Mechanism of Lenalidomide Activity in Multiple Myeloma
Authors: Krönke Jan 
Published: October 2019

Abstract
Lenalidomide is a derivative of thalidomide, a drug developed in the 1950s as a sedative and treatment for morning sickness that became infamous for causing limb deformations (phocomelia) and other birth defects when used by pregnant women. Following the discovery in the 1990s that thalidomide inhibits the release of tumor necrosis factor (TNF) and blocks angiogenesis, researchers began studying thalidomide in other diseases, including cancer. Thalidomide demonstrated high in vitro activity in multiple myeloma and high response rates in clinical trials, leading to its accelerated approval by the FDA in 2006. In addition to the direct antiproliferative effects on multiple myeloma cells, thalidomide and its more potent derivatives, lenalidomide and pomalidomide, have pleiotropic effects on immune cells and are therefore called immunomodulatory drugs (IMiDs). IMiDs enhance the release of interleukin-2 (IL-2) and interferon-γ (IFN-γ) from activated T cells, inhibit the immunosuppressive activity of regulatory T cells, and increase natural killer (NK) cell-mediated cytotoxicity. In peripheral blood monocytes (PBMCs) IMiDs inhibit the release of TNF and other cytokines including interleukin-6 (IL-6), a critical growth factor for multiple myeloma cells. Recently, cereblon (CRBN) was identified as the common primary target for all IMiDs. CRBN forms an E3 ubiquitin ligase together with DNA damage-binding protein 1 (DDB1), cullin 4A (CUL4A), and regulators of cullins (ROC1) CRBN-CRL4. Interaction with this enzymatic complex has been shown to be essential for most properties of IMiDs including teratogenicity, antiproliferative effects in multiple myeloma and some of the immunomodulatory properties. More recently, it was demonstrated that lenalidomide and its analogues activate the CRBN-CRL4 E3 ligase to ubiquitinate and degrade two members of the Ikaros family of zinc finger transcription factors: Ikaros (IKZF1) and Aiolos (IKZF3). IKZF1 and IKZF3 are key regulators in lymphopoiesis and essential for lymphoid progenitor differentiation into effector cells. While IKZF1 and IKZF3 deletions and loss of function mutations are frequent in acute lymphoblastic leukemia, mature B-cell lymphomas like multiple myeloma and chronic lymphocytic leukemia have high IKZF1 and IKZF3 expression. Inactivation of IKZF1 and IKZF3 results in growth inhibition in multiple myeloma. Conversely, over-expression of IKZF1 or IKZF3 confers lenalidomide resistance, demonstrating that degradation of IKZF1 and IKZF3 is responsible for the direct cytotoxic effects of lenalidomide in multiple myeloma. One of the transcriptional targets of IKZF1 and IKZF3 is interferon regulatory factor 4 (IRF4), a transcription factor that is essential for proliferation and survival of multiple myeloma cells that is down-regulated after lenalidomide-induced degradation of IKZF1 and IKZF3. At the IL-2 locus, IKZF3 is a transcriptional repressor that is de-repressed after lenalidomide-induced degradation of IKZF3; explaining one of the immunomodulatory properties of lenalidomide. While IKZF1 and IKZF3 degradation is likely involved in the other effects of lenalidomide on T cell subsets and NK cells, it is unlikely that this accounts for all of its properties. Limb deformations, for instance, do not occur in mice with germline genetic inactivation of IKZF1 or IKZF3. Similarly, it is unlikely that degradation of the lymphoid transcription factors IKZF1 and IKZF3 accounts for the specific activity of lenalidomide in myelodysplastic syndrome with chromosome 5q deletion. Since most of these effects have been shown to depend on CRBN, it is conceivable that they result from lenalidomide-induced alteration of other substrates of the CRBN-CRL4 E3 ubiquitin ligase. Future studies aiming to identify the substrates responsible for each of the biological effects of IMiDs could enable the development of more specific drugs that modify ubiquitination of different sets of proteins with fewer side effects.

Disclosures
No relevant conflicts of interest to declare.