Discovery of mobocertinib, a potent, oral inhibitor of EGFR exon 20 insertion mutations in non-small cell lung cancer.

In the treatment of non-small cell lung cancer (NSCLC), patients harboring exon 20 insertion mutations in the epidermal growth factor receptor (EGFR) gene (EGFR) have few effective therapies because this subset of mutants is generally resistant to most currently approved EGFR inhibitors. This report describes the structure-guided design of a novel series of potent, irreversible inhibitors of EGFR exon 20 insertion mutations, including the V769_D770insASV and D770_N771insSVD mutants. Extensive structure-activity relationship (SAR) studies led to the discovery of mobocertinib (compound 21c), which inhibited growth of Ba/F3 cells expressing the ASV insertion with a half-maximal inhibitory concentration of 11 nM and with selectivity over wild-type EGFR. Daily oral administration of mobocertinib induced tumor regression in a Ba/F3 ASV xenograft mouse model at well-tolerated doses. Mobocertinib was approved in September 2021 for the treatment of adult patients with advanced NSCLC with EGFR exon 20 insertion mutations with progression on or after platinum-based chemotherapy.

Mobocertinib (TAK-788): A Targeted Inhibitor of EGFR Exon 20 Insertion Mutants in Non-Small Cell Lung Cancer.

Most EGFR exon 20 insertion (EGFRex20ins) driver mutations in non-small cell lung cancer (NSCLC) are insensitive to approved EGFR tyrosine kinase inhibitors (TKI). To address the limitations of existing therapies targeting EGFR-mutated NSCLC, mobocertinib (TAK-788), a novel irreversible EGFR TKI, was specifically designed to potently inhibit oncogenic variants containing activating EGFRex20ins mutations with selectivity over wild-type EGFR. The in vitro and in vivo activity of mobocertinib was evaluated in engineered and patient-derived models harboring diverse EGFRex20ins mutations. Mobocertinib inhibited viability of various EGFRex20ins-driven cell lines more potently than approved EGFR TKIs and demonstrated in vivo antitumor efficacy in patient-derived xenografts and murine orthotopic models. These findings support the ongoing clinical development of mobocertinib for the treatment of EGFRex20ins-mutated NSCLC. SIGNIFICANCE: No oral EGFR-targeted therapies are approved for EGFR exon 20 insertion (EGFRex20ins) mutation-driven NSCLC. Mobocertinib is a novel small-molecule EGFR inhibitor specifically designed to target EGFRex20ins mutants. Preclinical data reported here support the clinical development of mobocertinib in patients with NSCLC with EGFR exon 20 insertion mutations.See related commentary by Pacheco, p. 1617.This article is highlighted in the In This Issue feature, p. 1601.

The Potent ALK Inhibitor Brigatinib (AP26113) Overcomes Mechanisms of Resistance to First- and Second-Generation ALK Inhibitors in Preclinical Models.

PURPOSE: Non-small cell lung cancers (NSCLCs) harboring ALK gene rearrangements (ALK+) typically become resistant to the first-generation anaplastic lymphoma kinase (ALK) tyrosine kinase inhibitor (TKI) crizotinib through development of secondary resistance mutations in ALK or disease progression in the brain. Mutations that confer resistance to second-generation ALK TKIs ceritinib and alectinib have also been identified. Here, we report the structure and first comprehensive preclinical evaluation of the next-generation ALK TKI brigatinib. EXPERIMENTAL DESIGN: A kinase screen was performed to evaluate the selectivity profile of brigatinib. The cellular and in vivo activities of ALK TKIs were compared using engineered and cancer-derived cell lines. The brigatinib-ALK co-structure was determined. RESULTS: Brigatinib potently inhibits ALK and ROS1, with a high degree of selectivity over more than 250 kinases. Across a panel of ALK+ cell lines, brigatinib inhibited native ALK (IC50, 10 nmol/L) with 12-fold greater potency than crizotinib. Superior efficacy of brigatinib was also observed in mice with ALK+ tumors implanted subcutaneously or intracranially. Brigatinib maintained substantial activity against all 17 secondary ALK mutants tested in cellular assays and exhibited a superior inhibitory profile compared with crizotinib, ceritinib, and alectinib at clinically achievable concentrations. Brigatinib was the only TKI to maintain substantial activity against the most recalcitrant ALK resistance mutation, G1202R. The unique, potent, and pan-ALK mutant activity of brigatinib could be rationalized by structural analyses. CONCLUSIONS: Brigatinib is a highly potent and selective ALK inhibitor. These findings provide the molecular basis for the promising activity being observed in ALK+, crizotinib-resistant patients with NSCLC being treated with brigatinib in clinical trials. Clin Cancer Res; 22(22); 5527-38. ©2016 AACR.

Discovery of Brigatinib (AP26113), a Phosphine Oxide-Containing, Potent, Orally Active Inhibitor of Anaplastic Lymphoma Kinase.

In the treatment of echinoderm microtubule-associated protein-like 4 (EML4)-anaplastic lymphoma kinase positive (ALK+) non-small-cell lung cancer (NSCLC), secondary mutations within the ALK kinase domain have emerged as a major resistance mechanism to both first- and second-generation ALK inhibitors. This report describes the design and synthesis of a series of 2,4-diarylaminopyrimidine-based potent and selective ALK inhibitors culminating in identification of the investigational clinical candidate brigatinib. A unique structural feature of brigatinib is a phosphine oxide, an overlooked but novel hydrogen-bond acceptor that drives potency and selectivity in addition to favorable ADME properties. Brigatinib displayed low nanomolar IC50s against native ALK and all tested clinically relevant ALK mutants in both enzyme-based biochemical and cell-based viability assays and demonstrated efficacy in multiple ALK+ xenografts in mice, including Karpas-299 (anaplastic large-cell lymphomas [ALCL]) and H3122 (NSCLC). Brigatinib represents the most clinically advanced phosphine oxide-containing drug candidate to date and is currently being evaluated in a global phase 2 registration trial.

Synergistic activity of the mTOR inhibitor ridaforolimus and the antiandrogen bicalutamide in prostate cancer models.

Although androgen ablation therapy is the foundation of current prostate cancer treatment, most patients ultimately develop castration-resistant disease. One proposed mechanism to account for androgen receptor (AR) activity in the castrate environment is via crosstalk with other signaling pathways. Specifically, reciprocal interactions between the AKT/mTOR and AR pathways have been implicated in prostate cancer progression. Here, we used the potent inhibitor ridaforolimus to target mTOR signaling alone and in combination with AR blockade by bicalutamide to examine the effect of abrogating these signaling pathways. Ridaforolimus treatment inhibited the proliferation of all six prostate cancer cell lines examined with the greatest sensitivity associated with loss of PTEN and elevated AKT/mTOR pathway activity. Dual inhibition of the AR and mTOR signaling pathways provided further benefit with the ridaforolimus-bicalutamide combination producing synergistic antiproliferative effects in prostate cancer cells in vitro when compared with each agent alone. Pharmacodynamic analysis confirmed that combination treatment resulted in full inhibition of each of the respective pathways. Importantly, the ridaforolimus-bicalutamide combination exhibited potent antitumor activity with parallel reductions in plasma PSA levels in vivo. Taken together, ridaforolimus exhibited potent antiproliferative and antitumor activity in prostate cancer models and the addition of bicalutamide represents a potentially effective combination strategy for patient therapy.

Crizotinib-resistant mutants of EML4-ALK identified through an accelerated mutagenesis screen.

Activating gene rearrangements of anaplastic lymphoma kinase (ALK) have been identified as driver mutations in non-small-cell lung cancer, inflammatory myofibroblastic tumors, and other cancers. Crizotinib, a dual MET/ALK inhibitor, has demonstrated promising clinical activity in patients with non-small-cell lung cancer and inflammatory myofibroblastic tumors harboring ALK translocations. Inhibitors of driver kinases often elicit kinase domain mutations that confer resistance, and such mutations have been successfully predicted using in vitro mutagenesis screens. Here, this approach was used to discover an extensive set of ALK mutations that can confer resistance to crizotinib. Mutations at 16 residues were identified, structurally clustered into five regions around the kinase active site, which conferred varying degrees of resistance. The screen successfully predicted the L1196M, C1156Y, and F1174L mutations, recently identified in crizotinib-resistant patients. In separate studies, we demonstrated that crizotinib has relatively modest potency in ALK-positive non-small-cell lung cancer cell lines. A more potent ALK inhibitor, TAE684, maintained substantial activity against mutations that conferred resistance to crizotinib. Our study identifies multiple novel mutations in ALK that may confer clinical resistance to crizotinib, suggests that crizotinib's narrow selectivity window may underlie its susceptibility to such resistance and demonstrates that a more potent ALK inhibitor may be effective at overcoming resistance.

Antitumor activity of ridaforolimus and potential cell-cycle determinants of sensitivity in sarcoma and endometrial cancer models.

Ridaforolimus is a nonprodrug rapamycin analogue that potently inhibits mTOR and has shown significant activity in patients with metastatic sarcoma and endometrial cancer, two diseases where high unmet need remains. Here, we evaluated the activity of ridaforolimus in preclinical models of these tumor types and used these models to explore molecular correlates of sensitivity. The in vitro sensitivity of a panel of sarcoma and endometrial cancer cell lines was established by measuring the effect of ridaforolimus on cell proliferation rate, revealing broad inhibition at low nanomolar concentrations. Additional benefit was found when ridaforolimus was combined with agents used to treat sarcoma and endometrial cancer patients. In vivo, potent antitumor activity of ridaforolimus associated with inhibition of mTOR signaling was observed in sarcoma and endometrial xenograft models. Immunoblot analysis was conducted to assess the expression and activation state of multiple signaling proteins in the phosphoinositide-3-kinase/AKT/mTOR and cell-cycle pathways. In endometrial but not sarcoma cell lines, the absence of PTEN or elevated levels of phosphorylated or total AKT was associated with greater sensitivity. However, in both tumor types, the proportion of cells in the G(0)-G(1) phase before treatment correlated significantly with ridaforolimus sensitivity. Consistent with this, expression of several G(1) phase cell-cycle proteins, notably p21 and p27, was higher in more sensitive lines. These results underscore the promise of ridaforolimus as a single agent or combination treatment of these tumor types and suggest novel potential predictive biomarkers of sensitivity to an mTOR inhibitor based on cell-cycle status.

Ridaforolimus (AP23573; MK-8669), a potent mTOR inhibitor, has broad antitumor activity and can be optimally administered using intermittent dosing regimens.

The mTOR pathway is hyperactivated through oncogenic transformation in many human malignancies. Ridaforolimus (AP23573; MK-8669) is a novel rapamycin analogue that selectively targets mTOR and is currently under clinical evaluation. In this study, we investigated the mechanistic basis for the antitumor activity of ridaforolimus in a range of human tumor types, exploring potential markers of response, and determining optimal dosing regimens to guide clinical studies. Administration of ridaforolimus to tumor cells in vitro elicited dose-dependent inhibition of mTOR activity with concomitant effects on cell growth and division. We showed that ridaforolimus exhibits a predominantly cytostatic mode of action, consistent with the findings for other mTOR inhibitors. Potent inhibitory effects on vascular endothelial growth factor secretion, endothelial cell growth, and glucose metabolism were also observed. Although PTEN and/or phosphorylated AKT status have been proposed as potential mTOR pathway biomarkers, neither was predictive for ridaforolimus responsiveness in the heterogeneous panel of cancer cell lines examined. In mouse models, robust antitumor activity was observed in human tumor xenografts using a series of intermittent dosing schedules, consistent with pharmacodynamic observations of mTOR pathway inhibition for at least 72 hours following dosing. Parallel skin-graft rejection studies established that intermittent dosing schedules lack the immunosuppressive effects seen with daily dosing. Overall these findings show the broad inhibitory effects of ridaforolimus on cell growth, division, metabolism, and angiogenesis, and support the use of intermittent dosing as a means to optimize antitumor activity while minimizing systemic effects.