59, EA02-64 and EA02-65) that are MGMT and MMR independent in terms of inducing cell kill in vitro. These compounds are promising leads for future development. In conclusion, this thesis has demonstrated that interfering with the metabolic phenotype of cancer can enhance the activity of existing drugs and identified novel analogues of TMZ that circumvent drug resistance mechanisms that hamper the efficacy of TMZ.

Introdução: Os inibidores tirosino-quinase (ITQs) constituem uma nova terapia molecular para o carcinoma medular da tireoide (CMT). O vandetanibe, um ITQ que atua contra os receptores VEGFR, EGFR e RET, inibe a transformação e o crescimento do tumor no CMT. No entanto, os ITQs têm importantes efeitos adversos, incluindo o hipotireoidismo. O aumento da expressão da iodotironina desiodase do tipo 3 (D3/DIO3), uma enzima chave na inativação dos hormônios da tireoide, pode ser um possível mecanismo de indução do hipotireoidismo por estas drogas. Objetivo: Investigar os efeitos dos inibidores tirosino-quinase na expressão da D3 em células derivadas do CMT. Métodos: Estudo experimental in vitro, utilizando linhagem de células humanas oriundas de CMT (células TT). As células foram cultivadas em meio específico e tratadas com diferentes doses do ITQ vandetanibe (0,25; 0,5 e 1μM) ou com DMSO. A proliferação celular foi determinada por contagem em câmara de Neubauer. A expressão do mRNA foi avaliada por meio de PCR em tempo real, a expressão proteica por meio de Western Blot e a atividade da D3 foi avaliada por meio da técnica de cromatografia em colunas de Sephadex LH-20. Resultados: A adição do vandetanibe ao meio de cultura causou diminuição do número de células e seu efeito foi tempo e dose dependente, apresentando uma redução máxima (77%) após 6 dias de tratamento na dose de 1μM. Como esperado, o tratamento com vandetanibe inibiu a fosforilação do ERK. Não foram observadas alterações significativas dos níveis de mRNA da DIO3 após 3 (0,02 vs. 0,02 vs. 0,01 vs. 0,01; P = 0,34) ou 6 dias (0,02 vs. 0,02 vs. 0,03 vs. 0,02; P = 0,33) de tratamento. Consequentemente, a expressão proteica da D3 não aumentou nos grupos tratados. No entanto, observou-se um aumento de 2 a 5 vezes na atividade da D3 após 3 dias de tratamento e um aumento de 1,5 a 2,15 vezes em 6 dias de tratamento. Conclusões: O tratamento com vandetanibe não foi associado com níveis aumentados de expressão do mRNA e da proteína da D3 em células derivadas de CMT, embora tenha sido observado um aumento na sua atividade enzimática. / Background: Tyrosine kinase inhibitors (TKIs) constitute a novel molecular therapy for medullary thyroid carcinoma (MTC). Vandetanib, a TKI that acts against the VEGFR, EGFR and RET receptors, inhibits tumor transformation and growth in MTC. However, TKIs have important adverse effects, including hypothyroidism. Increases in the expression of type 3 iodothyronine deiodinase (D3/DIO3), a key enzyme in the inactivation of thyroid hormones, may be a possible mechanism of induction of hypothyroidism by these drugs. Objective: To investigate the effects of vandetanib on D3 expression in MTC-derived cells. Methods: In vitro experimental study using human MTC cell line (TT cells). Cells were cultured in specific medium and treated with different doses of vandetanib (0.25, 0.5 and 1μM) or DMSO. Cell proliferation was determined by counting in Neubauer's chamber. Expression of mRNA was evaluated by real-time PCR, protein expression by Western Blot and D3 activity was evaluated by Sephadex LH-20 column chromatography. Results: The addition of vandetanib to the culture medium caused a time and dose-dependent decrease in the number of cells, with a maximum reduction (77%) after 6 days of treatment at 1μM dose. As expected, vandetanib treatment inhibited ERK phosphorylation. No significant changes in DIO3 mRNA levels were observed after 3 (0.02 vs. 0.02 vs. 0.01 vs. 0.01; P = 0.34) or 6 days (0.02 vs. 0.02 vs. 0.03 vs. 0.02; P = 0.33) of treatment. Accordingly, D3 protein expression did not increase in treated groups. However, we observed a 2 to 5-fold increase in D3 activity after 3 days of treatment and a 1.5 to 2.15-fold increase in 6 days of treatment. Conclusions: Treatment with vandetanib was not associated with increased DIO3 mRNA and D3 protein expression levels in MTC-derived cells, although an increase in enzyme activity has been observed.

Multidrug resistance (MDR), a phenomenon in which tumors that were initially sensitive, recur and start showing resistance not only to the initial chemotherapeutic agent but also to various anticancer drugs that are structurally and functionally different from the initial drug, constitutes one of the main reasons for the failure of chemotherapy. An important mechanism of MDR is the enhanced cellular efflux of anticancer agents due to an overexpression of ATP-binding cassette (ABC) transporters (i.e. efflux transporters), especially P-glycoprotein (Pgp), Multidrug Resistance-associated Protein 1 (MRP1) and Breast Cancer Resistance Protein (BCRP), in cancer cells. In order to reverse this resistance, there has been a lot of emphasis on the development of Pgp, MRP1 and BCRP inhibitors. Although this search has been ongoing for three decades, there are still no clinically available efflux transporter modulators. Tyrosine kinase inhibitors (TKIs) are a novel, rapidly growing class of anticancer agents that have a target-based mechanism of action, and their use transformed cancer chemotherapy due to higher specificity and enhanced safety profiles compared to conventional chemotherapeutic agents. Despite their tremendous success in treating various types of tumors, patients develop resistance to TKIs over time. Most of the FDA- approved TKIs are substrates of Pgp and/or BCRP, and as a result, these efflux transporters are also an important cause of conferred resistance against TKIs in cancer cells. Additionally, none of the 31 approved TKIs have an indication for use in brain tumors and interestingly, this may also due to the presence of Pgp and BCRP at the blood-brain barrier (BBB) and in the tumor cells, which prevent the TKI from crossing the BBB and reaching its target tumor site. Since Pgp- and BCRP- mediated TKI efflux has been shown to be involved in TKI resistance, the inhibition of these transporters could represent a potential TKI resistance reversal strategy. Over the last three decades, a large number of Pgp and/or BCRP inhibitors have been identified, but none of them have successfully made it to the clinic. It was observed that most drugs identified as inhibitors were either unable to achieve Pgp and BCRP inhibitory concentrations in-vivo without imparting severe toxicity, or did not possess adequate bioavailability and tissue distribution profiles in order to reach the tumor site. From these identified candidate inhibitors, after much thought and consideration, we chose to investigate TKIs and methylated flavones as modulators of efflux transporter-mediated TKI resistance. The overall goal of this project was to investigate the promising chemosensitizing potential of TKIs and methylated flavones in efflux transporter-mediated TKI resistance, both in-vitro and in-vivo. To identify potent efflux transporter inhibitor TKIs, we evaluated the effect of various TKIs on the accumulation of afatinib, the model TKI substrate, in Pgp- and BCRP- overexpressing cell lines. Afatinib was chosen as the model TKI substrate for our study because it undergoes very minimal metabolism in several species. Afatinib is a substrate of both Pgp and BCRP, but is not a substrate of uptake transporters. Therefore, it was anticipated that an in-vivo efflux transporter-mediated interaction with afatinib would most likely not be confounded or masked by other factors influencing its disposition. From the in-vitro cell uptake studies, we found that nilotinib is a potent inhibitor of both Pgp and BCRP, and it reversed Pgp- and BCRP- mediated afatinib efflux. Subsequently, an in-vivo study was carried out in mice to investigate the interaction between afatinib and nilotinib; and also the impact of nilotinib on the pharmacokinetics and tissue distribution of afatinib. Afatinib exposure in the plasma and in most tissues, namely liver, lung, kidney, heart, muscle, fat, and skin, was found to be significantly increased when nilotinib was coadministered with afatinib. Further, the nilotinib concentrations in most mice tissues was above that needed for Pgp and BCRP inhibition. These results showed that nilotinib could be a potent chemosensitizing agent for Pgp- and BCRP- mediated TKI resistance. Additionally, a significant increase in afatinib brain exposure was observed in the mice which were administered afatinib in combination with nilotinib. This is an interesting and important finding that could potentially be very useful in the treatment of primary and metastasized brain tumors. We also developed a physiologically based pharmacokinetic model of afatinib to characterize its tissue disposition in mice organs, and this model was then scaled up to humans. The developed model accurately predicted afatinib plasma exposure in healthy volunteers and patients with solid malignant tumors, renal impairment, and hepatic impairment. To investigate the chemosensitizing potential of methylated flavones in efflux transporter-mediated TKI resistance, the Bcrp1 inhibitory effect of 5,7-DMF and its effect on sorafenib accumulation was evaluated in-vitro. 5,7- DMF was found to be a potent inhibitor of Bcrp1 and consequently, its impact on the pharmacokinetics and tissue distribution of sorafenib was evaluated in mice. Results showed that co-administration with 5,7-DMF led to significantly greater sorafenib exposure in plasma and in most tissues collected. This indicated that 5,7-DMF may represent a promising chemosensitizing agent for Bcrp1-mediated TKI resistance due to its low toxicity and potent Bcrp1 inhibition. Our results may have important clinical implications as TKIs are currently the most widely used anticancer agents. 5,7-DMF may show great potential in reversing MDR in tumors expressing BCRP. On the other hand, TKI-TKI combination therapy, especially with nilotinib as the perpetrator, is an attractive strategy to combat both Pgp- and BCRP-mediated TKI resistance. Additionally, since nilotinib has a wide volume of distribution and can reach various tissues at concentrations sufficient enough to inhibit Pgp and BCRP; it could potentially be used as a chemosensitizer in the treatment of numerous types of cancers. Furthermore, its chemosensitizing potential could particularly be useful in the treatment of primary and metastatic brain tumors. Further studies are warranted to assess the chemosensitizing effect of nilotinib in tumor xenograft models.