The IMiD-Target Cereblon Determines Transmembrane Protein Quality Control Promoting Tumor Metabolism

Introduction:

Cereblon (CRBN) is the target for immunomodulatory drugs (IMiDs) such as thalidomide and its derivatives lenalidomide and pomalidomide, which are key therapeutics for hematologic malignancies such as multiple myeloma (MM) and del(5q) myelodysplastic syndrome (MDS). We have previously described a ubiquitin-independent chaperone-like function of CRBN, which stabilizes the transmembrane proteins CD147 and MCT1. IMiDs interfere with this chaperone-like function of CRBN in a competitive manner to mediate both their anti-tumor and their teratotoxic effects (Eichner et al. Nature Medicine 2016). So far, the underlying mechanisms of transmembrane protein maturation, the global impact of CRBN on the cell surface proteome and the precise molecular mechanism of IMiDs, especially their clinically well-established synergy with proteasomal inhibitors remain unclear.

Methods:

Novel CRBN-clients were identified by cross-validation of the CRBN-interactome with a cell surface proteomic screen. Various molecular and cell biological methods including immunofluorescence, flow cytometry, immunoprecipitations, GST-pulldowns, amino acid transport and proliferation assays were used to decipher underlying mechanisms. In vitro assays and in vivo xenograft experiments were performed using MM cell lines and patient-derived CD138+ MM cells. ¹⁸FDG- and ¹⁸FET-PET was used for imaging of xenografted tumors.

Results:

Our unbiased screening approaches imply a global role of CRBN in transmembrane protein maturation. In particular, we identify the amino acid transporter LAT1 and its functional subunit CD98hc as novel CRBN client proteins, which are frequently overexpressed in MM to drive cell proliferation. CD98hc/LAT1 become destabilized and inactivated upon IMiD treatment, which attenuates MM cell proliferation, tumor formation and perturbs the uptake of essential amino acids, thereby further linking IMiD-activity to tumor-metabolism. CD98hc/LAT1 destabilization only occurs in IMiD-sensitive patients and cell lines, thus being a potential biomarker to predict IMiD-response. Moreover, inhibition of LAT1 is cytotoxic in both IMiD-sensitive and -resistant cells, which makes it an attractive therapeutic option for IMiD-resistant and -refractory patients. Mechanistically, we show CRBN to function as a new selective co-chaperone of HSP90, which facilitates transmembrane protein maturation in a ubiquitin-independent way, which is impaired by IMiD-treatment.

Conclusion:

We establish CRBN as a transmembrane protein-specific co-chaperone for HSP90 and identify modulation of the CRBN-CD98hc/LAT1 axis as crucial means by which IMiDs mediate their anti-tumor activity. Notably, we specify CD98hc/LAT1 as valuable biomarkers for IMiD-response and druggable targets for IMiD-resistant and -refractory MM patients and beyond. Furthermore, this ubiquitin-independent mechanism solves the paradox regarding the well documented synergistic anti-myeloma activity of IMiDs and proteasomal inhibitors.