The precise molecular mechanism of action and targets through which thalidomide and related immunomodulatory drugs (IMiDs) exert their antitumor effects remains unclear. We investigated the role of cereblon (CRBN), a primary teratogenic target of thalidomide, in the antimyeloma activity of IMiDs. CRBN depletion is initially cytotoxic to human myeloma cells, but surviving cells with stable CRBN depletion become highly resistant to both lenalidomide and pomalidomide, but not to the unrelated drugs bortezomib, dexamethasone, and melphalan. Acquired deletion of CRBN was found to be the primary genetic event differentiating isogenic MM1.S cell lines cultured to be sensitive or resistant to lenalidomide and pomalidomide.

• CC-122 is a novel agent for DLBCL with antitumor and immunomodulatory activity.• CC-122 binds CRBN and degrades Aiolos and Ikaros resulting in a mimicry of IFN signaling and apoptosis in DLBCL.Cereblon (CRBN), a substrate receptor of the Cullin 4 RING E3 ubiquitin ligase complex, is the target of the immunomodulatory drugs lenalidomide and pomalidomide. Recently, it was demonstrated that binding of these drugs to CRBN promotes the ubiquitination and subsequent degradation of 2 common substrates, transcription factors Aiolos and Ikaros.Here we report that CC-122, a new chemical entity termed pleiotropic pathway modifier, binds CRBN and promotes degradation of Aiolos and Ikaros in diffuse large B-cell lymphoma (DLBCL) and T cells in vitro, in vivo, and in patients, resulting in both cell autonomous as well as immunostimulatory effects. In DLBCL cell lines, CC-122-induced degradation or short hairpin RNA-mediated knockdown of Aiolos and Ikaros correlates with increased transcription of interferon (IFN)-stimulated genes independent of IFN-a, -b, and -g production and/or secretion and results in apoptosis in both activated B-cell (ABC) and germinal center B-cell DLBCL cell lines. Our results provide mechanistic insight into the cell-of-origin independent antilymphoma activity of CC-122, in contrast to the ABC subtype selective activity of lenalidomide. (Blood. 2015;126(6):779-789)

Proteasome inhibition with bortezomib is a validated approach to the treatment of multiple myeloma, but drug resistance often emerges and limits its utility in the retreatment setting. To begin to identify some of the mechanisms involved, we developed bortezomib-resistant myeloma cell lines that, unlike previously reported models, showed no ␤5 subunit mutations. Instead, up-regulation of the insulinlike growth factor (IGF)-1 axis was identified, with increased autocrine and paracrine secretion of IGF-1, leading to increased activation of the IGF-1 receptor (IGF-1R). Exogenous IGF-1 reduced cellular sensitivity to bortezomib, whereas pharmacologic or small hairpin RNAmediated IGF-1R suppression enhanced bortezomib sensitivity in cell lines and patient samples. In vitro studies with OSI-906, a clinically relevant dual IGF-1R and insulin receptor inhibitor, showed it acted synergistically with bortezomib, and potently resensitized bortezomib-resistant cell lines and patient samples to bortezomib. Importantly, OSI-906 in combination with bortezomib also overcame bortezomib resistance in an in vivo model of myeloma. Taken together, these data support the hypothesis that signaling through the IGF-1/IGF-1R axis contributes to acquired bortezomib resistance, and provide a rationale for combining bortezomib with IGF-1R inhibitors like OSI-906 to overcome or possibly prevent the emergence of bortezomib-refractory disease in the clinic. (Blood. 2012;120(16):3260-3270) IntroductionMultiple myeloma is a malignancy of immunoglobulin-secreting clonal plasma cells that is most often found in the bone marrow. 1,2 Modulation of the activity of the ubiquitin-proteasome pathway with the small molecule proteasome inhibitor bortezomib (VEL-CADE) has been validated as a rational therapeutic strategy for this disease 3,4 both in the front-line and relapsed/refractory settings. Despite these and other advances, myeloma remains an incurable disease characterized by decreasing response durations with each subsequent salvage therapy. 5 This is mediated in part through both intrinsic and acquired drug resistance, the latter of which emerges during and after bortezomib therapy. 6 Response rates in patients with previously bortezomib-sensitive disease are typically decreased on drug rechallenge 7-9 and may be as low as 23% among patients who had achieved at least a partial remission previously. 7 These findings indicate a need for an understanding of the molecular basis for bortezomib resistance.Proteasome inhibition acutely activates multiple inducible chemoresistance pathways that reduce the efficacy of bortezomib. One example is the antiapoptotic Akt pathway that can be activated by proteasome inhibitors, 10 and suppression of this pathway can induce chemosensitization to bortezomib. [11][12][13] Another possible mechanism aiding in acquired resistance to bortezomib may be the development of mutations in the bortezomib-binding pocket of the ␤5 proteasome subunit, or increased expression of ␤5 itself. [14][15][16] However, ␤5 proteasome sub...

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.

Lenalidomide plays an important role in our chemotherapeutic armamentarium against multiple myeloma, in part by exerting direct anti-proliferative and pro-apoptotic effects. Unfortunately, long-term exposure leads to the development of drug resistance through unknown mechanisms, and we therefore sought to identify pathways that could be responsible for this phenotype. Chronic drug exposure produced myeloma cell lines that were tolerant of the direct effects of lenalidomide, with a degree of resistance of up to 2,500-fold. Gene expression profiling and pathway analysis identified dysregulation of the Wnt/␤-catenin pathway as a consistent change across four independent cell isolates, and a pair of primary plasma cell samples. Acute drug treatment also increased ␤-catenin transcription by 3-fold or more, and both acute and chronic exposure resulted in enhanced accumulation of ␤-catenin protein by up to 20-fold or more. This produced Wnt/␤-catenin pathway activation, as judged by increased activity of a lymphoid enhancer factor/T-cell factor promoter reporter, and enhanced accumulation of the downstream targets cyclin D1 and c-Myc. Components of the ␤-catenin destruction complex were also impacted by lenalidomide, which suppressed casein kinase 1␣ expression while augmenting glycogen synthase kinase 3␣/␤ phosphorylation. Stimulation of Wnt/␤-catenin signaling with recombinant Wnt-3a, or by overexpression of ␤-catenin, reduced the anti-proliferative activity of lenalidomide. Conversely, suppression of ␤-catenin with small hairpin RNAs restored plasma cell sensitivity to lenalidomide. Together, these findings support the hypothesis that lenalidomide mediates activation of Wnt/␤-catenin signaling in plasma cells as a mechanism of inducible chemoresistance through effects at the transcriptional and post-translational levels.

Resistance of myeloma to lenalidomide is an emerging clinical problem, and though it has been associated in part with activation of Wnt/β-catenin signaling, the mediators of this phenotype remained undefined. Lenalidomide-resistant models were found to overexpress the hyaluronan (HA)-binding protein CD44, a downstream Wnt/β-catenin transcriptional target. Consistent with a role of CD44 in cell adhesion-mediated drug-resistance (CAM-DR), lenalidomide-resistant myeloma cells were more adhesive to bone marrow stroma and HA-coated plates. Blockade of CD44 with monoclonal antibodies, free HA, or CD44 knockdown reduced adhesion and sensitized to lenalidomide. Wnt/β-catenin suppression by FH535 enhanced the activity of lenalidomide, as did interleukin-6 neutralization with siltuximab. Notably, all-trans-retinoic acid (ATRA) down-regulated total β-catenin, cell-surface and total CD44, reduced adhesion of lenalidomide-resistant myeloma cells, and enhanced the activity of lenalidomide in a lenalidomide-resistant in vivo murine xenograft model. Finally, ATRA sensitized primary myeloma samples from patients that had relapsed and/or refractory disease after lenalidomide therapy to this immunomodulatory agent ex vivo. Taken together, our findings support the hypotheses that CD44 and CAM-DR contribute to lenalidomide-resistance in multiple myeloma, that CD44 should be evaluated as a putative biomarker of sensitivity to lenalidomide, and that ATRA or other approaches that target CD44 may overcome clinical lenalidomide resistance.

No abstract

No abstract