Background
Treatment modalities seek to achieve the cancer free state by inducing tumor cell death whether through small molecule inhibitors, cellular, radiation, immuno or chemo, therapies. Tumors evade the induction of cell death through competing survival pathways. Even when executed to completion, the classical programmed cell death of apoptosis induces immune tolerance impairing further immune mediated tumor cell removal. Leveraging advances in gene therapy vehicles, gene expression data and immune and bioengineering principals we have designed a chimeric cell death protein, MLKL-TfR that induces unavoidable, immunogenic cell death. Therapeutically, expression of this potent cell death molecule is achieved through plasmid DNA encoded tumor specific promoter control of the construct, delivered to the tumor by a self-assembling polymeric lipid nanoparticle PPS-PPDP2.
Methods
In vitro, to evaluate the efficiency and features of the MLKL-TfR construct we utilize ROSA26 CRISPR/CAS9 and doxycycline inducible DNA construct approaches to control the expression of MLKL-TfR in phenotypically normal (EPH4) epithelial cell line as well as MC38 colorectal tumors. We employ these cell lines in Damage Associated Molecular Pattern (DAMP) assays along with live cell dual phase/fluorescence imaging to determine the features of MLKL-TfR induced cell death. Utilizing Lenti-viral constructs we confirm MLKL-TfR potency in a human tumor cell line (K562). In vivo, we employ Nod.Scid Common Gamma Deficient Mice (NCG, n =12 ) and WT mice to demonstrate construct expression induced immune mediated tumor control. Lastly, to model therapeutic deployment, we utilize Balbc mice bearing established (35mm2) CT26 colorectal tumors and deliver pDNA encoding a tumor specific promoter controlling expression of MLKL-TfR through the PPS-PPDP2 nanoparticle delivery system.
Results
In vitro, extracellular release assays demonstrate induction of MLKL-TfR leads to rapid and significant release of the DAMP molecules extracellular ATP and HMGB1. In addition, live cell dual phase/fluorescence microscopy approaches characterizing the plasma and nuclear membrane features and kinetics of induced cell death demonstrate MLKL-TfR expression induces rapid necrosis like cell death, uninhibitable by any Caspase or RIPK inhibitors. In vivo experiments demonstrate an enhancement of tumor control through collaboration between the immune system and construct expression. Nanoparticle delivery of pDNA encoding MLKL-TfR (n =10) to established CT26 tumors under the control of a tumor specific promoter demonstrate significant control of tumor growth (p=.0027) and overall survival (p=.0156) compared to delivery of control plasmid (CMV- GFP).
Conclusions
MLKL-TfR potently induces unavoidable, immunogenic tumor cell death and represents a novel platform for cancer gene therapy applications.