New mechanism of lenalidomide activity

Abstract

Lenalidomide is an immunomodulatory agent (IMiD) that has activity in hematologic cancer (e.g., multiple myeloma). The immunomodulatory and apoptotic properties are readily apparent in therapy. However, the exact mechanism of action has been difficult to quantify until recently when it was shown that another IMiD, thalidomide, binds to an E3 ubiquitin ligase complex constituent, CRBN.¹ The article by Kronke et al. demonstrates that, by binding to CRBN and altering its selectivity, lenalidomide potentiates the ubiquitination and proteolysis of 2 specific proteins, IKZF1 and IKZF3. An article in the same issue, by Lu et al., supports these observations.^2,3 IKZF1 and IKZF3 are transcription factors that are necessary for multiple myeloma, and repression of these transcription factors is a likely mechanism for lenalidomide activity in this disease.

Keywords: :

• lenalidomide
• immunomodulatory agent
• IKZF1
• IKZF3
• CRBN
• IKZF1/3
• IMiD

This article refers to:

Using proteomic analysis, Kronke et al. first showed that IKZF1 and IKZF3 were rapidly degraded by the proteasome in the presence of lenalidomide; concurrently, they showed that thalidomide had a similar effect. The authors then confirmed that exposure to lenalidomide accelerated ubiquitination and degradation of luciferase-linked IKZF1 and IKZF3 constructs, and endogenous IKZF1 and IKZF3 in cell lines and patient-derived primary multiple myeloma samples. Lu et al. also demonstrated increased degradation of IKZF1 and IKZF3 in the presence of lenalidomide, that this mechanism is dependent on ubiquitination, and that lenalidomide activity requires CRBN.³

To confirm that lenalidomide potentiates the binding between IKZF1/3 and CRBN, the authors next coimmunoprecipitated these proteins from MM1S cell lysates. Both IKZF proteins coimmunoprecipitated with CRBN, and in vitro ubiquitination reactions demonstrated that this binding conferred greater E1 and E2 ubiquitin ligase activity. Introduction of shRNA against CRBN and mutations that prevent CRBN-lenalidomide binding introduced lenalidomide resistance in MM1S cells. Finally, using deletion of critical IKZF3 sequences, and multiple sequence alignment of ikaros proteins, the authors showed that a 30 amino acid sequence is essential for lenalidomide sensitivity. A single amino acid in this sequence (Q174) could be mutated to significantly reduce the proteolysis of IKZF3 following lenalidomide treatment. These data support the selectivity of the CRBN for IKZF1 and IKZF3, which is increased in the presence of lenalidomide.

Finally, to confirm that IKZF1/3 and CRBN alterations are responsible for the activity of lenalidomide, the authors studied these proteins in lenalidomide-sensitive and -insensitive MM cell lines infected with lentivirus-expressing IKZF1 or IKZF3 shRNAs and green fluorescent protein. Lenalidomide-sensitive cells were highly sensitive to IKZF1/3 shRNA treatment whereas insensitive cell lines were either insensitive, or partially sensitive. These results were confirmed using dominant negative IKZF3, and IKZF3-overexpressing cell lines were resistant to lenalidomide. The repression of IKZF3 on IL-2 transcription was also abrogated by lenalidomide, such that IL-2 expression was increased by 3.3-fold in lenalidomide-treated T cells expressing control shRNA. Similarly, IL-2 transcriptional repression was sustained in cells infected with CRBN shRNA. Therefore, lenalidomide activity is closely linked with Ikaros proteins and CRBN.

Lenalidomide has become a mainstay of MM therapy, and IMiDs have been useful in a variety of other tumor types and diseases. Conversely, iMID drugs are responsible for teratogeneicity. Until recently, numerous mechanisms have been proposed to explain the activity of these drugs. These include modulation of cytokines (e.g., TNFA, IL6, IL8, IL2, etc.), NFKB inactivation, cyclooxygenase inhibition, activation of NK and T cells, and antiangiogenesis. Although such mechanisms have been proposed, few of these studies ever clarified exactly how these drugs mediate these activities at the transcriptional level, nor were any true drug binding targets known. Therefore, the present studies represent a significant “step forward” in the understanding of the activities of iMID drugs, and identification of IKZF transcription factors as useful drug targets allows for the optimization of drugs that hit these targets.

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.