Volume 24, Issue 7, Pages 892-906.e5 (July 2017) Selectivity and Kinetic Requirements of HDAC Inhibitors as Progranulin Enhancers for Treating Frontotemporal Dementia  Angela She, Iren Kurtser, Surya A. Reis, Krista Hennig, Jenny Lai, Audrey Lang, Wen-Ning Zhao, Ralph Mazitschek, Bradford C. Dickerson, Joachim Herz, Stephen J. Haggarty  Cell Chemical Biology  Volume 24, Issue 7, Pages 892-906.e5 (July 2017) DOI: 10.1016/j.chembiol.2017.06.010 Copyright © 2017 Elsevier Ltd Terms and Conditions

Cell Chemical Biology 2017 24, 892-906. e5DOI: (10. 1016/j. chembiol Copyright © 2017 Elsevier Ltd Terms and Conditions

Figure 1 Profiling Hydroxamic Acid HDAC Inhibitor Effects on GRN mRNA and PGRN Protein Expression in Human iPSC-Derived NPCs and Neurons (A) Model and experimental scheme for measuring the HDAC inhibitor effect on progranulin expression, from mRNA (Assay 1, qPCR) to intracellular protein (Assay 2, western blot and ELISA) to secreted protein (Assay 3, ELISA). With the HDAC complex, Sn = subunit and TF = transcription factor. (B–E) Chemical structures for fast-on/-off HDAC inhibitors: (1) SAHA, (2) crebinostat, and (3) panobinostat (Herman et al., 2006). IC50 values from Bradner et al. (2010) for (1), Fass et al. (2013) for (2), and Khan et al. (2008) for (3). All increased GRN gene expression and PGRN protein levels in human NPCs (black) and 18-day neurons (gray) after 24 hr as shown in (C) (SAHA, 10 μM), (D) (crebinostat, 2.5 μM), and (E) (panobinostat, 0.5 μM); vehicle = DMSO. Dividing lines on western blots indicate samples from separate gels. Protein quantification was measured by ELISA. Each condition shown is the mean + SEM of n = 3 biological replicates × 3 technical replicates with significance calculated by unpaired t test. ***p < 0.001, ****p < 0.0001. Cell Chemical Biology 2017 24, 892-906.e5DOI: (10.1016/j.chembiol.2017.06.010) Copyright © 2017 Elsevier Ltd Terms and Conditions

Figure 2 HDAC6- and HDAC8-Selective Inhibitor Effects on GRN mRNA and PGRN Protein Expression in Human iPSC-Derived NPCs and Neurons (A) Chemical structures for HDAC6-selective inhibitors (4) ACY-1215 and (5) tubastatin A, and HDAC8-selective inhibitor (6) PCI-34051. IC50 values are from Santo et al. (2012) for (4), Butler et al. (2010) for (5), and Balasubramanian et al. (2008) for (6). (B) ACY-1215 (5 μM) increases GRN and PGRN expression in human NPCs (black) and 18-day neurons (gray) after 24 hr. Tubastatin A (10 μM) was not effective. (C) PCI-34051 (10 μM) does not increase GRN and PGRN expression in human NPCs (black) or 18-day neurons (gray) after 24 hr. Vehicle is DMSO. Protein quantification was measured by ELISA. Each condition shown is the mean + SEM of n = 3 biological replicates × 3 technical replicates with significance calculated by unpaired t test. Page: 1 ***p < 0.001, ****p < 0.0001. Cell Chemical Biology 2017 24, 892-906.e5DOI: (10.1016/j.chembiol.2017.06.010) Copyright © 2017 Elsevier Ltd Terms and Conditions

Figure 3 Apicidin and Valproate Increase GRN mRNA and PGRN Protein Expression in Human iPSC-Derived NPCs and Neurons (A) Chemical structure for the tetrapeptide Apicidin (7), with IC50 values from Huber et al. (2011). (B) Apicidin (2.5 μM) and positive control SAHA (10 μM) increase GRN mRNA and PGRN intracellular and secreted protein expression human NPCs (black) and 18-day neurons (gray) compared with vehicle (DMSO). (C) Chemical structure for the carboxylic acid valproate (8), with IC50 values from Fass et al. (2011). (D) Valproate (5 mM) significantly increases GRN mRNA and PGRN intracellular and secreted protein expression human NPCs (black) and 18-day neurons (gray) compared with vehicle (ddH2O). Protein quantification was measured by ELISA. Each condition shown is the mean + SEM of n = 3 biological replicates × 3 technical replicates with significance calculated by unpaired t test. *p < 0.05, ****p < 0.0001. Cell Chemical Biology 2017 24, 892-906.e5DOI: (10.1016/j.chembiol.2017.06.010) Copyright © 2017 Elsevier Ltd Terms and Conditions

Figure 4 Profiling ortho-Aminoanilide HDAC Inhibitors on GRN mRNA and PGRN Protein Expression in Human iPSC-Derived NPCs and Neurons (A) Chemical structures for slow-on/off ortho-aminoanilide HDAC inhibitors CI-994 (9) and Cpd-60 (10). IC50 values are from Bradner et al. (2010) for (9) and Schroeder et al. (2013) for (10). (B) Neither CI-994 nor Cpd-60 had a significant effect on GRN mRNA expression or PGRN protein expression in human NPCs (black) or 18-day neurons (gray). (C) Despite longer treatment times, CI-994 did not increase GRN mRNA and in fact, decreased PGRN secreted protein expression in NPCs compared with vehicle (see p values below); 2 biological × 3 technical replicates. (D) CI-994 and Cpd-60 increase H3K9 acetylation in 18-day neurons despite not enhancing PGRN protein levels (see also Figure S1). (E) Slow-on/off HDAC inhibitors CI-994 and Cpd-60 increase frataxin (FXN) mRNA expression in 18-day neurons, while fast-on/-off inhibitor SAHA does not (see also Figure S3). Cells were treated for 24 hr, unless otherwise indicated, with vehicle (DMSO), SAHA (10 μM), CI-994 (10 μM), or Cpd-60 (5 μM). Protein quantification was measured by ELISA. All graphs are shown as mean + SEM, with significance relative to vehicle calculated by unpaired t test. *p < 0.05, **p < 0.01, ****p < 0.0001. Cell Chemical Biology 2017 24, 892-906.e5DOI: (10.1016/j.chembiol.2017.06.010) Copyright © 2017 Elsevier Ltd Terms and Conditions

Figure 5 Optoepigenetic Probe BG47 Upregulates PGRN in the Presence of Light (A) Activation schematic for optoepigenetic probe BG47 (11). BG47 is activated to its active cis isomer by 470 nm light and engages target within microseconds, whereupon it stays bound for hours. (B) BG47 (10 μM) enhanced PGRN in NPCs after 16 hr of treatment with light (25 ms on/75 ms off). This effect is not seen without light. n = 4 biological replicates. Graph shows mean + SEM with significance calculated by unpaired t test. ****p < 0.0001. (C) Model for HDAC inhibitor-mediated GRN enhancement based on kinetics of binding of the HDAC inhibitor. If the on-rate of the HDAC inhibitor is slower than the HDAC complex-chromatin association rate, the HDAC inhibitor will be unable to upregulate GRN. Cell Chemical Biology 2017 24, 892-906.e5DOI: (10.1016/j.chembiol.2017.06.010) Copyright © 2017 Elsevier Ltd Terms and Conditions

Figure 6 Comparison of GRN mRNA and PGRN Protein Levels across Cell Types Correlation between effects of HDAC inhibitors on GRN mRNA expression, PGRN intracellular protein expression, and PGRN-secreted protein in (A) NPCs and (B) 18-day differentiated neurons, with representative compounds CI-994 and panobinostat labeled. Data show a positive correlation between mRNA expression, protein expression, and protein secretion with correlation coefficient, r, and p value enumerated. Cell Chemical Biology 2017 24, 892-906.e5DOI: (10.1016/j.chembiol.2017.06.010) Copyright © 2017 Elsevier Ltd Terms and Conditions

Figure 7 ChIP-qPCR of H3K27 Acetylation on GRN Promoter/Enhancer Region (A) Visualization of the promoter/enhancer region of GRN in two human brain regions and human embryonic stem cell H9-derived NPCs and neurons, with measured H3K27 acetylation marks in the brain regions and imputed H3K27 acetylation in the H9-derived cell lines as compiled by the WashU EpiGenome Browser (Zhou et al., 2011). TSS, transcriptional start site. (B) Promoter/enhancer region of GRN, showing H3K27 acetylation and binding sites for selected ENCODE transcription factors for up to seven cell lines (indicated above). Image was modified from UCSC Genome Browser (Kent et al., 2002) and shown to scale with (A). H3K27 acetylation data is layered, so some colors may not be reflected in the legend. More information may be found on the UCSC Genome Browser. Transcription factors were selected based on binding affinity for HDAC complexes or known HDAC-modulated genes. Transcription factors are color coded in grayscale; the darkness of the box is proportional to maximum ChIP data value seen in any cell line in the region. The ChIP regions denote regions of interest for ChIP-qPCR. (C) H3K27 acetylation ChIP-qPCR data in NPCs for the regions on the GRN promoter denoted in (A) and (B). Cells were treated with vehicle (DMSO), panobinostat (0.5 μM), or CI-994 (10 μM) for 24 hr. Each condition is shown as the mean + SEM of 3 technical replicates with significance calculated by unpaired t test. **p < 0.01, ***p < 0.001. Cell Chemical Biology 2017 24, 892-906.e5DOI: (10.1016/j.chembiol.2017.06.010) Copyright © 2017 Elsevier Ltd Terms and Conditions

Figure 8 TFEB Protein Expression and Occupancy on the GRN Promoter after HDAC Inhibitor Treatment (A) TFEB, pTFEB(Ser142), and PGRN protein levels in NPCs when treated with HDAC inhibitors. Graph shows best-fit curve with 95% confidence interval (dotted line). NPCs were treated with vehicle (ddH2O for valproate, DMSO for all others), SAHA (10 μM), panobinostat (0.5 μM), crebinostat (2.5 μM), ACY-1215 (5 μM), tubastatin A (10 μM), PCI-34051 (10 μM), apicidin (2.5 μM), CI-994 (10 μM), Cpd-60 (5 μM), or valproate (5 mM) for 24 hr. Quantification was done with ImageJ. (B) Potential TFEB binding sites on the GRN promoter/enhancer region shown relative to H3K27 acetylation in seven cell lines from the ENCODE project and the UCSC Genome Browser (Kent et al., 2002). H3K27 acetylation data is layered, so some colors may not be reflected in the legend. More information may be found on the UCSC Genome Browser. E-box sites (5′-CANNTG-3′) are shown with blue lines. CLEAR motifs (5′-TCACG-3′) are shown in orange. These sites are overlaid with the ENCODE transcription factor binding regions (yellow boxes). Portions of the genome where both E-box sites and CLEAR motifs occur near known transcription factor binding sites are shown, with the start site of GRN 5′ UTR marked in bold, highlighted in purple. (C) TFEB ChIP-qPCR data in NPCs for the regions on the GRN promoter denoted in (B). Cells were treated with vehicle (DMSO), panobinostat (0.5 μM), or CI-994 (10 μM) for 24 hr. Each condition is shown as the mean + SEM of 2 replicates with significance calculated by unpaired t test. *p < 0.05, ***p < 0.001. Cell Chemical Biology 2017 24, 892-906.e5DOI: (10.1016/j.chembiol.2017.06.010) Copyright © 2017 Elsevier Ltd Terms and Conditions