# Discovering the mechanisms-of-action of mistargeted anti-cancer agents

> **NIH NIH R01** · COLD SPRING HARBOR LABORATORY · 2020 · $269,280

## Abstract

Project Summary
Cancer cells require the proteins encoded by certain genes in order to proliferate. These “genetic dependencies”
are promising targets for therapeutic intervention, as drugs that block the function of a dependency can induce
apoptosis and durable tumor regression. The discovery and characterization of genetic dependencies and the
drugs that can inhibit them are key goals of preclinical cancer research.
My laboratory has investigated multiple putative genetic dependencies using CRISPR/Cas9 mutagenesis. We
have found that verified mutagenesis of many cancer drug targets fails to recapitulate published results obtained
when these genes were knocked down with RNAi. Moreover, we find that multiple “targeted inhibitors” currently
in clinical trials continue to kill cancer cells harboring CRISPR-induced null mutations in their reported targets,
demonstrating pervasive off-target cell killing among clinical inhibitors. These results – coupled with the
observation that 97% of drug-indication pairs that enter clinical trials in oncology fail to receive FDA approval -
suggest the existence of fundamental shortcomings in how cancer genetic dependencies are identified and
studied. In this work, we will develop a robust, preclinical target validation pipeline to characterize both the
consequences of loss-of-function alterations in potential drug targets and to validate on-target activity of putative
clinical inhibitors. In particular, we will select genes that are reported to be cancer dependencies and that are
targeted by small-molecule inhibitors, and we will study the cellular consequences of their deletion or inhibition
(Aim 1). Next, we will use cells harboring CRISPR-induced knockouts of these putative drug targets to investigate
the chemical inhibitors that had been used to target them (Aim 2). If these reagents continue to kill cells that
totally lack their reported targets, then this would indicate that they induce cell death through an off-target
mechanism. Then, we will deploy both spontaneous- and CRISPR-directed mutagenesis in order to generate
mutations that confer resistance to these small-molecule inhibitors, thereby helping to identify their true cellular
targets (Aim 3). Finally, by isolating drug-resistance mutations, we have discovered that one mischaracterized
anti-cancer drug is in fact the first potent and specific inhibitor of the CDK11B kinase to be described. Using this
knowledge, we will seek to identify biomarkers that can predict therapeutic responses to this drug (Aim 4). In
total, these experiments will delineate a robust preclinical pipeline for target validation, shed light on the genetic
architecture that underlies cancer-essential genes, and allow drug re-purposing studies of multiple clinical
inhibitors by uncovering their true targets.

## Key facts

- **NIH application ID:** 9886861
- **Project number:** 1R01CA237652-01A1
- **Recipient organization:** COLD SPRING HARBOR LABORATORY
- **Principal Investigator:** Jason Sheltzer
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2020
- **Award amount:** $269,280
- **Award type:** 1
- **Project period:** 2020-05-01 → 2025-04-30

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/9886861

## Citation

> US National Institutes of Health, RePORTER application 9886861, Discovering the mechanisms-of-action of mistargeted anti-cancer agents (1R01CA237652-01A1). Retrieved via AI Analytics 2026-05-27 from https://api.ai-analytics.org/grant/nih/9886861. Licensed CC0.

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