Lenalidomide Promotes CRBN-Mediated Ubiquitination and Degradation of IKZF1 and IKZF3

Lenalidomide is a highly effective drug for the treatment of multiple myeloma and has activity in additional B cell lymphomas. Lenalidomide has been shown to bind the CRBN-DDB1 E3 ubiquitin ligase, but it is unknown how lenalidomide alters the activity of this enzyme complex, and how this leads to therapeutic efficacy.  We used a combination of quantitative proteomic approaches to demonstrate that lenalidomide acts by a novel mechanism of action for a therapeutic agent: in multiple myeloma cells, lenalidomide increases the binding of two substrates, IKZF1 (Ikaros) and IKZF3 (Aiolos), to the CRBN substrate adaptor; increases the ubiquitination of these substrates; and causes the targeted degradation of these transcription factors that are essential for the differentiation and survival of plasma cells including multiple myeloma cells.

To identify targets of the CRBN-DDB1 ubiquitin ligase that are altered by lenalidomide, we applied SILAC (stable isotope labeling by amino acids in cell culture)-based quantitative mass spectrometry studies to globally assess changes in ubiquitination and proteome levels in the multiple myeloma cell line MM1S. Two members of the Ikaros transcription factor family, IKZF1 and IKZF3, were differentially ubiquitinated and decreased after lenalidomide treatment.  Subsequent validation experiments in various cell lines demonstrated that lenalidomide, thalidomide, and pomalidomide cause a decrease of endogenous and ectopically expressed IKZF1 and IKZF3 protein levels but not mRNA levels. Furthermore, we confirmed that IKZF1 and IKZF3 bind CRBN in the presence of lenalidomide, supporting CRBN’s role as a substrate adaptor. Consistent with this, shRNA mediated knockdown or overexpression of a CRBN mutant (CRBN^YWAA) that does not bind lenalidomide abrogated lenalidomide’s effect on IKZF1 and IKZF3. Moreover, CRBN promoted IKZF3 ubiquitination in vitro in the presence of lenalidomide, demonstrating that it is an enzymatic substrate.

Using deletion mutants of IKZF3 we identified a 58-amino-acid degron in the N-terminal zinc finger domain that is sufficient for lenalidomide-induced degradation. Based on sequence alignment of that region between lenalidomide responding Ikaros proteins IKZF1 and IKZF3 vs. non-responding IKZF2, IKZF4 and IKZF5 we substituted a single amino acid (IKZF3^Q147H) that prevented binding of IKZF3 to CRBN and conferred resistance to lenalidomide induced degradation.

IKZF1 and IKZF3 are essential transcription factors for terminal B cell differentiation. We evaluated the biological effects of IKZF1 and IKZF3 loss using shRNAs in a variety of cell lines. IKZF1 and IKZF3 specific shRNAs inhibited the growth of multiple myeloma cell lines while lenalidomide insensitive cell lines derived from other hematopoietic neoplasms were unaffected. Similarly, a dominant negative IKZF3 mutant resulted in growth inhibition of MM1S cells. In contrast, expression of IKZF3^Q147Hconferred lenalidomide resistance to MM1S cells.

Lenalidomide induces IL-2 expression and release in T cells. We found that lenalidomide induced a dose-dependent decrease of IKZF1 and IKZF3 protein levels in primary human T cells. Previous studies have shown that IKZF3 is a transcriptional repressor of IL-2. To further evaluate the effect of IKZF3 loss, we transduced primary human T cells with shRNAs targeting either IKZF3 or control. IL2 RNA levels increased 3.3 fold after lenalidomide treatment in T cells expressing control shRNAs. In contrast, the baseline IL2 RNA level in T cells transduced with IKZF3 specific shRNAs was 3.7 fold higher compared to controls and this effect could not be further stimulated by lenalidomide.

In conclusion, selective targeting of two lymphoid transcription factors, IKZF1 and IKZF3, explains lenalidomide’s selective growth inhibition in multiple myeloma and likely other B cell lymphomas as well as its immunomodulatory effects in T cells. Furthermore, selective ubiquitination and degradation of specific targets provides a novel mechanism of therapeutic activity for proteins that are not otherwise amenable to small-molecule inhibition.