Rate of CRL4(CRBN) substrate Ikaros and Aiolos degradation underlies differential activity of lenalidomide and pomalidomide in multiple myeloma cells by regulation of c-Myc and IRF4.

Recent discoveries suggest that the critical events leading to the anti-proliferative activity of the IMiD immunomodulatory agents lenalidomide and pomalidomide in multiple myeloma (MM) cells are initiated by Cereblon-dependent ubiquitination and proteasomal degradation of substrate proteins Ikaros (IKZF1) and Aiolos (IKZF3). By performing kinetic analyses, we found that the downregulation or proteasomal degradation of Ikaros and Aiolos led to specific and sequential downregulation of c-Myc followed by IRF4 and subsequent growth inhibition and apoptosis. Notably, to ensure growth inhibition and cell death, sustained downregulation of Ikaros and Aiolos, c-Myc or IRF4 expression was required. In addition, we found that the half-maximal rate, rather than the final extent of Ikaros and Aiolos degradation, correlated to the relative efficacy of growth inhibition by lenalidomide or pomalidomide. Finally, we observed that all four transcription factors were elevated in primary MM samples compared with normal plasma cells. Taken together, our results suggest a functional link between Ikaros and Aiolos, and the pathological dysregulation of c-Myc and IRF4, and provide a new mechanistic understanding of the relative efficacy of lenalidomide and pomalidomide based on the kinetics of substrate degradation and downregulation of their downstream targets.

Cellular effects of leishmanial tubulin inhibitors on L. donovani.

To aid our investigation of tubulin as an antileishmanial drug target, the effects of the mammalian antimicrotubule agents ansamitocin P3, taxol, and hemiasterlin on Leishmania donovani promastigotes were described. These drugs affected the assembly of purified leishmanial tubulin and inhibited the growth of L. donovani promastigotes at micromolar concentrations. When promastigotes were treated with these agents, mitotic partitioning of nuclear DNA and cytokinesis were usually inhibited. The spatial orientation of kinetoplasts was often disturbed, suggesting a role for microtubules in the segregation of these organelles during mitosis. Aberrant cell types produced in drug-treated cultures included parasites with one nucleus and two geometrically distinct kinetoplasts, parasites with multiple kinetoplasts, and cytoplasts containing a kinetoplast but no nucleus. A subset of unique cell types, parasites containing two nuclei, a spindle fiber, and two geometrically distinct kinetoplasts, were observed in hemiasterlin-treated cultures. Flow cytometric analysis of L. donovani promastigotes treated with these three drugs indicated a dramatic shift toward the G2 + M phase of the cell cycle, with some cells containing four times the amount of DNA present in G1. These results were used to evaluate the cellular effects of WR85915, an aromatic thiocyanate with in vitro antileishmanial and anti-tubulin activity, on L. donovani. Treatment of parasites with WR85915 did not produce the unusual cell types described above and did not cause the accumulation of parasites in G2 + M, suggesting that WR85915 acts on target(s) in Leishmania in addition to tubulin. These studies validate tubulin as a suitable antileishmanial drug target and provide criteria to assess the cellular mechanism of action of new candidate antileishmanial agents.