Intracellular Accumulation Of Light Chains Caused By Lenalidomide, and Mediated Via CRBN, Causes Major ER Stress and Is Implicated In The Synergistic Combination Of Imids and Proteasome Inhibitors
Authors: Sebastian Sinto , Zhu Yuan Xiao , Braggio Esteban , Panchabhai Sonali , Shi Chang-Xin , Ahmann Gregory , Bergsagel Leif , Fonseca Rafael 
Published: October 2019

Background
The precise molecular mechanism and targets of lenalidomide specific anti-Multiple Myeloma (MM) activity remain largely unknown, although, cereblon (CRBN) has been identified as a central mediator of activity with IRF-4 acting as a downstream molecule. Lenalidomide resistance in MM cells which, despite CRBN depletion, are able to restore their IRF-4 levels suggests the existence of alternative pathways.

Methods
To measure apoptosis and gene expression analysis 1x 106cells were incubated with 10μm to 50μm of lenalidomide for 24 to 96 hours before they were examined by annexin-PI and FACS analysis. Gene and protein expression were measured by RT-PCR, western blot, and immunohistochemistry. For lenalidomide followed bortezomib serial studies, cells were incubated with 10μm of lenalidomide for 48 hours and later treated with bortezomib for 24 to 48 hrs. CRBN and immunoglobulin light chain knock-down were performed by lentiviral mediated shRNA.

Results
We found lenalidomide induces Endoplasmic reticulum stress (ERS) via accumulation of intracellular immunoglobulin proteins, but only in the presence of CRBN in a panel of MM cell lines. This accumulation of intracellular proteins resulted in ERS as documented by western blot and RT-PCR in OPM2 and MMIS cell lines. We next utilized OPM2 cells knocked down for CRBN and control, non-target (NT), and treated with different doses of lenalidomide. After three days of treatment an ERS response was occurring in OPM2-NT cells but not in CRBN knockdown cells. Using Western blot we found decreased XBP-1u and increased XBP-1s, ERS marker proteins, in CRBN positive (NT) cells after 72 hours of lenalidomide treatment, but not in CRBN knock-down cells. Another ERS marker GRP78/BiP was also clearly accumulated after lenalidomide treatment in CRBN positive cells.
Inactivation of the tumor suppressor p53 by degradation is a mechanism utilized by cells to adapt to ERS has been reported. We also found that p53 protein down regulated after lenalidomide treatment in CRBN positive OPM2 and MMIS cells. We have analyzed other isogenic cell lines MM1.S (lenalidomide-sensitive) and isogenic MM1.Sres (lenalidomide-resistant) HMCLs and found that lenalidomide induces ERS in MM1.S but not MM1.Sres cells. CRBN is important for lenalidomide to induce immunoglobulin protein accumulation and ERS was further proved by over-expressing CRBN in OCI-My5 MM cell line and subsequent lenalidomide treatment. To test the effect of light chain accumulation as ER stressors, we knocked down immunoglobulin light chain lambda in human MM cell line OPM2. Knock-down of lambda light chain in OPM2 cells resulted in lenalidomide resistance and it induced endoplasmic reticulum stress mediated anti-myeloma activity.
Our preliminary data reveals that lenalidomide mediated progressive ERS can positively enhance bortezomib induced apoptosis in an in-vitro MM model. We pre-treated myeloma cell lines with lenalidomide and subsequently treated with bortezomib. MM cells pre-treated with lenalidomide for two days and there after treated with bortezomib clearly shown increased sensitivity to bortezomib induced apoptosis than non-pre-lenalidomide treated cells.

Conclusion
Here we show that lenalidomide causes accumulation of immunoglobulin light chains, inducing progressive ER stress, resulting in anti-myeloma activity and that effect depends on the presence of CRBN. Proteasome inhibitors such as bortezomib also sensitize MM cells to endoplasmic reticulum stress-mediated apoptosis. Our data demonstrated the synergistic effect of lenalidomide and proteasome inhibitors, likely due to the augmented protein stress. This data support the use of serial therapy utilizing pre-lenalidomide treatment followed by bortezomib or carfilzomib treatment.

Disclosures:
Fonseca: Medtronic: Consultancy; Otsuka: Consultancy; Celgene: Consultancy; Genzyme: Consultancy; BMS: Consultancy; Lilly: Consultancy; Onyx: Consultancy, Research Funding; Binding Site: Consultancy; Millennium: Consultancy; AMGEN: Consultancy; Cylene: Research Funding; Prognostication of MM based on genetic categorization of the disease: Prognostication of MM based on genetic categorization of the disease, Prognostication of MM based on genetic categorization of the disease Patents & Royalties.