GM604 Down-Regulates SOD1 and Alters Expression of 89 Genes Associated with Amyotrophic Lateral Sclerosis William R
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GM604 down-regulates SOD1 and alters expression of 89 genes associated with amyotrophic lateral sclerosis William R. Swindell1, Krzysztof Bojanowski2, Mark Kindy3,4, Tony Shum5, Raymond Chau5, Dorothy Ko5 1Ohio University, Heritage College of Osteopathic Medicine, Athens, OH, USA; 2Sunny BioDiscovery, Inc., Santa Paula, CA, USA; 3Department of Pharmaceutical Sciences, College of Pharmacy, University of South Florida, Tampa, FL, USA; 4James A. Haley VAMC, Tampa, FL, USA; 5Genervon Pharmaceuticals LLC, Pasadena, CA, USA. INTRODUCTION Source ALS Genes GM6 ▲ GM6 ▼ Test 1* Test 2** † Amyotrophic lateral sclerosis (ALS) is an incurable disease of unknown ALSoD 78 35 8 P = 0.004 P = 0.152 etiology characterized by motor neuron death leading to muscle paralysis. GWAS Catalog 156 49 19 P = 0.002† P = 0.859 While the exact cause of ALS remains unclear, possible pathogenic OMIM 21 14 1 P = 0.001† P = 0.04† processes include glutamate toxicity, mutations and/or post-translational DisGeNET 198 61 37 P = 0.304 P = 0.003† modifications affecting superoxide dismutase 1 (SOD1), mutations † impacting DNA or RNA binding proteins (e.g., TDP-43, TUS/TLS), and MeSH 609 178 125 P = 0.184 P = 0.003 protein mislocalization or aggregate formation. Table 2. ALS-associated genes identified from multiple database sources exhibit unique expression responses to GM6 in SH-SY5Y cells. *Directional test. Over the past 145 years no curative drug for ALS has been discovered. P < 0.05† indicates that ALS-associated genes were more likely to be increased by Numerous ALS drug candidates have failed in clinical trials in recent GM6 as compared to all other genes expressed by SH-SY5Y cells (n = 5000 decades. GM604 (GM6) is a synthetic oligopeptide containing 6 amino acids simulation trials). **Non-directional test. P < 0.05† indicates that ALS-associated developed by Genervon Biopharmaceuticals. It has been proposed that genes were more likely to be altered (increased or decreased) by GM6 as compared to all other genes expressed by SH-SY5Y cells (5000 simulation trials). GM6 targets multiple pathways key to neuronal development during the embryonic stage. However, these pathways and their associated signaling ALS-associated genes MAT2A, OSBP, HIF1A, UBAP2L, PPP2R5D, CNTN4, DNM1L, FGF2, CEP250, cascades are not yet fully elucidated. strongly increased by HSPD1, PAK1, DPYSL3, ADAM10, PSEN1, SMC3, FOXO3, ADD1, TBK1, ALS2, TNPO1, HUS1, HDAC4, PTH2R, DAG1, MFN1, RBMS3, INTS6, CAT, GM6 (FDR < 0.10, FC > DCTN1, HDAC6, ALCAM, LIG4, SS18L1, SPAST, SLC18A3, ATP2B2, MAP3K7, We used DNA microarray analysis to identify genes altered by GM6 in SH- 2.00) MATR3, CASP3, DCTN5, SQLE, TCP1, BRAP SY5Y neuroblastoma cells. This report summarizes key findings from this Figure 2. Genes associated with ALS in GWAS studies and their response to ALS-associated genes NPY, PARK7, RCC1, ARHGAP18, PFDN2, TXN, PPIH, CST3, NES, PFDN5, strongly decreased by NPEPPS, TAF15, FIS1, APRT, NDUFA2, SOD1, BAX, INS, CREBBP, LAGE3, analysis and evaluates associations with genes of known importance to GM6 in SH-SY5Y cells. (A) ALS-associated genes most strongly increased by GM6. EXOSC1, SUMO3, B2M, CYP1A2, GAP43, LGALS1, NRGN, FADD, DDIT3, ALS. A tentative mechanism of action for GM6 in the setting of ALS is (B) ALS-associated genes most strongly decreased by GM6. (C, D) Simulation GM6 (FDR < 0.10, FC < EIF3K, GSTO1, KHSRP, SRP14, POLR2H, LY6E, PABPN1, PNO1, IGFBP2, proposed. analyses. Sets of 156 SH-SY5Y-expressed genes were sampled at random. In (C), 0.50) NDUFB3, CTDSP1, S100A6, KIN, RAB5A, TH, SNCG, CD163 the histogram shows the average FC among sets of 156 randomly sampled genes Table 3. 89 ALS-associated genes strongly altered by GM6. Genes were (arrow: observed average FC among 156 ALS-associated genes). In (D), the associated with ALS by at least 1 of 5 database sources (see Table 2). Genes are RESULTS histogram shows the average value of 2abs[log2(FC)] for each randomly sampled gene ranked such that the first genes listed were most strongly altered by GM6 (e.g., set, representing the average non-directional change in gene expression (arrow: MAT2A and NPY). DNA microarrays were used to compare gene expression in GM6- and observed value among 156 ALS-associated genes). control-treated SH-SY5Y cells (n = 2 replicates per treatment). Cells were The 89 ALS-associated GM6-regulated genes were functionally associated To evaluate whether genes altered by GM6 might also be involved in the treated with GM6 for 48 hours prior to extraction of total RNA. The microarray with neuronal generation, regulation of neuron death, and regulation of pathogenesis of ALS, we cross-referenced our results with genes that have platform included probes associated with 15597 human genes, of which oxidative stress-induced death (Figure 4). 13452 were expressed above background and included in statistical analyses. been associated with ALS by previous genome-wide associated studies Differential expression analyses identified > 1000 genes modulated by GM6. (GWASs). The NHGRI-EBI GWAS catalog was used to identify ALS- The exact number of GM6-regulated genes varied depending upon the fold- associated genes. From this source, 156 ALS-associated genes expressed by change (FC) and false discovery rate (FDR) threshold applied (Table 1). The SH-SY5Y cells were identified (49 GM6-increased; 19 GM6-decreased; FDR 581 genes strongly elevated by GM6 (FC > 2) were associated with cell < 0.10; Figure 2). On average, the 156 ALS-associated genes were increased division and mitosis (Figure 1A). In contrast the 678 genes most strongly by 12.5% in GM6-treated cells (average FC = 1.125). This average FC was decreased by GM6 were highly associated with the mitochondrial envelope significantly larger than observed among randomly sampled sets of 156 (Figure 1B). genes (P = 0.002; 5000 simulation trials; Figure 2C). ALS-associated genes thus exhibited a unique expression response to GM6 and were more likely Strong increase (FDR < 0.10, FC > 2.00) 581 genes than other genes to be GM6-increased. Notably, SOD1 was the ALS-associated gene most strongly decreased Mild increase (FDR < 0.10, FC > 1.50) 1418 genes by GM6 (P = 6.98 × 10-6; Figure 2B). Using RT-PCR, we confirmed that Figure 4. Functional associations of ALS-associated genes altered by GM6. Weak increase (FDR < 0.10, FC > 1.00) 1600 genes decreased SOD1 mRNA expression is an early response of SH-SY5Y cells to Gene ontology (GO) biological process (BP) terms enriched among the 43 ALS- Strong decrease (FDR < 0.10, FC < 0.50) 678 genes GM6 and occurs as early as 2 hrs. post-treatment (2 hrs: FC = 0.53; 4 hrs: FC associated GM6-increased genes (left) and 46 ALS-associated GM6-decreased genes (right) are shown. Larger font sizes indicate a stronger statistical association = 0.48). We next measured SOD1 protein abundance in patients treated with Mild decrease (FDR < 0.10, FC < 0.67) 789 genes between the GO BP term and the set of ALS-associated genes (conditional Weak decrease (FDR < 0.10, FC < 1.00) 998 genes six doses of GM6 (n = 8) or placebo (n = 8) during a 2 week period. There hypergeometric test). was a trend towards decreased SOD1 in CSF from GM6-treated patients (P = No change (FDR > 0.10) 7388 genes 0.123), while plasma SOD1 was significantly reduced by GM6 treatment (P = Table 1. 13452 genes with detectable expression were categorized based 0.009) (Figure 3). Summary upon the degree to which expression was altered by GM6. These analyses demonstrate that GM6 down-regulates SOD1 and has unique effects on the expression of genes implicated in the pathogenesis of ALS. Compared to other SH-SY5Y-expressed genes, ALS-associated genes were more frequently up- or down-regulated by GM6 treatment (Table 2). This result is consistent with the idea that GM6 alters signaling pathways involved in the pathogenesis of ALS. Our findings suggest a tentative mechanism of action (MOA) by which GM6 could prolong motor neuron survival in ALS patients. We propose a “tripartate mechanism”. First, by reducing SOD1 expression (Figures 2B and 3), GM6 may block accumulation of pathologic SOD1 aggregates in Figure 3. SOD1 protein in CSF and plasma of GM6- and control-treated patients motor neurons. Second, by reducing mitochondrial gene expression and (Phase 2A clinical trial). A phase 2A trial was carried out in which 8 ALS patients were potentially mitochondrial abundance (Figure 1B), GM6 may disrupt the treated with GM6 and 4 ALS patients were treated with placebo. SOD1 was measured mitochondrial (intrinsic) apoptotic pathway. Third, GM6 appears to in cerebrospinal fluid (CSF) and plasma at baseline (visit 1) and following 6 doses of GM6 over 2 weeks (visit 6). In (A) and (B), each point represents a single ALS patient. activate developmental/mitotic pathways (Figures 1A and 4), which may Patients below the diagonal showed decreased SOD1 in CSF or plasma post- promote cellular repair, axonogenesis, and neuron projection. treatment. P-values (lower right) were generated from a one-tailed t-test comparing change in SOD1 (visit 6 – visit 1) between GM6- and placebo-treated patients. In a phase 2A trial, short-term GM6 treatment favorably altered forced vital capacity and the biomarker profile of ALS patients (n = 8 GM6- Bioinformatic analyses were repeated with ALS-associated genes identified treated; n = 4 placebo). Changes in SOD1 protein following GM6 from 4 other database sources, including the ALS Online Genetics treatment are shown in Figure 3 and demonstrate GM6-induced declines Database (ALSoD), Online Mendelian Inheritance in Man (OMIM), in plasma SOD1 (P = 0.009) with a similar trend in CSF (P = 0.123). DisGeNET database, and Medical Subject Headings (MeSH) database These clinical findings appear consistent with our microarray/RT-PCR Figure 1.