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)

Abstract

Background: 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.

Methods: 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.

Results: 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.

Conclusions: The current findings provide strong preclinical evidence in support of the potential for LBH589 to overcome osimertinib resistance in the clinic.

Keywords: LBH589; acquired resistance; apoptosis; epidermal growth factor receptor (EGFR); histone deacetylase (HDAC); lung cancer; osimertinib.

Fig. 1.. The combination of LBH589 and osimertinib synergistically decreases the survival (A) and inhibits colony formation and growth (B) of osimertinib-resistant EGFR-mutant NSCLC cell lines.

A, The indicated cell lines seeded in 96-well plates were treated the next day with the given concentrations of AZD9291 alone, LBH589 alone or their combinations. After 72 hours, cell numbers were estimated using the SRB assay. The numbers inside the graphs are CIs for the given combinations. B. The indicated cell lines were seeded in 12-well cell culture plates. On the second day, the cells were treated with fresh medium containing DMSO, 5 nM (PC-9/AR) or 8 nM (HCC827/AR) LBH589 alone, 200 nM osimertinib alone and LBH589 plus osimertinib and the treatment was repeated every 3 days for a total of 12 days. The data are means ± SDs of four replicates (A) or triplicate (B) determinations. *** P < 0.001 at least compared with other treatments. Osim, osimertinib.

Fig. 2.. The combination of LBH589 and osimertinib enhances induction of apoptosis in osimertinib-resistant EGFR-mutant NSCLC cell lines.

The indicated cell lines were exposed to DMSO, 40 nM (PC-9/AR and PC-93M) or 60 nM (PC-9/GR/AR) LBH589, 0.5 μM osimertinib or LBH589 plus osimertinib for 48 h (A) or 40 h (B) and then harvested for detection of apoptosis with annexin V/flow cytometry (A) and for detection of PARP and caspase cleavage with Western blotting (B). The data in A are means ± SDs of triplicate determinations. *** P < 0.001 at least compared with other treatments. CF, cleaved form; Osim, osimertinib.

Fig. 3.. The combination of LBH589 and osimertinib enhances Bim elevation (A and B) accompanied with augmented suppression of ERK and Akt (A) through stabilizing Bim protein (C) in osimertinib-resistant cells.

A and B, The indicated cell lines were exposed to 0.5 μM osimertinib alone, 40 nM LBH589 alone or their combination for 8 h (A) or for different times as indicated (B) and then harvested for preparation of whole-cell protein lysates and subsequent Western blot analysis. C, PC-9/GR/AR cells were treated with DMSO, 40 nM LBH589, 0.5 μM osimertinib or LBH589 and osimertinib combination for 8 h followed by addition of 10 μg/ml CHX. At the indicated times post CHX, the cells were harvested for preparation of whole-cell protein lysates and subsequent Western blot analysis. Band intensities were quantified by NIH image J software and Bim levels presented as a percentage of levels at 0 time post CHX treatment. Osim, osimertinib.

Fig. 4.. Bim knockout compromises the ability of the LBH589 and osimertinib combination to enhance the induction of apoptosis in osimertinib-resistant cells.

The indicated cell lines were exposed to 0.5 μM osimertinib alone, 40 nM LBH589 alone or their combination for 40 h (A) or 48 h (B) and then harvested for detection of Bim (A) and for measurement of apoptotic cells with Annexin V/flow cytometry (B). The data in B are means ± SDs of duplicate determinations. Osim, osimertinib.

Fig. 5.. The combination of LBH589 and osimertinib effectively inhibits the growth of PC-9/AR xenografts in vivo.

PC-9/AR xenografts were treated (once a day for 5 days/week) with vehicle control, osimertinib, LBH589 and their combination starting on the same day after grouping for 3 weeks. Tumor sizes (A) were measured as indicated. Each measurement is mean ± SEM (n = 6). At the end of the treatment, the mice were sacrificed to remove tumors, which were weighed (B). Mouse body weights were also compared (C). * P < 0.05, **P < 0.01 and *** P < 0.001 at least compared with all other groups. Osim, osimertinib.

Fig. 6.. The combination of SAHA and osimertinib synergistically decreases the survival (A) and induces apoptosis (B) of osimertinib-resistant NSCLC cell lines.

A, The indicated cell lines seeded in 96-well plates were treated the next day with the given concentration of osimertinib alone, SAHA alone or their combinations. After 72 hours, cell numbers were estimated using the SRB assay. The numbers inside the graphs are CIs for the given combinations. B-D. PC-9/GR/AR cells were exposed to DMSO, 6 μM SAHA, 0.5 μM osimertinib or SAHA plus osimertinib for 48 h (B), 40 h (C) or 12 h (D) and then harvested for detection of apoptosis with annexin V/flow cytometry (B) and for detection of different proteins with Western blotting (C and D). The data in B are means ± SDs of duplicate determinations. CF, cleaved form; Osim, osimertinib.