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Restore the Secretion and Activity of Factor VIII Mutants By Proteostasis Regulator

Quantitative or qualitative deficiency in factor VIII (FVIII) result in hemophilia A. FVIII deficiency is caused by broad spectrum mutations, and missense mutation is the most common type of mutation, which lead to various defects of FVIII from decreased secretion to impaired function. Cellular protein homeostasis is disturbed by FVIII mutants, and thus excessive misfolding and degradation of FVIII mutant result in FVIII deficiency. We hypothesized that folding and/or trafficking of FVIII can be enhanced by proteostasis regulators, leading to increased coagulation factor levels sufficient to restore hemostasis. We performed a cell-based screening using a FDA-approved drug library and a natural compound library looking for compounds that enhance secretion of FVIII with a B domain deletion. We identified SAHA (Suberanilohydroxamic acid) as a compound that increased both wild-type (WT) and a missense mutant FVIII. SAHA is a histone deacetylase inhibitor that was also reported to enhance proteostaisis of mutant CFTR, a-1 antitrypsin, lysosomal glucocerebrosidase and the GABA receptor. Here we show that SAHA can increase the secretion and activity of FVIII mutants by enhancing their folding and trafficking as well transcription.
 To confirm the ability of SAHA to enhance FVIII mutant secretion, we first used the WT FVIII A2 domain and the M614T mutant A2 domain to detect the proper working time and concentration of SAHA in HEK 293T cells. M614T is a missense mutation that was shown to cause secretion defect of FVIII in our previous study. Western blot showed a dose-dependent increase in levels of both A2-WT and A2-M614T in cell culture media between 0 and 2.5ug/ml of SAHA after 24-hour treatment. Levels of both WT and mutant A2 domain dropped when SAHA concentration exceeded 5 ug/ml due to cellular toxicity. SAHA enhanced the secretion of A2-M614T much more than that of A2-WT. Of note, protein levels of both A2-WT and A2-M614T in cell lysate were also slightly elevated by SAHA. Time course study showed that the maximum effect on secretion was achieved after 24 hours of SAHA treatment.
 We next studied the effect of SAHA on activity and secretion of several full-length FVIII mutants (A469S, R527W, R531G, I566T, N582D, S584T, R593C, M614T, R1997G, R2150C), which were shown to decrease FVIII secretion due to various defects in intracellular trafficking in our previous studies. Except for N582D, activity levels of all FVIII mutants in cell culture media were elevated 3-14 folds after a 24-hour SAHA treatment (P<0.05), whereas the activity level of FVIII WT only increased by ~2 folds (P<0.05). Antigen levels of FVIII mutants in both cell lysates and cell culture media increased by ~3 folds following SAHA treatment (P<0.05). The increase in activities of several FVIII mutants (I566T, S584T, R593C, R1997G, R2150C) is higher than the increase in antigen levels of the same mutants in cell culture media, which indicates that, in addition to enhancing secretion, SAHA treatment also increased the proportions of functional proteins in secreted FVIII mutants in cell culture media.
 To investigate the mechanism, we studied the interactions of WT FVIII and several responsive mutant FVIII (I566T, S584T, R593C and R1997C) with endoplasmic reticulum (ER) chaperones BiP and calreticulin by co-Immunoprecipitation. The protein levels of BiP and calreticulin in HEK 293T cells were not affected by SAHA. However, the interactions of BiP with both WT FVIII and FVIII mutants were remarkably enhanced by SAHA, while no significant differences in the interactions of calreticulin with FVIII WT or FVIII mutants were observed. The effect of SAHA on FVIII secretion can be blocked by simultaneously treating cells with a BiP inhibitor SubAB. We next measured mRNA levels of BiP as well as FVIII in cells expressing FVIII WT and FVIII mutants with or without SAHA treatment via real time PCR. Similar to protein levels, BiP mRNA levels were not changed by SAHA. However, FVIII mRNA levels were elevated by 1.2 to 3.1 folds (P<0.05) by SAHA, in keeping with the histone deacetylase inhibitor activity of SAHA.
 In summary, we have shown that SAHA increases FVIII antigen and activity levels in cell culture media by increasing FVIII mRNA levels and by enhancing its folding and trafficking via specifically promoting its interaction with BiP. SAHA is a potential proteostaisis regulator that can increase the secretion and restore the function of certain FVIII missense mutants.
 
 
 No relevant conflicts of interest to declare.
