6‐Aryl and Heterocycle Quinazoline Derivatives as Potent EGFR Inhibitors with Improved Activity toward Gefitinib‐Sensitive and ‐Resistant Tumor Cell Lines

A group of novel anilinoquinazoline derivatives with variable aryl and heterocyclic substituents at position 6 were synthesized and tested for their EGFR‐inhibitory activity. Aryl and heterocyclic rings were attached to the quinazoline scaffold through different linkages such as imine, amide, and thiourea. Most of the aryl and heterocyclic derivatives showed potent inhibition of wild‐type EGFR with IC₅₀ values in the low nanomolar range. Among these, thiourea derivatives 6 a , 6 b and compound 10 b also retained significant activity toward the gefitinib‐insensitive EGFR^T790M/L858R mutant, displaying up to 24‐fold greater potency than gefitinib. In addition, cell growth inhibitory activity was tested against cancer cell lines with wild‐type (KB cells) and mutant EGFR (H1975 cells). Several compounds including 6 a were found to be more potent than the reference compound gefitinib toward both cell lines, as was the case for compound 10 b against H1975 cells. Therefore, compounds 6 a and 10 b in particular may serve as new leads for the development of inhibitors effective against wild‐type EGFR as well as gefitinib‐resistant mutants.     

EGFR(T790M)抑制剂的设计、合成及活性评价

表皮生长因子受体(EGFR)的T790M突变最为频发,也是肺癌临床治疗失败的主要原因之一。鉴于先导化合物B6优良的抗H1975细胞系和异植瘤活性,对其进行了EGFR(T790M)激酶抑制活性的确认,并使用Autodock软件确认了两者的相互作用。以EGFR(T790M)为靶点对B6进行定向结构修饰,所得目标化合物经NMR和MS表征后,联合体外激酶、细胞生物活性与Autodock软件解释它们的构效关系。结果表明,3-(苯并[d][1,3]二氧杂-5-基)-1-(1-(乙烯基磺酰基)哌啶-4-基)-1H-吡唑并[3,4-d]嘧啶-4-胺的抗H1975细胞增殖活性(IC₅₀=(1.16±0.24)μmol/L)与B6(IC₅₀=(0.91±0.36)μmol/L)相似,尽管其对EGFR(T790M)的抑制活性(IC₅₀=(148.2±7.2)nmol/L)不如B6(IC₅₀=(22.0±2.6)nmol/L)。以7H-吡咯并[2,3-d]嘧啶-4-胺为母核,取代基分别为胡椒环基和4-取代哌啶基者可开发活性更优的EGFR(T790M)抑制剂,指导后期研究。     

EGFR Mutations in Surgically Resected Fresh Specimens from 697 Consecutive Chinese Patients with Non-Small Cell Lung Cancer and Their Relationships with Clinical Features

We aimed to reveal the true status of epidermal growth factor receptor (EGFR) mutations in Chinese patients with non-small cell lung cancer (NSCLC) after lung resections. EGFR mutations of surgically resected fresh tumor samples from 697 Chinese NSCLC patients were analyzed by Amplification Refractory Mutation System (ARMS). Correlations between EGFR mutation hotspots and clinical features were also explored. Of the 697 NSCLC patients, 235 (33.7%) patients had tyrosine kinase inhibitor (TKIs) sensitive EGFR mutations in 41 (14.5%) of the 282 squamous carcinomas, 155 (52.9%) of the 293 adenocarcinomas, 34 (39.5%) of the 86 adenosquamous carcinomas, one (9.1%) of the 11 large-cell carcinomas, 2 (11.1%) of the 18 sarcomatoid carcinomas, and 2 (28.6%) of the 7 mucoepidermoid carcinomas. TKIs sensitive EGFR mutations were more frequently found in female patients (p < 0.001), non-smokers (p = 0.047) and adenocarcinomas (p < 0.001). The rates of exon 19 deletion mutation (19-del), exon 21 L858R point mutation (L858R), exon 21 L861Q point mutation (L861Q), exon 18 G719X point mutations (G719X, including G719C, G719S, G719A) were 43.4%, 48.1%, 1.7% and 6.8%, respectively. Exon 20 T790M point mutation (T790M) was detected in 3 squamous carcinomas and 3 adenocarcinomas and exon 20 insertion mutation (20-ins) was detected in 2 patients with adenocarcinoma. Our results show the rates of EGFR mutations are higher in all types of NSCLC in Chinese patients. 19-del and L858R are two of the more frequent mutations. EGFR mutation detection should be performed as a routine postoperative examination in Chinese NSCLC patients.     

Acquired Resistance to Osimertinib in EGFR-Mutated Non-Small Cell Lung Cancer: How Do We Overcome It?

Osimertinib is currently the preferred first-line therapy in patients with non-small cell lung cancer (NSCLC) with common epidermal growth factor receptor (EGFR) mutation and the standard second-line therapy in T790M-positive patients in progression to previous EGFR tyrosine kinase inhibitor. Osimertinib is a highly effective treatment that shows a high response rate and long-lasting disease control. However, a resistance to the treatment inevitably develops among patients. Understanding the secondary mechanisms of resistance and the possible therapeutic options available is crucial to define the best management of patients in progression to osimertinib. We provide a comprehensive review of the emerging molecular resistance mechanism in EGFR-mutated NSCLC pre-treated with osimertinib and its future treatment applications.     

Treatment of Brain Metastases of Non-Small Cell Lung Carcinoma

Lung cancer is one of the most common malignant neoplasms. As a result of the disease’s progression, patients may develop metastases to the central nervous system. The prognosis in this location is unfavorable; untreated metastatic lesions may lead to death within one to two months. Existing therapies—neurosurgery and radiation therapy—do not improve the prognosis for every patient. The discovery of Epidermal Growth Factor Receptor (EGFR)—activating mutations and Anaplastic Lymphoma Kinase (ALK) rearrangements in patients with non-small cell lung adenocarcinoma has allowed for the introduction of small-molecule tyrosine kinase inhibitors to the treatment of advanced-stage patients. The Epidermal Growth Factor Receptor (EGFR) is a transmembrane protein with tyrosine kinase-dependent activity. EGFR is present in membranes of all epithelial cells. In physiological conditions, it plays an important role in the process of cell growth and proliferation. Binding the ligand to the EGFR causes its dimerization and the activation of the intracellular signaling cascade. Signal transduction involves the activation of MAPK, AKT, and JNK, resulting in DNA synthesis and cell proliferation. In cancer cells, binding the ligand to the EGFR also leads to its dimerization and transduction of the signal to the cell interior. It has been demonstrated that activating mutations in the gene for EGFR-exon19 (deletion), L858R point mutation in exon 21, and mutation in exon 20 results in cancer cell proliferation. Continuous stimulation of the receptor inhibits apoptosis, stimulates invasion, intensifies angiogenesis, and facilitates the formation of distant metastases. As a consequence, the cancer progresses. These activating gene mutations for the EGFR are present in 10–20% of lung adenocarcinomas. Approximately 3–7% of patients with lung adenocarcinoma have the echinoderm microtubule-associated protein-like 4 (EML4)/ALK fusion gene. The fusion of the two genes EML4 and ALK results in a fusion gene that activates the intracellular signaling pathway, stimulates the proliferation of tumor cells, and inhibits apoptosis. A new group of drugs—small-molecule tyrosine kinase inhibitors—has been developed; the first generation includes gefitinib and erlotinib and the ALK inhibitor crizotinib. These drugs reversibly block the EGFR by stopping the signal transmission to the cell. The second-generation tyrosine kinase inhibitor (TKI) afatinib or ALK inhibitor alectinib block the receptor irreversibly. Clinical trials with TKI in patients with non-small cell lung adenocarcinoma with central nervous system (CNS) metastases have shown prolonged, progression-free survival, a high percentage of objective responses, and improved quality of life. Resistance to treatment with this group of drugs emerging during TKI therapy is the basis for the detection of resistance mutations. The T790M mutation, present in exon 20 of the EGFR gene, is detected in patients treated with first- and second-generation TKI and is overcome by Osimertinib, a third-generation TKI. The I117N resistance mutation in patients with the ALK mutation treated with alectinib is overcome by ceritinib. In this way, sequential therapy ensures the continuity of treatment. In patients with CNS metastases, attempts are made to simultaneously administer radiation therapy and tyrosine kinase inhibitors. Patients with lung adenocarcinoma with CNS metastases, without activating EGFR mutation and without ALK rearrangement, benefit from immunotherapy. This therapeutic option blocks the PD-1 receptor on the surface of T or B lymphocytes or PD-L1 located on cancer cells with an applicable antibody. Based on clinical trials, pembrolizumab and all antibodies are included in the treatment of non-small cell lung carcinoma with CNS metastases.     

C6‐Unsubstituted Pyrazolo[3,4‐d]pyrimidines Are Dual Src/Abl Inhibitors Effective against Imatinib Mesylate Resistant Chronic Myeloid Leukemia Cell Lines

Docking simulations were used to predict the most favorable interaction between the T315I mutated form of Abl (invariably associated with resistance to the tyrosine kinase inhibitor imatinib mesylate, IM) and C6‐unsubstituted and substituted pyrazolo[3,4‐d]pyrimidines previously found to be dual Src/Abl inhibitors. Two C6‐unsubstituted ( 1 and 2 ) and eight C6‐substituted compounds ( 3 – 10 ) were selected and assayed for their effects on the Ba/F3 cell line transducing the wild‐type p210Bcr–Abl construct, which is IM‐sensitive, or three of the most common mutations associated with IM resistance in vivo (T315I, Y253F, and E255K), and driven to drug resistance by saturating doses of IL‐3 or by the expression of the Bcr–Abl construct coding for the p185 protein of acute lymphoblastic leukemia. Compounds 1 and 2 were active against all cell lines assayed (LD₅₀ range: 0.7–4.3 μM ), whereas C6‐substituted compounds exhibited lower activity (LD₅₀～8 μM for compound 3 toward the T315I mutant). Notably, 1 and 2 were also effective toward the T315I mutation, which is insensitive to dual Src/Abl inhibitors. The cytotoxic effects of 1 and 2 on IM‐sensitive and IM‐resistant Ba/F3 cells were attributable, at least in part, to their pro‐apoptotic activity. Taken together, such findings suggest that C6‐unsubstituted pyrazolo[3,4‐d]pyrimidines may represent useful inhibitors to target IM‐resistant chronic myeloid leukemia.     

Technical Validation and Clinical Implications of Ultrasensitive PCR Approaches for EGFR-Thr790Met Mutation Detection in Pretreatment FFPE Samples and in Liquid Biopsies from Non-Small Cell Lung Cancer Patients

In pretreatment tumor samples of EGFR-mutated non-small cell lung cancer (NSCLC) patients, EGFR-Thr790Met mutation has been detected in a variable prevalence by different ultrasensitive assays with controversial prognostic value. Furthermore, its detection in liquid biopsy (LB) samples remains challenging, being hampered by the shortage of circulating tumor DNA (ctDNA). Here, we describe the technical validation and clinical implications of a real-time PCR with peptide nucleic acid (PNA-Clamp) and digital droplet PCR (ddPCR) for EGFR-Thr790Met detection in diagnosis FFPE samples and in LB. Limit of blank (LOB) and limit of detection (LOD) were established by analyzing negative and low variant allele frequency (VAF) FFPE and LB specimens. In a cohort of 78 FFPE samples, both techniques showed an overall agreement (OA) of 94.20%. EGFR-Thr790Met was detected in 26.47% of cases and was associated with better progression-free survival (PFS) (16.83 ± 7.76 vs. 11.47 ± 1.83 months; p = 0.047). In LB, ddPCR was implemented in routine diagnostics under UNE-EN ISO 15189:2013 accreditation, increasing the detection rate of 32.43% by conventional methods up to 45.95%. During follow-up, ddPCR detected EGFR-Thr790Met up to 7 months before radiological progression. Extensively validated ultrasensitive assays might decipher the utility of pretreatment EGFR-Thr790Met and improve its detection rate in LB studies, even anticipating radiological progression.     

Gene Mutation Analysis in EGFR Wild Type NSCLC Responsive to Erlotinib: Are There Features to Guide Patient Selection?

Tyrosine kinase inhibitors (TKIs) are very efficacious in non-small-cell lung cancer (NSCLC) patients harboring activating Epidermal Growth Factor Receptor (EGFR) mutations. However, about 10% of EGFR wild type (wt) patients respond to TKI, with unknown molecular mechanisms of sensitivity. We considered a case series of 34 EGFR wt NSCLC patients responsive to erlotinib after at least one line of therapy. Responsive patients were matched with an equal number of non-responsive EGFR wt patients. A panel of 26 genes, for a total of 214 somatic mutations, was analyzed by MassARRAY^® System (Sequenom, San Diego, CA, USA). A 15% KRAS mutation was observed in both groups, with a prevalence of G12C in non-responders (80% vs. 40% in responders). NOTCH1, p53 and EGFR-resistance-related mutations were found more frequently in non-responders, whereas EGFR-sensitizing mutations and alterations in genes involved in proliferation pathways were more frequent in responders. In conclusion, our findings indicate that p53, NOTCH1 and exon 20 EGFR mutations seem to be related to TKI resistance. KRAS mutations do not appear to influence the TKI response, although G12C mutation is more frequent in non-responders. Finally, the use of highly sensitive methodologies could lead to the identification of under-represented EGFR mutations potentially associated with TKI sensitivity.     

Dexmedetomidine-Induced Aortic Contraction Involves Transactivation of the Epidermal Growth Factor Receptor in Rats

In this study, we examined whether aortic contraction, induced by the alpha-2 adrenoceptor agonist dexmedetomidine, is involved in the transactivation of the epidermal growth factor receptor (EGFR) in isolated endothelium-denuded rat aortas. Additionally, we aimed to elucidate the associated underlying cellular mechanisms. The effects of the alpha-2 adrenoceptor inhibitor rauwolscine, EGFR tyrosine kinase inhibitor AG1478, Src kinase inhibitors PP1 and PP2, and matrix metalloproteinase inhibitor GM6001 on EGFR tyrosine phosphorylation and c-Jun NH₂-terminal kinase (JNK) phosphorylation induced by dexmedetomidine in rat aortic smooth muscles were examined. In addition, the effects of these inhibitors on dexmedetomidine-induced contraction in isolated endothelium-denuded rat aorta were examined. Dexmedetomidine-induced contraction was inhibited by the alpha-1 adrenoceptor inhibitor prazosin, rauwolscine, AG1478, PP1, PP2, and GM6001 alone or by a combined treatment with prazosin and AG1478. AG1478 (3 × 10⁻⁶ M) inhibited dexmedetomidine-induced contraction in isolated endothelium-denuded rat aortas pretreated with rauwolscine. Dexmedetomidine-induced EGFR tyrosine and JNK phosphorylation were inhibited by rauwolscine, PP1, PP2, GM6001, and AG1478. Furthermore, dexmedetomidine-induced JNK phosphorylation reduced upon EGFR siRNA treatment. Therefore, these results suggested that the transactivation of EGFR associated with dexmedetomidine-induced contraction, mediated by the alpha-2 adrenoceptor, Src kinase, and matrix metalloproteinase, caused JNK phosphorylation and increased calcium levels.     

Recognition by nonaromatic and stereochemical subunit-containing polyamides of the four Watson-Crick base pairs in the DNA minor groove

In order to develop an optimal subunit as a T‐recognition element in hairpin polyamides, 15 novel chirality‐modified polyamides containing (R)‐α,β‐diaminopropionic acid (^Rβ), (S)‐α,β‐diaminopropionic acid (^Sβ), (1R,3S)‐3‐aminocyclopentanecarboxylic acid (^RSCp), (1S,3R)‐3‐amino‐cyclopentanecarboxylic acid (^RSCp), (1R,3R)‐3‐aminocyclopentanecarboxylic acid (^RRCp) and (1S,3S)‐3‐amino‐cyclopentanecarboxylic acid (^SSCp) residues were synthesized. Their binding characteristics to DNA sequences 5′‐TGC N CAT‐3′/3′‐ACG N′ GTA‐5′ ( N?N′ =A ? T, T ? A, G ? C and C ? G) were systemically studied by surface plasmon resonance (SPR) and molecular simulation (MSim) techniques. SPR showed that polyamide 4 , AcIm‐^Sβ‐ImPy‐γ‐ImPy‐β‐Py‐βDp (β/^Sβ pair), bound to a DNA sequence containing a core binding site of 5′‐TGC A CAT‐3′ with a dissociation equilibrium constant (K_D) of 4.5×10^?8 m. This was a tenfold improvement in specificity over 5′‐TGCTCAT‐3′ (K_D=4.5×10^?7 M ). MSim studies supported the SPR results. More importantly, for the first time, we found that chiral 3‐aminocyclopentanecarboxylic acids in polyamides can be employed as base readers with only a small decrease in binding affinity to DNA. In particular, SPR showed that polyamide 9 (^RRCp/β pair) had a 15‐fold binding preference for 5′‐TGCTCAT‐3′ over 5′‐TGCACAT‐3′. A large difference in standard free energy change for A ? T over T ? A was determined (ΔΔG^o=5.9 kJ mol^?1), as was a twofold decrease in interaction energy by MSim. Moreover, a 1:1 stoichiometry ( 9 to 5′‐TGC T CAT‐3′/3′‐ACG A GTA‐5′) was shown by MSim to be optimal for the chiral five‐membered cycle to fit the minor groove. Collectively, the study suggests that the (S)‐α‐amino‐β‐aminopropionic acid and (1R,3R)‐3‐aminocyclopentanecarboxylic acid can serve as a T‐recognition element, and the stereochemistry and the nature of these subunits significantly influence binding properties in these recognition events. Subunit (1R,3R)‐3‐aminocyclopentanecarboxylic acid broadens our scope to design novel polyamides.     

The Therapeutic Potential of the Restoration of the p53 Protein Family Members in the EGFR-Mutated Lung Cancer

Despite the recent development of precision medicine and targeted therapies, lung cancer remains the top cause of cancer-related mortality worldwide. The patients diagnosed with metastatic disease have a five-year survival rate lower than 6%. In metastatic disease, EGFR is the most common driver of mutation, with the most common co-driver hitting TP53. EGFR-positive patients are offered the frontline treatment with tyrosine kinase inhibitors, yet the development of resistance and the lack of alternative therapies make this group of patients only fit for clinical trial participation. Since mutant p53 is the most common co-driver in the metastatic setting, therapies reactivating the p53 pathway might serve as a promising alternative therapeutic approach in patients who have developed a resistance to tyrosine kinase inhibitors. This review focuses on the molecular background of EGFR-mutated lung cancer and discusses novel therapeutic options converging on the reactivation of p53 tumor suppressor pathways.     

Using multiple site-directed modification of epoxide hydrolase to significantly improve its enantioselectivity in hydrolysis of rac-glycidyl phenyl ether

The epoxide hydrolase gene(SpEH) from Sphingomonas sp. HXN-200 was synthesized and expressed in robust Escherichia coli cells that had a dual protection system. The enantioselectivity(E-value) of the recombinant SpEH was 7.7 and the yield of the remaining(R)-PGE was 24.3% for the hydrolysis of racemic phenyl glycidyl ether(rac-PGE). To improve the catalytic properties of SpEH, the site-directed mutagenesis was carried out based on homology modeling, sequence alignment and molecular docking. Six residues(V195, V196, F218,N226, Q312, and M332) near the active site were mutated to hydrophobic amino acids and the positive mutations were selected for combinatorial mutation. The optimal mutant SpEH~(V196A/N226A/M332A) had an enhanced E-value of 21.2 and a specific activity of 4.57 U·mg~(-1)-wet cells, which were 2.8-, and 2.3-fold higher than those of wild-type SpEH. The optimal temperature and p H for purified Sp EHV196 A/N226 A/M332 Ato catalyze the hydrolysis of rac-PGE were 25 ℃ and 7.0 with 200 U·mg~(-1). The enantioselectivity and yield of the remaining(R)-PGE of E. coli_SpEH~(V196A/N226A/M332A)increased from 7.7 to 21.2 and 24.3% to 40.9%, respectively. The molecular docking and kinetic parameter analyses showed that SpEH~(V196A/N226A/M332A) has a greater affinity toward(S)-PGE than(R)-PGE, and that it was more difficult for the O-atom of ASP170 to achieve the nucleophilic attack on the Cα of(R)-PGE, resulting in its improved enantioselectivity.     

GmIAA27 Encodes an AUX/IAA Protein Involved in Dwarfing and Multi-Branching in Soybean

Soybean plant height and branching affect plant architecture and yield potential in soybean. In this study, the mutant dmbn was obtained by treating the cultivar Zhongpin 661 with ethylmethane sulfonate. The dmbn mutant plants were shorter and more branched than the wild type. The genetic analysis showed that the mutant trait was controlled by a semi-dominant gene. The candidate gene was fine-mapped to a 91 kb interval on Chromosome 9 by combining BSA-seq and linkage analysis. Sequence analysis revealed that Glyma.09g193000 encoding an Aux/IAA protein (GmIAA27) was mutated from C to T in the second exon of the coding region, resulting to amino acid substitution of proline to leucine. Overexpression of the mutant type of this gene in Arabidopsis thaliana inhibited apical dominance and promoted lateral branch development. Expression analysis of GmIAA27 and auxin response genes revealed that some GH3 genes were induced. GmIAA27 relies on auxin to interact with TIR1, whereas Gmiaa27 cannot interact with TIR1 owing to the mutation in the degron motif. Identification of this unique gene that controls soybean plant height and branch development provides a basis for investigating the mechanisms regulating soybean plant architecture development.     

Synthesis and SAR of Tetracyclic Inhibitors of Protein Kinase CK2 Derived from Furocarbazole W16

The serine/threonine kinase CK2 modulates the activity of more than 300 proteins and thus plays a crucial role in various physiological and pathophysiological processes including neurodegenerative disorders of the central nervous system and cancer. The enzymatic activity of CK2 is controlled by the equilibrium between the heterotetrameric holoenzyme CK2α₂β₂ and its monomeric subunits CK2α and CK2β. A series of analogues of W16 ((3aR,4S,10S,10aS)-4-{[(S)-4-benzyl-2-oxo-1,3-oxazolidin-3-yl]carbonyl}-10-(3,4,5-trimethoxyphenyl)-4,5,10,10a-tetrahydrofuro[3,4-b]carbazole-1,3(3aH)-dione ((+)- 3 a )) was prepared in an one-pot, three-component Levy reaction. The stereochemistry of the tetracyclic compounds was analyzed. Additionally, the chemically labile anhydride structure of the furocarbazoles 3 was replaced by a more stable imide ( 9 ) and N-methylimide ( 10 ) substructure. The enantiomer (−)- 3 a (K_i=4.9 μM) of the lead compound (+)- 3 a (K_i=31 μM) showed a more than sixfold increased inhibition of the CK2α/CK2β interaction (protein-protein interaction inhibition, PPII) in a microscale thermophoresis (MST) assay. However, (−)- 3 a did not show an increased enzyme inhibition of the CK2α₂β₂ holoenzyme, the CK2α subunit or the mutated CK2α′ ^C336S subunit in the capillary electrophoresis assay. In the pyrrolocarbazole series, the imide (−)- 9 a (K_i=3.6 μM) and the N-methylimide (+)- 10 a (K_i=2.8 μM) represent the most promising inhibitors of the CK2α/CK2β interaction. However, neither compound could inhibit enzymatic activity. Unexpectedly, the racemic tetracyclic pyrrolocarbazole (±)- 12 , with a carboxy moiety in the 4-position, displays the highest CK2α/CK2β interaction inhibition (K_i=1.8 μM) of this series of compounds.     

State-of-the-Art Molecular Oncology of Lung Cancer in Taiwan

Lung cancers are life-threatening malignancies that cause great healthcare burdens in Taiwan and worldwide. The 5-year survival rate for Taiwanese patients with lung cancer is approximately 29%, an unsatisfactorily low number that remains to be improved. We first reviewed the molecular epidemiology derived from a deep proteogenomic resource in Taiwan. The nuclear factor erythroid 2-related factor 2 (NRF2)antioxidant mechanism was discovered to mediate the oncogenesis and tumor progression of lung adenocarcinoma. Additionally, DNA replication, glycolysis and stress response are positively associated with tumor stages, while cell-to-cell communication, signaling, integrin, G protein coupled receptors, ion channels and adaptive immunity are negatively associated with tumor stages. Three patient subgroups were discovered based on the clustering analysis of protein abundance in tumors. The first subgroup is associated with more advanced cancer stages and visceral pleural invasion, as well as higher mutation burdens. The second subgroup is associated with EGFR L858R mutations. The third subgroup is associated with PI3K/AKT pathways and cell cycles. Both EGFR and PI3K/AKT signaling pathways have been shown to induce NRF2 activation and tumor cell proliferation. We also reviewed the clinical evidence of patient outcomes in Taiwan given various approved targeted therapies, such as EGFR-tyrosine kinase inhibitors and anaplastic lymphoma kinase (ALK)inhibitors, in accordance with the patients’ characteristics. Somatic mutations occurred in EGFR, KRAS, HER2 and BRAF genes, and these mutations have been detected in 55.7%, 5.2%, 2.0% and 0.7% patients, respectively. The EGFR mutation is the most prevalent targetable mutation in Taiwan. EML4-ALK translocations have been found in 9.8% of patients with wild-type EGFR. The molecular profiling of advanced NSCLC is critical to optimal therapeutic decision-making. The patient characteristics, such as mutation profiles, protein expression profiles, drug-resistance profiles, molecular oncogenic mechanisms and patient subgroup systems together offer new strategies for personalized treatments and patient care.     

Selective Delivery of Doxorubicin to EGFR+ Cancer Cells by Cetuximab–DNA Conjugates

Doxorubicin is a hydrophobic anticancer drug that has poor selectivity, due to the lack of active targeting capability. Here, learning lessons from the success of antibody–drug conjugates, we have designed a new doxorubicin delivery system without conjugating doxorubicin to antibody directly. In this setup, cetuximab, an antibody that targets the epidermal growth factor receptor (EGFR) in cancer cells, was conjugated to a single-stranded DNA with a carefully designed sequence in a site-selective manner by using the DNA-templated protein conjugation (DTPC) method. The DNA duplex in the conjugates serves as a carrier of doxorubicin through noncovalent intercalation, and cetuximab functions as the targeting agent; this could drastically decrease systemic toxicity and potentially avoid under- or overdosing. The size of conjugates loaded with doxorubicin was about 8.77 or 16.61 nm when characterized by dynamic light scattering and atomic force microscopy, respectively. In vitro cytotoxicity and selective cancer cell killing was investigated against two EGFR⁺ cell lines (KB and MDA-MB-231) and one EGFR⁻ cell line (NIH-3T3). Cytotoxicity and flow cytometry data showed that doxorubicin loaded in cetuximab–DNA conjugates was more potent in terms of cell cytotoxicity than free doxorubicin in EGFR-overexpressed cell lines, thus suggesting that the conjugates were more selectively and easily taken up into cells, followed by rapid release of doxorubicin from the system into the cytoplasm from endosomes.