Bortezomib (BTZ) is a first-generation proteasome inhibitor with anti-tumor properties for multiple myeloma and mantle cell lymphoma. Increasing evidence has shown that BTZ exhibits toxic effects on diverse tumor cells, including non-small cell lung cancer (NSCLC) cells. However, the mechanism has not been fully evaluated. Here, we examined the regulatory effect of BTZ on cellular senescence, a potent tumor suppressive mechanism, in NSCLC cell lines. SA-β-gal staining assay showed that BTZ caused a significant increase in β-Gal positive A549 cells. BTZ also induced cell cycle arrest on G0/G1 phase in A549 cells. Furthermore, telomerase activity was markedly reduced in A549 cells treated with BTZ. BTZ reduced the expression levels of hTERT, and the key proteins binding to telomeric DNA, including POT1 and TIN2. It also induced the expressions of the cell cycle-associated tumor suppressors p53 and p21 in A549 cells. Moreover, hTERT overexpression abolished the effects of BTZ on A549 cells. These results show that BTZ induced cellular senescence by stimulating telomere shortening. Our results provide experimental data for the potential clinical application of BTZ in NSCLC treatment.

3138 Background: Actionable muts in EGFR and ALK define two molecular subtypes sensitive to EGFR-TKIs and ALK-TKIs, respectively. Although generally mutually exclusive, they did co-exist in some cases. However, when and how do they co-exist are not well understood. Methods: Pts with concurrent actionable muts in ALK and EGFR were selected from our database. Their mutation profiles and treatment histories were analyzed. PFS was estimated using Kaplan-Meier method. Results: Among 341 ALK-positive ( ALK-pos) and 3804 EGFR-positive ( EGFR-pos) pts, 9 (2.6% of ALK-pos, 0.2% of EGFR-pos) had concurrent EGFR and ALK actionable muts, including 3 EX19Indel + EML4-ALK, 2 EX19Indel + STRN-ALK, 2 L858R + L1152R, 1 L858R + EML4-ALK, and 1 G719C + S768I + STRN-ALK. All 9 pts had lung cancer. One pt with EX19Indel + EML4-ALK was treatment naïve. The other 8 pts have taken ≥ 1 EGFR-TKIs. The mPFS of these pts on first-generation EGFR-TKIs was 22 mo (95% CI: 11 - NR). Except for 1 pt who progressed on Gefitinib and subsequently on Osimertinib had a T790M+C797G, the other 7 EGFR-TKI resistance pts had no common known resistance muts. 3 pts ordered NGS tests before taking EGFR-TKIs. None of them had ALK muts at that time. Later, 1 pt (19Indel) gained an STRN-ALK after 15 mo on Osimertinib, 1 pt (L858R) gained an EML4-ALK after 5 mo on Gefitinib, and 1 pt (L858R) gained an L1152R after 10 mo on Afatinib. Therefore, ALK muts were likely developed as resistance mechanisms during EGFR-TKIs therapies in these 3 pts. Unfortunately, with no information on ALK status before EGFR-TKI therapies, we can not tell if the ALK muts were also developed during and conferred resistance to EGFR-TKI therapies in the other 5 pts. Both STRN-ALK and ALK L1152R were recorded 4 times in our database, and they concurred with EGFR actionable muts in 3 and 2 of the 4 records, respectively. Conclusions: ALK and EGFR actionable muts concurred at a relatively low frequency in our pts. In some cases, ALK muts were developed during EGFR-TKI therapies. Developed either together or sequentially, some combinations of EGFR and ALK muts, such as L858R with L1152R and EX19Indel with ALK fusion, may form more easily or may be preferable than other combinations for the development or evoluation of tumors.

Natural killer (NK) cells recognize stress-activated NK group 2, member D (NKG2D) ligands in tumors. In the present study, the expression levels of NKG2D ligands were examined in four lung cancer cell lines (A549, PLA801D, NCI-H157 and NCI-H520). In the A549 cells, the expression of MHC class I polypeptiderelated sequence (MIC)A/B and UL16 binding protein (ULBP)1 was weak, the expression of ULBP2 was typical, and neither ULBP3 nor ULBP4 were expressed. The mechanism underlying the regulatory effect of a cancer treatment agent on the expression of NKG2D ligands was investigated using the proteasome inhibitor MG132. Following treatment for 8 h with MG132, the transcription levels of MICB and ULBP1 were upregulated 10.62- and 11.09-fold, respectively, and the expression levels of MICB and ULBP1 were increased by 68.18 and 23.65%, respectively. Notably, MICB exhibited significant time-dependent change. MG132 increased the transcription of MICB by acting at a site in the 480-bp MICB upstream promoter. The activity of the MICB promoter was upregulated 1.77-fold following treatment with MG132. MG132 treatment improved the cytotoxicity of NK cells, which was partially blocked by an antibody targeting NKG2D, and more specifically the MICB molecule. The expression of MICB induced by MG132 was inhibited by KU-55933 [ataxia telangiectasia mutated (ATM) kinase inhibitor], wortmannin (phosphoinositide 3 kinase inhibitor) and caffeine (ATM/ATM-Rad3-related inhibitor). The phosphorylation of checkpoint kinase 2 (Chk2), an event associated with DNA damage, was observed following treatment with MG132. These results indicated that MG132 selectively upregulates the expression of MICB in A549 cells, and increases the NKG2D-mediated cytotoxicity of NK cells. The regulatory effect of MG132 may be associated with the activation of Chk2, an event associated with DNA damage. The combination of MG132 with NK cell immunotherapy may have a synergistic effect that improves the therapeutic effect of lung cancer treatment.