Volume 26, Issue 1, Pages 157-170.e7 (July 2017) Inhibition of IKKɛ and TBK1 Improves Glucose Control in a Subset of Patients with Type 2 Diabetes  Elif A. Oral, Shannon M. Reilly, Andrew V. Gomez, Rasimcan Meral, Laura Butz, Nevin Ajluni, Thomas L. Chenevert, Evgenia Korytnaya, Adam H. Neidert, Rita Hench, Diana Rus, Jeffrey F. Horowitz, BreAnne Poirier, Peng Zhao, Kim Lehmann, Mohit Jain, Ruth Yu, Christopher Liddle, Maryam Ahmadian, Michael Downes, Ronald M. Evans, Alan R. Saltiel  Cell Metabolism  Volume 26, Issue 1, Pages 157-170.e7 (July 2017) DOI: 10.1016/j.cmet.2017.06.006 Copyright © 2017 Elsevier Inc. Terms and Conditions

Cell Metabolism 2017 26, 157-170.e7DOI: (10.1016/j.cmet.2017.06.006) Copyright © 2017 Elsevier Inc. Terms and Conditions

Figure 1 Study Design and Insulin Sensitivity Measurements from the Open-Label Study (A) Summary of the study protocols. † indicates protocol elements that only apply to the open-label study. (B) Baseline characteristics of the six open-label study participants. (C–E) Metabolic data of the six patients included in the study at baseline and 12 weeks are shown: (C) hemoglobin A1c % (HbA1c), (D) fasting glucose, and (E) fasting insulin. (F) Insulin resistance calculated by the homeostatic model assessment linear estimation HOMA-IR. (G–I) Hyperinsulinemic euglycemic clamp: (G) tate of glucose disappearance (Glucose Rd), (H) body-weight-normalized glucose Rd, and (I) hepatic glucose production at fasting state. (J) Percent fat in liver, as determined via MRI using the Dixon method. (K) Glucose (left), insulin (middle), and FFAs (right) during the mixed-meal test. Data are presented as individual values (in C–H) or mean ± SEM (in K). n = 6, except in (G) and (H), as the 6th patient did not achieve steady state during the clamp performed at 12 weeks, where n = 5. ∗ indicates p value ≤ 0.05 and # indicates p value ≤ 0.10 (two-tailed paired t test). DEXA, dual-emission x-ray absorptiometry; t.i.d., ter in die, three times a day. See also Figure S1 and Table S1. Cell Metabolism 2017 26, 157-170.e7DOI: (10.1016/j.cmet.2017.06.006) Copyright © 2017 Elsevier Inc. Terms and Conditions

Figure 2 Weight Loss and Gene Expression Changes in the Open-Label Study Subjects (A) Body weight (left) and body weight represented as percentage of baseline body weight (right) during 12-week treatment and 4-week washout observation period. (B) Relative expression of UCP family of genes encoding mitochondrial inner-membrane protein. (C) Correlation of relative UCP1 expression with weight loss after 12 weeks of treatment. (D) Relative expression of the ADRB family of genes encoding β-adrenergic receptors. (E–G) Relative expression of beige fat markers: (E) FGF21, (F) DIO2, (G) PPARGC1A, PPARGC1B, PRDM16, and ELOVL3. (F) shows the correlation of DIO2 expression and weight loss (bottom). (H) Relative expression of IL4. (I) Relative expression of ADIPOQ, the gene encoding adiponectin. n = 6. ∗∗ indicates p value ≤ 0.01; ∗ indicates p-value ≤ 0.05; # indicates p value ≤ 0.10 (two-tailed paired t test). In (C) and (F) Pearson’s correlation with 95% CI is used. AU, arbitrary unit. Cell Metabolism 2017 26, 157-170.e7DOI: (10.1016/j.cmet.2017.06.006) Copyright © 2017 Elsevier Inc. Terms and Conditions

Figure 3 Measures of Glycemic Control in the Double-Blind, Placebo-Controlled Trial (A) Consort diagram: Schematic of randomized, double-blind, placebo-controlled trial. For details on evaluable n of secondary and exploratory end-point analyses, refer to Table S2. (B and C) Difference from baseline in (B) hemoglobin A1c and (C) fructosamine in the placebo- and amlexanox-treated patients, left; on right are baseline and 12-week values for each patient connected with a line. (D and E) Insulin (D) and glucose (E) during the mixed-meal test. (F) Insulin ratio (week 12 divided by baseline area under the curve for insulin measured during the mixed-meal test). In (B), n = 18 placebo, n = 20 amlexanox; in (C–F), n = 17 placebo, n = 20 amlexanox (n = 7 A-R). ∗∗ indicates p value ≤ 0.01 and ∗ indicates p value ≤ 0.05; two-tailed t test; (B), (C), and (E) unpaired, (D) paired. Data are presented as individual data and mean ± SEM (in B, C, and F) or mean ± SEM only (in D and E). See also Figures S2–S4 and Tables S3 and S4. Cell Metabolism 2017 26, 157-170.e7DOI: (10.1016/j.cmet.2017.06.006) Copyright © 2017 Elsevier Inc. Terms and Conditions

Figure 4 Baseline Differences in Responders Versus Nonresponders (A and B) Serum levels of (A) amlexanox and (B) one of its metabolites at baseline and 12 weeks. n = 18 placebo, 7 amlex-R (Amlexanox-treated, responder) and 13 amlex-NR (amlexanox treated, nonresponder). See also Table S5. (C) Genes whose expression was significantly different in responders versus nonresponders at baseline in RNA-sequencing analysis. Values presented as log (fold change) in responders relative to nonresponders (n = 4 per group). (D) Baseline CRP values in the placebo-controlled trial in the placebo- and drug-treated patients. The latter group is further divided into responders and nonresponders. Baseline CRP values for one of the placebo-treated patients were excluded as they was obviously compromised. n = 17 placebo, 7 amlex-R, 13 amlex-NR. (E) Correlation of baseline CRP values with change in HbA1c from baseline to 12 weeks in amlexanox-treated patients (left, n = 20) and placebo controls (right, n = 17). Note the significant inverse relationship in the drug-treated group compared to lack of correlation in the placebo-treated group. (F) Correlation between baseline BMI and serum CRP at baseline in all patients (left; p = 0.031, Pearson’s r = 0.36, n = 37) and FMI (fat mass index) versus serum CRP in all patients (right) at baseline (p = 0.0003, Pearson’s r = 0.56, n = 37). (G) Correlation between liver fat and serum CRP at baseline in patients who were evaluated for liver fat (p = 0.035, Pearson’s r = 0.49, n = 19). # indicates p value ≤ 0.10; ∗ indicates p value ≤ 0.05 (two-tailed t test). Data are presented as individual data and mean ± SEM or 95% CI. CRP: C-reactive protein. BMI: body mass index. See also Figure S4. Cell Metabolism 2017 26, 157-170.e7DOI: (10.1016/j.cmet.2017.06.006) Copyright © 2017 Elsevier Inc. Terms and Conditions

Figure 5 Responder-Specific Changes in Serum IL-6 and Subcutaneous Fat Gene Expression (A) Serum IL-6 levels over the course of the trial: 12 weeks of treatment and 4 weeks of observation period post treatment. (B–E) Relative expression of genes in the subcutaneous fat at baseline and after 12 weeks of treatment. Relative expression of beige-fat-associated genes: (B) UCP1 (Ct values in the nonresponder [baseline: 35.1, 12-week: 35.9] and placebo [baseline: 34.7 12-week: 34.4] were not changed after treatment, while the Ct value in the responder was reduced [baseline: 35.9 12-week: 33.8]; while these high Ct values indicate low abundance of UCP1, good linearity was observed in the standard curve from Ct 30 to Ct 35), (C) DIO2, (D) PRDM16 (left), FGF21 (right), and (E) COX5B. (F) Relative expression of CCL2 in the subcutaneous fat at baseline and after 12 weeks of treatment. (G) Heatmap illustrating the genes that were significantly changed from baseline to 12 weeks in a paired analysis of the RNA-sequencing data within each of the three groups. (H) Heatmap illustrating genes from different pathways that were differentially regulated by treatment in the responders (∗HSPB1 was also significantly upregulated in the nonresponder group). (I) Relative expression in the subcutaneous fat at baseline and after 12 weeks of treatment of LIPE (top) and FASN (bottom). See also Tables S6 and S7. In (A), n = 17 placebo, 7 amlex-R and 13 amlex-NR; In (C)–(F) and (I), n = 15 placebo, 7 amlex-R and 13 amlex-NR; in (G) and (H), n = 4 per group. ∗ indicates p value ≤ 0.05; # indicates p value ≤ 0.10; two-tailed t test (in A, unpaired; in B–I, paired). Data are presented as mean ± SEM/ Amlex-R: amlexanox-treated responder, Amlex-NR: amlexanox-treated, nonresponder. Cell Metabolism 2017 26, 157-170.e7DOI: (10.1016/j.cmet.2017.06.006) Copyright © 2017 Elsevier Inc. Terms and Conditions