Therapeutic strategies targeting EGFR T790M

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The EGFR T790M occurs in about 50%–60% of EGFR TKI-resistant tumors.39–42 This gatekeeper mutation, and consequent amino acid change, interferes with reversible EGFR TKIs binding through steric hindrance and is analogous to L1196M in ALK and T315I in the BCR-ABL fusion gene conferring resistance to corresponding TKIs. The presence of T790M can also restore the affinity of mutant receptor for ATP, thus reducing the potency of competitive inhibitors.53 The identification of T790M prompted the development of different therapeutic strategies to overcome resistance. Second-generation EGFR TKIs, including afatinib, dacomitinib and neratinib, are irreversible, non-selective EGFR inhibitors showing greater potency compared with first-generation TKIs in inhibiting T790M kinase activity in preclinical studies.54–56

However, clinical data suggested a rather limited activity of these drugs in the setting of acquired resistance to first-generation TKIs.38,57–60 These results have been largely attributed to the fact that the high rate of toxicities, mainly represented by skin and gastrointestinal adverse events (AEs), due to non-selective inhibition of wild-type EGFR, limited the ability to administer in vivo doses sufficient to effectively inhibit the T790M mutation. Based on promising preclinical data, the combination of afatinib and cetuximab was tested in Phase Ib study in heavily pretreated patients with EGFR-mutated NSCLC and in those resistant to gefitinib or erlotinib; the results showed significant clinical activity, with ORR of 29% and median PFS of 4.7 months.61 Results were comparable in both subgroups of patients with T790M-positive and T790M-negative tumors. However, a high incidence of grade ≥3 AEs were observed (mainly skin and gastrointestinal toxic effects), with 13% of patients discontinuing study drugs due to treatment-related AEs.61 To overcome pharmacodynamic shortcomings of second-generation EGFR TKIs, third-generation, mutant EGFR-selective TKIs have been designed, with preferential activity toward sensitizing mutations, as well as T790M resistance mutation over the wild-type receptor, thus likely reducing on-target toxicities. The first-described compound directed at EGFRT790M was WZ4002, identified by screening an irreversible kinase inhibitor library for drugs that bound EGFR T790M. The drug binds irreversibly to mutant EGFR at the C797 residue and demonstrated 100-fold less potency against WT EGFR and 30–100 fold more potency against EGFR T790M.62,63 While WZ4002 has not progressed to clinical development, other drugs with similar characteristics were developed and taken into early phase clinical trials. Among these, CO-1686 (rociletinib) and AZD9291 (osimertinib) were the first to demonstrate pronounced clinical activity as single agent in EGFR-mutated NSCLC patients with acquired resistance to erlotinib, gefitinib or afatinib.64–67 However, while clinical development of rociletinib was interrupted due to some concerns regarding its benefit–risk profile,22,68 osimertinib development proceeded and it was granted US Food and Drug Administration (FDA) approval in November 2015 for the treatment of patients with metastatic EGFR T790M mutation-positive NSCLC who have progressed on or after EGFR TKI therapy.69


Design and mechanism of action

Osimertinib (TAGRISSOTM, AZD9291; AstraZeneca) is a mono-anilino-pyrimidine compound that irreversibly and selectively targets EGFR TKI-sensitizing- and T790M resistance-mutant forms of EGFR, while sparing wild-type EGFR (Figure 1).65 Osimertinib binds to the EGFR kinase by targeting the cysteine-797 residue in the ATP-binding site via covalent bond formation. While WZ4002 and rociletinib share a number of common structural features, osimertinib has a distinct, unique chemical structure. In EGFR recombinant enzyme assays, osimertinib had nearly 200 times greater potency against L858R/T790M than wild-type EGFR, thus confirming its selectivity for mutant EGFR.65 Osimertinib showed a wide therapeutic window against EGFR T790M- resistant mutations in an in vitro model established to assess the mutation specificity of different EGFR TKIs.70 In murine in vivo studies, the metabolism of osimertinib produced at least 2 metabolites, AZ5104 and AZ7550; of these 2 metabolites, only AZ7550 had comparable potency and selectivity to the original compound, while AZ5104 was more powerful against mutant and wild-type EGFR forms, at the expense of minor selectivity. When tested across multiple other kinases, osimertinib showed minimal off-target kinase activity. At 1 μM, the compound showed inhibition >60% of a limited number of additional kinases, including ErbB2/4, ACK1, ALK, BLK, BRK, MLK1 and MNK2, thereby supporting the overall selectivity of osimertinib. Interestingly, osimertinib did not show activity in vitro against the IGF-1R and insulin receptor, which also have a methionine gatekeeper in their kinase domains and this observation was confirmed in in vivo studies, thus suggesting a low risk of dose-limiting toxicities related to hyperglycemia, in contrast to rociletinib.65

Osimertinib showed similar potency to early generation reversible TKIs in inhibiting EGFR phosphorylation in EGFR cell lines harboring sensitizing EGFR mutations. However, unlike first-generation TKIs, it inhibited phosphorylation of EGFR in T790M mutant cell lines (H1975: L858R/T790M and PC-9VanR: ex19del/T790M) with higher potency (mean IC50 <15 nM) than wild-type EGFR (mean IC50: 480–1865 nM). In mutant EGFR cell lines, osimertinib also induced inhibition of downstream signaling substrates (pAKT, pERK) more potently compared with EGFR wild-type cell lines. In vitro studies on cell lines transfected with cDNAs encoding other rare EGFR mutations and HER2mutations, which have been also correlated with EGFR TKIs acquired resistance, showed that AZ5104 had some activity against the EGFRvIII mutant, while osimertinib was effective in inhibiting the growth of cells harboring HER2 mutations (VC insertion at G776 in exon 20 and the most common exon 20 YVMA 776-779ins). Therefore, depending on the clinical exposures achieved, osimertinib and AZ5104 may also inhibit these targets.65

In mutant EGFR xenograft models, once daily dosing of osimertinib induced significant dose-dependent regression in both TKI-sensitizing and T790M-resistant EGFR-mutant disease models. Significant tumor shrinkage was observed at low doses. When osimertinib was administered as long-term daily oral dosing, complete and durable responses were observed in these EGFR-mutant xenografts, with no evidence of tumor progression out to 200 days of treatment and with good tolerability in the animals, showing <5% of starting body weight loss.65 Finally, osimertinib demonstrated its antitumor activity by inducing significant tumor shrinkage in in vivo transgenic mouse tumor models harboring EGFR L858R or L858R+T790M mutants. In these models, the pharmacodynamic properties of osimertinib to inhibit its target and downstream pathway were further confirmed.

Based on promising results from preclinical studies, in which osimertinib largely demonstrated its role as a mutant-selective EGFR inhibitor, with sustained antitumor activity, subsequent clinical trials were conducted to develop this drug and identify its best use in therapy.