DGAT2 Inhibition Alters Aspects of Triglyceride Metabolism in Rodents but Not in Non-human Primates

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1 Article DGAT2 Inhibition Alters Aspects of Triglyceride Metabolism in Rodents but Not in Non-human Primates Graphical Abstract Authors David G. McLaren, Seongah Han, Beth Ann Murphy,..., Stephen F. Previs, Jason E. Imbriglio, Shirly Pinto Correspondence (D.G.M.), (S.H.), (S.P.) In Brief DGAT2 catalyzes the final step in triglyceride synthesis and regulates VLDL production. Using a variety of methods and preclinical models, McLaren et al. show that while DGAT2 inhibition is effective at correcting dyslipidemia in murine models of obesity, these beneficial effects are not translated in vivo in rhesus primates. Highlights d Inhibition of DGAT2 in rodents yields submaximal suppression of VLDL-TG secretion d d DGAT1 compensates for loss of DGAT2 activity supporting hepatocyte TG secretion Inhibition of DGAT2 in rhesus does not affect plasma triglycerides or VLDL-apoB McLaren et al., 2018, Cell Metabolism 27, June 5, 2018 ª 2018 Elsevier Inc.

2 Cell Metabolism Article DGAT2 Inhibition Alters Aspects of Triglyceride Metabolism in Rodents but Not in Non-human Primates David G. McLaren, 2,6,8, * Seongah Han, 1,6, * Beth Ann Murphy, 2 Larissa Wilsie, 1 Steven J. Stout, 2 Haihong Zhou, 1 Thomas P. Roddy, 1 Judith N. Gorski, 2 Daniel E. Metzger, 2 Myung K. Shin, 3 Dermot F. Reilly, 3 Heather H. Zhou, 1 Marija Tadin-Strapps, 3 Steven R. Bartz, 4 Anne-Marie Cumiskey, 2 Thomas H. Graham, 5 Dong-Ming Shen, 5 Karen O. Akinsanya, 4 Stephen F. Previs, 1 Jason E. Imbriglio, 5 and Shirly Pinto 1,7, * 1 Division of Cardio Metabolic Disease, Merck & Co., Inc., Kenilworth, NJ 07033, USA 2 Pharmacology, Merck & Co., Inc., Kenilworth, NJ 07033, USA 3 Genetics and Pharmacogenomics, Merck & Co., Inc., Boston, MA 02115, USA 4 Business Development and Licensing, Merck & Co., Inc., Kenilworth, NJ 07033, USA 5 Discovery Chemistry, Merck & Co., Inc., Kenilworth, NJ 07033, USA 6 These authors contributed equally 7 Present address: Kallyope Inc., 430 East 29th Street, New York, NY 10016, USA 8 Lead Contact *Correspondence: david.mclaren@merck.com (D.G.M.), seongah_han@merck.com (S.H.), shirly@kallyope.com (S.P.) SUMMARY Diacylglycerol acyltransferase 2 (DGAT2) catalyzes the final step in triglyceride (TG) synthesis and has been shown to play a role in regulating hepatic very-low-density lipoprotein (VLDL) production in rodents. To explore the potential of DGAT2 as a therapeutic target for the treatment of dyslipidemia, we tested the effects of small-molecule inhibitors and gene silencing both in vitro and in vivo. Consistent with prior reports, chronic inhibition of DGAT2 in a murine model of obesity led to correction of multiple lipid parameters. In contrast, experiments in primary human, rhesus, and cynomolgus hepatocytes demonstrated that selective inhibition of DGAT2 has only a modest effect. Acute and chronic inhibition of DGAT2 in rhesus primates recapitulated the in vitro data yielding no significant effects on production of plasma TG or VLDL apolipoprotein B. These results call into question whether selective inhibition of DGAT2 is sufficient for remediation of dyslipidemia. INTRODUCTION Despite the protective effect of statins and the multiple glucoselowering agents that have become available over the past decades, patients diagnosed with type 2 diabetes are still at higher risk for cardiovascular arterial diseases (Ginsberg, 2000). Some of the characteristics of these patient populations include increased very-low-density lipoprotein (VLDL) production, increased plasma triglyceride (TG) levels, hepatic steatosis, reduced levels of high-density lipoprotein (HDL) and apolipoprotein-a1, and increased small, dense low-density lipoprotein (LDL) particles (Adiels et al., 2008a, 2008b). Diabetic dyslipidemia, characterized by increased plasma TG and insulin resistance, is an important factor underlying increased risk of atherosclerosis in these individuals. It has been postulated that reducing VLDL-TG secretion by attenuating hepatic TG synthesis could lead to lowering of apolipoprotein B (apob) release and, ultimately, a reduction in the number of circulating LDL particles. Correspondingly, inhibition of hepatic TG synthesis in combination with statin treatment might lead to an increased benefit in terms of cardiovascular protection. The final, committed step in TG biosynthesis is catalyzed by the diacylglycerol acyltransferase enzyme of which there are two known isoforms, DGAT1 and DGAT2 (Chen and Farese, 2000). Although both DGAT1 and DGAT2 esterify diacylglycerol to triacylglycerol, the enzymes share no homology at the protein level and also differ with regard to their pattern of tissue expression (Yen et al., 2008). Through pharmacological and genetic manipulation, it has been demonstrated that the primary site of action of DGAT1 is in the gastrointestinal (GI) system where it acts as the primary enzyme responsible for the esterification of dietary fat. Indeed, DGAT1 deletion in rodents has been shown to have multiple beneficial effects with regard to obesity and diabetes phenotypes (Smith et al., 2000), principally arising through alteration of lipid absorption and commensurate stimulation of incretin release (Liu et al., 2013). Given these findings and the apparent promise of DGAT1 as a therapeutic target, multiple pharmaceutical companies discovered and developed small-molecule inhibitors of the enzyme. Proof of biology was achieved in the clinic; however, significant adverse effects related to GI intolerability were associated with inhibition of DGAT1 (DeVita and Pinto, 2013). It later emerged that DGAT1 loss-of-function mutations were associated with rare occurrences of congenital diarrheal disorders (Haas et al., 2012). Given these clinical findings it is significant to note that these adverse effects were not observed during preclinical development of DGAT1 inhibitors in rodents. In contrast to the successful manipulation of DGAT1, genetic deletion of DGAT2 was shown to be lethal with knockout mice dying shortly after birth and presenting with severe and systemic 1236 Cell Metabolism 27, , June 5, 2018 ª 2018 Elsevier Inc.

3 fatty acid substrate (e.g., exogenous versus de novo-synthesized fatty acids) and subcellular localization (Wilfling et al., 2013, 2014). DGAT1 has been localized to the ER whereas DGAT2 has also been detected in lipid droplets. A study using an in vitro hepatic system demonstrated that DGAT1 and DGAT2 can compensate for each other and together are responsible for hepatic TG synthesis (Li et al., 2015). Given the ambiguity around a dominant influence for DGAT2 in hepatic lipid metabolism, evidence to support that findings in rodents are translatable to higher preclinical species, and ultimately to humans, is of critical importance to the continued development of DGAT2 inhibitors as potential therapeutics. Herein we report the results of our investigations into the effects of DGAT2 inhibition on lipid metabolism using a variety of translational models including mice and non-human primates. In these studies we capitalized on the use of both pharmacological and genetic manipulation of DGAT2 activity using cellular and whole-body models of metabolic flux. Wherever possible, particular attention was directed toward comparing and contrasting the roles of DGAT2 and DGAT1 by addressing questions surrounding translational physiology, including the impact of nutritional status. RESULTS Figure 1. Structure, Pharmacokinetic, Physicochemical, Activity, and Selectivity Properties of Compound 2 reductions in TG (Stone et al., 2004). In the face of this perinatal lethal phenotype, the majority of our understanding regarding the metabolic role of DGAT2 has come from work utilizing antisense oligonucleotides (ASO) in rodents. Several chronic studies demonstrated beneficial effects related to hepatic lipid metabolism, including reductions in liver steatosis and VLDL-TG secretion. Additionally, reductions in plasma insulin, TG, free fatty acid (FFA), and total cholesterol were also observed, all closely associated with improvements in insulin sensitivity (Choi et al., 2007; Liu et al., 2008; Yu et al., 2005). These effects seem to be unique to inhibition of DGAT2, as ASO against DGAT1 did not lead to similar beneficial effects. Collectively these data support the notion that inhibition of DGAT2 activity could have a beneficial effect on dyslipidemia and insulin sensitivity and, similarly to the case for DGAT1, this has led to the development of several diverse small-molecule inhibitors of DGAT2 as potential therapeutics (Futatsugi et al., 2015, 2017; Kim et al., 2013). Although the liver is one of the primary sites for DGAT2 expression, DGAT1 is also expressed in the liver and the respective roles of the enzymes with regard to hepatic TG synthesis have not been well defined, especially across different species. It has been proposed that DGAT1 and DGAT2 may differ in their respective preferences for pools of DGAT2-Mediated TG Production Is Dependent upon Nutritional Status We have previously reported on the discovery and characterization of Compound 16, a selective and potent small-molecule inhibitor of DGAT2 (Imbriglio et al., 2015). Here we disclose a structurally distinct DGAT2 inhibitor, Compound 2, with comparable potency and selectivity against the enzyme (Figure 1). To test the efficacy of these DGAT2 inhibitors in vivo we administered [ 13 C 18 ]oleic acid intravenously to lean, wild-type mice that had been handled under conditions of different nutritional status. This tracer acts as a substrate for lipid synthesis (McLaren et al., 2011, 2013) and allowed us to measure the appearance of 13 C 18 -labeled TG 52:2 and 54:3 in plasma (Figures 2A and 2B, respectively). A selective inhibitor of microsomal triglyceride transfer protein (MTPi; CP ), which does not interfere with lipid synthesis but completely blocks packaging of TG into nascent lipoprotein particles and ultimately prevents secretion from the liver into the blood (Chandler et al., 2003), was included as a positive control. Acute treatment with either Compound 2 or Compound 16 showed effective reduction of newly synthesized TG appearing in plasma, and this effect was accentuated in overnight fasted and 4-hr refed mice as compared with a shorter 4-hr fasting condition. Appearance of TG in VLDL was also significantly reduced by treatment with Compound 2 in the fast-refeed paradigm (Figure 2D). Titration of Compound 2 revealed a leftward shift in the response curve and an increase in maximal effect under the fast-refeed paradigm compared with the 4 hr fast paradigm (Figure 2E). As anticipated, the overnight fasted and refed condition showed elevated plasma insulin (Figure 2F) suggestive of an increased window for hepatic lipogenesis and demonstrating overall a greater sensitivity to DGAT2 inhibition in the refed metabolic state. The fact that the plasma concentrations of Compound 2, Compound 16, and the MTP inhibitor were similar between the two different feeding paradigms (Figure 2C) suggests it is unlikely Cell Metabolism 27, , June 5,

4 Figure 2. The Appearance of Newly Synthesized Triglyceride in Plasma after Treatment with DGAT2 Inhibitors in an Acute Rodent Model (A and B) Newly synthesized triglycerides appearing in plasma in 4-hr-fasted or overnight-fasted + 4-hr-refed (O/N Fast; ReFed) mice administered Vehicle, Compound 2 (30 mg/kg, per os [p.o.]), Compound 16 (30 mg/kg, p.o.), or MTP inhibitor (3 mg/kg, p.o.). (C) The test compound concentrations measured from plasma. (D) Newly synthesized triglycerides measured in VLDL from O/N Fast; ReFed mice were treated with Vehicle, Compound 2 (30 mg/kg, p.o.), or MTP inhibitor (3 mg/kg, p.o.). (E) Titration of Compound 2 on TG 54:3 synthesis in 4-hr-fasted 1, 3, and 10 mg/kg (intravenously) or O/N-fasted/refed condition 0.1, 0.3, 1, 3, 10, 30, 50, and 100 mg/kg (p.o.). (F) Circulating insulin levels in O/N-fasted/refed compared with 4-hr-fasted mice. The levels of newly synthesized TG 52:2 or 54:3 of compound-treated mice was compared with their corresponding vehicle control. Percent inhibition of new TG synthesis of compound-treated mice relative to their respective vehicle is shown above the corresponding bar. **p < 0.01; error bars represent SEM. that a pharmacokinetic effect of nutritional status played any role in mediating the differences in efficacy observed. The Effects of DGAT2 Inhibition on Lipid and Glucose Metabolism in Obese Mice Having demonstrated the direct effects of DGAT2 inhibition on the appearance of TG in plasma, we set out to test the hypothesis that chronic attenuation of DGAT2 activity would improve dyslipidemia, hepatosteatosis, and insulin sensitivity. To explore this question, we tested AAV-short hairpin RNA (shrna)-mediated hepatic DGAT2 knockdown and in-feed administration of Compound 2 in a growing diet-induced obese (gdio) mouse model for 87 and 30 days, respectively. There was no change in body weight between AAV-shDGAT2 or DGAT2 inhibitor-treated animals when compared with control groups throughout the duration of the study (Figures 3A and 3B). Hepatic DGAT2 mrna expression levels were reduced by 88% after 87 days of AAV-shDGAT2 treatment (Figure S1A). The appearance of newly synthesized TG 54:3 in plasma was significantly reduced in both AAV-shDGAT2- and DGAT2 inhibitor-treated gdio mouse groups compared with control groups (Figure 3C), suggesting that DGAT2 activity was successfully 1238 Cell Metabolism 27, , June 5, 2018

5 Figure 3. Chronic DGAT2 Inhibition Suppresses TG Synthesis and Hepatic TG Contents in an Obese Mouse Model (A) Body weight growth of mice on a high-fat diet treated with PBS, AAV-shCon, and AAV-shDGAT2. (B) Body weight growth of mice on a high-fat diet treated with vehicle and Compound 2 dosed in feed, 100 mg/kg/day. (C) Newly synthesized plasma TG 54:3 at the end of treatment period with AAV-shDGAT2 or Compound 2. (D and E) Hepatic TG content of AAV-shDGAT2-treated or Compound 2-treated mice at the end of the study. Data presented are mean ± SEM (n = 9 10). Twotailed unpaired Student s t test: AAV-shDGAT2 versus AAV-shCon; Compound 2 versus vehicle or chow fed. *p < 0.05, **p < 0.01, ***p < See also Figure S1. attenuated by both treatments. In addition, there was no further reduction in TG 54:3 in an AAV-shDGAT2 + Compound 2 combination treatment group, suggesting that there was no significant residual DGAT2 activity following AAV-shDGAT2 silencing. Total liver TGs were also significantly reduced by chronic AAVshDGAT2 silencing (Figure 3D) and chronic treatment with Compound 2 (Figure 3E) supporting the expectation that, unlike the MTP inhibitor, there is a direct effect of DGAT2 inhibition on attenuating TG synthesis. Both AAV-shRNA and pharmacologically mediated hepatic DGAT2 inhibition led to reduced total plasma TG levels as well as reductions in total LDL and HDL cholesterol (Table 1). In addition, expression of other genes involved in lipid synthesis and metabolism including FASN, SCD-1, and PNPLA3 was reduced in AAV-shDGAT2- and DGAT2 inhibitor-treated animals compared with controls, suggesting a possible negative regulation of SREBP tone by DGAT2 inhibition (Figures S1C and S1D). Interestingly, neither DGAT2 silencing nor inhibition had any statistically significant effect on hepatic expression of DGAT1 (Figures S1A and S1B). Despite many of the beneficial changes in lipid profile, we observed no change in plasma insulin or glucose sensitivity (as assessed by a glucose tolerance test) in either the AAVshRNA- or DGAT2 inhibitor-treated groups when compared with controls (Table 1; Figures S2E and S2F). Cell Metabolism 27, , June 5,

6 Table 1. Metabolic Characteristics of Obese Mice (gdio) Treated with PBS, AAV-shCon, AAV-shDGAT2, Vehicle, DGAT2 Inhibitor (Compound 2), or Chow-Fed Lean Mice PBS AAV-shCon AAV-shDGAT2 Vehicle (Veh) DGAT2i (Compound 2) Chow Fed Total cholesterol, mg/dl ± ± ± 5.4*** ± ± 4.6*** ± 2.3*** LDL-C, mg/dl 32.2 ± ± ± 2.1* 20.6 ± ± 0.8*** 7 ± 1.6*** HDL-C, mg/dl ± ± ± 4.9*** ± ± 5.2*** ± 2.9*** Triglycerides, mg/dl ± ± ± 2.5** 96.3 ± ± 2.6*** 86.3 ± 5.3 Glucose, mg/dl a ± ± ± ± ± 5.1*** NA Insulin, ng/ml a 15.8 ± ± ± ± ± 0.2 NA n = Two-tailed unpaired Student s t test: *p < 0.05, **p < 0.01, ***p < 0.001, AAV-shDGAT2 versus AAV-shCon (control); Compound 2 versus vehicle (Veh) or chow fed. NA, no data available. a Plasma glucose and insulin levels were measured 26 days after Cpd2 treatment. DGAT2 and DGAT1 Act Synergistically to Affect TG Secretion from Primary Hepatocytes The ability of DGAT2 inhibitors to affect TG secretion in primary hepatocytes from multiple species was tested in vitro. Treatment with Compound 16 or Compound 2 led to a reduction of newly synthesized TG being released from mouse primary hepatocytes (Figure 4A). In contrast, there was a complete lack of effect in primary human and non-human primate (NHP; rhesus and cynomolgus) hepatocytes following selective inhibition of DGAT2 with Compound 16 (Figures 4B 4D). Similarly, treatment with the selective DGAT1 inhibitor PF (DGAT1i [Dow et al., 2011]) in the human and NHP cells also did not perturb TG secretion in these systems. Interestingly, simultaneous inhibition of both DGAT2 and DGAT1 in the primary hepatocytes from these higher species was required in order to observe a robust suppression of TG secretion. This finding can be partially explained by the relative expression levels of DGAT2 versus DGAT1 enzymes in liver cells and tissue from the different species. The ratio of DGAT2 to DGAT1 mrna in liver shows predominantly DGAT2 expression in rodents versus a more balanced expression of DGAT2 and DGAT1 in higher species (Figures 4E and 4F). These results suggest that DGAT2 may play a more dominant role in hepatic TG synthesis in rodents and that DGAT1 may be more able to compensate for the loss of DGAT2 activity across higher species, consistent with the data obtained from the primary hepatocyte cell culture systems. DGAT2 and DGAT1 Act Synergistically to Reduce Hepatic TG Secretion in Wild-Type Mice To further explore the effects of selective and combination inhibition of DGAT2 and DGAT1 in vivo, we carried out a P-407 injection study in 18-hr-fasted, lean, wild-type mice. P-407 blocks the removal of lipoproteins from the circulation and thus the rate of accumulation of TG measured in plasma reflects the rate of hepatic TG secretion (Millar et al., 2005). These studies revealed a reduction in the plasma TG secretion rate (Figure 5A) and in the levels of total plasma TG (Figure 5B) or VLDL-TG (Figure 5C) at 6 hr following treatment with Compound 16 alone. Treatment with the DGAT1 inhibitor alone did not result in any measurable reduction in TG secretion under the same paradigm (Figures 5A 5C). Again, co-administration of the DGAT2 and DGAT1 inhibitors resulted in greater reductions in the levels of plasma and VLDL-TG when compared with DGAT2 inhibition alone (Figures 5A 5C). This observation highlights that compensatory and residual effects of DGAT1 do exist in wild-type mice. Acute Inhibition of DGAT2 Activity on Systemic Triglyceride and VLDL-ApoB Production in Rhesus The discrepancies observed between species on the relative contributions of DGAT2 toward TG secretion in vitro highlight the potential for a translation gap when trying to bridge results between preclinical species and ultimately in making projections to man. To further explore this, we evaluated the acute effects of both Compound 2 and Compound 16 on plasma TG and apob synthesis in a fit-for-purpose, fructose-fed rhesus primate model. This short-term, fructose-challenged model was designed to mimic the fast-refeed paradigm found to enhance sensitivity to DGAT2 inhibition in mice. Feeding rhesus fructose for 3 consecutive days changed the metabolic status of the animals, including a tendency toward elevated plasma insulin, increased circulating PCSK9, and reduced FFA levels (Figure S2A). Three days of fructose feeding was also found to significantly enhance plasma TG and VLDL apob synthesis (Figures S2B and S2C). To validate methods for measuring systemic TG and VLDL apob production in this rhesus model, we conducted a study whereby animals were treated with either vehicle or a single dose of the MTP inhibitor (5 mg/kg). Animals were given an oral bolus dose of [ 2 H] 2 O for the purpose of measuring VLDL apob production and a separate, intravenous bolus of [ 2 H 5 ]glycerol for measuring systemic TG production. [ 2 H 5 ]Glycerol was used as a tracer for TG synthesis instead of [ 13 C 18 ]oleic acid to avoid potential confounding effects that the Intralipid 20 dosing vehicle used in administering the fatty acid tracer could exert over apob measurements. The single dose of MTP inhibitor significantly reduced the plasma concentrations of TG 54:3 and VLDL apob as well as the appearance of newly synthesized TG 54:3 labeled with the [ 2 H 5 ]glycerol tracer (Figures S3A, S3E, and S3B, respectively). This single dose of the MTP inhibitor did not significantly affect the production rate of plasma TG 54:3 (Figures S3C and S3D). A similar experimental paradigm was used to evaluate the effects of both DGAT2 inhibitors on TG and VLDL apob production (Figure6A). Because of the limited oral bioavailability of the DGAT2 compounds in rhesus (Figure 1), an intravenous infusion paradigm was employed to maintain effective concentrations of compound in plasma. During the 4-hr infusion of Compound 16, 1240 Cell Metabolism 27, , June 5, 2018

7 Figure 4. Effects of DGAT2 and DGAT1 Inhibitors on TG Secretion in Primary Hepatocytes and Comparison with Relative mrna Levels across Species (A D) Secretion of newly synthesized triglycerides from primary hepatocytes treated with DGAT2 inhibitors or DGAT1 plus DGAT2 inhibitors. Mouse (A), human (B), rhesus monkey (C), and cynomolgus monkey (D) are shown. Primary hepatocytes were treated with inhibitors for hr. The cell culture media were analyzed for TGs using LC-MS as described in STAR Methods. (E and F) The ratio of DGAT2 to DGAT1 mrna expression by real-time qpcr in primary hepatocytes (E) or liver tissue (F) from mouse, human, cynomolgus monkey, or rhesus monkey. Data presented are mean ± SEM, n = 3-5. a modest but statistically significant reduction in the production rate of TG54:3 was observed (Figure 6E) and judged to be notable based on the preceding observation that this specific TG also appeared to be the most sensitive marker of DGAT2 inhibition in rodents (Figures 2B versus 2A). In contrast, the production rates for the three additional TGs measured were not significantly affected by treatment with the DGAT2 inhibitors, and therefore the aggregate TG production rate was not found to be different from control treatment (Figure S5). Consistent with the lack of effect on aggregate TG production, we found no change in the plasma concentration or deuterium labeling of VLDL apob for either of the DGAT2 inhibitors when compared with control treatment (Figures 6F and 6G). Pharmacokinetic analysis from this study showed that intravenous infusion of both DGAT2 inhibitors achieved steady-state concentrations for the duration of the study, and these were significantly greater than the EC 90 (plasma exposure required to achieve maximal efficacy in vivo) determined from the rodent studies (Figures S4A and S4B). The levels of compound achieved thus suggest that sufficient inhibition of DGAT2 was maintained over the duration of the study. Chronic Silencing of Hepatic DGAT2 in Rhesus Using sirna To explore the question of whether more chronic attenuation of DGAT2 activity could have an effect on TG secretion in higher species, we employed a small interfering RNA (sirna)-mediated silencing approach in chow-fed rhesus primates. Rhesus Cell Metabolism 27, , June 5,

8 Figure 5. DGAT2 and DGAT1 Inhibitors Act Synergistically to Reduce Hepatic Triglyceride Secretion and VLDL-TG Levels in Mice (A) Circulating TG levels in 18-hr-fasted mice administered Pluronic detergent (P-407) at 0 hr. The mice were administered Compound 16 subcutaneously and oral DGAT1 inhibitor either alone or in combination at 18 hr and 1 hr prior to the P-407 injection. Circulating plasma TG levels were measured at 0-, 2-, 4-, and 6-hr time points. Mice administered non-compound-containing vehicle (Veh) or oral MTP inhibitor (3 mg/kg) served as the negative and positive controls, respectively. (B and C) Area under the curve (AUC) of the plasma TG (B) and plasma VLDL TG (C) time course from 0 to 6 hr post Pluronic injection. Data are presented as mean ± SEM. Treatment comparisons were assessed by one-way ANOVA and Bonferroni s multiple comparison test to determine individual group differences. n = 6 8 per treatment group. *p < monkeys were administered a single dose of DGAT2, APOB, or non-targeting control sirna, and fasted plasma samples were collected for lipid profiling 14 days after sirna administration. To characterize the effect of hepatic DGAT2 knockdown on TG secretion, we also dosed animals with the [ 13 C 18 ]oleic acid tracer on day 14 of the study (30 min after the fasted sample had been collected) and obtained blood samples 30 min and 24 hr later. Although we observed a marked inhibition of TG secretion in animals treated with the apob sirna, only a modest trend toward reduction in TG synthesis and secretion was detected in animals following DGAT2 sirna treatment (18% 25% reduction, depending on the TG species, p = 0.54 to 0.19, respectively, Figures 7A and 7B). In addition, there was a slight trend toward an increase in phospholipid production (20%, p = 0.45, Figure 7C). Since inhibition of DGAT2 could affect the partitioning of fatty acid between TG and phospholipids, we also evaluated the change in the ratio of TG/phospholipid. Although these effects were not significant, the trend in the ratio is suggestive of movement of fatty acid trafficking in response to DGAT2 knockdown (31% decrease in the ratio of TG/phospholipid, p = 0.13, Figure 7D). Analysis of DGAT2 and APOB expression in liver biopsies collected on day 15 confirmed that sirna-mediated silencing of both genes was highly effective in this rhesus model, with 80% and 93% mrna knockdown achieved, respectively (Figure 7E). Neither silencing of DGAT2 nor APOB was found to have a significant effect on expression of DGAT1. Additionally, knockdown of DGAT2 in this NHP model did not lead to any significant changes in total, LDL, or HDL cholesterol or in total plasma TG levels, whereas knockdown of APOB resulted in significant reductions in total cholesterol, LDL cholesterol, and plasma TG after 15 days (data not shown). DISCUSSION Metabolic abnormalities that associate with disturbances in the production and clearance of plasma lipoproteins represent a high risk for atherosclerosis and cardiovascular diseases. It has been proposed that inhibition of DGAT2 could have potential therapeutic value in mitigating cardiovascular risk based on the hypothesis that alteration of hepatic TG synthesis and subsequent reductions in VLDL production could ultimately lead to lower levels of atherogenic ApoB. We sought to test this hypothesis by evaluating the effects of the DGAT2 inhibitors Compounds 2 and 16 in a variety of translational models. Acute treatment with both compounds in 4-hr-fasted lean mice resulted in significant reductions in newly made TG appearing in plasma; however, the effects achieved by selective DGAT Cell Metabolism 27, , June 5, 2018

9 Figure 6. Acute Effects of DGAT2 Inhibitors in Rhesus Monkeys (A) Study design showing the relative timing of experimental procedures. The administration of [ 2 H 5 ] glycerol tracer defines Time 0 hr (square). Monkeys received three bolus doses of oral fructose dissolved in water (open circles). On study day 3 they were administered an additional oral bolus of fructose dissolved in [ 2 H] 2 O (closed circle). Rates of TG and apob synthesis were measured from blood samples collected throughout the study (triangles). (B) Plasma concentration of endogenous TG 54:3. (C) Plasma concentration of TG 54:3 labeled with [ 2 H 5 ]glycerol. (D) Production rate for TG 54:3; the data for each treatment were fit using a one-phase decay linear regression model (dotted line) to calculate the turnover rate (k; pools per hour). (E) DGAT2 inhibitors reduce TG 54:3 production rate compared with vehicle (one-way ANOVA F(2, 33) = 4.09, p = 0.03). The rates for compounds 2 and 16 were compared with vehicle using Dunnett s post hoc test. p values for the individual comparisons are noted on the graph (*p < 0.05). (F) Plasma concentration ratio (normalized to internal standard) for VLDL apob. (G) Deuterium labeling of VLDL apob. Error bars represent the SEM. See also Figures S4 and S5. Cell Metabolism 27, , June 5,

10 Figure 7. Chronic Effects of sirna-mediated DGAT2 Suppression on TG Synthesis in Rhesus Monkeys (A D) Levels of newly synthesized plasma TG 52:2 (A) and TG 54:3 (B) measured on day 14 of the study, 30 min after administration of [ 13 C 18 ]oleic acid tracer; (C) concentration of [ 13 C 18 ]oleate-labeled plasma phospholipid, 24 hr after administration of the [ 13 C 18 ]oleic acid tracer; (D) ratio of newly synthesized TG to newly synthesized phospholipid in PBS, sirna-apob, sirna-control (Con), and sirna-dgat2 treated rhesus monkeys. n = 3 6, **p < 0.01 compared with sirna-con (ANOVA; Dunnett s test). (E) Percentage of DGAT2, APOB, and DGAT1 mrna expression after 15 days of a single sirna treatment at day 0. Error bars represent SEM. inhibition initially approached only 50% of the effect achieved with an MTP inhibitor. The effects of both Compound 2 and Compound 16 were augmented in mice that were subjected to an overnight fast followed by a 4-hr high carbohydrate diet refeeding period (fast-refeed), with reductions in newly made plasma TG approaching, but not equaling, that of MTP inhibition. Although the effects of DGAT2 and MTP inhibition as measured by the appearance of newly synthesized TG in the plasma appear similar in these experiments, the mechanisms by which this occurs are distinct. It is well known that the reductions in plasma TG effected by inhibition of MTP are the result of abrogated secretion, not altered synthesis, and this is accompanied by increased lipid levels in tissue (Chandler et al., 2003). Given this, one can reconcile that inhibition of MTP will lead to a reduction in the plasma concentration of TG, but would not be expected to change the inherent production rate of the lipid; i.e., TG is produced and stored but not secreted. Indeed, this is precisely what we observed in our studies with the MTP inhibitor in rhesus. Conversely, inhibition of DGAT2 results in diversion of fatty acid substrate away from TG synthesis at the level of diglyceride, and this is partially reflected by an increase in phospholipid synthesis (Imbriglio et al., 2015), an effect which was recapitulated in the present studies in mice (data not shown). Indeed, gene silencing of DGAT2 using sirna has been shown to partially remediate the increase in liver TG caused by gene silencing of MTP in LDLr +/ /CETP +/ mice (Tep et al., 2012) Cell Metabolism 27, , June 5, 2018

11 Cumulatively, these data support the conclusion that DGAT2 plays a significant role in hepatic TG synthesis in mice, particularly during a state of high nutrient flux to the liver. Our studies in high-fat diet-induced obese mice also demonstrated that chronic inhibition of murine DGAT2 leads to measurable benefits in lipid profile, independent of changes in body weight gain, including reductions in plasma TG, LDL cholesterol, and total cholesterol, and extending even to complete normalization of hepatic TG levels. The results of these studies also demonstrated complete target engagement and maximal inhibition of DGAT2 as evidenced by the similar effects demonstrated on TG synthesis when the AAV-treated mice were treated in combination with the small-molecule inhibitor. These data further demonstrate that similar results are achieved via either inhibition of systemic DGAT2 by a small molecule or via more selective inhibition of hepatic DGAT2 by the AAV-expressing shrna-silencing approach, suggesting that the liver is the primary site of action for DGAT2 in regard to TG synthesis. Interestingly, despite the profound correction of hepatic steatosis observed in these rodent studies, glucose homeostasis and insulin sensitivity were largely unaffected, thus highlighting the importance of the underlying mechanisms by which correction of hepatic steatosis occurs. On the basis of these results in rodents it is certainly tantalizing to postulate that the beneficial effects of DGAT2 inhibition on dyslipidemia could extend to higher species. However, there are both encouraging and cautionary notes that can be gleaned from what we have learned regarding translation through pharmacological manipulation of DGAT1. Our own studies with the DGAT1 inhibitor PF demonstrate that postprandial TG excursion can be acutely and effectively inhibited in both lean mice and rhesus NHPs (Figure S6). These results have been recapitulated in humans (Maciejewski et al., 2013), including a study in patients suffering from familial chylomicronemia whereby a dose of the DGAT1 inhibitor pradigastat as low as 20 mg per day for 21 days was sufficient to significantly reduce both fasting and postprandial triglycerides (Meyers et al., 2015). Although these effects on TG reduction were found to successfully translate from rodents to humans, it is important to note that the adverse GI events reported in the clinic upon inhibition of DGAT1 (Denison et al., 2014) were not observed in any rodent studies that have been reported. This has been explained by the fact that DGAT2 expression is high in the intestine of rodents whereas its expression is essentially absent in the human intestine (Haas et al., 2012). As such, DGAT2 may be able to play enough of a compensatory role in rodents to nullify the adverse effects of intestinal DGAT1 inhibition that were observed in humans. Perhaps foreshadowed by this knowledge, the in vitro data that we present here suggest that DGAT1 acts synergistically with DGAT2 to control TG secretion from hepatocytes, especially in those of the higher species tested. In vivo data in the mouse also demonstrate that combined inhibition of DGAT2 and DGAT1 is required to achieve an effect on VLDL secretion equivalent to that obtained through inhibition of MTP. In light of these results, we felt it was imperative to explore the effects of selective DGAT2 inhibition in vivo in primates in order to obtain a clear understanding of its relative value as a therapeutic target for dyslipidemia. The rhesus model we initially employed for this purpose proved to be appropriately responsive to manipulations known to affect VLDL-TG secretion. In the acute setting, rhesus fed with fructose for 3 consecutive days showed increased levels of PCSK9 consistent with increased SREBP tone. Furthermore, these animals also displayed increased circulating insulin and decreased FFA levels, consistent with a state of decreased lipolysis and increased de novo lipogenesis (Hillgartner et al., 1995). In this regard the model bears similarity to the rodent fasted-refed model, which demonstrated enhanced sensitivity to DGAT2 inhibition. Administration of 5 mg/kg of the MTP inhibitor to these fructose-fed rhesus primates also led to drastic decreases in VLDL apob and suppressed the appearance of newly made and total plasma TG. It is noteworthy that the reductions in TG 54:3 observed in our MTP study (20% at 4 hr compared with vehicle control) are similar to the total TG lowering observed in humans (25% at 4 hr compared with baseline) for a comparable dose (Chandler et al., 2003; Zhou et al., 2012). Given that an MTP inhibitor (lomipatide) has been approved by the US Food and Drug Administration for treating familial hypercholesterolemia and improving metabolic function, recapitulation of the pharmacodynamics of MTP inhibition in our rhesus model suggests that the sensitivity is sufficient to detect clinically meaningful changes in TG and VLDL ApoB synthesis. Because the oral bioavailability of both Compound 2 and Compound 16 is extremely limited in rhesus, we carefully selected intravenous infusion doses for each inhibitor so that the steady-state plasma exposures achieved during the study period would be similar to or greater than the plasma exposures measured in the rodent studies where we observed maximal inhibition of TG secretion. Based on the rodent studies for Compound 2, the EC 90 is 1.47 mm. The plasma exposure of Compound 2 in the fructose-fed NHPs reached levels roughly 8- to 15-fold over this threshold, ranging between 16 and 30 mm during the period of the study. For Compound 16, we achieved plasma exposures 5- to 7-fold over those required to achieve maximal efficacy in the rodent studies. Taking into account that the plasma unbound fraction for these compounds is similar in rodents and in NHP and that the potencies against the enzyme are relatively similar, we conclude that DGAT2 inhibition with these two structurally diverse compounds was greater than 90% in the NHP study. Further supporting this, we observed a very modest but significant reduction in the amount of newly made triolein (TG 54:3), the same discrete TG that seemed most sensitive to DGAT2 inhibition in rodent models. More chronic silencing of DGAT2 using sirna in lean rhesus yielded similar findings. Although robust knockdown of 80% of DGAT2 mrna was sustained for 15 days, we observed no effect on TG synthesis and secretion even after this prolonged period despite the fact that in the same setting, knockdown of APOB resulted in robust TG lowering. It is also worth noting that a lesser degree of DGAT2 knockdown for a comparable duration in mice was sufficient to lead to a significant reduction in plasma TG and a trend toward reduced hepatic TG (Tep et al., 2012), further highlighting species-specific differences in the role of DGAT2. It seems that the results obtained across species from in vitro primary hepatocytes are predictive of the outcome of in vivo experiments. Our results demonstrate effective translation from cells to in vivo models in the mouse and also show that the lack of effect of selective DGAT2 inhibition on TG secretion Cell Metabolism 27, , June 5,

12 observed in both human and NHP hepatocytes ultimately correlated with the in vivo data obtained in rhesus. The differentiation in response to DGAT2 inhibition between rodents and higher species seems as though it can be at least partially explained by the relative distribution of hepatic DGAT1 and DGAT2. The much higher relative expression of DGAT2 in rodents compared with higher species is suggestive of a primary role for DGAT2 in rodent TG synthesis, and this appears consistent with the robust effects observed following DGAT2 inhibition in our mouse experiments. We recognize that a study in NHP to evaluate the combination of DGAT2 and DGAT1 inhibition would likely provide us with definitive proof of a compensatory effect. Ultimately, however, there are practical barriers to conducting such a study. We anticipate that simultaneous inhibition of DGAT2 with DGAT1 would likely result in adverse GI events in NHP that are comparable with, or even exacerbated from, those reported in humans, and we did not feel that the potential insights to be gained from testing the combination hypothesis outweighed the risks to tolerability. Collectively, the data we have presented here support the conclusion that DGAT2 does not exert a controlling influence over hepatic TG synthesis leading to VLDL apob secretion in higher species, even though it appears to do so in the rodent. As such, it seems unlikely that selective inhibition of DGAT2 would provide a useful means of correcting dyslipidemias in a clinical setting. Recent data from an exome-wide study of plasma lipids in more than 300,000 individuals have also yielded valuable insight into this question (Liu et al., 2017). Within the cohort of subjects profiled there were 489 who were determined to be heterozygous for a nonsense Y285X mutation in the DGAT2 gene. The expected consequence of this mutation is that 50% of DGAT2 protein produced would be non-functional and quickly degraded in these individuals, leading to an overall reduction in activity. The only significant association reported in that study between this DGAT2 mutation and the plasma lipid profile was a reduction in HDL cholesterol; there was no significant association between the mutation and levels of plasma TG (data reproduced in Table S1). As a final note, we do feel it is important to point out that our studies have not systematically addressed any potential role for DGAT2 in mediating hepatic TG storage in higher species, such as may be dysregulated in non-alcoholic fatty liver disease (NAFLD) or non-alcoholic steatohepatitis (NASH). Indeed, certain results from our studies in obese mice could be interpreted as being supportive of such a role. For example, key genes involved in TG production were reduced following DGAT2 inhibition, and this is consistent with data reported in vitro showing suppression of SREBP1 following attenuation of DGAT2 activity (Li et al., 2015). The reduction of PNPLA3 mrna levels following DGAT2 inhibition is intriguing and could be considered supportive of a hypothesis for DGAT2 involvement in mediating NASH. It will not be lost on the reader, however, that our results demonstrating a lack of translatability from mouse to higher species with regard to the role of DGAT2 in controlling TG synthesis leading to VLDL secretion could be equally suggestive of a lack of translation for any potential role in mediating NAFLD/NASH. Unfortunately, the path for testing such translation preclinically is not entirely clear. A recent publication from colleagues (Shang et al., 2017) demonstrated that dysmetabolic NHPs have marked defects in adipose and skeletal muscle tissues but only mild hepatic alterations in insulin signaling. Despite being classified as dysmetabolic, on autopsy the animals had no sign of excess liver fat, suggesting that NHP may not be an ideal model to examine NASH/ NAFLD endpoints related to DGAT2 inhibition. Further experiments would be warranted to fully examine the potential therapeutic benefits that inhibition of DGAT2 may have for the treatment of indications other than dyslipidemia. Limitations of the Study In our studies incorporation of [ 13 C 18 ]oleate or [ 2 H 5 ]glycerol into the specific molecular triglycerides 52:2, 52:3, 54:3, and 54:4 was used as the measure of synthesis and secretion of plasma TG. This approach assumes that changes in the production of these specific triglycerides are reflective of changes in the total pool of all plasma TG, and this can be considered a limitation. A further limitation is that we measured the synthesis and secretion of plasma TG only and not VLDL-TG in our rhesus studies. While we can confidently say that inhibition of DGAT2 in rhesus did not have a significant effect on the production of VLDL apob or our selected plasma TG, it should be noted that a modest effect on these triglycerides in VLDL specifically cannot be conclusively ruled out. STAR+METHODS Detailed methods are provided in the online version of this paper and include the following: d KEY RESOURCES TABLE d CONTACT FOR REAGENT AND RESOURCE SHARING d METHOD DETAILS B Study Approval B Hepatic TG Production in Mice B Systemic TG Production in Mice Using [ 13 C 18 ] Oleic Acid B Assessment of Lipid and Glucose Metabolism in Diet- Induced Obese Mice B In Vitro Studies in Primary Hepatocytes B The Effects of sirna Mediated DGAT2 Inhibition on TG Synthesis in Rhesus Monkey B Development of a Fit for Purpose Rhesus Model of Stimulated VLDL Production B Effect of MTPi and DGAT2i on TG and apob Synthesis in Rhesus Monkeys B Plasma Glucose, Insulin, Lipid, and Hepatic Lipid Determination B Generation of Recombinant Adenoviral Associated Viral Vector Expressing shrna against Mouse Dgat2 B Real-Time Quantitative PCR Analysis B Effects of the DGAT1 Inhibitor PF on Postprandial TG Excursion in Rhesus Monkeys d QUANTIFICATION AND STATISTICAL ANALYSIS SUPPLEMENTAL INFORMATION Supplemental Information includes six figures and one table and can be found with this article online at Cell Metabolism 27, , June 5, 2018

13 ACKNOWLEDGMENTS The authors would like to express their sincere gratitude to the following individuals for their support and assistance: Brian Hubbard and Andrew Taggart for early contributions; Oksana Palyha for assistance with the hepatocyte assays; Duncan Brown for sirna design; Lily Luo and Walter Strapps for assistance with sirna screening; RNA Tx oligo synthesis and formulations teams for providing reagents for NHP studies; the Sirna PM Team, New Iberia Research Center, Kenny Wong, Alison Kulick, Marcie Donnelly, and Robert DeVita for NHP study design, management, and execution. Collaborative work with Prof. Sek Kathiresan and the Global Lipid Genetics Consortium on the DGAT2 nonsense variant is also gratefully acknowledged. All funding for the studies reported herein was provided by Merck & Co., Inc., Kenilworth, NJ. AUTHOR CONTRIBUTIONS Conceptualization, D.G.M., S.H., B.A.M., L.W., S.F.P., T.P.R., J.E.I., and S.P.; Methodology, D.G.M., S.H., B.A.M., L.W., S.F.P., A.-M.C., M.T.-S., S.R.B., and S.P.; Investigation, D.G.M., S.H., B.A.M., L.W., S.F.P., S.J.S., H.Z., J.N.G., D.E.M., D.F.R., and A.-M.C.; Resources, M.K.S., H.H.Z., M.T.-S., S.R.B., T.H.G., D.-M.S., K.O.A. and J.E.I.; Writing Original Draft, D.G.M., S.H., B.A.M., S.F.P., M.T.-S., J.E.I., and S.P.; Writing Review and Editing, D.G.M., S.H., B.A.M., S.F.P., and S.P.; Supervision, B.A.M., T.P.R., J.E.I., and S.P. DECLARATION OF INTERESTS Merck & Co., Inc. (Kenilworth, NJ) has filed patent applications for Compound 2, Compound 16, and related DGAT2 inhibitors (patent application number WO ; others pending). All authors were employees of Merck & Co., Inc. (Kenilworth, NJ) at the time this research was conducted. T.P.R. is presently employed by Agios Pharmaceuticals (Cambridge, MA). 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