The lysosome integrates anabolic signaling and nutrient sensing to regulate intracellular growth pathways. The leucine-rich repeat-containing 8 (LRRC8) channel complex forms a lysosomal anion channel and regulates PI3K-AKT-mTOR signaling, skeletal muscle differentiation, growth, and systemic glucose metabolism. Here, we define the endogenous LRRC8 subunits localized to a subset of lysosomes in differentiated myotubes. We show that LRRC8A affects leucine-stimulated mTOR; lysosome size; number; pH; expression of lysosomal proteins LAMP2, P62, and LC3B; and lysosomal function. Mutating an LRRC8A lysosomal targeting dileucine motif sequence (LRRC8A-L706A;L707A) in myotubes recapitulates the abnormal AKT signaling and altered lysosomal morphology and pH observed in LRRC8A knockout cells. In vivo, LRRC8A-L706A;L707A knock-in mice exhibit increased adiposity, impaired glucose tolerance and insulin resistance associated with reduced skeletal muscle PI3K-AKT-mTOR signaling, glucose uptake, and impaired incorporation of glucose into glycogen. These data reveal a lysosomal LRRC8-mediated metabolic signaling function regulating lysosomal function, systemic glucose homeostasis, and insulin sensitivity.

ABSTRACT:Platelet shape and volume changes are early mechanical events contributing to platelet activation and thrombosis. Here, we identify single-nucleotide polymorphisms in leucine-rich repeat-containing 8 (LRRC8) protein subunits that form the volume-regulated anion channel (VRAC), which are independently associated with altered mean platelet volume. LRRC8A is required for functional VRAC in megakaryocytes (MKs) and regulates platelet volume; adhesion; and agonist-stimulated activation, aggregation, adenosine triphosphate (ATP) secretion, and calcium mobilization. MK-specific LRRC8A conditional knockout mice have reduced laser injury-induced cremaster arteriolar thrombus formation and prolonged FeCl3 induced carotid arterial thrombosis without prolonged bleeding times. Mechanistically, platelet LRRC8A mediates swell-induced cytosolic ATP release to amplify agonist-stimulated calcium-phosphoinositide 3-kinase-protein kinase B signaling. Small-molecule LRRC8 channel inhibitors recapitulate defects observed in LRRC8A-null platelets in vitro and in vivo. These studies identify the mechanoresponsive LRRC8 channel complex as an ATP release channel in platelets, which positively regulates platelet function and thrombosis, providing a proof of concept for a novel antithrombotic drug target.

During activation, platelets undergo both shape and volumetric changes which are mechanical events essential for thrombosis. Volume-Regulated Anion Channels (VRACs) are ubiquitously expressed mechanoresponsive, heterohexameric anion channels comprised of leucine-rich repeat containing protein 8 (LRRC8) A (essential for channel activity) in combination with B, C, D and/or E subunits. In this study, we identify single-nucleotide polymorphisms (SNPs) in three LRRC8 subunits (LRRC8A, C, and D) which are expressed in human platelets and are independently associated with altered mean platelet volume in human genetic studies – implicating the volume-sensing LRRC8 channel complex as a regulator of platelet function in humans. We show that LRRC8A is required for functional LRRC8/VRAC in megakaryocytes (MKs). MK-specific LRRC8A conditional knockout (cKO) mice give rise to LRRC8A-null platelets with larger mean volumes. LRRC8A-null platelets and CRISPR-edited human LRRC8A KO MKs (hiPSC MKs) have reduced agonist-stimulated P-selectin exposure and aIIbb3 integrin activation. LRRC8A-null platelets exhibit impaired adhesion to collagen-coated surfaces and reduced agonist-stimulated aggregation and ATP secretion. Additionally, VRAC in MKs mediate ATP currents which are abolished in LRRC8A-null MKs. In vivo, MK-specific LRRC8A cKO mice have reduced platelet thrombus formation in laser-induced cremaster arteriolar thrombosis, and prolonged occlusion times in FeCl 3 -induced carotid arterial thrombosis, compared to control mice, without affecting tail bleeding times. Mechanistically, platelet LRRC8A mediates swell-induced ATP release to amplify agonist-stimulated calcium influx and platelet aggregation. Small-molecule LRRC8 channel inhibitors largely recapitulate the defects observed in LRRC8A-null platelets. These studies identify the mechanoresponsive LRRC8 channel complex as an ATP release channel in platelets which regulates platelet function and arterial thrombosis, providing a proof-of-concept for a novel anti-thrombotic drug target.

Though mitochondria have their own genome and protein synthesis machineries, the majority of the mitochondrial proteins are actually encoded by the nuclear genome. Most of these mitochondrial proteins are imported into specific compartments of the mitochondria due to their mitochondrial target sequence (MTS). Unlike the nuclear target sequence, the MTS of most of the mitochondrial localized proteins remain poorly understood, mainly due to their variability, heterogeneity, unconventional modes of action, mitochondrial potential-dependent transport, and other complexities. Recently, we reported that transient receptor potential vanilloid subtype 4 (TRPV4), a thermosensitive cation channel, is physically located at the mitochondria. Here we characterize a small segment (AA 592-630) located at the TM4-loop4-TM5 segment of TRPV4 that acts as a novel MTS. The same region remains highly conserved in all vertebrates and contains a large number of point mutations each of which causes an diverse spectrum of diseases in human. Using confocal and super-resolution microscopy, we show that this MTS of TRPV4 or its mutants localizes to the mitochondria independently and also induces functional and quantitative changes in the mitochondria. By using conformal microscopy, we could detect the presence of the MTS region within the isolated mitochondria. These findings may be important to understand the complexity of MTS and TRPV4-induced channelopathies better.

Supplementary Figure 2. (A) Colony forming ability (progenitor cell content) per equal number of Linlow GFP+ cells in HSPCs transduced with EV,MycWT,MycP59Q, MycT58N. 10,000 GFP+ cells per genotype were plated in M3434 media and enumerated 7-10 days later. 10,000 cells recovered from each round were plated again and enumerated 7-10 days later for 3 subsequent rounds. Bar graphs represent average and standard deviation. One-way ANOVA, n = 3 replicate per genotype, *p<0.05, ***p<0.001. (B) Boxplot representing AUCell (y-axis) expression for the 247 genes of cluster 3 in RNA sequencing from AML cells with MYC mutations vs. AML cells without MYC mutations ***FDR<0.001. (C) Heatmap representing the DEGs at bulk RNA sequencing between GFP+ GMP EV control cells vs. Mycoverexpressing cells (Supplementary Data S5). (D) Scatterplot of all terms in the KEGG_2019_Mouse gene set library. Genes upregulated and downregulated by in the bulk RNA sequencing analysis of Myc overexpressing cells are highlighted by red and blue circles respectively

Supplementary Data S6 ChIP-seq MACS peaks

Supplementary Figure 7. (A) UMAP projection of 36085 cells from MycT58N/+ and Myc+/+ mice colored by genotype, as indicated in the legend. (B) UMAP projection of 36085, split by genotype and colored by the combined expression of Mpo (Myeloperoxidase) and Elane (Elastase, Neutrophil Expressed) transcripts. In yellow are GMP cells, that strongly express both transcripts. (C) UMAP projection of 36085 cells split by genotype and colored by the combined expression of Cd177 and Ngp (neutrophil granule protein). In yellow are granulocytopoietic progenitors, that express high levels of both transcripts. (D) Trajectory analysis of 36085 showing the temporo-spatial expression of Myc (cells that express Myc are in shades of red). (E) Venn diagram showing the percentage overlap in scRNA-seq DEGs between the overexpression and the knockin mouse model. Comparisons are: Empty vector*GMP vs. MycT58N*GMP and MycT58N/+* GMP vs. Myc+/+*GMP for the overexpression and the knockin, respectively. 55% of the DEGs (8334 genes) are shared between the two models. (F) Schematic summary of the nucleocytoplasmic transport components’ transcripts that are upregulated >2 folds in MycT58N/+ versus Myc+/+(created with BioRender.com) (G) Enrichment plots (top) and heatmaps of the leading-edge genes in each pathway (bottom), showing the differential enrichment on Ribosome Biogenesis, Pyrimidine metabolism and Pluripotency pathways in MycT58N/+ GMP-like cells. (H) Enrichment plots (top) and heatmaps of the leading-edge genes in each pathway (bottom), showing the enrichment of Hematopoietic cell lineage and Immune pathways in Myc+/+ GMP-like cells.

Differentially expressed genes in single cell RNA sequencing and Enrichr pathway analysis

Supplementary Figure 1. Lollipop plots for AML-associated MYC mutations as reported in Ferraro et al.

Supplementary Data S7 Proteomic Normalized Spectral Counts

Supplementary Data S8 Graph Based Cluster Biomarkers

Platelet shape and volume changes are early mechanical events contributing to platelet activation and thrombosis. Here, we identify single-nucleotide polymorphisms in Leucine-Rich Repeat Containing 8 (LRRC8) protein subunits that form the Volume-Regulated Anion Channel (VRAC) which are independently associated with altered mean platelet volume. LRRC8A is required for functional VRAC in megakaryocytes (MKs) and regulates platelet volume, adhesion, and agonist-stimulated activation, aggregation, ATP secretion and calcium mobilization. MK-specific LRRC8A cKO mice have reduced arteriolar thrombus formation and prolonged arterial thrombosis without affecting bleeding times. Mechanistically, platelet LRRC8A mediates swell-induced ATP/ADP release to amplify agonist-stimulated calcium and PI3K-AKT signaling via P2X1, P2Y 1 and P2Y 12 receptors. Small-molecule LRRC8 channel inhibitors recapitulate defects observed in LRRC8A-null platelets in vitro and in vivo . These studies identify the mechanoresponsive LRRC8 channel complex as an ATP/ADP release channel in platelets which regulates platelet function and thrombosis, providing a proof-of-concept for a novel anti-thrombotic drug target.

Differentially Expressed Genes in Bulk RNA sequencing

Platelet shape and volume changes are early mechanical events in platelet activation proposed to contribute to arterial thrombosis. Here, we identify single-nucleotide polymorphisms (SNPs) in four leucine-rich repeat containing protein subunits (LRRC8A, B, C, and D) expressed in human platelets that are independently associated with increased mean platelet volume in human genetic studies - implicating the volume-sensing LRRC8 channel complex as regulating platelet function in humans. We show LRRC8A is required for functional LRRC8/VRAC in megakaryocytes (MKs). MK-specific LRRC8A conditional knockout mice give rise to LRRC8A-null platelets with larger mean volumes. LRRC8A-null platelets and CRISPR-edited human LRRC8A KO MKs (hiPSC MKs) have reduced agonist -stimulated P-selectin exposure, and αIIbβ3 integrin activation. LRRC8A-null platelets exhibit impaired adhesion to collagen-coated surfaces, agonist-induced platelet aggregation and ATP secretion. MK-specific LRRC8A conditional knockout mice have reduced platelet thrombus formation in laser-induced cremaster arteriolar thrombosis and prolonged occlusion times in FeCl 3 -induced carotid arterial thrombosis, compared to control mice, without affecting tail bleeding times. Mechanistically, LRRC8A mediates swelling-induced ATP release to amplify agonist-stimulated calcium influx and aggregation in platelets. Treatment of platelets with small-molecule LRRC8 channel inhibitors recapitulate the defects in LRRC8A-null platelets. These studies identify the mechanoresponsive LRRC8 channel complex as a regulator of platelet function and thrombosis - providing a proof-of-concept for a novel anti-thrombotic drug target.

Gene Ontology analysis of Cluster 3 genes

Supplementary Figure 4. (A) Histogram plot showing the viability (as measured by Acridine Orange/Propidium Iodine stain read on a Cellometer instrument) of control cells, MYCWT, MYCP59Q and MYCT58N expressing cells at before (time 0) and 8 hours after treatment with DRB. None of the difference are statistically significant. (B) Schematic overview of the nascent proteomic analysis experimental setup. (C) Volcano plots of the fold changes in spectral counts of MYC overexpressing (doxycycline treated) vs. control (vehicle treated) cells (Supplementary Data S7). Black dashed lines indicate significance (ANOVA, Dox vs Vehicle, fold changes ± 2, adjp<0.05) (D-E) Volcano plots showing the trends in spectral counts fold changes (x-axis) over 30 minutes of OPP nascent polypeptide labeling for cluster 3 genes (D) and for the genes in figure 4A (E). Comparisons are MBI MYCMUT*Dox vs MYCWT*Dox. Red dashed lines indicate significance.

Supplementary Figure 6. (A-B) Bar graphs of the distribution of white blood cells (WBC) counts (1000/microliter), hemoglobin (Hb, grams/deciliter) and platelets (1000/microliter) in MycT58N/+ and Myc+/+mice at 8 and 12 weeks of age. None of the differences are statistically significant. (C) Bar graphs of the distribution of weight in 8-12 weeks old males (left panel) and females (right panel) MycT58N/+and Myc+/+showing no significant differences across genotype. (D-E) Bar graphs of the distribution of stem and progenitor cells in the bone marrow compartment of 6-8 weeks old MycT58N/+ and Myc+/+ as determined by flow cytometry analysis. None of the differences are statistically significant. (F) Confocal images of HSPCs isolated from healthy, 6 months old, MycT58N/+ and littermate controls mice. The blue is DAPI (nuclei), the red is LAMININ A/C (Alexa Fluor 647, nuclear envelope), and the green is MYC (Alexa Fluor 488). Scalebar 30 microns (µm). (G) MYC staining Mean Fluorescence Intensity (MFI) average in nuclear and cytoplasmic compartments of MycT58N/+and Myc+/+ mice showing increase cytoplasmic MYC signal in MycT58N/+ (two-tail t test, ***p<0.001). (H) Cytoplasmic to nuclear ratio of MFI MYC signal from (G) (two-tail t-test, **p<0.01). (I) Bar-graph of aggregation propensity of MYCT58N proteins compared to MYC wildtype (two-tail t-test, **p<0.01), as detected using PROTEOSTAT. (J-L) Number of stem cells and progenitor cells per femur of MycT58N/+and Myc+/+detected by flow cytometry in the bone marrow of moribund MycT58N/+mice at takedown. Age-matched littermate controls were taken down and analyzed side-by-side for comparison (*=p<0,05, all other comparison not significant). (M) Kaplan-Meier curve for MycT58N/+ secondary tumors transplant (n = 5 from 2 separate donors). (D-F) Flow cytometry analysis showing the distribution of hematopoietic progenitor cells.

Purpose: Somatic missense mutations in the phosphodegron domain of the MYC gene (MYC Box I or MBI) are detected in the dominant clones of a subset of patients with acute myeloid leukemia (AML), but the mechanisms by which they contribute to AML are unknown. Experimental Design: To investigate the effects of MBI MYC mutations on hematopoietic cells, we employed a multi-omic approach to systematically compare the cellular and molecular consequences of expressing oncogenic doses of wild type, threonine-58 and proline-59 mutant MYC proteins in hematopoietic cells, and we developed a knockin mouse harboring the germline MBI mutation p.T58N in the Myc gene. Results: Both wild-type and MBI mutant MYC proteins promote self-renewal programs and expand highly selected subpopulations of progenitor cells in the bone marrow. Compared with their wild-type counterparts, mutant cells display decreased cell death and accelerated leukemogenesis in vivo, changes that are recapitulated in the transcriptomes of human AML-bearing MYC mutations. The mutant phenotypes feature decreased stability and translation of mRNAs encoding proapoptotic and immune-regulatory genes, increased translation of RNA binding proteins and nuclear export machinery, and distinct nucleocytoplasmic RNA profiles. MBI MYC mutant proteins also show a higher propensity to aggregate in perinuclear regions and cytoplasm. Like the overexpression model, heterozygous p.T58N knockin mice displayed similar changes in subcellular MYC localization, progenitor expansion, transcriptional signatures, and develop hematopoietic tumors. Conclusions: This study uncovers that MBI MYC mutations alter RNA nucleocytoplasmic transport mechanisms to contribute to the development of hematopoietic malignancies.