Bortezomib Improves Endothelial Thromboresistance Via Induction of KLF Transcription Factors.
Authors: Hiroi Toyoko , Deming Clayton B , Zhao Haige , Hansen Baranda S , Arkenbout Elisabeth K , Myers Thomas J , Riordan William J , McDevitt Michael A , Rade Jeffrey J 
Published: April 2020

Abstract
Background: Patients with multiple myeloma (MM) are at high risk for venothromboembolic events (VTE). Recent studies, however, suggest that MM patients treated with bortezomib, an approved proteasome inhibitor with potent NF-kB inhibitory effects, appear to have a lower risk of VTE compared to those treated with other therapies. We hypothesize that this could be due to a beneficial effect of bortezomib on endothelial thromboresistance.
Methods and Results: Human umbilical vein endothelial cells (HUVECs) were incubated with bortezomib for 20 hours and changes in the expression of a panel of coagulation and inflammation-related genes measured by qPCR. Bortezomib stimulated baseline expression of anticoagulant genes (thrombomodulin (TM), eNOS and tissue factor pathway inhibitor), suppressed baseline expression of pro-coagulant genes (vWF and protease activated receptor-1) and suppressed cytokine-mediated induction of E-selectin, VCAM-1 and tissue factor. Most pronounced, was the dose-dependent upregulation of TM, a member of the protein C anticoagulant pathway (229 ± 15% and 341 ± 7% of control, at 5 nM and 10 nM bortezomib, respectively, p &lt;0.0001). Induction of TM gene expression was paralleled by a significant upregulation of TM protein expression, assessed by Western blot analysis, and by an increased capacity to generate activated protein C (205 ± 5% of control with 5 nM bortezomib, p &lt;0.0001). Bortezomib-induced TM upregulation was blocked by cycloheximide, suggesting that induction of a transcriptional pathway, and not simply inhibition of the NF-kB pathway, was required. We therefore examined the effects of bortezomib on the expression of several Krüppel like transcription factors (KLFs) that are known to be important regulators of TM expression and endothelial thromboresistance. Bortezomib significantly upregulated the expression of KLF2, KLF4 and KLF6 in HUVECs (18 ± 1, 8 ± 1 and 2 ± 0.1-fold of control, respectively, p &lt;0.0001 for each) following a 5 nM exposure for 20 hours. Knock-down experiments using small interfering RNAs revealed that KLF2 and KLF4, but not KLF6, play critical and synergistic roles in mediating bortezomib-induced TM upregulation. To determine the in vivo significance of these findings, mice were administered ascending doses of bortezomib for 7 days and TM expression measured in selected tissue. Compared to controls, a dose of 0.8 mg/kg bortezomib ip increased TM gene and protein expression in the liver by 7.0 ± 1.1 and 9.5 ± 2.9-fold, respectively (p &lt;0.0001 for each) and TM gene expression in the kidney by 2.5 ± 0.2-fold (p &lt;0.0001). There was no significant change in TM expression observed in heart and lung tissue. Paralleling changes in TM expression, expression of the KLF2 and KLF4 genes was also increased in the liver (2.1 ± 0.3 and 6.2 ± 1.5-fold of controls, respectively, p &lt;0.001 for each) and kidney (1.9 ± 0.2 and 2.9 ± 0.3-fold of controls, respectively, p &lt;0.01 for each), but not in heart or lung tissue.
Conclusions: Bortezomib markedly stimulates endothelial TM expression, both in vitro and in vivo in a tissue-specific manner. TM upregulation appears dependent on the induction of KLF2 and KLF4 transcription factors rather than by inhibition of the NFkB pathways. Our findings provide a rationale for further studies of bortezomib-induced enhancement of endothelial thromboresistance in patients with MM and may help explain why these patients are reduced risk for VTE.