Biology subjects

Luberto, C.

Publications and source records attributed to Luberto, C..

3 recordsLinked to original sources

bioRxiv2025

Critical Evaluation of Sphingolipids Detection by MALDI-MSI

The increasing interest in the role of sphingolipids in (patho)physiology has led to the demand for visualization of these lipids within tissue samples (both from animal models and patient specimens) using techniques such as matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI). While increasingly adopted, detection of sphingolipids with MALDI-MSI is challenging due to: i) the significant structural variations of sphingolipid molecules, ii) the potential breakdown of the more complex molecules into structurally simpler species which may confound the analysis, and iii) the great difference in levels among sphingolipid classes and subspecies, with the low-abundant ones often being close to the detection limit. In this study, we adopted a multi-pronged approach to establish a robust pipeline for the detection of sphingolipids by MALDI-MSI and to establish best practices and limitations of this technology. First, we evaluated the more commonly adopted methods [2,5-Dihydroxyacetophenon (DHA) or 2,5-Dihydroxybenzoic acid (DHB) matrix in positive ion mode and 1,5-Diaminonaphthalene (DAN) matrix in negative ion mode] using MALDI-MS on reference standards. These standards were used at ratios similar to their relative levels in biological samples to evaluate signal artifacts originating from fragmentation of more complex sphingolipids and impacting low level species. Next, by applying the most appropriate protocol for each sphingolipid class, MALDI-MSI signals were validated in cell culture by modulating specific sphingolipid species using sphingolipid enzymes and inhibitors. Finally, the optimized parameters were utilized on breast cancer tissue from the PyMT mouse model. We report the optimal signal for sphingomyelin (SM) and, for the first time, Sph in DHB positive ion mode (in cells and PyMT tissue), and the validated detection of ceramides and glycosphingolipids in DAN negative ion mode. We document the extensive fragmentation of SM into sphingosine-1-phosphate (S1P) and even more so into ceramide-1-phosphate (C1P) using DAN in negative ion mode and its effect in generating an artifactual C1P tissue signal; we also report the lack of detectable signal for S1P and C1P in biological samples (cells and tissue) using the more suitable DHB positive ion mode protocol.

cell biology↗

bioRxiv2025

Fenretinide targets GATA1 to induce cytotoxicity in GATA1 positive Acute Erythroid and Acute Megakaryoblastic Leukemic cells.

Patients with Acute Myeloid Leukemia (AML) subtypes, acute erythroleukemia and acute megakaryocytic leukemia (M6 and M7 AMLs, respectively) have a median survival of only a few months with no targeted effective treatment. Our gene expression analysis using the Cancer Cell Line Encyclopedia and CRISPR screen from DepMap showed that M6/M7 AMLs have high levels of the transcription factor GATA1 and depend on GATA1 for survival. While GATA1 was shown to support AML cell proliferation and resistance to chemotherapy, GATA1 has long been considered "undruggable". Here, we identify the small molecule N-(4-hydroxyphenyl)retinamide (4-HPR, Fenretinide) as a novel GATA1 targeting agent in M6 and M7 AML cells, with nM to low M concentrations of 4-HPR causing loss of GATA1. In M6 AML OCIM1 cells, knock-down of GATA1 induced cytotoxicity similarly to low doses 4-HPR while overexpression of GATA1 significantly protected cells from 4-HPR-induced cytotoxicity. In M6 AML cells resistant to current standard-of-care (SOC) Azacytidine plus Venetoclax, 4-HPR synergized with SOC overcoming cell resistance to the drugs. As single-agent, 4-HPR outperformed SOC. In M6 AML cells sensitive to SOC, 4-HPR enhanced and prolonged the growth inhibitory effect of SOC. 4-HPR is a synthetic derivative of vitamin A, and numerous clinical trials have supported its safe profile in cancer patients; therefore, targeted use of 4-HPR against M6 and M7 AMLs may represent a novel therapeutic window. Key Points- Fenretinide (4-HPR) targets the transcription factor GATA1, which was previously thought to be "undruggable" and induces GATA1 loss. - M6 and M7 Acute Myeloid Leukemias (AML) have enriched expression of GATA1 and they can be considered GATA1 positive. - Loss of GATA1 contributes significantly to 4-HPR cytotoxicity in M6 OCIM1 cells. - 4-HPR treatment overcomes chemotherapeutic resistance in M6 Acute Myeloid Leukemia cells, synergizes with standard-of-care and outperforms standard-of-care as a single agent.

cancer biology↗

bioRxiv2025

Regulation of cell cycle by the novel GATA1/TAL1/Sphingomyelin Synthase 1 (SGMS1) transcriptional axis. Implications for anti-leukemic strategies.

Sphingomyelin Synthase 1 (gene name: SGMS1) participates in regulation of sphingolipid levels by synthesizing sphingomyelin from ceramide and phosphatidylcholine. Evidence have supported SGMS1s functions in regulating proliferation, cell cycle, cell death and migration. While its functions have begun to be explored, very little is known about upstream regulators. Here, we demonstrate that SGMS1 is a direct gene target of the GATA1-TAL1 transcriptional complex in K562 erythroleukemic cells. A predicted GATA1 consensus DNA binding sequence was identified with in a newly characterized alternative SGMS1 promoter (TSS-7 promoter) and ChIP analysis confirmed GATA1 occupancy on the promoter. Down-regulation of GATA1 in K562 cells significantly decreased SGMS1 expression and enzymatic activity, and ChIP-Seq analysis from ENCODE showed colocalized peaks for GATA1 and TAL1 (a well-established GATA1 DNA binding partner) on the SGMS1 gene. Analysis of publicly available datasets shows that elevated GATA1, TAL1 and SGMS1 expression not only clusters GATA1 positive chronic myelogenous leukemia cells (like K562), but also selectively identifies acute erythrocytic and megakaryocytic leukemias (M6 and M7 AML, respectively). Microarray gene expression analysis after down-regulation of SGMS1 in M6 AML Hel cells revealed alteration of genes regulating G2/M check point and mitotic spindle formation. This phenotype was functionally confirmed by the significant delay in G2/M cell cycle progression of cells with SGMS1 downregulation and sensitization to the clinically relevant anti-mitotic agent, Taxol. Altogether, these results identify SGMS1 as a novel target of GATA1/TAL1 transcriptional complex and they support a role for the GATA1/TAL1/SGMS1 axis in regulating transit through G2/M. Importantly, results also point to combination of anti-mitotic agents and inhibition of SGMS1 as a potential novel therapeutic approach against the aggressive and resilient M6 AMLs. KEY POINTSO_LIThe Sphingomyelin Synthase 1 gene (SGMS1) is a novel direct target of GATA1 and TAL1. C_LIO_LIHigh SGMS1 levels are associated with high GATA1/TAL1 expression and regulate cell cycle progression through the G2/M checkpoint in GATA1+ erythroleukemic Acute Myeloid Leukemia Hel cells. C_LIO_LIHigh SGMS1 is associated with lower probability of survival of patients with Acute Myeloid Leukemia and down-regulation of SGMS1 co-operates with microtubule targeting agents to induce cytotoxicity in GATA1 positive AML Hel cells. C_LI

cancer biology↗