(M1030-02-09) Combinational Approach Using AKT/ERK Dual Inhibitors for Pancreatic Cancer

Purpose: Fast metabolism and ineffective transport to the desmoplastic pancreatic cancer (PC) render poor therapeutic efficacy of Gemcitabine (GEM). Hedgehog (Hh) signaling activates pancreatic stellate cells (PSCs) and is crucial for the growth of cancer stem cells (CSCs) and the development of desmoplasia. KRAS is frequently mutated in pancreatic cancer, yet it is still a challenging gene to target. We suggest combining GEM with ONC201, an AKT/ERK dual inhibitor, to successfully treat PC since mTORC1/2 inhibition promotes ERK phosphorylation. TNF-related apoptosis-inducing ligand (TRAIL)-mediated apoptosis is induced by ONC201, which also reduces cell proliferation. Using multifunctional nanomedicine composed of GEM, and ONC201, the long-term impact is to create novel approaches for reducing desmoplasia-induced chemoresistance in PC.

Methods: We have determined the cytotoxic effects of GEM and ONC201 using MTT assay in MIA PaCa-2 cells. To check the pro-apoptotic effect of a single treatment compared to a combination approach, we have used cell migration assay, colony formation assay, and cell apoptosis assay. We determined the effect of GEM and ONC201 combination therapy on cell cycle arrest. We have generated an orthotopic pancreatic tumor-bearing mouse model to determine whether systemic administration of GEM (40 mg/kg) and ONC201 (30 mg/kg), and their combination could reduce tumor burden. We established orthotopic tumors with MIA PaCa-2 cells. Mice received drug treatment with intravenous injections every alternate day for 2 weeks. We took bioluminescent images of tumors in mice treated with vehicle, GEM, ONC201, or their combination. We also took images of excised tumors. We performed immunohistochemistry of p-AKT, p-mTOR, and cleaved caspase3 staining.

Results: GEM and ONC201 resulted in a dose-dependent cell killing when incubated with MIA PaCa-2 cells at different doses, with IC50 values of 2.5 μM and 10 μM, respectively for GEM and ONC201. GEM and ONC201 showed a combinational approach in the Transwell membrane method of cell migration assay, significantly inhibiting the migration compared to DMSO control. These results demonstrate that GEM and ONC201 combination strongly controls aberrant cell proliferation and hinders cell migration efficiently in PC. By colony formation assay, we determined the effect of GEM and ONC201 at IC₅₀ values on the tumorigenic potential of MIA PaCa-2 cells. The Annexin V assay determined apoptotic death with the total apoptotic cell population of GEM, ONC-201, and combination 19%, 10% and 33%, respectively. GEM arrested the cells in the S phase; cells were arrested in the G1 phase when incubated with ONC201, while the combination arrested MIA PaCa-2 cells in the G1 phase. In our in-vivo experiments, mice bearing pancreatic tumors generated by injecting MIA PaCa-2 cells orthotopically into the pancreas showed a significant decrease in tumor burden after treatment with GEM and ONC201. GEM and ONC201 combination treatment decreased p-PI3K and p-mTOR staining while increasing cleaved caspase 3 staining.

Conclusion: Our results confirm the improved efficacy of the treatment with our combination approach. Various assays confirmed the dose-dependent cell killing of GEM and ONC201 along with the combination. The cell cycle analysis confirmed the S phase arrest with GEM as it incorporates into DNA replication, thus inhibiting chain elongation of DNA and causing cell death by apoptosis. Similarly, ONC201 is a TRAIL-inducing compound that can induce high levels of Sub-G1 DNA content in tumor cells. Our in-vivo results establish a mice-bearing orthotopic pancreatic tumor model; decrease in the burden confirms the effective treatment strategy. Also, the increased cleaved caspase 3 marker validates cell DNA fragmentation during apoptosis.