Fig 2: Gene re-expression representative of photoreceptor-rich retinoblastoma in MYCN-knockdown cell models.a Volcano plot shows the MYCN-RB signature of 244 genes derived from the overlap of previously identified MYCN targets and sleuth-modeled MYCN-knockdown expression pattern (Supplementary Data 25). Red dots correspond to significantly regulated MYCN targets (Storey-Tibshirani adjusted p ≤ 0.1). Orange dots correspond to other MYCN targets (Storey-Tibshirani adjusted p > 0.1). Black dots represent non-MYCN targets significantly downregulated in sleuth-modeled MYCN-knockdown expression pattern. b GSEA showing genes overexpressed in cluster A, genes representative of photoreceptor-rich retinoblastoma12,13 and the cluster B signature in the sleuth-modeled MYCN-knockdown expression pattern. Y-axis indicates enrichment score (ES). X-axis shows pathway genes. The dual-colored band represents the degree of correlation in the expression of these genes with MYCN-knockdown (red, MYCN-knockdown; blue, control). c Box plot (left) showing aggregated log1p-transformed transcript tpm values for OPTN expression and combined Storey-Tibshirani-adjusted p in 52 retinoblastomas. OPTN protein expression (right) in MYCN-knockdown retinoblastoma cell lines 48 h after doxycycline treatment. Dot plots show ratios of median fluorescence intensity (MFI) for OPTN in shNTC-expressing (doxycycline-treated cells vs non-treated cells) and shMYCN-expressing (doxycycline-treated cells vs non-treated cells) retinoblastoma from three independent experiments. Data are from 3 independent experiments, with average values (blue bots) indicated and p-values calculated with Welch t-test reported. Representative plots of flow cytometry for OPTN are shown in Supplementary Fig. S7c. Venn diagrams showing the pathways (FDR adjusted p ≤ 0.05) associated with downregulated genes (d) and upregulated genes (e) in MYCN-knockdown retinoblastoma cell lines. The 9 pathways depleted in all 4 cell lines are listed. f List of genes upregulated in both RB1−/− cell lines. Box plots showing the expression scores of the MYCN-RB signatures (g), and upregulated genes (h) after MYCN-knockdown in each of the 3 retinoblastoma clusters. Boxes show upper and lower quantile and median. Whiskers extend from the hinge to ±1.5 times the interquartile range.

Fig 3: Molecular network analysis of the ERK/MAPK pathway and associated canonical pathways. Integrated molecular network of ERK/MAPK signaling, autophagy, and apoptotic execution pathways in non-treated (A) NT (SC3KO vs. WT) and MK-8353 treated groups (B) (MK: SC3KO vs. WT). Molecular network analysis was performed using Ingenuity Pathway Analysis (IPA®). Node color indicates relative expression (red: upregulated; green: downregulated), and edges represent curated molecular relationships from the IPA knowledgebase. Legend indicates the function of each protein and the interactions between them. Significant proteins are determined by Log2FC ≤ 1.5 and p ≤ 0.05. (C) Protein–protein interaction network of SCAMP3-regulated phosphoproteins within the ERK1/2 cascade (yellow) and autophagy (red) (NT: SC3KO vs. WT). The network was generated using the STRING 2024 database with a confidence cutoff of 0.400. Interactions with scores < 0.400 are included and labeled with their numerical confidence values. Predicted interactions with SCAMP3, EGFR, MAP1LC3B, and mTOR are also highlighted. Gene names displayed in the network correspond to the following proteins: ACIN1, apoptotic chromatin condensation inducer in the nucleus; ELF4, E74 like ETS transcription factor 4; LMNB1, Lamin-B1; MAP1LC3B, microtubule-associated protein 1 light chain 3 beta; MAP2K2, dual specificity mitogen-activated protein kinase kinase 2; MAPK1, mitogen-activated protein kinase 1; MAPK3, MAP kinase-activated protein kinase 3; mTOR, mammalian target of rapamycin; OPTN, optineurin; PAK4, serine/threonine-protein kinase PAK 4; PI4K2A, phosphatidylinositol 4-kinase type 2-alpha; PLEC, Plectin; PPP1R10, serine/threonine-protein phosphatase 1 regulatory subunit 10; PRKCD, Protein kinase C delta type; RAF1, RAF proto-oncogene serine/threonine-protein kinase: SQSTM1, Sequestosome-1; TBC1D5, TBC1 domain family member 5; TJP1, tight junction protein ZO-1, and TJP2, tight junction protein ZO-2. In IPA networks, an asterisk (*) next to a protein name means the node is representative for a group of molecules/isoforms/aliases, not just a single, unique protein.

Fig 4: optineurin antibody screening by western blot.Lysates of U2OS WT and OPTN KO were prepared, and 35 μg of protein were processed for western blot with the indicated optineurin antibodies. The Ponceau stained transfers of each blot are presented to show equal loading of WT and KO lysates and protein transfer efficiency from the 4-20% Tris-Glycine polyacrylamide gels to the nitrocellulose membrane. Antibody dilutions were chosen according to the recommendations of the antibody supplier. Antibody dilutions used: ab213556** at 1/5000, ab242146** at 1/1000, NBP3-19900** at 1/1000, 70928** at 1/1000, 60293-1-Ig* at 1/5000, Z-OPTN-10** at 1/1000, 702766** at 1/200, 711879** at 1/5000, were titrated as the signal was too weak or too strong when following the supplier’s recommendations. Predicted band size: 66 kDa. ** = recombinant antibody, * = monoclonal antibody.

Fig 5: Phospho-proteomics defines the impact of TBK1 function on endo-lysosomal pathways(A) Schematic of global phospho-proteome-wide profiling. Briefly, phospho-peptides were enriched using titanium dioxide (TiO2) before being fractionated into 50 fractions for subsequent mass spectrometry analysis.(B) Volcano plots representing differential abundance of phospho-peptides of isogenic cell lines, following normalization to the global-proteomics data. A total of i = 50,872 normalized phospho-peptides were quantified. Phospho-peptides of selective autophagy cargo receptors (SACRs; in red) and autophagy factors (in yellow) were decreased in abundance in TBK1 KO cells compared to control, and Gene Ontology analysis demonstrated enrichment for the autophagy pathway. No significant pathway enrichment was found in either the TBK1 E696K or OPTN KO lines.(C and D) Specific phospho-peptides with phosphoserine residues consistent with TBK1 kinase consensus sites in autophagy proteins, including several SACRs (C) and key regulators of autophagy and endo-lysosomal pathways (D). Statistical significance: *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.(E) Heatmap representing a selection of phospho-peptides from autophagy factors, ALS-associated proteins, SACRs, and novel targets of interest.(F) Volcano plots representing the differential abundance of phospho-peptides normalized to the global proteomics data from TBK1 G217R hiPSC lines SNV/WT and SNV/SNV compared to REV/WT (isogenic CRISPR-revertant control). A total of i = 30,810 normalized phospho-peptides were quantified. Phosphorylation of the SARC p62/SQSTM1 (highlighted in red) was decreased in TBK1 G217RSNV/SNV compared to REV/WT.(G) Quantification of selected phospho-peptides with phosphoserine residues consistent with TBK1 kinase consensus sites of p62/SQSTM1, OPTN, GABARAPL2, and RAB7A across genotypes, demonstrating a variant-dosage effect on the phosphorylation of specific residues of key TBK1 substrates. Data are presented as mean ± s.e.m.; each dot represents an independent biological replicate (n = 4). Pairwise comparisons among genotypes were performed using a one-way ANOVA with Tukey’s multiple comparisons post hoc test. Statistical significance: *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.