Overcoming acquired resistance of EGFR-mutant NSCLC cells to the third generation EGFR inhibitor, osimertinib, with the natural product honokiol.

The development of acquired resistance to osimertinib (Osim) (AZD9291 or TAGRISSOTM ), an FDA-approved third-generation epidermal growth factor receptor (EGFR) inhibitor for the treatment of EGFR-mutant nonsmall cell lung cancer (NSCLC), limits the long-term benefits for patients. Thus, effective treatment options are urgently needed. To this end, we explored whether honokiol (HNK), a natural product with potential antitumor activity, may be used to overcome Osim resistance. The combination of HNK and Osim synergistically decreased the survival of several Osim -resistant cell lines with enhanced effects on inhibiting cell colony formation and growth and on inducing apoptosis. This combination also showed greater growth suppression of Osim-resistant xenograft tumors including those with 19del, T790M, and C797S triple mutations in nude mice. Mechanistically, the augmented induction of apoptosis by the combination is largely due to enhanced Mcl-1 reduction through facilitating its degradation. A synthetic HNK derivative exerted similar effects with greater efficacy. Our findings warrant further study of HNK and its derivatives in overcoming Osim resistance in the clinic.

Zang H ，Qian G ，Arbiser J ，Owonikoko TK ，Ramalingam SS ，Fan S ，Sun SY ... -  《-》

ERK inhibition effectively overcomes acquired resistance of epidermal growth factor receptor-mutant non-small cell lung cancer cells to osimertinib.

Osimertinib (AZD9291), a third-generation, mutation-selective epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor (EGFR-TKI), is an approved drug for patients who have non-small cell lung cancer (NSCLC) with activating EGFR mutations or those harboring a resistant T790M mutation. Unfortunately, all patients eventually relapse and develop resistance to osimertinib. The current study addressed whether ERK inhibition exerts effects similar to those produced by MEK inhibition in overcoming acquired resistance to osimertinib. Drug effects on cell and tumor growth were assessed by measuring cell number alterations and colony formation in vitro and with xenografts in nude mice in vivo. Apoptosis was assessed with annexin V/flow cytometry and protein cleavage. Protein alterations in cells were detected with Western blot analysis. Gene overexpression and knockout were achieved with lentiviral infection and CRISPR/Cas9, respectively. The combination of osimertinib with an ERK inhibitor synergistically decreased the survival of osimertinib-resistant cell lines with enhanced induction of apoptosis and effectively inhibited the growth of osimertinib-resistant xenografts in nude mice. Moreover, the combination of an MEK or ERK inhibitor with a first-generation (eg, erlotinib) or second-generation (eg, afatinib) EGFR-TKI also very effectively inhibited the growth of osimertinib-resistant cells in vitro and of tumors in vivo, although these cell lines were cross-resistant to first-generation or second-generation EGFR-TKIs. The current findings emphasize the importance of targeting MEK/ERK signaling in maintaining the long-term benefit of osimertinib through overcoming acquired resistance to osimertinib, warranting further investigation of this therapeutic strategy to improve the therapeutic efficacy of osimertinib in the clinic.

Li Y ，Zang H ，Qian G ，Owonikoko TK ，Ramalingam SR ，Sun SY ... -  《-》

Overcoming acquired resistance of epidermal growth factor receptor-mutant non-small cell lung cancer cells to osimertinib by combining osimertinib with the histone deacetylase inhibitor panobinostat (LBH589).

The major clinical obstacle that limits the long-term benefits of treatment with osimertinib (AZD9291) in patients with epidermal growth factor receptor-mutant non-small cell lung cancer is the development of acquired resistance. Therefore, effective strategies that can overcome acquired resistance to osimertinib are urgently needed. The authors' current efforts in this direction have identified LBH589 (panobinostat), a clinically used histone deacetylase inhibitor, as a potential agent in overcoming osimertinib resistance. Cell growth and apoptosis in vitro were evaluated by measuring cell numbers and colony formation and by detecting annexin V-positive cells and protein cleavage, respectively. Drug effects on tumor growth in vivo were assessed with xenografts in nude mice. Alterations of tested proteins in cells were monitored with Western blot analysis. Gene knockout was achieved using the CRISPR/Cas9 technique. The combination of LBH589 and osimertinib synergistically decreased the survival of different osimertinib-resistant cell lines, including those harboring C797S mutations, with greater inhibition of cell colony formation and growth. The combination enhanced the induction of apoptosis in osimertinib-resistant cells. Importantly, the combination effectively inhibited the growth of osimertinib-resistant xenograft tumors in nude mice. Mechanistically, the combination of LBH589 and osimertinib enhanced the elevation of Bim in osimertinib-resistant cells. Knockout of Bim in osimertinib-resistant cells substantially attenuated or abolished apoptosis enhanced by the LBH589 and osimertinib combination. These results collectively support a critical role of Bim elevation in the induction of apoptosis of osimertinib-resistant cells for this combination. The current findings provide strong preclinical evidence in support of the potential for LBH589 to overcome osimertinib resistance in the clinic.

Zang H ，Qian G ，Zong D ，Fan S ，Owonikoko TK ，Ramalingam SS ，Sun SY ... -  《-》

Activation of insulin-like growth factor-1 receptor confers acquired resistance to osimertinib in non-small cell lung cancer with EGFR T790M mutation.

Osimertinib (AZD9291) is a third-generation EGFR-tyrosine kinase inhibitor (TKI) that selectively inhibits the activating EGFR mutation and T790M mutation, and is currently used globally to treat EGFR-mutant non-small cell lung cancer (NSCLC). However, acquired resistance to osimertinib is inevitable. We established osimertinib-resistant cells (PC9/T790M/AZDR and H1975/AZDR) derived from EGFR-mutant NSCLC cells harboring T790M mutation, and investigated the mechanism of acquired resistance to osimertinib by whole-exome sequencing and multiple phospho-receptor tyrosine kinase (RTK) array. A tumor specimen from an EGFR-mutant NSCLC patient with acquired resistance to osimertinib was also subjected to immunohistochemical analysis. Whole-exome sequencing analysis demonstrated that genetic alterations, such as acquisition of EGFR C797S, loss of T790M mutation, MET amplification, or mutated KRAS, MEK, BRAF, PIK3CA, were not detected. Analysis of phospho-RTK array revealed that insulin-like growth factor-1 receptor (IGF1R) was activated in PC9/T790M/AZDR and H1975/AZDR cells. Knockdown of IGF1R by siRNA as well as inhibition of IGF1R activation by linstinib (IGF1R inhibitor) significantly restored the sensitivity to osimertinib. Immunohistochemical analysis revealed that the expression level of phosphorylated IGF1R was higher in the tumor specimen from the EGFR-mutant NSCLC patient with acquired resistance to osimertinib than in the specimen collected prior to the treatment. IGF1R activation could occur following treatment with osimertinib in EGFR-mutant NSCLC with T790M mutation, and might be one of the mechanisms underlying osimertinib resistance. Combined treatment of osimertinib and IGF1R inhibitor might be effective in overcoming the acquired resistance to osimertinib induced by IGF1R activation. Significant findings of the study: Using osimertinib-resistant cells, we found that IGF1R activation induced by osimertinib treatment in EGFR-mutant NSCLC with T790M mutation is involved in resistance. Increased phosphorylation of IGF1R was observed in the tumor specimen from an EGFR-mutant NSCLC patient with acquired osimertinib resistance. IGF1R activation might be one of the mechanisms of osimertinib resistance. A combination therapy with osimertinib and an IGF1R inhibitor might be an optimal approach for overcoming the acquired resistance to osimertinib induced by IGF1R activation.

Hayakawa D ，Takahashi F ，Mitsuishi Y ，Tajima K ，Hidayat M ，Winardi W ，Ihara H ，Kanamori K ，Matsumoto N ，Asao T ，Ko R ，Shukuya T ，Takamochi K ，Hayashi T ，Suehara Y ，Takeda Nakamura I ，Ueno T ，Kohsaka S ，Mano H ，Takahashi K ... -  《-》

Third generation EGFR inhibitor osimertinib combined with pemetrexed or cisplatin exerts long-lasting anti-tumor effect in EGFR-mutated pre-clinical models of NSCLC.

La Monica S ，Minari R ，Cretella D ，Flammini L ，Fumarola C ，Bonelli M ，Cavazzoni A ，Digiacomo G ，Galetti M ，Madeddu D ，Falco A ，Lagrasta CA ，Squadrilli A ，Barocelli E ，Romanel A ，Quaini F ，Petronini PG ，Tiseo M ，Alfieri R ... -  《JOURNAL OF EXPERIMENTAL & CLINICAL CANCER RESEARCH》