Fig 2: PIP5K1A mediates oncogenic KRAS signaling. a Representative Crystal Violet staining, b intensity of Crystal Violet staining (mean ± SE, triplicate samples, three experiments, normalized to vector control), and c immunoblot detection of phosphorylated (P-) ERK and AKT and PIP5K1A in cells expressing the indicated RASG12V proteins in the absence (vector) or presence of up to three separate PIP5K1A sgRNAs. *p = 0.05, one-sided Student’s t test. n = 2. d Immunoblot detection of P-AKT in Capan-1 cells stably transduced with a vector encoding Cas9 and no sgRNA (-) or PIP5K1A_1 sgRNA (+) and another vector encoding no transgene (vector), wild-type PIP5K1A, or PIP5K1AKM (detected with an a-V5 antibody). n = 2. e GO classifications showing fold enrichment and significance (p values, Fisher’s exact test) based on GO enrichment analysis of proteins biotinylated by BirA*-KRASG12V in the absence or in the presence of PIP5K1A. Where indicated, total (T-) ERK and AKT as well as ß-actin serve as loading controls

Fig 3: PIP5K1A loss is additive with a MEK inhibitor in KRAS-mutant cancer cells. Percent cell viability (mean ± SD, triplicate samples, two experiments, normalized to vector control cells treated with DMSO) of the indicated KRAS-mutant human cancer cell lines stably infected with a lentiviral vector encoding Cas9 and no transgene (vector) or one of up to three independent sgRNA targeting PIP5K1A (sgPIP5K1A-1 to -3) after treatment with vehicle (DMSO) or 25 nM of trametinib for 72 h. *p = 0.05, two-way ANOVA test

Fig 4: PIP5K1A binds specifically to KRASG12V. a Plots of proximity labeling by myc-tagged BirA* fused to (top), and sgRNA enrichment scores in cells transformed with (bottom), KRASG12V (black triangle), NRASG12V (orange square), or BirA*-HRASG12V (blue circle) for the indicated 19 kinases. b, c Immunoblot detection of the indicated immunoprecipitated (IP) and co-immunoprecipitated (CO-IP) myc-RASG12V and PIP5K1A-V5 proteins. n = 2. d Immunoblot detection of IP endogenous KRAS with CO-IP endogenous PIP5K1A from SW620 cells. n = 1. e Recombinant GST-PIP5K1A and His10-KRASG12V co-elution (eluates 2–4: E2, E3, and E4) from a Ni-charged column, as detected by Coomassie staining (bottom) or immunoblot (top). INPUT, flow through (FT), and the final wash (W) confirm protein mixtures and retention of proteins on the column. n = 2. f, g Immunoblot detection of IP recombinant f His10-KRASG12V or g His10-HRASG12V with CO-IP recombinant GST-PIP5K1A. n = 2. h Schematic representation of myc-KRASG12V unaltered, when the HVR is replaced with that of HRAS (HHVR), and when the indicated seven lysine residues in the HVR are mutated to alanine (7A). i, j Immunoblot detection of IP myc-KRASG12V and either i myc-KRASG12V-HHVR (n = 2) or j myc-KRASG12V-7A (n = 1) CO-IP with PIP5K1A-V5. Where indicated, INPUT lysates serve to confirm appropriate expression of the indicated proteins, the absence (−) of PIP5K1A-V5 or RAS isoforms serve as a control for the specificity of the CO-IP (note that the α-V5 INPUT immunoblot in c was run on a separate gel processed at the same time), and IgG or no antibody serve as a negative control

Fig 5: Upregulation of ABCA1 in NPC1 Disease Decreases PtdIns(4,5)P2 and Leads to Hyperexcitability(A) Schematic of PtdIns(4,5)P2 synthesis. Relevant enzymes are in red.(B) Blot stained for PtdIns(4,5)P2 metabolizing enzymes PIP5K1A, SYNJ1, and PLCß1 from cerebellums collected from NPC1I1061T and WT littermate mice.(C) Summary of fold change of protein abundance in NPC1I1061T cerebellum relative to WT (n = 4 blots from different mice).(D) Schematic of hypothesis: upregulation of ABCA1 reduces PtdIns(4,5)P2 levels.(E) Confocal micrographs of healthy (left) and NPC1I1061T (right) fibroblasts transfected with PHPLCd1. Some healthy fibroblasts were treated with 1 µM T0901317, an LXR agonist, overnight (center). Insets show the reduced intensity of PHPLCd1 at the PM from healthy fibroblasts treated with LXR agonist.(F) Comparison of the ratio of PHPLCd1 fluorescence intensity at the PM relative to the Cyt in healthy (empty, n = 13), healthy treated with LXR agonist (yellow, n = 15), and NPC1I1061T fibroblasts (red, n = 10).(G) Inverted confocal micrographs of HeLa cells with endogenous levels of ABCA1 (WT, left) and overexpressed levels of ABCA1 (ABCA1, right) transfected with PHPLCd1. Insets show the reduced intensity of PHPLCd1 at the PM of HeLa cells with upregulated levels of ABCA1.(H) Comparison of the ratio of PHPLCd1 fluorescence intensity at the PM to the Cyt in WT (n = 40) and ABCA1 HeLa cells (n = 33).(I) Top: blot stained for ABCA1 from cerebellum collected from NPC1I1061T and WT littermate mice. Bottom: summary of fold change of ABCA1 abundance in NPC1I1061T cerebellum relative to WT (n = 4 blots from different mice).(J) Schematic of the hypothesis: inhibition of ABCA1 in NPC neurons rescues current density and excitability.(K) Inset: protocol used to isolate KCNQ2/3 current. Comparison of KCNQ2/3 current recordings from neurons isolated from WT (black) and NPC1I1061T (red) mice. Some neurons isolated from NPC1I1061T were treated with 10 µM probucol (pink). Dotted line: 0 pA. Bottom: summary of KCNQ2/3 current density from WT (n = 8, empty), NPC1I1061T (n = 6, red), and NPC1I1061T treated with probucol (n = 8, pink).(L) Top: schematic of current injection protocol. Center: family of voltage recordings shows a probucol-treated NPC1I1061T neuron firing 2 APs with 40 pA current injection (black trace). Dotted line: -65 mV. Bottom: comparison of the number of APs induced with increasing current injections in probucol-treated NPC1I1061T neurons (pink, n = 9) and WT and NPC1I1061T neurons (same data as Figure 2).See also Figures S4, S5, S6, and S7.