Biology subjects

Cifani, P.

Publications and source records attributed to Cifani, P..

At least 19 recordsLinked to original sources

bioRxiv2026

PTP1B supports survival and regulates CFL1 and HSP90a abundance in trastuzumab-resistant HER2-positive breast cancer.

Trastuzumab resistance limits the treatment of HER2-positive breast cancer. Here, we identify protein tyrosine phosphatase 1B (PTP1B) as a survival dependency in models of intrinsic and acquired trastuzumab resistance, including cells with endogenous ERBB2 kinase domain mutations (L755S and V777L), a patient-derived tumor organoid model, and orthotopic xenografts. PTP1B expression was elevated in multiple trastuzumab-resistant models, accompanied by increased PI3K-AKT and ERK signaling in several of these. The PTP1B inhibitor DPM-1003 suppressed growth of trastuzumab-resistant cells and organoids, inhibited xenograft progression, and induced apoptosis. Genetic ablation of PTP1B reduced growth and restored trastuzumab responsiveness in multiple resistance models, although JIMT1 cells remained resistant. Quantitative phosphoproteomics, phosphotyrosine immunoprecipitation, and substrate-trapping assays identified cofilin-1 (CFL1) and HSP90 as substrates of PTP1B. Loss of PTP1B increased their tyrosine phosphorylation, reduced their abundance, and increased their recovery in ubiquitin pull-downs. Proteasome inhibition restored substrate abundance, and PTP1B re-expression restored protein levels in PTP1B-null cells. These findings support a model in which PTP1B-dependent dephosphorylation limits turnover of the substrates CFL1 and HSP90. Genetic or pharmacological targeting of PTP1B suppressed survival-associated signaling and increased apoptotic markers. Together, these results identify PTP1B as a therapeutic vulnerability in trastuzumab-resistant HER2-positive breast cancer and implicate regulation of CFL1 and HSP90 abundance in the accompanying signaling response.

cancer biology↗

bioRxiv2025

Macropinocytosis enables metabolic recycling of extracellular DNA in cancer cells

Avid nutrient consumption is a metabolic hallmark of cancer and leads to regional depletion of key metabolites within the tumor microenvironment (TME). Cancer cells consequently employ diverse strategies to acquire the fuels needed for growth, including bulk uptake of the extracellular medium by macropinocytosis. Here, we show that breast and pancreatic cancer cells macropinocytically internalize extracellular DNA (exDNA), an abundant component of the TME, and deliver it to lysosomes for degradation. This provides a supply of nucleotides that sustains growth when de novo biosynthesis is impaired by glutamine restriction or pharmacological blockade. Mechanistically, this process is dependent on the non-redundant lysosomal equilibrative nucleoside transporter SLC29A3 (ENT3), which mediates the export of nucleosides from the lysosomal lumen into the cytosol. Accordingly, genetic ablation of SLC29A3 or pharmacological disruption of lysosomal function prevents exDNA scavenging and potently sensitizes breast tumors to antimetabolite chemotherapy in vivo. These findings reveal a previously unrecognized nutrient acquisition pathway through which cancer cells recycle exDNA into metabolic building blocks and highlight SLC29A3 as a mediator of metabolic flexibility and a potential target to improve chemotherapy response.

cancer biology↗

bioRxiv2025

MYC-Driven Activation of USP39 Enhances SRSF1 Stability and Promotes PDAC Progression

Pancreatic ductal adenocarcinoma (PDAC) is a highly lethal malignancy primarily driven by oncogenic KRAS signaling. The splicing factor SRSF1 plays a key oncogenic role in PDAC, where its tightly regulated expression constrains KRAS-driven signaling under normal conditions, while its upregulation promotes tumorigenesis. SRSF1 expression is regulated in part by proteostasis. However, the precise mechanisms remain unclear. Here, we identify USP39 as a deubiquitinase that interacts with SRSF1 in an RNA-independent manner and stabilizes it by reducing ubiquitination. USP39 expression is elevated in PDAC tumors and precancerous lesions, and correlates with poor patient survival. USP39 knockdown suppresses PDAC cell proliferation and migration, effects that are partially rescued by SRSF1 overexpression. Mechanistically, we show that MYC directly activates USP39 transcription via E-box motifs within its exon 1b promoter, linking MYC-driven transcriptional regulation to post- translational stabilization of SRSF1. Together, these findings define a MYC-USP39-SRSF1 regulatory axis that integrates transcriptional and post-translational mechanisms in PDAC and highlight USP39 as a potential therapeutic target.

cancer biology↗

bioRxiv2025

Aerobic glycolysis and lactate regulate histone H3K18Lactylation occupancy to fine-tune gene expression in developing and mature retina

High aerobic glycolysis in retinal photoreceptors, as in cancer cells, is implicated in mitigating energy and metabolic demands. Lactate, a product of glycolysis, plays a key role in epigenetic regulation through histone lactylation in cancer. Here, we demonstrate that increased ATP production during retinal development is achieved primarily through augmented glycolysis. Histone lactylation, especially H3K18La, parallels enhanced glycolysis and lactate in developing retina and in retinal explants. Multi-omics analyses, combined with confocal imaging, reveal the localization of H3K18La near H3K27Ac in euchromatin at promoters of active retinal genes. H3K18La and gene expression are also concordant with glucose metabolism in retinal explants. Evaluation of accessible chromatin at H3K18La marked promoters uncovers an enrichment of GC-rich motifs for transcription factors of SP, KMT and KLF families, among others, indicating specificity of H3K18La-mediated gene regulation. Our results highlight glycolysis/lactate/H3K18La as a regulatory axis in fine-tuning gene expression in developing and mature retina.

molecular biology↗

bioRxiv2025

ER-tethering directs TREX1 penetration of a BAF-dependent barrier at micronuclei

Micronuclei are membrane-encapsulated nuclear aberrations that form following chromosome segregation errors. Micronuclear membrane collapse permits access of the pattern recognition receptor cGAS and its antagonist, the TREX1 exonuclease. TREX1 carboxy-terminal domain mediated endoplasmic reticulum tethering association is essential for invasion into ruptured micronuclei, however the mechanisms underlying this dependency are unknown. Here, we identify barrier-to-autointegration nuclear assembly factor 1 (BAF) as a key regulator of TREX1 activity at micronuclei. BAF accumulates on micronuclei following membrane collapse and augments TREX1 recruitment in a manner that depends on BAF interactions with membrane-associated LEM-domain proteins. Despite delayed entry, TREX1 exhibits enhanced micronuclear DNA degradation and independence from ER-tethering in BAF-deficient cells. In accordance, recombinant BAF protein inhibits TREX1-mediated DNA degradation in vitro in a manner that depends on BAF DNA-binding. BAF similarly outcompetes cGAS for micronuclear DNA interaction and reduces cGAS activation at micronuclei. These findings reveal, a BAF-dependent protective barrier to diffusive entry of DNA binding proteins at ruptured micronuclei, explaining the requirement of TREX1 ER-tethering for micronuclear localization and suppression of productive cGAS DNA substrate interactions that activate innate immune responses in chromosomally unstable cells.

molecular biology↗

bioRxiv2025

Signal peptide-independent secretion of keratin-19 by pancreatic cancer cells

The exclusion of T cells causes immune escape of pancreatic ductal adenocarcinoma (PDA). T cell exclusion is mediated by the interaction between CXCR4 on T cells and its ligand, CXCL12, which is complexed to keratin-19 (KRT19) on the surface of PDA cells. KRT19 secretion by PDA cells is essential to this process but is unusual because KRT19 lacks an endoplasmic reticulum (ER)-directing signal peptide (SP). By using biotinylation by an ER-restricted TurboID system and a split-GFP assay in PDA cells, we demonstrate that KRT19 enters the ER via its "head" domain. Additionally, KRT19 is shown to interact with the signal recognition particle and its secretion is sensitive to canonical protein secretion inhibitors. In vivo, mouse tumors formed with ER-TurboID-expressing PDA cells contain biotinylated KRT19. In contrast, keratin-8 (KRT8), which colocalizes with KRT19 on the surface of PDA cells, does not enter the ER. Rather, KRT8 is externalized via secretory autophagy possibly in a complex with KRT19. Thus, despite lacking a classical SP, PDA cells secrete KRT19 to capture CXCL12 and protect against immune attack. Significance StatementPancreatic ductal adenocarcinoma (PDA) is resistant to immunotherapy because T cells are excluded from cancer cell nests. Cancer cells capture cancer associated fibroblast sourced CXCL12, which ligates T cell CXCR4, to exclude T cells from cancer cell nests. CXCL12 is captured by cancer cells via the externalization of the normally intracellular intermediate filament keratin-19 (KRT19). We studied the unconventional secretion of KRT19 and found it is secreted by signal peptide independent entry into the endoplasmic reticulum, as well as via secretory autophagy. Thus, PDA externalized immunosuppressive KRT19 through two unconventional means.

cell biology↗

bioRxiv2024

The molecular basis of Human FN3K mediated phosphorylation of glycated substrate

Glycation, a non-enzymatic post-translational modification occurring on proteins, can be actively reversed via site-specific phosphorylation of the fructose-lysine moiety by FN3K kinase, to impact the cellular function of target protein. A regulatory axis between FN3K and glycated protein targets has been associated with conditions like diabetes and cancer. However the molecular basis of this relationship has not been explored so far. Here, we determined a series of crystal structures of HsFN3K in apo-state, and in complex with different nucleotide analogs together with a sugar substrate mimic to reveal the features important for its kinase activity and substrate recognition. Additionally, the dynamics in sugar substrate binding during the kinase catalytic cycle provide important mechanistic insights into HsFN3K function. Our structural work provides the molecular basis for rationale small molecule design targeting FN3K.

biochemistry↗

bioRxiv2024

MARK2/MARK3 kinases are catalytic co-dependencies of YAP/TAZ in human cancer

The Hippo signaling pathway is commonly dysregulated in human cancer, which leads to a powerful tumor dependency on the YAP/TAZ transcriptional coactivators. Here, we used paralog co-targeting CRISPR screens to identify the kinases MARK2/3 as absolute catalytic requirements for YAP/TAZ function in diverse carcinoma and sarcoma contexts. Underlying this observation is direct MARK2/3-dependent phosphorylation of NF2 and YAP/TAZ, which effectively reverses the tumor suppressive activity of the Hippo module kinases LATS1/2. To simulate targeting of MARK2/3, we adapted the CagA protein from H. pylori as a catalytic inhibitor of MARK2/3, which we show exerts anti-tumor activity in vivo. Together, these findings reveal MARK2/3 as powerful co-dependencies of YAP/TAZ in human cancer; targets that may allow for pharmacology that restores Hippo pathway-mediated tumor suppression.

cancer biology↗

bioRxiv2023

Plasma cells in human pancreatic ductal adenocarcinoma secrete antibodies to self-antigens

Intratumoral B cell responses are associated with more favorable clinical outcomes in human pancreatic ductal adenocarcinoma (PDAC). However, the antigens driving these B cell responses are largely unknown. We sought to discover these antigens by using single-cell RNA sequencing (scRNA-Seq) and immunoglobulin (Ig) sequencing of tumor-infiltrating immune cells from seven primary PDAC samples. We identified activated T and B cell responses and evidence of germinal center reactions. Ig sequencing identified plasma cell (PC) clones expressing isotype-switched and hyper-mutated Igs, suggesting the occurrence of T cell-dependent B cell responses. We assessed the reactivity of 41 recombinant antibodies that represented the products of 235 PCs and 12 B cells toward multiple cell lines and PDAC tissues, and observed frequent staining of intracellular self-antigens. Three of these antigens were identified: the filamentous actin (F-actin), the nucleic protein, RUVBL2, and the mitochondrial protein, HSPD1. Antibody titers to F-actin and HSPD1 were elevated in the plasma of PDAC patients, and also detectable in healthy donors. Thus, PCs in PDAC produce auto-antibodies reacting with intracellular self-antigens, which may result from promotion of pre-existing, autoreactive B cell responses. These observations indicate that the chronic inflammatory microenvironment of PDAC can support the adaptive immune response.

cancer biology↗

bioRxiv2023

Integrative proteogenomics using ProteomeGenerator2

Recent advances in nucleic acid sequencing now permit rapid and genome-scale analysis of genetic variation and transcription, enabling population-scale studies of human biology, disease, and diverse organisms. Likewise, advances in mass spectrometry proteomics now permit highly sensitive and accurate studies of protein expression at the whole proteome-scale. However, most proteomic studies rely on consensus databases to match spectra to peptide and proteins sequences, and thus remain limited to the analysis of canonical protein sequences. Here, we develop ProteomeGenerator2 (PG2), based on the scalable and modular ProteomeGenerator framework. PG2 integrates genome and transcriptome sequencing to incorporate protein variants containing amino acid substitutions, insertions, and deletions, as well as non-canonical reading frames, exons, and other variants caused by genomic and transcriptomic variation. We benchmarked PG2 using synthetic data and genomic, transcriptomic, and proteomic analysis of human leukemia cells. PG2 can be integrated with current and emerging sequencing technologies, assemblers, variant callers, and mass spectral analysis algorithms, and is available open-source from https://github.com/kentsisresearchgroup/ProteomeGenerator2.

bioinformatics↗

bioRxiv2022

Translesion DNA synthesis polymerase kappa functions in the nucleolar stress response

DNA polymerase kappa (Pol{kappa}) has multiple cellular roles such as translesion DNA synthesis, replication of repetitive sequences and nucleotide excision repair. However, the mechanisms regulating Pol{kappa}s cellular activities are unknown. Since all polymerases insert the canonical deoxynucleotide triphosphates, it is difficult to determine which polymerase is active at a particular genomic site. To counter this difficulty, we utilized the selective Pol{kappa} substrate, N2-(4-ethynylbenyl)-2-deoxyguanosine (EBndG), as bait to capture proteins associated with Pol{kappa} synthesized DNA. Here we show that, Pol{kappa} is active in the nucleolus and Pol{kappa} synthesized DNA are enriched with nucleolar proteins. Exposure of cells to benzo[a]pyrene diol epoxide (BPDE) induced hallmarks of nucleolar stress, increased Pol{kappa} stability and nucleolar activity. Other agents that induce nucleolar stress such as mitomycin C, cisplatin and actinomycin D also increased Pol{kappa}s nucleolar activity. The Pol{kappa} activity was cell-cycle independent and dependent on PCNA ubiquitination. In addition, we identified that the expression and activity of Pol{kappa} is regulated by the polycomb complex protein Ring Finger Protein 2 (RNF2) and by poly(ADP)-ribose polymerase 1 (PARP1) catalyzed PARylation. This study provides insight into the novel role of Pol{kappa} in ribosomal RNA synthesis, in maintaining ribosomal DNA integrity after DNA damage thus protecting the cells from nucleolar stress.

molecular biology↗

bioRxiv2022

Quantitative Cell Proteomic Atlas: Pathway-scale targeted mass spectrometry for high-resolution functional profiling of cell signaling

In spite of extensive studies of cellular signaling, many fundamental processes such as pathway integration, cross-talk and feedback remain poorly understood. To enable integrated and quantitative measurements of cellular biochemical activities, we have developed the Quantitative Cell Proteomics Atlas (QCPA). QCPA consists of panels of targeted mass spectrometry assays to determine the abundance and stoichiometry of regulatory post-translational modifications of sentinel proteins from most known physiologic and pathogenic signaling pathways in human cells. QCPA currently profiles 1,913 peptides from 469 effectors of cell surface signaling, apoptosis, stress response, gene expression, quiescence, and proliferation. For each protein, QCPA includes triplets of isotopically labeled peptides covering known post-translational regulatory sites to determine their stoichiometries and unmodified protein regions to measure total protein abundance. The QCPA framework incorporates analytes to control for technical variability of sample preparation and mass spectrometric analysis, including TrypQuant, a synthetic substrate for accurate quantification of proteolysis efficiency for proteins containing chemically modified residues. The ability to precisely and accurately quantify most known signaling pathways should enable improved chemoproteomic approaches for the comprehensive analysis of cell signaling and clinical proteomics of diagnostic specimens. QCPA is openly available at https://qcpa.mskcc.org.

biochemistry↗

bioRxiv2020

Discovery of protein modifications using high resolution differential mass spectrometry proteomics

Recent studies have revealed diverse amino acid, post-translational and non-canonical modifications of proteins in diverse organisms and tissues. However, their unbiased detection and analysis remain hindered by technical limitations. Here, we present a spectral alignment method for the identification of protein modifications using high-resolution mass spectrometry proteomics. Termed SAMPEI for Spectral Alignment-based Modified PEptide Identification, this open-source algorithm is designed for the discovery of functional protein and peptide signaling modifications, without prior knowledge of their identities. Using synthetic standards and controlled chemical labeling experiments, we demonstrate its high specificity and sensitivity for the discovery of sub-stoichiometric protein modifications in complex cellular extracts. SAMPEI mapping of mouse macrophage differentiation revealed diverse post-translational protein modifications, including distinct forms of cysteine itaconatylation. SAMPEIs robust parameterization and versatility are expected to facilitate the discovery of biological modifications of diverse macromolecules. SAMPEI is implemented as a Python package, and is available open-source from BioConda and GitHub (https://github.com/FenyoLab/SAMPEI).

biochemistry↗

bioRxiv2020

Therapeutic remodeling of CBP transcription factor complex controls oncogenic gene expression in acute myeloid leukemia

Dysregulated gene expression contributes to most prevalent features in human cancers. Here, we show that most subtypes of acute myeloid leukemia (AML) depend on the aberrant assembly of MYB transcriptional co-activator complex. By rapid and selective peptidomimetic interference with the binding of CBP/P300 to MYB, but not CREB or MLL1, we find that the leukemic functions of MYB are mediated by CBP/P300 co-activation of a distinct set of transcription factor complexes. These MYB complexes assemble aberrantly with LYL1, E2A, C/EBP family members, LMO2 and SATB1. They are organized convergently in genetically diverse subtypes of AML, and are at least in part associated with inappropriate transcription factor co-expression. Peptidomimetic remodeling of oncogenic MYB complexes is accompanied by specific proteolysis and dynamic redistribution of CBP/P300 with alternative transcription factors such as RUNX1 to induce myeloid differentiation and apoptosis. Thus, aberrant assembly and sequestration of MYB:CBP/P300 complexes provide a unifying mechanism of oncogenic gene expression in AML. This work establishes a compelling strategy for their pharmacologic reprogramming and therapeutic targeting for diverse leukemias and possibly other human cancers caused by dysregulated gene control.

cancer biology↗

bioRxiv2019

A cancer-associated missense mutation in PP2A-Aα increases centrosome clustering during mitosis

A single incidence of whole-genome doubling (WGD) is common early in tumorigenesis. In addition to increasing ploidy, WGD doubles centrosome number. In the ensuing mitoses, excess centrosomes form a multipolar spindle, resulting in a lethal multipolar cell division. To survive, cells must cluster centrosomes into two poles to allow a bipolar cell division. Cancer cells are typically more proficient at centrosome clustering than untransformed cells, but the mechanism behind increased clustering ability is not well understood. Heterozygous missense mutations in PPP2R1A, which encodes the alpha isoform of the A-subunit of protein phosphatase 2A (PP2A-A), positively correlate with WGD. To understand this correlation, we introduced a heterozygous hotspot mutation, P179R, in endogenous PP2A-A in human tissue culture cells. We find that PP2A-AP179R decreases PP2A assembly and targeting. Strikingly, when centrosome number is increased, either through cytokinesis failure or centrosome amplification, PP2A-A mutant cells are more proficient than WT cells at centrosome clustering, likely due to PP2A-A loss-of-function. PP2A-AP179R appears to enhance centrosome clustering by altering the interactions between centrosomes and the cell cortex. Thus, cancer-associated mutations in PP2A-A may increase cellular fitness after WGD by enhancing centrosome clustering.

cell biology↗

bioRxiv2018

Prohibitin is a prognostic marker of relapse and therapeutic target to block chemotherapy resistance in Wilms tumor

Wilms tumor (WT) is the most common childhood kidney cancer. To improve risk stratification and identify novel therapeutic targets for patients with WT, we used high-resolution mass spectrometry proteomics to identify urine tumor markers associated with WT relapse. We determined urine proteomes at diagnosis of 49 patients with WT, non-WT renal tumors, and age-matched controls, leading to the quantitation of 6,520 urine proteins. Supervised analysis revealed specific urine markers of renal rhabdoid tumors, kidney clear cell sarcomas, renal cell carcinomas, as well as those detected in cured and relapsed WT. In particular, urine prohibitin was significantly elevated at diagnosis in patients with relapsed as compared to cured WT. In a validation cohort of 139 patients, a specific urine prohibitin enzyme-linked immunosorbent assay demonstrated that prohibitin concentrations greater than 998 ng/mL at diagnosis were significantly associated with ultimate WT relapse. Immunohistochemical analysis revealed that prohibitin was highly expressed in primary WT specimens and associated with disease stage. Using functional genetic experiments, we found that prohibitin was required for the growth and survival of WT cells. Overexpression of prohibitin was sufficient to block intrinsic mitochondrial apoptosis and to cause resistance to diverse chemotherapy drugs, at least in part by dysregulating factors that control apoptotic cytochrome c release from mitochondrial cristae. Thus, urine prohibitin may improve therapy stratification, non-invasive monitoring of treatment response and early disease detection. In addition, therapeutic targeting of chemotherapy resistance induced by prohibitin dysregulation may offer improved therapies for patients with Wilms and other relapsed or refractory tumors.

cancer biology↗

bioRxiv2018

Optimized cross-linking mass spectrometry for in situ interaction proteomics

Recent development of mass spectrometer cleavable protein cross-linkers and algorithms for their spectral identification now permits large-scale cross-linking mass spectrometry (XL-MS). Here, we optimized the use of cleavable disuccinimidyl sulfoxide (DSSO) cross-linker for labeling native protein complexes in live human cells. We applied a generalized linear mixture model to calibrate cross-link peptide-spectra matching (CSM) scores to control the sensitivity and specificity of large-scale XL-MS. Using specific CSM score thresholds to control the false discovery rate, we found that higher-energy collisional dissociation (HCD) and electron transfer dissociation (ETD) can both be effective for large-scale XL-MS protein interaction mapping. We found that the density and coverage of protein-protein interaction maps can be significantly improved through the use of multiple proteases. In addition, the use of sample-specific search databases can be used to improve the specificity of cross-linked peptide spectral matching. Application of this approach to human chromatin labeled in live cells recapitulated known and revealed new protein interactions of nucleosomes and other chromatin-associated complexes in situ. This optimized approach for mapping native protein interactions should be useful for a wide range of biological problems.

systems biology↗

bioRxiv2017

ProteomeGenerator: A framework for comprehensive proteomics based on de novo transcriptome assembly and high-accuracy peptide mass spectral matching

Modern mass spectrometry now permits genome-scale and quantitative measurements of biological proteomes. However, analyses of specific specimens are currently hindered by the incomplete representation of biological variability of protein sequences in canonical reference proteomes, and the technical demands for their construction. Here, we report ProteomeGenerator, a framework for de novo and reference-assisted proteogenomic database construction and analysis based on sample-specific transcriptome sequencing and high-resolution and high-accuracy mass spectrometry proteomics. This enables assembly of proteomes encoded by actively transcribed genes, including sample-specific protein isoforms resulting from non-canonical mRNA transcription, splicing, or editing. To improve the accuracy of protein isoform identification in non-canonical proteomes, ProteomeGenerator relies on statistical target-decoy database matching augmented with spectral-match calibrated sample-specific controls. We applied this method for the proteogenomic discovery of splicing factor SRSF2-mutant leukemia cells, demonstrating high-confidence identification of non-canonical protein isoforms arising from alternative transcriptional start sites, intron retention, and cryptic exon splicing, as well as improved accuracy of genome-scale proteome discovery. Additionally, we report proteogenomic performance metrics for the current state-of-the-art implementations of SEQUEST HT, Proteome Discoverer, MaxQuant, Byonic, and PEAKS mass spectral analysis algorithms. Finally, ProteomeGenerator is implemented as a Snakemake workflow, enabling open, scalable, and facile discovery of sample-specific, non-canonical and neomorphic biological proteomes (https://github.com/jtpoirier/proteomegenerator).

bioinformatics↗