RESUMEN
Treatments with cytotoxic agents or viruses may cause Immunogenic Cell Death (ICD) that immunize tumor-bearing hosts but do not cause complete regression of tumor. We postulate that combining two ICD inducers may cause durable regression in immunocompetent mice. ICD was optimized in vitro by maximizing calreticulin externalization in human colorectal carcinoma (CRC) cells by exposure to mixtures of Oxaliplatin (OX) and human adenovirus (AdV). Six mm diameter CT26 or 4T1 carcinomas in flanks of BALB/c mice were injected once intratumorally (IT) with OX, AdV or their mixture. Tumor growth, Tumor-Infiltrating Lymphocytes (TIL), nodal cytotoxicity, and rejection of a viable cell challenge were measured. Tumors injected IT once with an optimum mixture of 80 µM OX - AdV 25 Multiplicity of Infection (MOI) in PBS buffer were 17-29% the volume of control tumors. When buffer was changed from PBS to 5% dextrose in water (D5W), volumes of tumors injected IT with 80 µM OX-AdV 25 MOI were 10% while IT OX or AdV alone were 32% and 40% the volume of IT buffer-treated tumors. OX-AdV IT increased CD3+ TIL by 4-fold, decreased CD8+ PD-1+ TIL from 79% to 19% and induced cytotoxicity to CT26 cells in draining node lymphocytes while lymphocytes from CT26-bearing untreated mice were not cytotoxic. OX-AdV IT in D5W caused complete regression in 40% of mice. Long-term survivors rejected a contralateral challenge of CT26. The buffer for Oxaliplatin is critical. The two ICD inducer mixture is promising as an agnostic sensitizer for carcinomas like colorectal carcinoma.
Asunto(s)
Neoplasias Colorrectales , Muerte Celular Inmunogénica , Adenoviridae/genética , Animales , Línea Celular Tumoral , Neoplasias Colorrectales/tratamiento farmacológico , Ratones , Ratones Endogámicos BALB C , OxaliplatinoRESUMEN
MCL-1, a member of the antiapoptotic BCL-2 family, is a prosurvival protein with an essential DNA repair function. This study aims to test whether inhibition of protein synthesis by mTOR complex (mTORC) inhibitors depletes MCL-1, suppresses homologous recombination (HR) repair, and sensitizes cancer cells to PARP inhibitors. Treatment with everolimus decreases MCL-1 in colorectal carcinomas and small cell lung cancer (SCLC) cells but not glioblastoma multiforme (GBM) cells with a PTEN mutational background. However, AZD2014, a dual mTORC inhibitor, depletes MCL-1 in GBMs. Further, we show that everolimus decreases 4EBP1 phosphorylation only in colorectal carcinoma, whereas AZD2014 decreases 4EBP1 phosphorylation in both colorectal carcinoma and GBM cells. Combination therapy using everolimus or AZD2014 with olaparib inhibits the growth of clone A and U87-MG xenografts in in vivo and decreases clonogenic survival in in vitro compared with monotherapy. Reintroduction of MCL-1 rescues the survival of cancer cells in response to combination of everolimus or AZD2014 with olaparib. Treatment of cells with mTORC inhibitors and olaparib increases γ-H2AX and 53BP1 foci, decreases BRCA1, RPA, and Rad51 foci, impairs phosphorylation of ATR/Chk1 kinases, and induces necroptosis. In summary, mTORC inhibitors deplete MCL-1 to suppress HR repair and increase sensitivity to olaparib both in in vitro and in xenografts. IMPLICATIONS: Targeting the DNA repair activity of MCL-1 in in vivo for cancer therapy has not been tested. This study demonstrates that depleting MCL-1 sensitizes cancer cells to PARP inhibitors besides eliciting necroptosis, which could stimulate antitumor immunity to improve the therapeutic intervention of cancers.