Characterization of a PERK Kinase Inhibitor with Anti-Myeloma Activity
Authors: Bagratuni Tina , Mavrianou Nefeli , Kastritis Efstathios , Liakos Christine , Terpos Evangelos , Dimopoulos Meletios A. 
Published: October 2019

Abstract
The malignant plasma cell of Multiple Myeloma (MM) is closely linked to dysregulated unfolded protein response in the ER due to increased production of immunoglobulin and the metabolic demands of malignant uncontrolled proliferation. Due to the immunoglobulin production, MM plasma cells are dependent on the unfolded protein response process (UPR), which controls protein production and ensures its proper translation and folding. A study by Michallet et al (2011) showed that knockdown of one of the three well-known arms of the UPR, PERK (protein kinase R (PKR)-like ER kinase) in MM cells resulted in autophagic cell death. This outcome indicated the important role of PERK activation for the metabolic shift of plasma cell to myeloma cell but it also showed that PERK knockdown could also impede the apoptotic effect. Therefore as PERK activity has been implicated in the survival of MM cells as well as with the apoptotic potential of the cells, targeting this pathway in MM may be an attractive therapeutic approach. In this study we used a small ATP competitive molecular inhibitor, GSK2606414, that acts by targeting PERK enzyme activity in its inactive DFG conformation at the ATP-binding region, while displaying ≥385 fold selectivity over c-kit, Aurora B, BRK and many other kinases.
To examine the expression of PERK in MM, we initially screened 25 CD138+ MM patients and 6 human myeloma cell lines (HMCLs) for PERK mRNA expression. Our results showed that PERK mRNA is highly expressed in almost all patients (5-10 fold higher than the mean PERK expression of HMCLs) whereas differentially expressed in HMCLs.
To test the effect of GSK2606414 on the proliferation of MM cells, 4 HMCLs were treated with different doses of GSK2606414 at two time points (24 and 48 hours). Treatment of cells with 3-30μM GSK2606414 resulted in a dose-dependent inhibition of cell proliferation in all HMCLs ranging for 20-95% reduction of proliferative activity, thus, indicating the dependency of these cells on PERK activity.
We further studied the induction of apoptosis in GSK2606414 treated HMCLs samples using Annexin-PI staining. Treatment with GSK2606414 at 20μM in H929 and L363 HMCLs for 24 hours resulted 25 % and 15 % increase in apoptotic cells respectively compared to the untreated cells. However, the most important finding was a significant synergistic effect of GSK2606414 with bortezomib in these cells. Specifically when H929 and L363 cells were treated with 5nM bortezomib in combination with 20μM GSK2606414, the outcome resulted in a synergistic effect where apoptotic cells reached 99% and 77% respectively, whereas bortezomib only treated cells resulted in 87% and 42% Annexin-PI positively stained cells respectively. These finding show the ability of GSK2606414 to enhance the apoptotic effect of bortezomib in myeloma cells and act as an attractive therapeutic anti-myeloma strategy
Under ER stress conditions, the activation of ATF6 and PERK/eIF2α leads to the induction of ATF4 translation and results in the upregulation of CHOP. To determine the gene target effects of GSK2606414, ATF4 and CHOP mRNA expression levels were determined in H929 cell line after 24 hour of treatment. Treatment with GSK2606414 alone did not alter the expression levels of CHOP but reduced more than 50% the expression levels of ATF4. When combined with bortezomib CHOP and ATF4 levels were reduced 20% and 60% respectively while treatment with bortezomib alone increased the levels of CHOP and ATF4 by 50-100%.
To test the hypothesis that ATF4 and CHOP expression is also suppressed during sustained UPR response, two myeloma cell lines, H929 and L363, were pre-treated with GSK2606414 and then subjected to ER stress conditions by treatment with tunicamycin (TM). In the presence of exogeneous UPR inducers, such as TM GSK2656157 had a significant effect on the expression of the above genes at much lower IC50 range (≤100 nM) suggesting the in a more active UPR (sustained ER stress) the effects are more pronounced. Thus, the combination of GSK2606414 with bortezomib may inhibit one of the mechanisms of MM cells to escape ER stress induced by bortezomib.
In conclusion, given the on-target pharmacological effects of PERK inhibitor on MM, development of PERK inhibitors may offer a therapeutic advantage by acting on another node of UPR signaling which directly or indirectly would affect MM pathogenesis and treatment.

Disclosures
Dimopoulos: Genesis: Honoraria; Novartis: Honoraria; Onyx: Honoraria; Celgene: Honoraria; Janssen: Honoraria; Janssen-Cilag: Honoraria; Amgen: Honoraria.