Quantitative Proteomic Analysis of Relapsed Multiple Myeloma Identifies CDK6 Upregulation As a Potential Targetable Resistance Mechanism for Lenalidomide

Lenalidomide, an immunomodulatory drug (IMiD), is highly active and broadly used for the treatment of multiple myeloma. Despite high initial remission rates, patients frequently relapse and become resistant to the drug. Comprehensive analyses of gene mutations and RNA expression have identified inactivating mutations and RNA downregulation in cereblon (CRBN), the primary target of lenalidomide, in some of the resistant patients. However, the underlying resistance mechanism for the majority of cases remains unknown. Here, we performed quantitative tandem mass tag (TMT)-based proteomic analyses and RNA sequencing in five paired pre-treatment and relapse samples from multiple myeloma patients treated with drug combinations comprising lenalidomide to identify changes in protein expression associated with resistance. Using a stringent cut-off with an adjusted P value < 0.1 and log2 fold change (FC) > 2, we found 7 proteins to be significantly upregulated and 10 proteins to be downregulated in the relapsed versus pre-treatment multiple myeloma samples. Of these 17 deregulated proteins at relapse, only two were also found to be deregulated on the RNA expression level (adjusted P value < 0.1) as assessed by RNA sequencing. In general, correlation between protein expression levels and RNA expression levels were weak (median Pearson correlation coefficient r=0.35).

Among the top upregulated proteins in relapse samples was cyclin-dependent kinase 6 (CDK6) with an average log2 FC of 2.1. Protein and RNA levels of CDK6 showed only weak correlation (r=0.4) and CDK6 RNA was not differentially expressed between the relapse and pretreatment samples. To validate the findings of the proteomic analysis, we assessed CDK6 protein levels by western blot in additional patient samples obtained at diagnosis (N=4) and at relapse (N=9). This confirmed a high CDK6 protein expression in 6 of 9 relapse samples while CDK6 could not be detected in the 4 pre-treatment samples. In order to determine the impact of CDK6 on drug sensitivity, we overexpressed CDK6 using either a retro- or lentiviral vector system in multiple myeloma cell lines. In two multiple myeloma cell lines tested, MM.1S and OPM2, CDK6 overexpression reduced sensitivity to lenalidomide and pomalidomide, but not to melphalan, bortezomib, or dexamethasone.

To examine whether lowered IMiD-sensitivity can be overcome by CDK6 inhibition, we treated multiple myeloma cell lines either with the CDK6 inhibitor palbociclib, an IMiD-based CDK6-selective proteolysis targeting chimera (PROTAC) or a non-selective CDK6-PROTAC which is also capable of pomalidomide-mediated degradation of IKZF1 and IKZF3 (Brand et al., Cell Chem Biol 2019). Both palbociclib and CDK6-selective PROTAC as single treatments had only mild effects on the majority of multiple myeloma cells, implying that multiple myeloma cells are generally not dependent on CDK6. In contrast, the combination treatment of palbociclib with lenalidomide, or the non-specific CDK6/IKZF1/IKZF3-targeting PROTAC significantly inhibited proliferation, producing synergistic effects on the decrease of myeloma cell viability in 6 multiple myeloma cell lines, including those with a low IMiD sensitivity like RPMI-8226 and L363. This demonstrates that CDK6 inhibition or degradation enhances the cytotoxic effects of IMiDs. In order to investigate a potential mechanism for the synergistic effects of CDK6 inhibition and IMiDs, we analyzed protein levels in treated cells. CDK6 inhibition or degradation had no effect on CRBN protein levels nor on lenalidomide-induced degradation of IKZF1 and IKZF3. In contrast, combined degradation of CDK6, IKZF1, and IKZF3 revealed decreased protein levels of c-MYC, which was not observable in cells treated with palbocicilib, CDK6-selective PROTAC or pomalidomide alone.

In conclusion, quantitative proteomics in primary multiple myeloma samples identified new druggable candidates including CDK6 in relapse that were overlooked by RNA expression analyses. Inhibition of CDK6 by palbociclib or a PROTAC sensitizes multiple myeloma cells to IMiDs and results in synergism when used in combination.