BME PhD Prospectus Defense - Maren Eltze

Title: “Engineering Sensitive and Cancer-Selective Assays for Src Family Kinase Activity Profiling”

Advisory Committee: John Ngo, PhD – BME (Chair) Erica Pratt, PhD – BME (Advisor) Alex Green, PhD – BME Vickery Trinkaus-Randall, PhD – Biochemistry & Cell Biology

Abstract: Src family non-receptor tyrosine kinases (SFKs) are frequently dysregulated in cancer, supporting tumor progression and metastasis. Phosphorylated (active) Src has been inversely correlated with patient survival in several cancers, suggesting its potential as a functional biomarker. However, live-cell SFK profiling using tumor specimens is a challenge, as current methods require a large number of cells, high tumor purity, or extensive sample handling. To fulfill this need, cell-deliverable Affinity-Based Peptide Probes (cAfBPs) have been developed to report SFK activity in live cells with minimal sample handling. These probes initiate transduction into live cells and are phosphorylated by native SFKs. They can therefore dynamically report live-cell SFK activity. However, current cAfBP designs are cell-type agnostic, making them poorly suited for profiling rare subpopulations present in complex biospecimens. Furthermore, current readout methods require extensive sample processing or fluorescently labeled antibodies and substrates. In this proposed work, I will develop cAfBP-based assays for use in rare cell analysis and heterogeneous matrices. In Aim 1, I will engineer novel tumor-selective cAfBPs. I hypothesize that by using a cancer-specific transduction sequence, cAfBPs can selectively initiate transport into the target cell type, making the assay cell-type selective. In this Aim, I am using colorectal cancer as a model system, as SFK hyperactivity has been well characterized in this disease. I have designed and synthesized cAfBPs containing a colorectal cancer-specific cellular transduction sequence. My preliminary data suggests that this transduction sequence transports the cAfBP cargo selectively into colorectal cancer cells while still allowing for SFK modification of the cAfBP recognition sequence. I will rigorously evaluate cAfBP selectivity across a panel of colorectal cancer cell lines and profile SFK activity in live colorectal cancer cells. If successful, this novel cAfBP will enable selective live-cell SFK activity profiling in heterogeneous matrices. In Aim 2, I will develop a highly sensitive cAfBP readout to enable SFK activity profiling in low cell-count samples. I will detect cAfBP phosphorylation using proximity ligation in combination with digital PCR (digital PLA). I hypothesize that by detecting cAfBP phosphorylation by digital PLA, I can increase the sensitivity of the cAfBP assay. To test this hypothesis, I have designed a PLA system that forms an amplifiable duplex oligonucleotide reporter when the cAfBP is phosphorylated. I will evaluate this assay's sensitivity across a broad dynamic range of peptide copy numbers. I will then apply this assay to detect recombinant Src activity using a biochemical assay. Overall, this work will improve the sensitivity and selectivity of live-cell SFK activity profiling, paving the way for functional SFK analysis in clinical cell samples.