In the past decade, molecularly targeted therapy represented by epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKI) has brought great changes to the treatment of lung cancer, but drug resistance is still an unsolved problem [1].
Taking EGFR-TKI as an example, in patients with non-small cell lung cancer (NSCLC) carrying EGFR mutations, it is often effective at first, but often evolves into acquired resistance and loses its therapeutic effect, such as the T790M mutation that causes the first/second generation EGFR-TKI resistance and the C797S mutation that causes the third generation EGFR-TKI resistance, especially tumors that also contain L858R/T790M/C797S mutations. The existing EGFR-TKI is helpless against it [2, 3].
The mountains and rivers are doubtful and there is no way, and the willows are dark and the flowers are bright and another village. Researchers began experimenting with developing fourth-generation EGFR inhibitors to overcome EGFR-TKI resistance caused by EGFR mutations.
In 2016, the first EGFR allosteric inhibitor EAI045 was discovered, kicking off the appearance of the fourth generation of EGFR inhibitors. Its site of action is a unique site formed by L858R mutations that is different from the ATP competitive EGFR-TKI binding site and therefore does not affect the effects of acquired EGFR mutations that affect TKI efficacy, providing a new strategy for overcoming drug-resistant EGFR mutations [4]. However, EAI045 requires co-administration of EGFR monoclonal antibodies to disrupt asymmetric EGFR dimerization, allowing EAI045 to bind to two monomers of the EGFR dimer [4]. This is obviously not convenient for clinical use.
In 2019, a team led by Michael J. Eck, Nathanael S. Gray, and Pasi A. J nne of the Dana-Farber Cancer Research Center at Harvard Medical School reported a mutation-selective EGFR allosteric inhibitor, JBJ-04-125-02[ 5]. It has been shown to be effective against EGFR L858R/T790M/C797S mutations in vitro and in vivo, but unfortunately, JBJ-04-125-02 monotherapy is ineffective in patient-derived cell lines or xenograft models[5].
The good news is that the three professors, together with Professor David A. Scott's team from the same unit, improved JBJ-04-125-02 and discovered a new and more effective EGFR allosteric inhibitor, JBJ-09-063, and characterized it in vitro and in vivo.
Recently, their study found that JBJ-09-063 has better pharmacokinetics and efficacy than JBJ-04-125-02, and it has a good therapeutic effect on models that lead to EGFR-TKI resistance, such as EGFR T790M mutation and C797S mutation, and the relevant results are published in the prestigious journal Nature Cancer [6].
The improved pharmacological properties of JBJ-09-063 have taken a key step in the clinical development of fourth-generation EGFR inhibitors, which have a good inhibitory effect on EGFR-mutant tumors that lead to first/second-generation/TKI resistance, especially in combination with third-generation EGFR-TKI, which can be called a "big killer" for the treatment of NSCLC.
Screenshot of the first page of the paper
To identify a more effective allosteric inhibitor, the research team modified JBJ-04-125-02 and identified JBJ-09-063 as a highly effective EGFR allosteric inhibitor.
Enzymatic analysis showed that JBJ-09-063 had a stronger inhibitory effect on EGFR L858R/T790M and EGFR L858R/T790M and EGFR L858R/T790M/C797S than JBJ-04-125-02 and osimtinib (third generation EGFR-TKI), and was not affected by C797S mutation (osimtinib was affected by C797S mutation).
In the EGFR L858R/T790M and EGFR L858R/T790M/C797S BA/F3 cell lines, JBJ-09-063 has a stronger inhibition of cell growth (about 10-fold) and a stronger effect on EGFR phosphorylation inhibition than JBJ-04-125-02. In gefitinib-resistant EGFR L858R/T790M BA/F3 cells, JBJ-09-063 inhibits cell growth comparable to oxytinib, and in oshitinib-resistant EGFR L858R/C797S BA/F3 cells, JBJ-09-063 inhibits cell growth on gefitinib.
In cytological studies, JBJ-09-063 demonstrated better inhibition of mutant EGFR than JBJ-04-125-02 and osimertinib
Immediately afterward, the researchers compared the pharmacokinetic properties of JBJ-09-063 and JBJ-04-125-02. JBJ-09-063 was found to have higher intravenous clearance (5.0 ml/min/kg vs 15.7 ml/min/kg) and bioavailability (14.6% vs 3%). It is not difficult to see that the pharmacokinetics of JBJ-09-063 is better than that of JBJ-04-125-02 and has better pharmacological properties.
Therefore, JBJ-09-063 will theoretically have better in vivo efficacy.
The researchers constructed H1975 cells (human lung adenocarcinoma cells) containing EGFR L858R/T790M mutations and patient-derived DFCI52 cell xenograft tumor models to evaluate the role of JBJ-09-063 in vivo.
In the H1975 model, JBJ-09-063 results in dose-dependent reductions in tumor volume and is more effective than JBJ-04-125-02. It is worth noting that JBJ-09-063 at doses of 50 mg /kg and 100 mg/kg is comparable to the therapeutic effect of 25 mg /kg of osimtinib. In the DFCI52 model, the therapeutic effect of 50 mg/kg dose of JBJ-09-063 was comparable to that of osimertinib.
JBJ-09-063 has a good therapeutic effect on EGFR L858R/T790M mutant tumors in vivo
Similarly, JBJ-09-063 also had a significant therapeutic effect in xenograft tumor models constructed by DFCI52-C797S and H3255GR-C797S cells (resistant to osimtinib).
JBJ-09-063 has a good therapeutic effect on EGFR C797S mutant tumors in vivo
Surprisingly, DFCI52 cells and H3255GR cells in vitro (cells with EGFR T790M mutation and resistant to gefitinib) are not as sensitive to JBJ-09-063 as they are in vivo, and when the ATP competitive EGFR inhibitor gefitinib is combined with JBJ-09-063, it can significantly inhibit the growth of DFCI52 cells and H3255GR cells.
In vitro DFCI52 cells are resistant to JBJ-09-063, but when combined with gefitinib, cell growth is significantly inhibited
Similarly, H3255GR-C797S and DFCI52-C797S (cells with EGFR C797S mutation, resistant to oshitinib) cells that are sensitive to JBJ-09-063 in vivo are also resistant to JBJ-09-063 in vitro, and the combination of JBJ-09-063 and osimtinib is reversible.
The researchers hypothesized that when these cells were cultured in vitro, they must have changed the conformation of EGFR for some reason, making cells that were originally sensitive to JBJ-09-063 become resistant, and EGFR TKI can reverse this resistance.
After research, the researchers found that it was originally due to the binding of erbb family ligands in the medium (such as EGF and NRG1) to EGFR, resulting in dimerization of EGFR with itself or other members of the ERBB family, making JBJ-09-063 unable to inhibit phosphorylation of EGFR, resulting in drug resistance. The binding of EGFR-TKI to EGFR inhibits the formation of dimers, thereby restoring cell sensitivity to JBJ-09-063.
In addition to the common C797S resistance to oshitinib, there are a number of mutations that can lead to resistance to oshitinib, including L718Q mutations, L792F mutations, and G796S mutations[7], which are limited to ATP sites, affecting the binding of osimiminib to sites, but theoretically do not affect binding to allosteric inhibitors.
C797S mutations, L718Q mutations, L792F mutations, and G796S mutations affect the binding of osiminib to the site, but theoretically do not affect binding to allosteric inhibitors
To verify whether JBJ-09-063 was effective in the case of these mutations, the researchers constructed BA/F3 cells of EGFR LT/L718Q, EGFR LT/L792F, and EGFR LT/G796S, and compared the effects of JBJ-09-063 and oshitinib on cell growth and EGFR. The results confirmed that these mutant cells were indeed resistant to osimtinib, but were all more sensitive to JBJ-09-063, and JBJ-09-063 reduced the level of EGFR phosphorylation in these cells, thereby inhibiting EGFR signaling and cell growth. These experiments show that JBJ-09-063 is widely and effectively treating EGFR-mutant tumors that are resistant to oshitinib.
Although JBJ-09-063 is effective against the EGFR mutation that causes osimtinib resistance, it is likely that there is an EGFR mutation that mediates resistance to JBJ-09-063.
To identify JBJ-09-063 drug-resistant mutations, the researchers screened resistant cell lines with 1 μM JBJ-09-063, 1 μM osiminib, or a combination of both (900 cell clones were treated for each use). In the cell clones treated with JBJ-09-063 and osimtinib, there were 76 and 13 cell clones growing, respectively, and when the two drugs treated cells together, no cell clones grew.
Screening for JBJ-09-063 resistant cell lines
Sequencing the EGFR tyrosine kinase (TK) domain of 76 JBJ09-063 resistant cell clones found that of the 76 JBJ09-063 resistant clones, 3 (3.94%) contained L747S mutations and no EGFR TK region mutations were found in the other 73 JBJ-09-063 resistant clones.
Through modeling and analysis, the researchers found that the side chain of L747 and the benzene ring of JBJ-09-063 formed a good hydrophobic contact, while the L747S mutation reduced this exposure. In BA/F3 cells expressing EGFR LT/L747S, JBJ-09-063 was less effective, confirming that the L747S mutation could indeed lead to JBJ-09-063 resistance.
In this study, the researchers identified the fourth-generation EGFR inhibitor JBJ-09-063, which has a good inhibitory effect on EGFR mutations that lead to EGFR-TKI resistance.
The researchers also found that homologous or heterologous dimerism of EGFR with itself or other members of the ERBB family, as well as mutations in EGFR L747S, lead to JBJ-09-063 resistance, but this resistance can be well addressed in combination with EGFR-TKI.
Overall, JBJ-09-063 is a fourth-generation EGFR inhibitor with outstanding clinical potential, which can be used as a single drug or in combination with EGFR-TKIs to treat EGFR-mutant lung cancer, and it is expected to enter the clinic as soon as possible to make up for the lack of EGFR-TKIs treatment.
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