# Rapid and direct control of the proteome through a multiplexed tag system

> **NIH NIH F32** · CHILDREN'S HOSP OF PHILADELPHIA · 2020 · $65,310

## Abstract

Project Summary
Understanding and regulating the functions of the proteome is a primary goal in the biomedical sciences. Cancer
biology, in a particular, is a field that is currently greatly benefiting from high-throughput functional analysis of
genes and proteins. The selective reliance of cancer cells on common cell pathways, a phenomenon known as
non-oncogene addiction, is often studied via high-throughput screening approaches, thus uncovering new
potential therapies. Unfortunately, modern functional screens based on CRISPR and RNA interference, although
informative, cannot affect the proteome acutely and are thus not only technically limiting, but also especially
susceptible to compensation mechanisms that obscure protein roles. Additionally, in the time it takes to establish
genetic knockdown, many proteins essential for viability are lost and cannot be tested for interesting phenotypes
in non-viability screen formats. To facilitate a broader and more comprehensive study of the proteome, I
propose to develop a new screening paradigm based on rapid and direct modulation of proteins at a
genome-wide scale. This screening platform will initially be developed to uncover many new insights on non-
oncogenic addiction in cancer cells. The technology is based on a multiplexed cell library where each gene is
tagged with a ligand-binding, bioorthogonal protein, one cell at a time. Small-molecule ligands will bidirectionally
regulate the stability of tagged proteins. In the first aim, I will determine the rules required for efficient large-scale
protein tagging. Subsequently, in the second aim, I will construct a genome-wide multiplexed tag library in cancer
cells and perform a protein essentiality screen by rapid degradation, demonstrating the ability of the platform to
uncover novel hits. Essentiality screens performed with traditional genetic perturbation tools, like CRISPR and
RNA interference, are predicted to be much more susceptible to cell compensation mechanisms, and so I will
directly compare the results obtained from this screen to results obtained from other, comparable essentiality
screens. In the third aim, I will utilize the developed platform to comprehensively explore the role of proteostasis
in non-oncogene addiction. Specifically, I will uncover a subcellular map of acute responses to proteotoxic stress
in both cancerous and healthy cells. This will be achieved by using a subset of the multiplexed tag cell library to
induce compartment-specific protein destabilization, which will be analyzed by single-cell RNA sequencing. In
summary, I will develop a strategy to rapidly and directly modulate the proteome in a high-throughput manner,
facilitating studies that will greatly contribute to our understanding of non-oncogenic addiction in cancer cells and
leading to the development of novel cancer therapies. The success of this project will be greatly facilitated by its
environment, which provides access to facilities and insight from exper...

## Key facts

- **NIH application ID:** 9899088
- **Project number:** 5F32CA239499-02
- **Recipient organization:** CHILDREN'S HOSP OF PHILADELPHIA
- **Principal Investigator:** Yevgeniy Vladimirovich Serebrenik
- **Activity code:** F32 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2020
- **Award amount:** $65,310
- **Award type:** 5
- **Project period:** 2019-04-01 → 2022-03-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/9899088

## Citation

> US National Institutes of Health, RePORTER application 9899088, Rapid and direct control of the proteome through a multiplexed tag system (5F32CA239499-02). Retrieved via AI Analytics 2026-05-22 from https://api.ai-analytics.org/grant/nih/9899088. Licensed CC0.

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