mln-8237 and Melanoma

Antagonists of MDM2-p53 interaction are emerging anti-cancer drugs utilized in clinical trials for malignancies that rarely mutate p53, including melanoma. We discovered that MDM2-p53 antagonists protect DNA from drug-induced damage in melanoma cells and patient-derived xenografts. Among the tested DNA damaging drugs were various inhibitors of Aurora and Polo-like mitotic kinases, as well as traditional chemotherapy. Mitotic kinase inhibition causes mitotic slippage, DNA re-replication, and polyploidy. Here we show that re-replication of the polyploid genome generates replicative stress which leads to DNA damage. MDM2-p53 antagonists relieve replicative stress via the p53-dependent activation of p21 which inhibits DNA replication. Loss of p21 promoted drug-induced DNA damage in melanoma cells and enhanced anti-tumor activity of therapy combining MDM2 antagonist with mitotic kinase inhibitor in mice. In summary, MDM2 antagonists may reduce DNA damaging effects of anti-cancer drugs if they are administered together, while targeting p21 can improve the efficacy of such combinations.

Topics: Animals; Antineoplastic Combined Chemotherapy Protocols; Azepines; Cell Line, Tumor; Cyclin-Dependent Kinase Inhibitor p21; DNA Damage; DNA Replication; HCT116 Cells; Humans; Imidazoles; Melanoma; Mice; para-Aminobenzoates; Piperazines; Protein Binding; Proto-Oncogene Proteins c-mdm2; Pyrimidines; Pyrrolidines; Tumor Suppressor Protein p53; Xenograft Model Antitumor Assays

MITF (microphthalmia-associated transcription factor) is a frequently amplified lineage-specific oncogene in human melanoma, whose role in intrinsic drug resistance has not been systematically investigated. Utilizing chemical inhibitors for major signaling pathways/cellular processes, we witness MITF as an elicitor of intrinsic drug resistance. To search kinase(s) targets able to bypass MITF-conferred drug resistance, we employed a multi-kinase inhibitor-directed chemical proteomics-based differential affinity screen in human melanocytes carrying ectopic MITF overexpression. A subsequent methodical interrogation informed mitotic Ser/Thr kinase Aurora Kinase A (AURKA) as a crucial regulator of melanoma cell proliferation and migration, independent of the underlying molecular alterations, including TP53 functional status and MITF levels. Crucially, assessing the efficacy of investigational AURKA inhibitor MLN8237, we pre-emptively witness the procurement of a molecular program consistent with acquired drug resistance. This involved induction of multiple MAPK (mitogen-activated protein kinase) signaling pathway components and their downstream proliferation effectors (Cyclin D1 and c-JUN) and apoptotic regulators (MITF and Bcl-2). A concomitant AURKA/BRAF and AURKA/MEK targeting overcame MAPK signaling activation-associated resistance signature in BRAF- and NRAS-mutated melanomas, respectively, and elicited heightened anti-proliferative activity and apoptotic cell death. These findings reveal a previously unreported MAPK signaling-mediated mechanism of immediate resistance to AURKA inhibitors. These findings could bear significant implications for the application and the success of anti-AURKA approaches that have already entered phase-II clinical trials for human melanoma.

Topics: Apoptosis; Aurora Kinase A; Azepines; Cell Line, Tumor; Drug Resistance, Neoplasm; GTP Phosphohydrolases; Humans; Melanoma; Membrane Proteins; Microphthalmia-Associated Transcription Factor; Mitogen-Activated Protein Kinase Kinases; Proto-Oncogene Proteins B-raf; Pyrimidines; Signal Transduction; Tumor Suppressor Protein p53

Therapeutics that induce cancer cell senescence can block cell proliferation and promote immune rejection. However, the risk of tumor relapse due to senescence escape may remain high due to the long lifespan of senescent cells that are not cleared. Here, we show how combining a senescence-inducing inhibitor of the mitotic kinase Aurora A (AURKA) with an MDM2 antagonist activates p53 in senescent tumors harboring wild-type 53. In the model studied, this effect is accompanied by proliferation arrest, mitochondrial depolarization, apoptosis, and immune clearance of cancer cells by antitumor leukocytes in a manner reliant upon Ccl5, Ccl1, and Cxcl9. The AURKA/MDM2 combination therapy shows adequate bioavailability and low toxicity to the host. Moreover, the prominent response of patient-derived melanoma tumors to coadministered MDM2 and AURKA inhibitors offers a sound rationale for clinical evaluation. Taken together, our work provides a preclinical proof of concept for a combination treatment that leverages both senescence and immune surveillance to therapeutic ends.

Topics: Animals; Antineoplastic Combined Chemotherapy Protocols; Apoptosis; Aurora Kinase A; Azepines; Cell Proliferation; Humans; Imidazoles; Melanoma; Melanoma, Experimental; Mice, Inbred BALB C; Mice, Inbred C57BL; Mice, Nude; Piperazines; Protein Kinase Inhibitors; Proto-Oncogene Proteins c-mdm2; Pyrimidines

Preclinical studies show that inhibition of aurora kinases in melanoma tumors induces senescence and reduces tumor growth, but does not cause tumor regression. Additional preclinical models are needed to identify agents that will synergize with aurora kinase inhibitors to induce tumor regression.. We combined treatment with an aurora kinase A inhibitor, MLN8237, with agents that activate death receptors (Apo2L/TRAIL or death receptor 5 agonists) and monitored the ability of this treatment to induce tumor apoptosis and melanoma tumor regression using human cell lines and patient-derived xenograft (PDX) mouse models.. We found that this combined treatment led to apoptosis and markedly reduced cell viability. Mechanistic analysis showed that the induction of tumor cell senescence in response to the AURKA inhibitor resulted in a decreased display of Apo2L/TRAIL decoy receptors and increased display of one Apo2L/TRAIL receptor (death receptor 5), resulting in enhanced response to death receptor ligand/agonists. When death receptors were activated in senescent tumor cells, both intrinsic and extrinsic apoptotic pathways were induced independent of BRAF, NRAS, or p53 mutation status. Senescent tumor cells exhibited BID-mediated mitochondrial depolarization in response to Apo2L/TRAIL treatment. In addition, senescent tumor cells had a lower apoptotic threshold due to decreased XIAP and survivin expression. Melanoma tumor xenografts of one human cell line and one PDX displayed total blockage of tumor growth when treated with MLN8237 combined with DR5 agonist antibody.. These findings provide a strong rationale for combining senescence-inducing therapeutics with death receptor agonists for improved cancer treatment.

Topics: Animals; Antibodies, Monoclonal; Antineoplastic Agents; Apoptosis; Aurora Kinases; Azepines; Caspases; Cell Line, Tumor; Cellular Senescence; Disease Models, Animal; Drug Evaluation, Preclinical; Female; Humans; Melanoma; Mice; Protein Kinase Inhibitors; Pyrimidines; Receptors, Death Domain; Receptors, TNF-Related Apoptosis-Inducing Ligand; Receptors, Tumor Necrosis Factor, Member 10c; Signal Transduction; TNF-Related Apoptosis-Inducing Ligand; Tumor Suppressor Protein p53; Xenograft Model Antitumor Assays