Biology subjects

Perera, R. M.

Publications and source records attributed to Perera, R. M..

9 recordsLinked to original sources

bioRxiv2026

Myeloid-targeted RNA nanotherapeutics rewire cholesterol metabolism to unleash anti-tumor immunity in glioblastoma

Tumor-associated myeloid cells (TAMCs) dominate the glioblastoma (GBM) microenvironment and suppress anti-tumor immunity. Here, we identify cholesterol efflux via ABCA1 as a targetable metabolic checkpoint controlling TAMC immunosuppression in GBM. Reprogramming TAMC cholesterol metabolism using TAMC-targeting lipid nanoparticle encapsulating ABCA1 siRNA (ABCA1 LNP) converts TAMCs into potent antigen-presenting cells with enhanced pro-inflammatory activity and antigen-presenting capacity, thereby inducing T cell activation, expansion, and tumor infiltration. Mechanistically, ABCA1 blockade induces cholesterol accumulation in TAMC membranes, promoting lipid raft formation and enhancing MHC-I-mediated antigen presentation. In multiple preclinical GBM models, ABCA1 LNP treatment dramatically induces T cell priming, extends animal survival, and overcomes GBM resistance to radiotherapy and immune checkpoint therapy. This efficacy was well-maintained in stem-like and recurrent GBM models, GBM patient specimens, and a renal cell carcinoma model. Altogether, our work identifies cholesterol efflux as a targetable metabolic vulnerability in TAMCs to overcome therapy resistance in myeloid-rich, immunologically "cold" tumors.

immunology↗

bioRxiv2025

PCSK9 dependent cholesterol acquisition and utilization underlies metastatic organ preference in pancreatic cancer.

To grow at distant sites, metastatic cells must overcome major challenges posed by the unique cellular and metabolic composition of secondary organs1. Pancreatic ductal adenocarcinoma (PDAC) is an aggressive disease that metastasizes to the liver and lungs. Despite evidence of metabolic reprogramming away from the primary site, the key drivers that dictate the ability of PDAC cells to colonize the liver or lungs and survive there are undefined. We identified Proprotein Convertase Subtilisin/Kexin Type 9 (PCSK9) as predictive of colonization of the liver versus lungs by integrating datasets describing the metastatic tropism of human PDAC cell lines2, with in vivo metastasis modeling in mice and gene expression correlation analysis. PCSK9 is a negative regulator of low density lipoprotein(LDL)-cholesterol import and, accordingly, PCSK9-low PDAC cells exhibit strong preference for colonizing LDL-rich liver tissue. Cholesterol taken up by liveravid PCSK9-low cells is converted into the signaling oxysterol, 24(S)-hydroxycholesterol, which reprograms the surrounding microenvironment to induce nutrient release from neighboring hepatocytes. Conversely, PCSK9-high, lung-avid PDAC cells rely on transcriptional upregulation of the distal cholesterol synthesis pathway to generate intermediates, chiefly, 7dehydrodesmosterol and 7-dehydrocholesterol, with protective action against ferroptosis, a vulnerability in the oxygen-rich microenvironment of the lung. Increasing PCSK9 levels redirected liver-avid cells to the lung whereas its ablation drove lung-avid cells to the liver, thereby establishing PCSK9 as necessary and sufficient for secondary organ site preference. Our studies reveal PCSK9-driven differential utilization of the distal cholesterol synthesis pathway as a key and potentially actionable driver of metastatic growth in PDAC.

cancer biology↗

bioRxiv2024

Leucine Aminopeptidase LyLAP enables lysosomal degradation of membrane proteins

Proteolysis of hydrophobic helices is required for complete breakdown of every transmembrane protein trafficked to the lysosome and sustains high rates of endocytosis. However, the lysosomal mechanisms for degrading hydrophobic domains remain unknown. Combining lysosomal proteomics with functional genomic data mining, we identify Lysosomal Leucine Aminopeptidase (LyLAP; formerly Phospholipase B Domain-Containing 1) as the hydrolase most tightly associated with elevated endocytic activity. Untargeted metabolomics and biochemical reconstitution demonstrate that LyLAP is not a phospholipase, but a processive monoaminopeptidase with strong preference for N-terminal leucine - an activity necessary and sufficient for breakdown of hydrophobic transmembrane domains. LyLAP is upregulated in pancreatic ductal adenocarcinoma (PDA), which relies on macropinocytosis for nutrient uptake, and its ablation led to buildup of undigested hydrophobic peptides, which compromised lysosomal membrane integrity and inhibited PDA cell growth. Thus, LyLAP enables lysosomal degradation of membrane proteins, and may represent a vulnerability in highly endocytic cancer cells. One sentence summaryLyLAP degrades transmembrane proteins to sustain high endocytosis and lysosomal membrane stability in pancreatic cancer.

biochemistry↗

bioRxiv2024

Hypoxia induces extensive protein and proteolytic remodeling of the cell surface in pancreatic adenocarcinoma (PDAC) cell lines

The tumor microenvironment (TME) plays a crucial role in cancer progression. Hypoxia is a hallmark of the TME and induces a cascade of molecular events that affects cellular processes involved in metabolism, metastasis, and proteolysis. In pancreatic ductal adenocarcinoma (PDAC), tumor tissues are extremely hypoxic. Here, we leveraged mass spectrometry technologies to examine hypoxia-induced alterations in the abundance and proteolytic modifications to cell surface and secreted proteins. Across four PDAC cell lines, we discovered extensive proteolytic remodeling of cell surface proteins involved in cellular adhesion and motility. Looking outward at the surrounding secreted space, we identified hypoxia-regulated secreted and proteolytically-shed proteins that are responsible for regulating the humoral immune and inflammatory response and an upregulation of proteins involved in metabolic processing and tissue development. Combining cell surface N-terminomics and secretomics to evaluate the cellular response to hypoxia enabled us to identify significantly altered candidate proteins which may serve as potential biomarkers and therapeutic targets in PDAC. Furthermore, this approach provides a blue print for studying dysregulated extracellular proteolysis in other cancers and inflammatory diseases.

cancer biology↗

bioRxiv2024

A multi-subunit autophagic capture complex facilitates degradation of ER stalled MHC-I in pancreatic cancer.

Pancreatic ductal adenocarcinoma (PDA) evades immune detection partly via autophagic capture and lysosomal degradation of major histocompatibility complex class I (MHC-I). Why MHC-I is susceptible to capture via autophagy remains unclear. By synchronizing exit of proteins from the endoplasmic reticulum (ER), we show that PDAC cells display prolonged retention of MHC-I in the ER and fail to efficiently route it to the plasma membrane. A capture-complex composed of NBR1 and the ER-phagy receptor TEX264 facilitates targeting of MHC-I for autophagic degradation, and suppression of either receptor is sufficient to increase total levels and re-route MHC-I to the plasma membrane. Binding of MHC-I to the capture complex is linked to antigen presentation efficiency, as inhibiting antigen loading via knockdown of TAP1 or beta 2-Microglobulin led to increased binding between MHC-I and the TEX264-NBR1 capture complex. Conversely, expression of ER directed high affinity antigenic peptides led to increased MHC-I at the cell surface and reduced lysosomal degradation. A genome-wide CRISPRi screen identified NFXL1, as an ER-resident E3 ligase that binds to MHC-I and mediates its autophagic capture. High levels of NFXL1 are negatively correlated with MHC-I protein expression and predicts poor patient prognosis. These data highlight an ER resident capture complex tasked with sequestration and degradation of non-conformational MHC-I in PDAC cells, and targeting this complex has the potential to increase PDAC immunogenicity.

cancer biology↗

bioRxiv2024

Functional screen for mediators of onco-mRNA translation specificity

Oncogenic protein dosage is tightly regulated to enable cancer cells to adapt and survive. Whether this is regulated at the level of translational control and the key factors in cis and trans remain unknown. The Myc oncogene is a central paradigm of an exquisitely regulated oncogene and a major driver of pancreatic ductal adenocarcinoma (PDAC). Using a functional genome-wide CRISPRi screen in PDAC cells, we identified activators of selective MYC translation through its 5 untranslated region (5UTR) and validated four RNA binding proteins (RBPs), including epitranscriptome modifiers. Among these RBPs, our top hit was RBM42, which is highly expressed in PDAC and predicts poor survival. Combining polysome sequencing and CLIP-seq analyses, we find that RBM42 binds and selectively regulates the translation of MYC and a precise, yet vital suite of pro-oncogenic transcripts, including JUN and EGFR. Mechanistically, employing IP-mass spectrometry analysis, we find that RMB42 is a novel ribosome-associated protein (RAP). Using DMS-Seq and mutagenesis analysis, we show that RBM42 directly binds and remodels the MYC 5UTR RNA structure, facilitating the formation of the translation pre-initiation complex. Importantly, RBM42 is necessary for human PDAC cell growth and fitness and PDAC tumorigenesis in xenograft mouse models in a Myc-dependent manner in vivo. In PDAC patient samples, RBM42 expression is correlated with Myc protein levels and transcriptional activity. This work transforms our understanding of the translational code in cancer and offers a new therapeutic opening to target the expression of oncogenes.

cancer biology↗

bioRxiv2021

Lysosomal retargeting of Myoferlin mitigates membrane stress to enable pancreatic cancer growth

Lysosomes must maintain integrity of their limiting membrane to ensure efficient fusion with incoming organelles and degradation of substrates within their lumen. Pancreatic cancer cells upregulate lysosomal biogenesis to enhance nutrient recycling and stress resistance, but whether dedicated programs for maintaining lysosomal membrane integrity facilitate pancreatic cancer growth is unknown. Using proteomic-based organelle profiling, we identify the Ferlin family plasma membrane repair factor, Myoferlin, as selectively and highly enriched on the membrane of pancreatic cancer lysosomes. Mechanistically, lysosome localization of Myoferlin is necessary and sufficient for maintenance of lysosome health and provides an early-acting protective system against membrane damage that is independent from the endosomal sorting complex required for transport (ESCRT)-mediated repair network. Myoferlin is upregulated in human pancreatic cancer, predicts poor survival, and its ablation severely impairs lysosome function and tumour growth in vivo. Thus, retargeting of plasma membrane repair factors enhances pro-oncogenic activities of the lysosome.

cancer biology↗

bioRxiv2020

NPC1-mTORC1 signaling Couples Cholesterol Sensing to Organelle Homeostasis and is a Targetable Pathway in Niemann-Pick type C

Lysosomes promote cellular homeostasis through macromolecular hydrolysis within their lumen and metabolic signaling by the mTORC1 kinase on their limiting membranes. Both hydrolytic and signaling functions require precise regulation of lysosomal cholesterol content. In Niemann-Pick type C (NPC), loss of the cholesterol exporter, NPC1, causes cholesterol accumulation within lysosomes, leading to mTORC1 hyperactivation, disrupted mitochondrial function and neurodegeneration. The compositional and functional alterations in NPC lysosomes, and how aberrant cholesterol-mTORC1 signaling contributes to organelle pathogenesis are not understood. Through proteomic profiling of NPC lysosomes, we find pronounced proteolytic impairment compounded with hydrolase depletion and enhanced membrane damage. Genetic and pharmacologic mTORC1 inhibition restores lysosomal proteolysis without correcting cholesterol storage, implicating aberrant mTORC1 as a pathogenic driver downstream of cholesterol accumulation. Consistently, mTORC1 inhibition ameliorates mitochondrial dysfunction in a neuronal model of NPC. Thus, cholesterol-mTORC1 signaling controls organelle homeostasis and is a targetable pathway in NPC.

cell biology↗

bioRxiv2020

Oncogene Regulated Release of Extracellular Vesicles

Oncogenes can alter cellular structure, function, development and metabolism including changing the balance between anabolic and catabolic processes. However, how oncogenes regulate tumor cell biomass remains poorly understood. Using isogenic mammary breast epithelial cells transformed with a panel of ten oncogenes found commonly mutated, amplified or overexpressed in multiple cancers, we show that specific oncogenes reduce the biomass of cancer cells by promoting extracellular vesicle release. While MYC and AURKB elicited the highest number of EVs, each oncogene tested selectively altered the protein composition of released EVs. Likewise, miRNAs were differentially sorted into EVs in an oncogene-specific manner. MYC overexpressing cells require ceramide, while AURKB require ESCRT to release high levels of EVs. Finally, lysosome-associated genes are broadly downregulated in the context of MYC and AURKB, suggesting that cellular contents instead of being degraded, were released via EVs. Thus, oncogene mediated biomass regulation via differential EV release is a new metabolic phenotype which may have implications for cellular signaling and homeostasis.

cell biology↗