Relationship between EGFR inhibition in lung adenocarcinoma, cell surface expression of the placental antigen ALPP, and efficacy of ALPP-ADC therapy

e15018 Background: In normal tissues placental alkaline phosphatases (ALPP) and ALPPL2 are exclusively expressed in the placenta. Their discovery in the cancer surfaceome has opened the possibility of targeting ALPP/ALPPL2. However, their low expression in most cancer types limits their therapeutic potential. In this study, we report a novel mechanism of ALPP upregulation in cancer cells that renders these cells more susceptible to ALPP-targeting therapies, which we evidence in the context of lung adenocarcinoma. Methods: Surface expression of ALPP and ALPPL2 was determined following biotinylation tagging of the cancer surfaceome, isolation of biotinylated proteins using monomeric avidin and mass spectrometry of the biotinylated trypsin digested fraction. ALPP antibody was conjugated with Monomethylauristatin F (ALPP-ADC-MMAF). For orthotopic xenograft models of LUAD were injected. For gefitinib treatment, Mice xenotransplanted with H1650 were treated with gefitinib (50mg/kg) daily for 10 days starting from Day 17 post cancer cell inoculation. For the gefitinib/ALPP-ADC combinational treatment, mice received anti-ALPP-MMAF (5mg/kg) at Day 19 and Day 26. Results: We assessed ALPP and ALPPL2 surfaceome protein levels in 158 cancer cell lines and their mRNA expression levels in 10,967 tumors representing 32 cancer types from The Cancer Genome Atlas (TCGA), which revealed ALPP, and to a lesser extent ALPPL2, to be variably expressed in several cancer types including lung adenocarcinoma (LUAD). We further confirmed surface expression of ALPP by tissue microarray analysis of 204 lung tumors. We report that treatment of LUAD cells with EGFR inhibitors or induction of cancer cell quiescence greatly enhanced ALPP surface expression. Mechanistically, repression of EGFR signaling resulted in activation of the transcription factor FoxO3a, which triggered expression of ALPP. Using xenograft models of LUAD, we further demonstrate that gefitinib treatment significantly upregulated surface expression of ALPP in LUAD tumors but not normal tissue. Combination therapy with gefitinib and an ALPP-ADC-MMAF resulted in superior anti-cancer efficacy compared with gefitinib or ALPP-ADC-MMAF alone. Conclusions: Our study provides mechanistic insights into ALPP upregulation in cancer cells and establishes FoxO3a as a transcriptional regulator of ALPP. Importantly, our findings have yielded a novel ‘two-hit’ combinational therapeutic strategy with enhanced ALPP targeting for greater efficacy.