BLU-945, a potent and selective next-generation EGFR TKI, has antitumor activity in models of osimertinib-resistant non-small-cell lung cancer

Abstract

Introduction: Despite the availability of several epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs), most patients with non-small-cell lung cancer (NSCLC) eventually develop resistance to these agents. Notably, EGFR_C797S mutations confer resistance to the third-generation EGFR-TKI osimertinib and no approved post-osimertinib targeted pharmacology options are currently available. BLU-945 is a novel, reversible, and orally available next-generation EGFR-TKI that selectively targets EGFR-activating (EGFRm) and resistance mutations (including EGFR_C797S) with nanomolar potency while sparing wild-type EGFR in vitro.

Methods: In vitro activity of BLU-945 as a single agent and in combination with osimertinib was tested in engineered EGFR-mutant cell lines as well as patient-derived cells and patient-derived organoids. In vivo activity was evaluated in osimertinib-resistant patient-derived xenograft mouse models. Three patient cases from the global, first-in-human, phase I/II SYMPHONY trial (NCT04862780) demonstrating the clinical efficacy of BLU-945 were reported.

Results: In vitro BLU-945 demonstrated inhibited cell viability and growth of EGFR-mutant/osimertinib-resistant cell lines. BLU-945 demonstrated in vivo tumor shrinkage in osimertinib-resistant models of NSCLC (osimertinib second line: EGFR_L858R/C797S and third line: EGFR_ex19del/T790M/C797S and L858R/T790M/C797S) both as monotherapy and in combination with osimertinib. BLU-945 also demonstrated tumor shrinkage in patients from the SYMPHONY trial.

Conclusion: Our findings demonstrate the preclinical and early clinical activity of BLU-945 in EGFRm NSCLC progressing on previous EGFR-TKIs.

Keywords: BLU-945; EGFR; EGFR inhibitors; NSCLC; TKI; clinical trial design; drug resistance; osimertinib; patient-derived xenograft.

Figure 1.

In vitro inhibitory activity of BLU-945 as a second-line or third-line treatment in Ba/F3 cell lines with EGFR mutations. (a–d) Inhibition of pEGFR levels in engineered Ba/F3 cells expressing (a) EGFR_ex19del/C797S mutations, (b) EGFR_L858R/C797S mutations, (c) EGFR_ex19del/T790M/C797S mutations, and (d) EGFR_L858R/T790M/C797S mutations; all treated with BLU-945, gefitinib, and osimertinib. pEGFR levels were plotted relative to the vehicle-treated group (vehicle = 100%). Data plotted as mean ± SEM. (e, f) Western blot analysis of EGFR activity and downstream signaling molecules in Ba/F3 cell lysates expressing (e) EGFR_ex19del/T790M/C797S mutations and (f) EGFR_L858R/T790M/C797S respectively; cells were treated with vehicle or with 10 or 100 nM of osimertinib or BLU-945, respectively. Two bands are shown for pEGFR; the upper band has a short exposure while the lower band has a long exposure. (g, h) Cell viability at 72 h post-treatment with increasing concentrations of osimertinib or BLU-945, in Ba/F3 cells expressing (g) EGFR_ex19del/T790M/C797S mutations and (h) EGFR_L858R /T790M/C797S mutations. IC₅₀ values of BLU-945 were 15 and 6 nM, respectively, and IC₅₀ values of osimertinib were >1000 nM in both cell lines. Cell viability is plotted as mean ± SEM relative to the control-treated group (representing 100%). AKT, alpha serine/threonine-protein kinase; EGFR, epidermal growth factor receptor; ERK, extracellular-signal-regulated kinase; ex19del, exon 19 deletion; IC₅₀, half-maximal inhibitory concentration; osi, osimertinib; pEGFR, phosphorylated EGFR; S6K, S6 kinase; SEM, standard error of the mean; tEGFR, total EGFR.

Figure 2.

In vitro activity of BLU-945 as a second-line treatment in patient-derived preclinical models and cell lines with C797S mutation. (a) Establishment of a patient-derived cell line (YU-1182) from a patient with EGFR-mutant NSCLC who received prior osimertinib, harboring EGFR_L858R/C797S. The representative CT images (mediastinal window) display the status of lung cancer at the time of progression on osimertinib. Sanger sequencing performed in this patient shows the development of C797S mutation after first-line osimertinib. The patient therefore developed EGFR_L858R/C797S (establishment of YU-1182). (b) Cell viability at 72 h post-treatment with increasing concentrations of osimertinib or BLU-945 in YU-1182. YU-1182 showed resistance to osimertinib (IC₅₀ > 1000 nM) but was sensitive to BLU-945 with an IC₅₀ of 293 nM. Cell viability is plotted as mean ± SEM relative to the control-treated group (representing 100%). (c) Western blot analysis of EGFR activity and downstream signaling molecules in YU-1182 PDC lysates treated with osimertinib and BLU-945 (30 and 300 nM). (d) Colony formation assay investigating the dose-dependent effect of BLU-945 (10, 30, or 100 nM) and osimertinib (30 or 100 nM), either as single agents or in combination on YU-1182 cell survival. DMSO was used as a control. (e) Western blot analysis showing the dose-dependent effect on EGFR activity and downstream signaling molecules in YU-1182 cells following the administration of vehicle, BLU-945 (100 nM), or osimertinib (100 nM), either as single agents or in combination. AKT, alpha serine/threonine-protein kinase; BLU, BLU-945; CT, computerized tomography; DMSO, dimethyl sulfoxide; EGFR, epidermal growth factor receptor; ERK, extracellular-signal-regulated kinase; IC₅₀, half-maximal inhibitory concentration; NSCLC, non-small-cell lung cancer; osi, osimertinib; p, phosphorylated; PDC, patient-derived cell line; S6K, S6 kinase; SEM, standard error of the mean; t, total.

Figure 3.

In vitro activity of BLU-945 as a single agent and in combination with osimertinib as a third-line treatment in osimertinib-resistant patient-derived models. (a) Establishment of a PDO (YUO-143) from a patient with EGFR-mutant NSCLC who received prior gefitinib, dacomitinib, and osimertinib, harboring EGFR_ex19del/T790M/C797S mutation. The representative CT images (mediastinal window) display the status of the lung cancer at the time of progression to respective TKI (gefitinib, dacomitinib, and osimertinib). Sanger sequencing performed in this patient showed development of T790M mutation after first-line gefitinib, and subsequent development of C797S mutation after 21 months with osimertinib. The patient therefore developed EGFR_ex19del/T790M/C797S (establishment of YUO-143). (b) Cell viability at 120 h post-treatment with increasing concentrations of osimertinib or BLU-945 in YUO-143. YUO-143 showed resistance to osimertinib but was sensitive to BLU-945. Cell viability is plotted as mean ± SEM relative to the control treated group (representing 100%). (c) Western blot analysis of EGFR activity and downstream signaling molecules in YUO-143 cells treated with osimertinib and BLU-945. (d) Establishment of a PDC (YU-1097) from a patient with EGFR-mutant NSCLC who received prior gefitinib and osimertinib, harboring EGFR_ex19del/T790M/C797S. The representative CT images (mediastinal window) display the status of lung cancer at the time of progression to respective TKI (gefitinib and osimertinib). Sanger sequencing performed in this patient shows the development of T790M mutation after first-line gefitinib, and subsequent development of C797S mutation after osimertinib treatment. The patient, therefore, developed EGFR_ex19del/T790M/C797S (establishment of YU-1097). (e) Cell viability at 72 h post-treatment with increasing concentrations of osimertinib or BLU-945 in YU-1097. YU-1097 showed resistance to osimertinib (IC₅₀ > 1000 nM) but was sensitive to BLU-945 with an IC₅₀ of 108 nM. Cell viability is plotted as mean ± SEM relative to the control-treated group (representing 100%). (f) Western blot analysis of EGFR activity and downstream signaling molecules in YU-1097 PDC lysates treated with osimertinib and BLU-945. (g) Colony formation assay investigating the dose-dependent effect of BLU-945 (10, 30, or 100 nM) and osimertinib (30 or 100 nM), either as single agents or in combination on YU-1097 cell survival. DMSO was used as a control. (h) Western blot analysis showing the dose-dependent effect on EGFR activity and downstream signaling molecules in YU-1097 cells following the administration of vehicle, BLU-945 (100 nM), or osimertinib (100 nM), either as single agents or in combination. AKT, alpha serine/threonine-protein kinase; BLU, BLU-945; CT, computerized tomography; DMSO, dimethyl sulfoxide; EGFR, epidermal growth factor receptor; ERK, extracellular-signal-regulated kinase, ex19del, exon 19 deletion; IC₅₀, half-maximal inhibitory concentration; NSCLC, non-small-cell lung cancer; osi, osimertinib; p, phosphorylated; PDC, patient-derived cell line; PDO, patient-derived organoid; S6K, S6 kinase; SEM, standard error of the mean; t, total; TKI, tyrosine kinase inhibitor.

Figure 4.

In vivo antitumor activity of BLU-945 as a single agent in the YHIM-1094 osimertinib-resistant EGFR_ex19del/T790M/C797S patient-derived xenograft mouse model. (a) Chest CT scans (mediastinal window) of a patient with NSCLC captured after being treated with gefitinib and osimertinib. Sanger sequencing was performed and the presence of the EGFR_C797S mutation was detected 16 months following treatment with osimertinib. The patient, therefore, developed the EGFR_ex19del/T790M/C797S mutation (establishment of YHIM-1094). Significant difference from vehicle denoted as *** p < 0.0001. Significant difference from osimertinib 25 mg/kg denoted as ^##P = 0.002. (b, c) In vivo antitumor effects of compound BLU-945 in YHIM-1094 patient-derived tumor xenograft. Effects of vehicle (black line), osimertinib at 25 mg/kg (blue line), and BLU-945 at 100 mg/kg (red line) on the growth of YHIM-1094 patient-derived tumor xenograft models for 33 days. (b) Tumor volume during the administration period. (c) Body weight of mice. (d) pEGFR and Ki67 expression in the YHIM-1094 xenografted patient tumor analyzed by IHC. Staining xenografted YHIM-1094 patient tumor treated with vehicle, osimertinib, and BLU-945 (scale bar = 100 μm). H&E staining was also performed. N = 6 animals/group. Data plotted as mean ± SEM. CT, computerized tomography; EGFR, epidermal growth factor receptor; ex19del, exon 19 deletion; H&E, hematoxylin and eosin; IHC, immunohistochemistry; NSCLC, non-small cell lung cancer; osi, osimertinib; p, phosphorylated; SEM, standard error of the mean.

Figure 5.

In vivo activity of BLU-945 in combination with osimertinib in patient-derived preclinical models with EGFR_ex19del/T790M/C797S. In vivo antitumor effects of compound BLU-945 in YU-1097 patient-derived tumor xenograft mouse model expressing EGFR_ex19del/T790M/C797S. Effects of activity of vehicle (black line), osimertinib at 25 mg/kg QD (blue line), BLU-945 at 100 mg/kg BID (green line), and combination of BLU-945 at 100 mg/kg BID and osimertinib at 25 mg/kg QD (red line) in YU-1097 PDX. (a) Tumor volume during the administration period. (b) Body weight of mice. N = 12 animals/group. Data plotted as mean ± SEM for all available data at the indicated time point (days), with significance from vehicle marked as ***, p < 0.001 and significance from osimertinib 25 mg/kg marked as ^##p = 0.002 and ^###p <0.001. (c) pEGFR and Ki67 expression in the YU-1097 xenografted patient tumor analyzed by IHC. Xenografted YU-1097 patient tumor was treated with vehicle, osimertinib, and BLU-945 (scale bar = 100 μm). H&E staining was also performed. Data plotted as mean ± SEM with significance marked as ***p<0.001 and *p<0.05. EGFR, epidermal growth factor receptor; ex19del, exon 19 deletion; H&E, hematoxylin and eosin; IHC, immunohistochemistry; ns, non-significant; osi, osimertinib; p, phosphorylated; PDC, patient-derived cell line; PDX, patient-derived xenograft; SEM, standard error of the mean; TGI, tumor growth inhibition.

Figure 6.

Encouraging early clinical activity with BLU-945. (a) Chest CT scans (mediastinal window) in a 68-year-old woman with advanced adenocarcinoma of the lung harboring EGFR_ex19del/T790M/C797S (case 3) show marked decrease in the size of mass (arrow) after treatment with BLU-945 monotherapy. Serial CT images were taken at baseline, 4 weeks, 8 months, and 10 months after treatment initiation. Green arrows indicate tumor lesions in the sternum (left panel), fifth rib (middle panel), and adrenal gland (right panel). (b–d) Longitudinal ctDNA profiling via plasma NGS showcasing treatment-induced reductions or clearance of EGFR-activating and resistance mutant alleles in three different patients: (b) case 1, (c) case 2, and (d) case 3 at cycle 1 day 15 with detectable EGFR mutant allele levels (displayed as VAF (%)) at baseline. CT, computerized tomography; ctDNA, circulating tumor deoxyribonucleic acid DNA; EGFR, epidermal growth factor receptor; ex19del, exon 19 deletion; NGS, next-generation sequencing; rt, right; VAF, variant allele frequency.