Biology subjects

Peri, S.

Publications and source records attributed to Peri, S..

7 recordsLinked to original sources

bioRxiv2023

Automated workflow for the cell cycle analysis of non-adherent and adherent cells using a machine learning approach

Understanding the details of the cell cycle at the level of individual cells is critical for both cellular biology and cancer research. While existing methods using specific fluorescent markers have advanced our ability to study the cell cycle in cells that adhere to surfaces, there is a clear gap when it comes to non-adherent cells. In this study, we combine a specialized surface to improve cell attachment, the genetically-encoded FUCCI(CA)2 sensor, an automated image processing and analysis pipeline, and a custom machine-learning algorithm. This combined approach allowed us to precisely measure the duration of different cell cycle phases in non-adherent cells. Our method provided detailed information from hundreds of cells under different experimental conditions in a fully automated manner. We validated this approach in two different Acute Myeloid Leukemia (AML) cell lines, NB4 and Kasumi-1, which have unique cell cycle characteristics. Additionally, we tested the impact of drugs affecting the cell cycle in NB4 cells. Importantly, our cell cycle analysis system is freely available and has also been validated for use with adherent cells. In summary, this report introduces a comprehensive, automated method for studying the cell cycle in both adherent and non-adherent cells, offering a valuable tool for cancer research and drug development.

cell biology↗

bioRxiv2023

Unique vulnerability of RAC1-mutant melanoma to combined inhibition of CDK9 and immune checkpoints

RAC1P29S is the third most prevalent hotspot mutation in sun-exposed melanoma. RAC1 alterations in cancer are correlated with poor prognosis, resistance to standard chemotherapy, and insensitivity to targeted inhibitors. Although RAC1P29S mutations in melanoma and RAC1 alterations in several other cancers are increasingly evident, the RAC1-driven biological mechanisms contributing to tumorigenesis remain unclear. Lack of rigorous signaling analysis has prevented identification of alternative therapeutic targets for RAC1P29S-harboring melanomas. To investigate the RAC1P29S-driven effect on downstream molecular signaling pathways, we generated an inducible RAC1P29S expression melanocytic cell line and performed RNA-sequencing (RNA-seq) coupled with multiplexed kinase inhibitor beads and mass spectrometry (MIBs/MS) to establish enriched pathways from the genomic to proteomic level. Our proteogenomic analysis identified CDK9 as a potential new and specific target in RAC1P29S-mutant melanoma cells. In vitro, CDK9 inhibition impeded the proliferation of in RAC1P29S-mutant melanoma cells and increased surface expression of PD-L1 and MHC Class I proteins. In vivo, combining CDK9 inhibition with anti-PD-1 immune checkpoint blockade significantly inhibited tumor growth only in melanomas that expressed the RAC1P29S mutation. Collectively, these results establish CDK9 as a novel target in RAC1-driven melanoma that can further sensitize the tumor to anti-PD-1 immunotherapy.

cancer biology↗

bioRxiv2023

Ribosomal protein control of hematopoietic stem cell transformation through direct, non-canonical regulation of metabolism

We report here that expression of the ribosomal protein, RPL22, is frequently reduced in human myelodysplastic syndrome (MDS) and acute myelogenous leukemia (AML); and, reduced RPL22 expression is associated with worse outcomes. Mice null for Rpl22 display characteristics of an MDS-like syndrome and develop leukemia at an accelerated rate. Rpl22-deficient mice also display enhanced hematopoietic stem cell (HSC) self-renewal and obstructed differentiation potential, which arises not from reduced protein synthesis but from altered metabolism including increased fatty acid oxidation (FAO) and a striking induction of the stemness factor Lin28b in the resulting leukemia. Lin28b promotes a substantial increase in lipid content, upon which the survival of Rpl22-deficient leukemias depends. Altogether, these findings reveal that Rpl22 insufficiency enhances the leukemia potential of HSC through regulation of FAO and promotes leukemogenesis through Lin28b promotion of lipid synthesis. HighlightsO_LIRPL22 insufficiency is observed in MDS/AML and is associated with reduced survival C_LIO_LIRpl22-deficiency produces an MDS-like syndrome and facilitates leukemogenesis C_LIO_LIRpl22-deficiency does not impair global protein synthesis by HSC C_LIO_LIRpl22 controls leukemia survival through control of lipid synthesis C_LI eTOC: Rpl22 controls the function and transformation potential of hematopoietic stem cells through regulation of lipid metabolism.

cancer biology↗

bioRxiv2020

Identification of SARS-CoV2-mediated suppression of NRF2 signaling reveals a potent antiviral and anti-inflammatory activity of 4-octyl-itaconate and dimethyl fumarate

Antiviral strategies to inhibit Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV2) and the pathogenic consequences of COVID-19 are urgently required. Here we demonstrate that the NRF2 anti-oxidant gene expression pathway is suppressed in biopsies obtained from COVID-19 patients. Further, we uncover that NRF2 agonists 4-octyl-itaconate (4-OI) and the clinically approved dimethyl fumarate (DMF) induce a cellular anti-viral program, which potently inhibits replication of SARS-CoV2 across cell lines. The anti-viral program extended to inhibit the replication of several other pathogenic viruses including Herpes Simplex Virus-1 and-2, Vaccinia virus, and Zika virus through a type I interferon (IFN)-independent mechanism. In addition, induction of NRF2 by 4-OI and DMF limited host inflammatory responses to SARS-CoV2 infection associated with airway COVID-19 pathology. In conclusion, NRF2 agonists 4-OI and DMF induce a distinct IFN-independent antiviral program that is broadly effective in limiting virus replication and suppressing the pro-inflammatory responses of human pathogenic viruses, including SARS-CoV2. One Sentence SummaryNRF2 agonists 4-octyl-itaconate (4-OI) and dimethyl fumarate inhibited SARS-CoV2 replication and virus-induced inflammatory responses, as well as replication of other human pathogenic viruses.

microbiology↗

bioRxiv2020

Inactivation of p21-Activated Kinase 2 (Pak2) Inhibits the Development of Nf2-Deficient Malignant Mesothelioma

Malignant mesotheliomas (MM) show frequent somatic loss of the NF2 tumor suppressor gene. The NF2 product, Merlin, is implicated in several tumor-related pathways, including p21-activated kinase (PAK) signaling. Merlin is both a phosphorylation target for PAK and a negative regulator of this oncogenic kinase. Merlin loss results in PAK activation, and PAK inhibitors hold promise for the treatment of NF2-deficient tumors. To test this possibility in an in vivo genetic system, Nf2f/f;Cdkn2af/f mice were crossed to mice with conditional knockout of Pak2, a highly expressed group I Pak member. Cohorts of these animals were injected in either the thoracic or peritoneal cavities with adeno-Cre virus to delete floxed alleles in the mesothelial lining. Loss of Pak2 resulted in a markedly decreased incidence and delayed onset and progression of pleural and peritoneal MMs in Nf2;Cdkn2a-deficient (NC) mice, as documented by Kaplan-Meier survival curves and in vivo bioluminescent imaging. RNA-seq revealed that MMs from NC;Pak2-/- mice showed downregulated expression of genes involved in several oncogenic pathways (Wnt, Akt) when compared to MMs from mice retaining Pak2. Kinome profiling showed that, as compared to NC MM cells, NC;Pak2-/- MM cells had multiple kinase changes indicative of an epithelial to mesenchymal transition. Collectively, these findings suggest that NC;Pak2-/- MMs adapt by reprogramming their kinome and gene signature profiles to bypass the need for PAK activity via the activation of other compensatory oncogenic kinase pathways. The identification of such secondary pathways offers opportunities for rational combination therapies to circumvent resistance to anti-PAK drugs.Competing Interest StatementThe authors have declared no competing interest.View Full Text

cancer biology↗

bioRxiv2020

Kinome Profiling of Primary Endometrial Tumors Using Multiplexed Inhibitor Beads and Mass Spectrometry Identifies SRPK1 As Candidate Therapeutic Target

Protein kinases (collectively, termed the kinome) represent one of the most tractable drug targets in the pursuit of new and effective cancer treatments. However, less than 20% of the kinome is currently being explored as primary targets for cancer therapy, leaving the majority of the kinome untargeted for drug therapy. Chemical proteomics approaches such as Multiplexed Inhibitor Beads and Mass Spectrometry (MIB-MS) have been developed that measure the abundance of a significant portion of the kinome, providing a strategy to interrogate kinome landscapes and dynamics. Kinome profiling of cancer cell lines using MIB-MS has been extensively characterized, however, application of this method to measure tissue kinome(s) has not been thoroughly explored. Here, we present a quantitative proteomics workflow specifically designed for kinome profiling of tissues that pairs MIB-MS with a newly designed super-SILAC kinome standard. Using this workflow, we mapped the kinome landscape of endometrial carcinoma (EC) tumors and normal endometrial (NE) tissues and identified several kinases overexpressed in EC tumors, including Serine/Arginine-Rich Splicing Factor kinase, (SRPK1). Immunohistochemical (IHC) analysis of EC tumor TMAs confirmed MIB-MS findings and showed SRPK1 protein levels were highly expressed in endometrioid and uterine serous cancer (USC) histological subtypes. Querying large-scale genomics studies of EC tumors revealed high expression of SRPK1 correlated with poor survival. Inhibition of SRPK1 in USC cells altered mRNA splicing, downregulating several oncogenes including MYC and Survivin resulting in apoptosis. Taken together, we present a SILAC-based MIB-MS kinome profiling platform for measuring kinase abundance in tumor tissues, and demonstrate its application to identify SRPK1 as a plausible kinase drug target for the treatment of EC.

cancer biology↗

bioRxiv2019

Cholesterol deprivation induces TGFβ signaling to promote basal differentiation in pancreatic cancer

Oncogenic transformation alters the metabolism of cellular nutrients to sustain tumor growth. We here define a mechanism by which modifications in cholesterol metabolism control the formation of pancreatic ductal adenocarcinoma (PDAC). Disruption of distal cholesterol biosynthesis by means of conditional inactivation of Nsdhl in mice bearing a tumor-inducing Kras mutation (KrasG12D) prevented PDAC formation in the context of a heterozygous Trp53f/+genotype without impairing normal pancreatic development. In mice with pancreatic Nsdhl ablation and homozygous loss of Trp53, the emerging tumors presented with the aggressive basal (mesenchymal) phenotype as opposed to the classic (glandular) PDAC. This paralleled significantly reduced expression of cholesterol metabolic pathway genes in human basal PDAC subtype. Mechanistically, we demonstrate that genetic or metabolic cholesterol deprivation stabilizes the transforming growth factor beta (TGF{beta}) receptor to activate pro-mesenchymal effectors in human and murine PDAC, providing a direct mechanism by which cholesterol metabolism can condition tumor differentiation.

cancer biology↗