PROJECT SUMMARY

Lenalidomide is a transformative treatment for patients with myelodysplastic syndrome, inducing a complete cytogenetic response in approximately 50% of patients with deletions of chromosome 5q. Despite this dramatic efficacy, the mechanism of action of lenalidomide in MDS has not been elucidated, the mutations that cause greater sensitivity and resistance to the drug have not been characterized, and combinations of lenalidomide with other drugs that might have increased efficacy have not been identified.

In the past several years, we completed studies that elucidated the basic mechanism of action of lenalidomide, a finding that was recently published in Science in 2014, with acknowledgment of Evans Foundation support. We found that lenalidomide works by an entirely novel mechanism of action for a drug. Lenalidomide binds to the CRL4-CRBN ubiquitin ligase. Surprisingly, lenalidomide does not inhibit the enzyme, but rather causes it to degrade proteins. We then completed studies that elucidated the mechanism of action of lenalidomide in del(5q) MDS, published in Nature in 2015. We found that lenalidomide targets casein kinase 1A1 (CSNK1A1), encoded by a gene located on chromosome 5q, for degradation. We have now demonstrated that del(5q) MDS patients, who have 50% of the normal levels of CSNK1A1, are sensitized to treatment with lenalidomide, explaining the unique sensitivity of del(5q) MDS cells to this drug. In addition, we have shown that CSNK1A1 depletion kills cells via p53 activation, explaining why patients who become resistant to lenalidomide commonly develop p53 mutations.

We have discovered that lenalidomide does not have effects in murine cells due to a specific amino acid change between mice and humans in CRBN. In the past 6 months, we have generated a knock-in mouse with this amino acid change so that we can examine the biology of lenalidomide in vivo. We generated a novel mouse model (Fink et al., Blood 2018) that showed the first in vivo efficacy of lenalidomide in a pre-clinical model, facilitating the development of new drugs. In combination with an Rps14 conditional knockout mouse, we can now model both del(5q) MDS and its response to lenalidomide.

In addition, we have focused our attention on another line of investigation that was proposed in our grant, the determination of mechanisms of resistance to lenalidomide. We have performed a genome-wide CRISPR/Cas9 screen (120,000 small guide RNAs targeting all human genes) in MM1S cells in the presence or absence of lenalidomide. The screen worked extremely well, with CRBN scoring at the top of all genes, i.e. loss of CRBN results in lenalidomide activity as expected. In addition, 20 components of the CRL4-CRBN ubiquitin ligase scored in the screen, informing both the biology of the drug and mechanisms of resistance (Sievers et al., Blood 2018).

Finally, we have extended our findings to identify novel zinc finger proteins that can be degraded by lenalidomide analogs. We screened all the zinc fingers in the proteome and solved the crystal structure of two zinc finger proteins bound to CRBN and the drug. This study indicates that lenalidomide derivatives will be able to degrade a range of zinc finger transcription factors.