Lenalidomide, Pomalidomide and CC-220 Resistant Multiple Myeloma: Confirmation of Role of CRBN/IRF4 Axis and Identification of Multiple Disruption Mechanisms

The immunomodulatory drugs (IMiDs) thalidomide, lenalidomide and pomalidomide, play a pivotal role in the treatment of multiple myeloma (MM). However, while the majority of newly diagnosed MM patients respond to IMiD therapy, most patients eventually develop resistance.

Cereblon (CRBN) expression has been associated with IMiDs resistance. Using a MM-specific targeting sequencing panel, we previously reported acquired mutations of CRBN or other genes in the CRBN E3 ligase complex or in the downstream CRBN pathway in 12% and 10% of MM patients refractory to IMiDs respectively. Likely this is an underestimate of pathway disruption in resistant disease since structural variation was not assessed. Notwithstanding it is clear that some IMIDs resistant MM cases failed to demonstrate any abnormality in the CRBN pathway.

In this report, we describe the establishment of four isogenic human MM cell lines (HMCLs) respectively sensitive and resistant to lenalidomide, generated by culturing lenalidomide-responsive cell lines (MM.1S, KMS11, XG1 and OPM2) in the presence of lenalidomide for an extended time. Those cell lines were further tested for their responses to other IMiD members (pomalidomide and CC-220) and Bortezomib (as a control). All of the 4 isogenic resistant lines showed resistance to each of the IMiDs, but not to Bortezomib. To identify the underlying mechanisms for resistance, immunobotting was performed for expression of CRBN, IKZF1/3, IRF4, MYC, CD147, MCT1, CTNNB1 and ERK1/2. Three of the four resistant cell lines were identified as having a significant decrease or absence of CRBN expression compared to their isogenic IMiDs-sensitive lines. Consistently, CRBN mediated degradation of IKZF1 and IKZF3 and down-regulation of IRF4 and MYC upon lenalidomide treatment in those cells was abrogated or substantially reduced. Lenalidomide induced-CD147 and MCT1 destabilization was also inhibited in resistant cells. When wild type-CRBN was re-introduced, sensitivity to lenalidomide was completely restored, confirming that CRBN deficiency or dysfunction mediated the resistance. Genomic analysis of two resistant cell lines indicated that MM.1Sres carries both a CRBN deletion and a point mutation on the second allele, while KMS11res harbors a structural deletion of CRBN.

Surprisingly one of the resistant cell lines, XG1res, still has abundant CRBN expression. Accordingly, lenalidomide induced-degradation of IKZF1 and IKZF3 in XG1res is identical to its isogenic sensitive line XG1, and the introduction of exogenous CRBN into XG1res did not restore its sensitivity to lenalidomide. In both XG1 and XG1res, a truncated IRF4 resulting from a genomic inversion is identified. This truncated IRF4 is substantially decreased upon lenalidomide treatment in XG1 but not in XG1res. In addition, CD147 expression is upregulated in XG1res. To understand if CD147 expression contributes to acquired resistance, CRISPR-cas9 technology was used to knockout endogenous CD147 . Depletion of CD147 in XG1res and three additional HMCLs (including two IMiD sensitive cell lines, MM.1S and KMS11, and one IMiD resistant cell line, RPMI8226)did not impact survival nor did it restore (or change) lenalidomide sensitivity even though CD147 depletion also induced a substantial reduction of MCT1 expression. mRNAseq was performed in isogenic XG1 and XG1res. Two genes associated with myeloma growth, IL-6 and STAT3, were identified as highly upregulated in XG1res. We confirmed by increased and highly activated STAT3 in XG1res. XG1res was then treated with lenalidomide plus either a specific STAT3 inhibitor (PB-1-102) or an IL-6 antibody. Compared with each drug as single agent, the combination of PB-1-102 and lenalidomide demonstrated the highest anti-myeloma activity in XG1res on MTT assay and the most downregulated IRF4 expression on immunoblotting, suggesting that activation of STAT3 was the source of IMiDs resistance, perhaps as a consequence of its regulation of the truncated IRF4.

In summary, we confirm CRBN/ IKZF/IRF4/MYC pathway deficiency or dysfunction, by mutation or deletion, as the dominant mechanism associated with acquired IMIDs resistance. We also identify a novel indirect path to resistance mediated by upregulated IL-6 and STAT3 activation with consequent impact on IRF4 expression.