Sequence-dependent synergistic effect of aumolertinib-pemetrexed combined therapy on EGFR-mutant non-small-cell lung carcinoma with pre-clinical and clinical evidence

Abstract

Background Inevitably developed resistance of the third-generation epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor (TKI) limited its clinical benefit on non-small cell lung cancer (NSCLC). Upfront combination therapy is promising to prevent this resistance. Compelling clinical evidence indicated the failure of third-generation EGFR TKIs combined with either immunotherapy or antiangiogenic agents. In comparison, combined treatment of third-generation EGFR TKIs and chemotherapy might be a favorable choice. Herein, we systematically analyzed and compared the effects of pemetrexed and a novel third-generation EGFR TKI aumolertinib combined in different sequences, subsequently revealed the potential mechanisms and proved the optimal combination schedule with clinical retrospective study. Methods Three combination schedules involving pemetrexed and aumolertinib in different sequences were developed. Their inhibition effects on cell proliferation and metastasis were firstly compared upon three human NSCLC cell lines in vitro, by cell counting kit-8, colony formation, wound healing and transwell assays respectively. Further evaluation in vivo was proceeded upon H1975 and HCC827 xenograft model. Gene and protein expression were detected by Q-PCR and western blot. Drug concentration was determined by LC–MS/MS. VEGF secretion was determined by ELISA. Tumor vessel was visualized by immunofluorescence. Lastly, a clinical retrospective study was raised with 65 patients’ data. Results The combination of pemetrexed and aumolertinib exhibited a sequence-dependent and EGFR mutant-dependent synergistic effect in vitro and in vivo. Only treatment with aumolertinib following pemetrexed (P-A) exhibited synergistic effect with stronger anti-tumor growth and anti-metastasis ability than monotherapy and also other combination sequences. This synergism could exclusively be observed in H1975 and HCC827 but not A549. Pathway analysis showed that P-A significantly enhanced the suppression of EGFR pathway. In addition, our results intriguingly found an obvious reduction of VEGF secretion and the accompanying normalization of the intratumor vessel, consequently increasing intratumoral accumulation of pemetrexed in P-A group. Finally, the clinical retrospective study verified the synergistic effect of P-A combination by significantly superior tumor response than aumolertinib monotherapy. Conclusion Aumolertinib-pemetrexed combined therapy is promising for EGFR mutant NSCLC but only in right administration sequence. P-A could become an advantageous combination strategy in clinical with synergistic inhibition of tumor growth and metastasis.

ALK inhibitor and chemotherapy combinations in ALK translocated NSCLC models

“…Furthermore, even though in vivo models often re ect clinical setting more closely than in vitro, their translation to clinical setting is also always questionable. Our results are more hypothesis generating based on rationality derived from EGFR mutant NSCLC [6], [17], [18] and scarce clinical evidence of TKI and chemotherapy combination in ALK + NSCLC [13], [19].…”

Targeting GFPT2 to reinvigorate immunotherapy in EGFR-mutated NSCLC

“…GFPT2 distinctly regulates CXCL10 and VEGF, which are not glycosylated, by modifying their gene expression, in contrast to its effect on glycosylated immunosuppressive ligands (Fig.4h, Extended Data Fig.6e ) . In EGFR-mutated cells, the EGFR/AKT/IRF1 axis transcriptionally downregulates CXCL10 2,34 , while the EGFR/ERK/NRP1 pathway upregulates VEGF transcriptionally 35,36 . Absence of EGFR in H1975 cells negates GFPT2’s effect on CXCL10 and VEGF mRNA and secretion levels (Fig.4h, Extended Data Fig.8i) , yet does not affect PD-L1, CD276, PVR and CD73 expression (Extended Data Fig.8j) .…”

“…6e). In EGFR-mutated cells, the EGFR/AKT/IRF1 axis transcriptionally downregulates CXCL10 2,34 , while the EGFR/ERK/NRP1 pathway upregulates VEGF transcriptionally 35,36 . Absence of EGFR in H1975 cells negates GFPT2's effect on CXCL10 and VEGF mRNA and secretion levels (Fig.…”

GFPT2 controls immune evasion in EGFR-mutated non-small cell lung cancer

“…6e). In EGFR-mutated cells, the EGFR/AKT/IRF1 axis transcriptionally downregulates CXCL10 2,31 , while the EGFR/ERK/NRP1 pathway upregulates VEGF transcriptionally 32,33 . Absence of EGFR in H1975 cells negates GFPT2's effect on CXCL10 and VEGF mRNA and secretion levels (Fig.…”