CREB1 and TXNIP Regulate Autophagy and Unfolded Protein Response in Multiple Myeloma

Introduction:

CREB1 (cAMP responsive element binding protein 1) is a transcription factor involved in several aspects of cellular growth and metabolism. We previously demonstrated that CD56 (also known as neuronal cell adhesion molecule 1), a surface glycoprotein aberrantly expressed in more than seventy percent of patients with multiple myeloma (MM), promotes CREB1 phosphorylation and its binding to the DNA, leading to genomic transcription. CREB1 inhibitors are toxic towards MM cells and activate natural killer cell immunity by decreasing HLA-E expression. In this study, we are characterizing the role of CREB1 in modulating autophagy and unfolded protein response in MM.

Results:

In silico analysis of RNA-sequencing data from patients with MM in the CoMMpass database showed that patients with high CREB1 expression have increased activation of pathways involved in proteostasis and autophagy, such as the mTOR (Mammalian target of rapamycin) and unfolded protein response (UPR) pathways. Active mTOR in MM prevents activation of autophagy, limiting the ability of the cells to remove excess of protein aggregates; conversely, the UPR is activated in response to the accumulation of misfolded proteins (proteotoxic stress). Both pathways are relevant to MM survival, given the large amounts of produced immunoglobulins.

We first evaluated whether CREB1 was modulating the mTOR pathway. Indeed, the overexpression of CREB1 in U266 cells induced several markers of activation of the mTOR pathway, including phosphorylation of mTOR itself and its downstream targets, 4EBP1 and PS6. Conversely, treatment with the CREB1 inhibitor, 666-15, reduced phospho-4EBP1 and phospho-mTOR and increased markers of autophagy such as LC3, ATG5, and ULK1. Quantitative PCR in the same conditions increased mRNA levels of ATG3, ATG5, and ATG7, and of TSC1 and AMPK, negative regulators of mTOR. An increase in autophagy was confirmed via flow cytometry using the quantitative assay CYTO-ID autophagy detection kit in several treated MM cell lines (OPM-2, H929, and RPMI-8226 cells).

We then identified that CREB1 also regulates several proteins involved in the UPR. Overexpression of CREB1 in U266 cells upregulated PERK and downregulated IRE1-alpha expression. Conversely, CREB1 inhibition induced early increase at 4-6 h treatment of the total and phosphorylated form of IRE1-alpha, with normalization at 24 hours, and increase of the spliced form of XBP1, followed by reduction of both spliced and unspliced XBP1 forms at 24 hours. Treatment with the CREB1 inhibitor also led to a decrease in markers of the adaptive arm of UPR—ATF6 and PERK, indicating increased reliance on the apoptotic arm. Proteasome inhibitors (PIs) heighten ER stress by preventing the degradation of substrates and reducing stress signaling via PERK and XBP1 inhibition. Previous studies reported that CD56 expression, which is upstream to CREB1, is relevant to responses to PIs. Combining CREB1 inhibition with PIs, such as bortezomib and carfilzomib, was indeed additive in terms of cell death and statistically increased the expression of pro-apoptotic CHOP and GADD34.

TXNIP is a protein at the crossroads of UPR, autophagy, and reactive oxygen species (ROS) clearance. While downregulated in some cancers, TXNIP is markedly overexpressed in MM cells compared to normal plasma cells. TXNIP gene is located in the 1q cytoband, an area amplified in 30 percent of patients with MM. CREB1 inhibition downregulates TXNIP protein expression, by FBXO22 upregulation. Given the high expression of TXNIP in patients and its modulation by CREB1, we reasoned that TXNIP could be relevant for MM growth. Indeed, H929 and OPM-2 cells silenced for TXNIP grew slower than control cells and had increased apoptosis and ROS production. Differences in markers of UPR including PERK, GADD34, and CHOP were also noted in TXNIP silenced cells. Finally, we tested a specific TXNIP inhibitor, named SRI-7330, in a panel of MM cell lines. This inhibitor was able to reduce TXNIP expression, induce cell death, and activate apoptotic UPR by GADD34 and CHOP expression.

Conclusions:

In conclusion, our work describes a novel mechanism of regulation of UPR and autophagy by CREB1 in MM. We also identified TXNIP as a key regulator of this phenomenon, and show that CREB1 and TXNIP inhibition are both toxic in MM.