Zongertinib-tolerant cells enhanced sensitivity to Topo1 inhibition in Her2-positive NCSLC

Her2 aberration is found in approximately 2-4% of patients with non-small cell lung cancer (NSCLC). For the treatment of Her2 mutated/amplified NSCLC, the antibody-drug conjugate Trastuzumab deruxtecan, several Her2-selective inhibitors, such as zongertinib and sevabertinib, have shown beneficial efficacy in clinical settings. Although these Her2 inhibitors have exhibited durable clinical benefits, drug resistance is not inevitable, and tumor eventually relapse. To date, numerous resistance mechanisms to pan-ERBB family inhibitors have been identified, and combination strategies to overcome the resistance have been proposed. However, resistance mechanisms to Her2-selective inhibitor zongertinib have not been fully elucidated. In this study, we established zongertinib-tolerant Her2-positive NSCLC cells (zongerR) and investigated their therapeutic vulnerability. To establish the zongerR cells in vitro, Her2-amplified or Her2-mutated patient-derived cells were sequentially cultured with 100 nM zongertinib for 9 days, followed by a drug holiday. After 2-4 cycles, established zongerR cells demonstrated a more than 2-fold increase in cell viability at 100 nM zongertinib in vitro. Immunoblot analysis revealed that zongertinib suppressed Her2 phosphorylation, indicating that resistance mechanisms were related to bypass signaling. To assess the activating bypass signaling in zongerR cells, we performed inhibitor library screening with or without zongertinib. Drug screening revealed that zongerR cells exhibited sensitivity to the multi-tyrosine kinase inhibitor ponatinib and foretinib. Additionally, PI3K/AKT pathway inhibitors (PI3Ki) such as GDC0941 and mTOR inhibitor PP242 also markedly suppressed the survival of zongerR cells in combination with zongertinib. Notably, we identified a single treatment with the TopoI inhibitor SN38 suppressed cell viability in a zongerR cell derived from Her2-amplified H2170 cells (H2170zongerR), compared with H2170 parental cells. From the analysis of immunoblot and cell cycle, TopoI inhibitors induce DNA damage, leading to G2/M arrest and apoptosis, in H2170zongerR. These findings suggested that sequential treatment with zongertinib, followed by T-Dxd, might have been effective. Therefore, we evaluated the treatment efficacy in vivo. In a preliminary result, administration of zongertinib regressed tumor growth for several weeks, whereas tumor regrowth was observed. Subsequently, T-Dxd treatment showed tumor regression in contrast to the continuous treatment of the zongertinib group. Currently, zongertinib and T-Dxd have been approved for Her2-positive NSCLC, whereas treatment strategies to prolong the durable response have not been well-established. Further investigations were needed, but our findings suggested that T-DXd treatment after zongertinib may be a potential therapeutic strategy in a subset of Her2-positive NSCLC.