EMBO/soda-nel huggingface dataset:
Inhibition of pyrimidine biosynthesis targets protein translation in AML. (2022)
Figure 1

Figure 1. AG636 is an effective single agent therapy in MLL-rearranged AML.

A Frequency of MN leukemic cells expressing the immature marker cKit and mature myeloid marker Ly6G in the peripheral blood following 5 days of treatment (n = 9-10 mice/group; mice with < 2% tumor burden in any condition were censored from the analysis).

B Absolute number of MN cells in the peripheral blood quantified by flow cytometry. Grey bars denote treatment (n = 11-12 mice/group).

C Kaplan-Meier survival curve of leukemic mice (n = 11-12 mice/group, median survival is 16.5 for vehicle and not reached for AG636 and doxycycline, P < 0.0001 by Log-rank test).

D Representative FACS plots showing differentiation induced by AG636 and doxycycline.

E Spleen weights of MN tumor-bearing mice (n = 3-6 mice/group).

F Absolute number of MN cells in the bone marrow (n = 3-6 mice/group).

G Frequency (as percentage of all MN cells) of LSCs (CD11blowcKithighFcgR+) and differentiated cells (CD182+Ly6G+) in the bone marrow (n = 3-6 mice/group).

H May-Grunwald-Giemsa-stained cytospins of sorted MN cells showing myeloid differentiation.

I Quantification of functionally-define LSCs using a limiting dilution assay calculated by ELDA (n = 4-8 mice/group).

Data information: data in A and E-G are presented as mean ± s.d.; P values were calculated using a one-tailed Student's unpaired t-test. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001; Dox - doxycycline.
Inhibition of pyrimidine biosynthesis targets protein translation in AML. (2022)
Figure 2

Figure 2. DHODH inhibition induces differentiation and inhibits proliferation in multiple AML sub-types.

A Spleen weights of RUNX1-RUNX1T1 or I1DN tumor bearing mice treated with AG636 or vehicle for 4 days.

B-C Absolute number of leukemic cells in the bone marrow (B) and spleens (C) of RUNX1-RUNX1T1 or I1DN tumor bearing mice treated with AG636 or vehicle for 4 days.

D-E Frequency of leukemic cells expressing the immature marker cKit and mature myeloid marker CD11b in the bone marrow and spleens of RUNX1-RUNX1T1 (D) or I1DN (E) tumor bearing mice treated with AG636 or vehicle for 4 days (n = 6 mice/group).

Data information: n = 5-6 mice/group for RUNX1-RUNX1T1 model, n = 4 mice/group for I1DN model; data are presented as mean ± s.d.; P values were calculated using a one-tailed Student's unpaired t-test. *P < 0.05, **P < 0.01.
Inhibition of pyrimidine biosynthesis targets protein translation in AML. (2022)
Figure 5

Figure 5. Downregulation of YY1 protein contributes to altered gene expression in AML cells following DHODH inhibition.

A-B ATAC sequencing performed on cKithighCD11blow MN cells sorted from mice treated with AG636 or vehicle for 2 days (n = 3 mice/group). HOMER motif analysis showing transcription factor motifs enriched within regions of differential chromatin accessibility (A). HOMER motif analysis showing enrichment of YY1 motifs within regions of accessible chromatin associated with translation genes. All other regions of open chromatin were used as the background (B).

C Rank plots of ChIPseq enrichment scores of transcription factors at the promoter regions (+1000 bp to -50 bp from TSS) of genes within the indicated gene sets extracted from ENCODE 3 (n = 24 genes for translation genes, n = 707 genes for AG636 down-regulated genes, n = 4237 for the other genes).

D ChIPqPCR showing the binding of YY1 at the promoter regions of selected translation genes in MN cells treated with AG636 or DMSO. For each replicate enrichment was normalized to YY1 pull-down in DMSO treated cells (n = 2 biological replicates).

E Time-course showing global downregulation of O-GlcNAcylation and YY1 expression in MN cells treated with AG636. The experiment was repeated twice with similar results.

F Western blot of YY1 expression in human AML cell lines treated with AG636 for 24 hours.

G qPCR showing down-regulation of genes encoding ribosomal proteins that are putative YY1 targets in Cas9 expressing MN cells treated with AG636 or DMSO (left); or transduced with sgRNAs targeting YY1 or control sgRNAs (right) (n = 2 biological replicates).

H Barcode plots showing downregulation of the Reactome Translation gene set in YY1 knockout pro B cells and human melanoma cells upon YY1 knockdown (Varum et al, 2019; Kleiman et al, 2016).

Data information: data in D and G are presented as mean ± s.e.m.; P values were calculated using a two-tailed Student's unpaired t-test in D and a one-tailed Student's unpaired t-test in G; *P < 0.05, ***P < 0.001, ****P < 0.0001.
Loss of the mitochondrial i-AAA protease YME1L leads to ocular dysfunction and spinal axonopathy (2018)
Figure 1

Figure 1 - Loss of YME1L in the nervous system causes microphthalmia, cataracts and retinal inflammation.

A Representative images of eyes and lenses from 6-7 week old wildtype (WT) and nervous system-specific YME1L knockout (NYKO) mice. Orange dashed lines mark eye morphology. Scale bars 5 mm.

B Retinal sagittal cross-sections from 6-7 week old mice stained with hematoxylin and eosin. NFL = Nerve fiber layer, IPL = inner plexiform layer, INL = inner nuclear layer, OPL = outer plexiform layer, ONL = outer nuclear layer, R&C = Rods and cones. Scale bars 30 µm.

C Quantification of nuclei in OPL (area = 1000 µm2) from retina cross-sections of 6-7 week old WT (n = 4) and NYKO (n = 4) mice.

D Immunoblot analysis of retinal lysates from 6-7 week old WT and NYKO mice. GFAP was used as a marker for reactive astrogliosis and SDHA as a loading control.

E mRNA levels of proinflammatory cytokines and NF-κB target genes from 6-7 week old retinas (WT, n = 5; NYKO, n = 5). Transcript levels were normalized to Hprt mRNA levels.

F mRNA levels of Fgf21 from 6-7 week old retinas (WT, n = 5; NYKO, n = 5). Transcript levels were normalized to Hprt mRNA levels.

G Transmission electron micrographs of optic nerves from 6-7 week old WT and NYKO mice. Scale bars 2 µm.

Data were analyzed using unpaired t-test, *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001, ns = not significant. Data are means ± SEM.
Loss of the mitochondrial i-AAA protease YME1L leads to ocular dysfunction and spinal axonopathy (2018)
Figure 3

Figure 3 - The loss of YME1L in the nervous system does not trigger brain atrophy.

A Brain weights were monitored at 31-32 weeks of age (WT, n = 15; NYKO, n = 14).

B Nissl stainings of sagittal sections across brain regions from 31-32 week old WT and NYKO mice. 4x and 40x enlargements are shown in the left and right panels, respectively. Scale bars 4x = 500 µm, 40x = 50 µm.

C Morphometric analysis of different brain regions from 31-32 week old WT (n = 6) and NYKO (n = 6) mice. GCL = Granule cell layer, ML = Molecular layer.

D Immunoblot analysis of tissue lysates (FB = forebrain; CB = cerebellum) from 31-32 week old WT and NYKO mice, using GFAP-specific and, as a loading control, GAPDH-specific antibodies.

Unpaired t-test, ns = not significant. Data are means ± SEM.
Loss of the mitochondrial i-AAA protease YME1L leads to ocular dysfunction and spinal axonopathy (2018)
Figure 5

Figure 5 - YME1L facilitates mitochondrial trafficking in neurons.

A Fluorescence images of isolated cortical neurons from Yme1lfl/fl animals expressing CAG-GFP (WT) or CAG-Cre-IRES-GFP (Yme1l-/-) at DIV12 and stained with TOMM20 to visualize mitochondria. Scale bars 30 µm.

B - E Quantification of total axon length (WT, n = 13 neurons; Yme1l-/-, n = 13 neurons), total dendritic length (WT, n = 12 neurons; Yme1l-/-, n = 13 neurons), axonal branch points (WT, n = 12 neurons; Yme1l-/-, n = 13 neurons) and number of dendrites (WT, n = 12 neurons; Yme1l-/-, n = 13 neurons). Individual neurons originate from two independent preparations.

F, G Kymographs showing mitochondrial movements in representative axons. Vertical lines correspond to stationary mitochondria and diagonal lines to moving mitochondria. Mitochondria were stained using Mitotracker. In total 39 axonal segments were analyzed for WT control neurons and 37 axonal segments for Yme1l-/- neurons at DIV7. Data are means of three independent experiments ± SEM.

H, I Kymographs showing mitochondrial movements in representative axons. Vertical lines correspond to stationary mitochondria and diagonal lines to moving mitochondria. Mitochondria were stained using Mitotracker. In total 120 axonal segments were analyzed for WT control neurons and 107 axonal segments for Yme1l-/- neurons at DIV10-14. Data are means of three independent experiments ± SEM.

J Axonal area occupied by mitochondria in Yme1l-/- neurons relative to WT controls. In total 120 axonal segments were analyzed for WT control neurons and 107 axonal segments for Yme1l-/- neurons at DIV10-14. Data are means of three independent experiments ± SEM.

Unpaired t-test, ns = not significant, *P ≤ 0.05.
Loss of the mitochondrial i-AAA protease YME1L leads to ocular dysfunction and spinal axonopathy (2018)
Figure 8

Figure 8 - Loss of OMA1 does not restore ocular dysfunction and deteriorates axonal degeneration in the spinal cord of NYKO mice.

A Immunoblot analysis of retinal and spinal cord lysates from 6-7 week old WT, NYKO, NOKO and NYOKO mice (n = 3) using the indicated antibodies. SDHA was used as a loading control. * was not reproducibly detectable in all experiments.

B Frequency distribution of mitochondrial lengths in spinal cords of 6-7 week old NYOKO mice (176 mitochondria) compared to WT (as in 7B). Kruskal-Wallis test, ns = not significant.

C Representative images of eyes and lenses from 6-7 week old WT and mice lacking OMA1 (NOKO) or both YME1L and OMA1 (NYOKO) in the nervous system. Orange dashed lines mark eye morphology. Scale bars 2.5 mm.

D Immunoblot analysis of retina lysates of 6-7 week old WT, NYKO, NOKO and NYOKO mice (n = 3). SDHA was used to control for gel loading.

E Transverse semithin sections of spinal cords of 6-7 week old WT, NOKO and NYOKO mice. Sections were stained with toluidine blue. Orange arrows point to MDBs indicating degenerating neurons. Scale bars 25 µm.

F MDBs in dorso-lateral tracts of 6-7 week old WT (n = 3), NOKO (n = 3), and NYOKO (n = 3) mice.

G Walking beam test of 6-7 week old WT (n = 6), NOKO (n = 5) and NYOKO (n = 7) mice.

Data were analyzed using one-way ANOVA. ***P ≤ 0.001, ****P ≤ 0.0001, ns = not significant. Data are means ± SEM.

EXPANDED VIEW FIGURE LEGENDS
LRRK2 regulates endoplasmic reticulum-mitochondrial tethering through the PERK-mediated ubiquitination pathway (2019)
Figure 1

Figure 1. LRRK2 regulates mitochondrial energetics and vulnerability to ER stress

(A) Electron micrographs of MEFs . Scale bar; 1 μm. High-magnification images depicting representative electron-dense materials are shown. Scale bar; 0.2 μm.

(B) Citrate synthase activity (absorbance/min) was measured in MEFs . Error bars represent ±SD from eight independent experiments.

(C, D) Oxygen consumption rates (OCRs) of MEFs were measured on an XF24 Analyzer. (C) Oxygen consumption profiles for MEFs exposed sequentially to oligomycin (2 μg/mL) (Oligo), FCCP (2 μM), and rotenone (1 μM) plus actinomycin (2 μM) (R & A). (D) OCR (pmol O2/min) (n=8). Error bars represent ±SD from eight independent experiments.

(E) ATP production (pmol/min) (n=8). Error bars represent ±SD from eight independent experiments.

(F) Representative immunoblot of LC3 and p62 in MEFs of indicated genotype. MEFs were treated with tunicamycin (5 μg/ml) or vehicle control in the presence or absence of bafilomycin A1 (400 nM), and endogenous LC3 and p62 levels were measured by immunoblotting. Data represent the ratios of LC3-II to LC3-I and p62 to actin in the absence of bafilomycin, which were normalized LRRK+/+ MEFs. Error bars represent ±SD from four independent experiments.

(G) Survival rate of MEFs treated with tunicamycin (5 μg/ml), thapsigargin (1 μM), or hydrogen peroxide (100 μM). Error bars represent ±SD from four independent experiments.

Data information: For graph B, D, E, F and G, the P values was determined by a Mann-Whitney U test. ns = not significant, *P < 0.05, **P < 0.01.
LRRK2 regulates endoplasmic reticulum-mitochondrial tethering through the PERK-mediated ubiquitination pathway (2019)
Figure 2

Figure 2. LRRK2 regulates ER-mitochondria Ca2+ transfer and tethering

(A-D) MEFs were transfected with mitochondrially targeted Cameleon. Free Ca2+ dynamics in the mitochondrial matrix were visualized using FRET. Mitochondrial [Ca2+] ([Ca2+]m) was continuously monitored by FRET imaging data are represented as absolute [Ca2+] in μmol. (A) Absolute [Ca2+]m changes in MEFs of indicated genotype in response to bradykinin (2.5 μM). (B) Basal and peak values of Ca2+ transients (μM). (C) Peak values of Ca2+ transients in MEFs transfected with IP3R or shRNA against IP3R, or treated with 2-AP (20 mM). (D) Peak values of Ca2+ transients in MEFs transfected with VDAC1 or shRNA against VDAC1. Error bars represent ±SD from six independent experiments.

(E) Representative electron of MEFs . Full arrowheads designate the limits of the zone of intimate contact between ER and mitochondria 20 nm). Scale bar: 100 nm. Data represent percentage of ERmitochondrial contacts per mitochondria perimeter. Error bars represent ±SD from 40 images MEFs .

(F) In situ PLA images using anti-IP3R and anti-VDAC1 antibodies. Scale bar: 20 μm. Data represent fluorescent in MEFs normalized LRRK2+/+ MEFs. Error bars represent ±SD from six independent experiments.

(G) Peak values of Ca2+ transients in MEFs transfected with synthetic tethering protein (TOM-mRFP-ER) to induce artificial tethering of the ER and mitochondria. Error bars represent ±SD from six independent experiments.

Data information: For graph B, C, D, E, F and G, the P values was determined by a Mann-Whitney U test. ns = not significant, *P < 0.05, **P < 0.01
LRRK2 regulates endoplasmic reticulum-mitochondrial tethering through the PERK-mediated ubiquitination pathway (2019)
Figure 3

Figure 3. Physical and functional interaction between LRRK2 and E3 ubiquitin ligases

(A) Representative immunoblots of MAM components in MEFs of indicated genotype. MAM fraction was extracted from MEFs by the Percoll gradient method and immunoblotted with antibodies indicated at the right.

(B) Diagram showing full-length and deletion constructs of LRRK2(G2019S). ANK, Ankyrin-repeat domain; LRR, leucine-rich domain; ROC, Ras complex domain; COR, C terminus of Roc domain; Kinase, kinase domain.

(C) Peak values of Ca2+ transients in MEFs transfected with deletion constructs of LRRK2(G2019S). Error bars represent ±SD from six independent experiments.

(D) Binding assays to detect interactions between deletion constructs of LRRK2(G2019S) and E3 ubiquitin ligases. HEK293 cells were transfected with deletion constructs of LRRK2(G2019S) and E3 ubiquitin ligases, and cell lysates were immunoprecipitated with antibody against the V5 or HA epitope. Precipitated proteins were subjected to SDS/PAGE, and blots were stained with antibody against the V5 or HA epitope, as indicated the right of each panel.

(E, F) Peak values of Ca2+ transients in MEFs transfected with ligase-active MARCH5(WT), MULAN(WT), and Parkin(W403A) [Parkin(WA)] or dominant-negative forms of MARCH5(H43W) [MARCH(HW)], MULAN(C339A) [MULAN(CA)], Parkin(C431A) [Parkin(CA)] (E) or in MEFs transfected with active USP30 or inactive USP30(C77S) [USP(CS)] (F). Error bars represent ±SD from six independent experiments.

Data information: For graph C, E and F, the P values was determined by a Mann-Whitney U test. ns = not significant, *P < 0.05
LRRK2 regulates endoplasmic reticulum-mitochondrial tethering through the PERK-mediated ubiquitination pathway (2019)
Figure 4

Figure 4. Phosphorylation and activation of E3 ubiquitin ligases by LRRK2

(A) Immunoprecipitation/Immunoblot of phosphorylated E3 ubiquitin ligases in LRRK2+/+ and LRRK2(G2019S)-expressing MEFs treated with vehicle (Control) or tunicamycin (1 μg/ml). Endogenous PERK, MARCH5, MULAN and Parkin were immunoprecipitated with antibody, and precipitates were immunoblotted with antibody indicated at the right. Data represent the ratio of phosphorylated to total protein levels. Error bars represent ±SD from independent experiments.

(B) Immunoprecipitation/immunoblot of phosphorylated E3 ubiquitin ligases and mitofusin 2 in MEFs in the of tunicamycin (5 μg/ml). LRRK2(G2019S) MEFs were also pretreated with LRRK2-IN-1 (1 μM). MEFs were transfected with each E3 ubiquitin ligase and biquitin and lysates were immunoprecipitated with antibody against the Myc or HA epitope. Precipitated proteins were subjected to SDS/PAGE, and blots were stained with antibody against the Myc epitope, phosphoserine or mitofin2 as indicated to the right of each panel. Mfn2: mitofusin 2, Ub: ubiquitin. Data represent ratios of phosphorylated to total E3 ubiquitin ligase and mitofusin2 to actin. Error bars represent ±SD from four independent experiments.

Data information: For graph A and B, the P values was determined by a Mann-Whitney U test. ns = not significant, *P < 0.05, **P < 0.01
LRRK2 regulates endoplasmic reticulum-mitochondrial tethering through the PERK-mediated ubiquitination pathway (2019)
Figure 5

Figure 5. PERK phosphorylates E3 ubiquitin ligases

(A) Peak values of Ca2+ transients in MEFs treated with LRRK2-IN-1 (1 μM). Error bars represent ±SD from six independent experiments.

(B)(Upper panel) Diagram showing full-length PERK Myc at the N-terminus and FLAG at the C-terminus (M-PERK-F). S1P recognition sequence R33SLL is mutated to A33SLL (M-PERK(R33A)-F).

(Lower panel) Immunoblot of PERK and PERK(R33A) from transfected MEFs under tunicamycin. MAM fraction and cytosol were extracted from transfected MEFs by the Percoll gradient method. Lysates were immunoprecipitated with antibody against FLAG. Precipitated proteins were subjected to SDS/PAGE, and blots were stained with antibody as indicated to the right.

(C) In vitro kinase assay using isolated PERK, isolated E3 ubiquitin ligase and [γ-32P]ATP. Reaction mixture was subjected to SDS/PAGE. Blots were with antibody against Myc, FLAG or phosphoserine. [γ-32P]ATP-incorporated E3 ubiquitin ligases were visualized by autoradiography ( blot was exposed for 24 , and right blot for 36 ).

(D) In vitro ubiquitination assay using phosphorylated E3 ubiquitin ligases, HA-tagged Ubl, and His-tagged mitofusin2 in the presence of E1 enzyme, UbcH7 and ATP. E3 ubiquitin ligases were initially phosphorylated PERK or PERK(K618R) in vitro. Phosphorylated E3 ubiquitin ligases were in vitro ubiquitination. itofusin 2 with Ni-NTA subjected to SDS/PAGE. Blots were with antibody against HA or mitofusin2. Mfn2: mitofusin 2, Ub: ubiquitin.

(E) Immunoprecipitation/Immunoblot of phosphorylated E3 ubiquitin ligases and mitofusin 2 in LRRK2(G2019S)-expressing MEFs transfected with the increasing amounts of LRRK2-d1-V5 (1, 5, 25 μg/106 cells) treated with tunicamycin (1 μg/ml). Endogenous MARCH5, MULAN and Parkin were immunoprecipitated with antibody, and precipitates were immunoblotted with antibody indicated at the right. Endogenous mitofusin 2 was immunoblotted with anti-mitofusin 2 antibody. Data represent the ratio of phosphorylated to total protein levels of MARCH5, MULAN and Parkin, and the ratio of mitofusin 2 to actin. Error bars represent ±SD from independent experiments.

Data information: For graph A and E, the P values was determined by a Mann-Whitney U test. ns = not significant, *P < 0.05, **P < 0.01
LRRK2 regulates endoplasmic reticulum-mitochondrial tethering through the PERK-mediated ubiquitination pathway (2019)
Figure 6

Figure 6. LRRK2 regulates PERK phosphorylation of E3 ubiquitin ligases

(A) Immunoprecipitation/mmunoblot of E3 ubiquitin ligases by PERK and the binding to mutant LRRK2. HEK293 cells were transfected with PERK and either MARCH5, MULAN or Parkin, LRRK2, LRRK2(D1994A) or LRRK2(S1292D). Cell lysates were immunoprecipitated with antibody against Myc or V5, and precipitates were immunoblotted with antibody indicated the right.

(B) In vitro kinase assay for auto-phosphorylated LRRK2 using isolated LRRK2 mutants and [γ-32P]ATP with or without LRRK2-IN-1 (1 μM). Mutant LRRK2 precipitated with antibody against V5 was subjected to SDS/PAGE. Blots were with antibody against V5 or phosphoserine. [γ-32P]ATP-incorporated LRRK2 visualized by autoradiography.

(C) Binding assays to detect interactions between phosphomimetic LRRK2(S1292D) or phosphorylation-defective LRRK2(S1292A) and E3 ubiquitin ligases. HEK293 cells were transfected with LRRK2 mutants and E3 ubiquitin ligases lysates were immunoprecipitated with antibody against Myc or V5 epitope. Precipitated proteins were subjected to SDS/PAGE, for immunoblotwith antibody against V5 or Myc.

(D) Immunoprecipitation/mmunoblot of E3 ubiquitin ligases by PERK-ΔN in the presence of LRRK2(S1292A) or LRRK2(S1292D). HEK293 cells were transfected with MARCH5, MULAN, Parkin, and PERK-ΔN LRRK2(S1292A) or LRRK2(S1292D). Cell lysates were immunoprecipitated with antibody against Myc, and precipitates were immunoblotted with antibody indicated the right.
LRRK2 regulates endoplasmic reticulum-mitochondrial tethering through the PERK-mediated ubiquitination pathway (2019)
Figure 7

Figure 7. Suppression of PERK rescues ER-mitochondrial dysfunction by LRRK2(G2019S)

(A) Immunoblot of LC3 and p62. MEFs were transfected with PERK-ΔN or PERK shRNA. Endogenous LC3 and p62 levels were measured by immunoblotting. Data represent the ratios of LC3-II to LC3-I and p62 to actin normalized LRRK+/+ MEFs. Error bars represent ±SD from four independent experiments.

(B) Peak values of Ca2+ transients in MEFs transfected with PERK-ΔN or PERK shRNA. Error bars represent ±SD from six independent experiments.

(C) Schematic of the signaling pathway consisting of LRRK2, E3 ubiquitin ligase, PERK, MAM components, and the IP3R-VDAC1 complex. Un-phosphorylated LRRK2 binds to E3 ubiquitin ligases and blocks PERKphosphorylation and activation of ligase, resulting in accumulation of MAM components. Subsequently, the accumulated MAM components strengthen the ER-mitochondrial interaction, as evidenced by increased mitochondrial Ca2+ transfer. By contrast, when LRRK2 is autophosphorylated, it detaches from E3 ubiquitin ligases and promotes PERKphosphorylation and activation of ligase, resulting in increased ubiquitination/proteosomal degradation of MAM components and reduced ER-mitochondrial interaction.

Data information: For graph A, the P values was determined by a Mann-Whitney U test. ns = not significant, *P < 0.05.

EXPANDED VIEW FIGURE LEGENDS