# The molecular basis of IMiD induced neo-substrate recruitment to the CRL4CRBN ubiquitin E3 ligase.

> **NIH NIH R01** · DANA-FARBER CANCER INST · 2020 · $395,901

## Abstract

Thalidomide and its analogs lenalidomide and pomalidomide (IMiDs), are highly effective treatments for
hematologic malignancies such as multiple myeloma (MM) or del(5q) myelodysplastic syndrome (5q-MDS), as
well as an FDA approved treatment for erythema nodosom leprosum (ENL). Over decades IMiDs were found
to have broad but not necessary related effects, ranging from induction of oxidative stress, inhibition of
angiogenesis, to multiple effects on the immune system such as enhanced production of cytokine interleukin-2
(IL-2) and inhibition of cytokine tumor necrosis factor (TNF). A seminal breakthrough in understanding the
activity of IMiDs was the discovery that IMiDs bind CRBN, the substrate receptor of the CUL4-RBX1-DDB1-
CRBN (CRL4CRBN) E3 ubiquitin ligase, and to exhibit dual activity: 1) preventing CRL4CRBN from ubiquitinating
its native substrates, including MEIS2, and 2) to alter the specificity of the CRL4CRBN ubiquitin ligase to
ubiquitinate new targets, notably the lymphoid transcription factors Ikaros (IKZF1) and Aiolos (IKZF3), and
casein kinase 1 alpha (CK1α). Therefore IMiDs impart gain-of-function properties to the CRL4CRBN substrate
receptor that enable binding and ubiquitination of key therapeutic targets. While IKZF1/3 and Ck1α provide a
plausible explanation for IMiD efficacy, they fall short in explaining all of the cellular and clinical response
induced by IMiDs and other substrates likely exist. Moreover, the finding that IMiDs act by altering the
substrate repertoire of the CRL4CRBN ubiquitin ligase towards neo-substrates has transformative potential to
drug discovery; however, we do not understand the molecular basis of neo-substrate recruitment. Important
unresolved issues are 1) the complete substrate repertoire of IMiDs, 2) the structural features of the degron
recognized by a CRL4CRBN-IMiD complex, and 3) how on molecular grounds, modifications to IMiDs can confer
substrate selectivity. In Aim 1 we will validate a list of high-confidence substrate candidates generated with a
novel pulse-SILAC mass spectrometry approach. We will publish validated substrates as a resource to the
community, which will greatly facilitate the in-depth understanding of the clinical and cellular outcomes of IMiD
treatment. In Aim 2, we will use a combination of X-ray crystallography, biochemical reconstitutions and
cellular experiments to address the molecular basis of IMiD activity. In depth understanding of this novel drug
mechanism will guide development of future medicine. Leveraging our intricate molecular and structural
understanding, we will develop IMiD derivatives with altered substrate selectivity in Aim 3 of this proposal.
Through a set of quantitative assays developed in the lab, we are for the first time able to follow a structure and
mechanism guided approach to synthesize derivatives of IMiDs and explain how subtle chemical modifications
result in altered substrate specificity. Taken together, we will define the molecular fram...

## Key facts

- **NIH application ID:** 9899745
- **Project number:** 5R01CA214608-04
- **Recipient organization:** DANA-FARBER CANCER INST
- **Principal Investigator:** Eric Sebastian Fischer
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2020
- **Award amount:** $395,901
- **Award type:** 5
- **Project period:** 2017-03-01 → 2022-02-28

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/9899745

## Citation

> US National Institutes of Health, RePORTER application 9899745, The molecular basis of IMiD induced neo-substrate recruitment to the CRL4CRBN ubiquitin E3 ligase. (5R01CA214608-04). Retrieved via AI Analytics 2026-05-23 from https://api.ai-analytics.org/grant/nih/9899745. Licensed CC0.

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