Combination Organelle Mitochondrial Endoplasmic Reticulum Therapy (COMET) for Multidrug Resistant Breast Cancer

Abstract

It is time for the story of mitochondria and intracellular communication in multidrug resistant cancer to be rewritten. Herein we characterize the extent and cellular advantages of mitochondrial network fusion in multidrug resistant (MDR) breast cancer and have designed a novel nanomedicine that disrupts mitochondrial network fusion and systematically manipulates organelle fusion and function. Combination Organelle Mitochondrial Endoplasmic reticulum Therapy (COMET) is an innovative translational nanomedicine for treating MDR triple negative breast cancer (TNBC) that has superior safety and equivalent efficacy to the current standard of care (paclitaxel). Our study has demonstrated that the increased mitochondrial networks in MDR TNBC contribute to apoptotic resistance and network fusion is mediated by mitofusin2 (MFN2) on the outer mitochondrial membrane. COMET consists of three components; Mitochondrial Network Disrupting (MiND) nanoparticles (NPs) that are loaded with an anti-MFN2 peptide, tunicamycin, and Bam7. The therapeutic rationale of COMET is to reduce the apoptotic threshold in MDR cells with MiND NPs, followed by inducing the endoplasmic reticulum mediated unfolded protein response (UPR) by stressing MDR cells with tunicamycin, and finally, directly inducing mitochondrial apoptosis with Bam7 which is a specific bcl-2 Bax activator. MiND NPs are PEGylated liposomes with the 21 amino acid (2577.98 MW) anti-MFN2 peptide compartmentalized in the aqueous core. Hypoxia (0.5% oxygen) was used to create MDR derivatives of MDA-MB-231 cells and BT-549 cells. Mitochondrial networks were quantified using 3D analysis of 60× live cell images acquired with a Keyence BZ-X710 microscope and MiND NPs effectively fragmented mitochondrial networks in drug sensitive and MDR TNBC cells. The IC₅₀ values, combination index, and dose reduction index derived from dose response studies demonstrate that MiND NPs decrease the apoptotic threshold of both drug sensitive and MDR TNBC cells and COMET is a synergistic drug combination. Complex V (ATP synthase) extracted from bovine cardiac mitochondria was used to assess the effect of MiND NPs on OXPHOS; both MiND NPs and anti-MFN2 peptide solution significantly decrease the activity of mitochondrial complex V and decrease the capacity of OXPHOS. A BacMam viral vector based fluorescent biosensor was used to quantify the unfolded protein response (UPR) at the level of the endoplasmic reticulum and tunicamycin specifically induces the UPR in drug sensitive and MDR TNBC cells. A caspase 3 colorimetric assay demonstrated that the synergistic triple drug combination of COMET increases the ability of Bam7 to specifically induce apoptosis. Dose limiting toxicity and off target effects are a significant challenge for current chemotherapy regimens including paclitaxel. COMET has significantly lower cytotoxicity than paclitaxel in human embryonic kidney epithelial cells and has the potential to fulfill the clinical need for safer cancer therapeutics. COMET is a promising early stage translational nanomedicine for treating MDR TNBC. Manipulating intracellular communication and organelle fusion is a novel approach to treating MDR cancer. The data from this study has rewritten the story of mitochondria, organelle fusion, and intracellular communication and by targeting this intersection, COMET is an exciting new chapter in cancer therapeutics that could transform the clinical outcome of MDR TNBC.

Keywords: COMET; Endoplasmic reticulum; Mitochondria; Multidrug resistance; Nanomedicine; Triple negative breast cancer.