(191ai) Targeted Protein Therapeutics As Powerful Tools for Understanding and Overcoming Drug Resistance in Cancer

Over the past few decades, protein-based therapies have emerged as the gold standard for targeted, potent anticancer treatment, often in combination with small molecule-based drugs. Despite remarkable progress in development of these protein drugs, intrinsic and acquired tumor resistance substantially limits the breadth and depth of their clinical efficacy. The issue of resistance is often studied or addressed only after a drug fails to elicit complete responses in patients. Protein drugs can be engineered for superior target specificity and tumor selectivity, making them ideal not only as therapeutics, but also as probes for interrogating the target of interest to reveal mechanisms of cancer drug resistance. Here, we present a broadly applicable platform for mapping the pathways of resistance to a targeted protein therapy. The results of this screen were used to rationally design and evaluate drug combinations to overcome resistance to the targeted protein therapeutic.

For proof-of-concept of this approach, we used a multivalent pro-apoptotic protein composed of engineered protein scaffold domains targeting death receptor 5, a clinically relevant target for colorectal cancers (CRCs). This death receptor agonist (DRA) induces apoptosis at picomolar concentrations in many CRC lines. However, the clinical relevance of this drug is severely limited due to its failure to induce apoptosis in a significant number of cell lines. In this work, we show that the efficacy of a targeted therapy can be resurrected through systematic evaluation and understanding of its limitations. We used a CRISPR/Cas9 knockout screen to identify genes that confer DRA drug sensitivity to DRA-resistant cell lines. We then identified the small molecule drugs targeting pathways and proteins corresponding to these genetic drivers of resistance, and tested them in combination with the DRA in vitro to select synergistic combinations for in vivo validation. Our rational approach elegantly provides optimal protein-small molecule drug combinations that elicit a robust anticancer response, exhibit minimal toxicity, and combat drug resistance.