Biology subjects

Ciccia, A.

Publications and source records attributed to Ciccia, A..

11 recordsLinked to original sources

bioRxiv2026

Mapping the genetic landscape of the DNA damage response with Cas12a-based combinatorial knockout screens

The DNA damage response (DDR) is a complex network of cellular pathways that ensures the faithful maintenance of our genomes upon a wide array of genomic insults. To elucidate the functional architecture of this network, we conducted unbiased genetic interaction screens using the Cas12a genome editor to disrupt 233 DDR genes frequently mutated in cancer and other genetic diseases, either individually or in pairwise combinations. This approach enabled us to assess the phenotypic effects induced by the disruption of >27,000 DDR gene pair combinations under unperturbed cell growth conditions. From this analysis, we identified over 750 high-confidence positive (buffering) or negative (synthetic lethal/sick) gene-gene interactions, along with multiple connections between previously unlinked DDR pathways and modules, allowing us to define novel aspects of the cellular response to spontaneous, DNA replication-associated DNA damage. Among the identified genetic interactions, we uncovered profound synthetic lethal interactions between genes encoding 1) the translesion polymerase REV1-Pol {zeta} complex and the MCM8-MCM9-HROB DNA helicase complex; 2) Fanconi Anemia (FA) proteins and the mitotic DNA repair factors GEN1, CIP2A, and RHINO; and 3) the DNA translocase SMARCAL1 and components of the FANCM complex, suggesting novel opportunities for targeted therapies in tumors carrying mutations in these genes. Additionally, we identified robust suppressor interactions between the DCLRE1B gene encoding the nuclease APOLLO and the core non-homologous end joining (NHEJ) genes XRCC4, LIG4, and NHEJ1, suggesting that NHEJ impairs the fitness of APOLLO-deficient cells. This work provides a functional map of the DDR network and demonstrates the power of Cas12a-based screens for identifying synthetic lethal and buffering interactions with therapeutic potential.

genetics↗

bioRxiv2026

SMARCAL1 is a candidate therapeutic target for ALT-positive tumors

A significant subset of tumors, including over 50% of osteosarcomas--an aggressive bone malignancy affecting children, adolescents, and young adults--relies on alternative lengthening of telomeres (ALT), a telomerase-independent, DNA repair-based mechanism for telomere elongation. The overall 5-year survival rate for osteosarcoma patients is [~]65%, underlying the need to develop novel targeted therapies. Through the Cancer Dependency Map, we identify SMARCAL1, a DNA translocase previously shown to remodel stalled replication forks, as a top selective dependency factor in telomerase-negative tumors. Using a panel of ALT-positive and ALT-negative cancer cell lines, as well as osteosarcoma patient-derived xenograft cells, we confirm that ALT-positive cells are uniquely sensitive to the loss of SMARCAL1, whose depletion exacerbates ALT-dependent phenotypes and telomeric DNA damage. Notably, we demonstrate that suppressing ALT abrogates their dependency on SMARCAL1. Mechanistically, we show that SMARCAL1 loss leads to telomeric ssDNA accumulation in ALT-positive cells, dependent in part on DNA repriming mediated by the DNA primase/polymerase PRIMPOL. Moreover, SMARCAL1s ssDNA annealing activity counteracts DNA unwinding by the BLM helicase, limiting telomeric ssDNA accumulation and DNA damage in ALT-positive cells. Importantly, SMARCAL1 depletion induces senescence in ALT-positive cancer cells, rendering them susceptible to treatment with senolytic agents. Together, these findings establish SMARCAL1 as a key regulator of ALT metabolism and highlight SMARCAL1 as a promising therapeutic target for ALT-positive tumors.

cancer biology↗

bioRxiv2025

Mechanistic Insights into Proteomic Mutation-Phenotype Linkages from Tiling Mutagenesis Screens

High-throughput mutagenesis screens are powerful tools for mapping mutations to phenotypes. However, deciphering the molecular mechanisms that link mutations to phenotypic outcomes remains a significant challenge. Here, we present ProTiler-Mut, a versatile computational framework that harnesses tiling mutagenesis screens, which introduce variants across entire protein sequences, to facilitate investigation of mutation-to-phenotype associations at multiple levels, including individual residues, protein substructures, and protein-protein interactions (PPIs). As demonstrated through our analyses of base editing (BE) screens targeting DNA Damage Response (DDR) proteins and T cell regulators, ProTiler-Mut provides novel insights into the mutation-phenotype linkages, including: i) refined classification of mutation that reveals separation-of-function (SOF) category beyond the conventional binary classification of loss-of-function (LOF) and gain-of-function (GOF); ii) definition of phenotype-associated hotspot substructures that enable the inference of the function of unscreened pathogenic mutations; and iii) identification of phenotype-associated PPIs disrupted by functional mutations. Through ProTiler-Mut analyses, we identified a substructure harboring pathogenic GOF mutations that disrupt interactions between the kinases MAPK1 and RSK1, leading to MAPK1 activation and elevated expression of the immune checkpoint receptor PD-1. Furthermore, we demonstrate the applicability of ProTiler-Mut to various mutagenesis screening platforms, highlighting its broad utility and generalizability.

bioinformatics↗

bioRxiv2025

Distinct mechanisms underlying extrachromosomal telomere DNA generation in ALT cancers

Alternative lengthening of telomeres (ALT) is a telomerase-independent telomere maintenance mechanism observed in 15% of human cancers. A hallmark of ALT cancers is the presence of C-circles, circular single-stranded DNAs (ssDNAs) enriched with cytosine-rich telomere (C-rich, CCCTAA) sequences. G-circles, containing guanosine-rich telomere (G-rich, GGGTTA) ssDNAs, also exist but are much less abundant. Recent studies indicate that excessive displacement of Okazaki fragments during lagging-strand synthesis is a unique feature of ALT telomeres and responsible for generating C-circles/C-rich ssDNAs. However, the distinct characteristics of C-circles compared to G-circles remain unclear. Here, we demonstrate that co-deficiency of the DNA translocases SMARCAL1 and FANCM leads to abundant generation of G-circle/G-rich ssDNAs. These G-rich ssDNAs mainly exist in linear form, ranging in size from 500 to 3000 nucleotides, which differs significantly from the structure and size of C-circle/C-rich ssDNAs. Mechanistically, both C-rich and G-rich ssDNAs originate from BLM/POLD-mediated excessive strand displacement; however, they differ in their origins and initiation mechanisms. Specifically, C-rich ssDNAs arise from lagging daughter strands initiated by the CST complex, whereas G-rich ssDNAs originate from leading daughter strands through RAD51-dependent G-strand synthesis. Our findings propose two distinct mechanisms for generating two different extrachromosomal telomere DNAs, C-and G-circles, during ALT-mediated telomere elongation.

biochemistry↗

bioRxiv2023

CRISPRmap: Sequencing-free optical pooled screens mapping multi-omic phenotypes in cells and tissue

Pooled genetic screens are powerful tools to study gene function in a high-throughput manner. Typically, sequencing-based screens require cell lysis, which limits the examination of critical phenotypes such as cell morphology, protein subcellular localization, and cell-cell/tissue interactions. In contrast, emerging optical pooled screening methods enable the investigation of these spatial phenotypes in response to targeted CRISPR perturbations. In this study, we report a multi-omic optical pooled CRISPR screening method, which we have named CRISPRmap. Our method combines a novel in situ CRISPR guide identifying barcode readout approach with concurrent multiplexed immunofluorescence and in situ RNA detection. CRISPRmap barcodes are detected and read out through combinatorial hybridization of DNA oligos, enhancing barcode detection efficiency, while reducing both dependency on third party proprietary sequencing reagents and assay cost. Notably, we conducted a multi-omic base-editing screen in a breast cancer cell line on core DNA damage repair genes involved in the homologous recombination and Fanconi anemia pathways investigating how nucleotide variants in those genes influence DNA damage signaling and cell cycle regulation following treatment with ionizing radiation or DNA damaging agents commonly used for cancer therapy. Approximately a million cells were profiled with our multi-omic approach, providing a comprehensive phenotypic assessment of the functional consequences of the studied variants. CRISPRmap enabled us to pinpoint likely-pathogenic patient-derived mutations that were previously classified as variants of unknown clinical significance. Furthermore, our approach effectively distinguished barcodes of a pooled library in tumor tissue, and we coupled it with cell-type and molecular phenotyping by cyclic immunofluorescence. Multi-omic spatial analysis of how CRISPR-perturbed cells respond to various environmental cues in the tissue context offers the potential to significantly expand our understanding of tissue biology in both health and disease.

genomics↗

bioRxiv2023

Massively parallel base editing screens to map variant effects on anti-tumor hallmarks of primary human T cells

Base editing enables generation of single nucleotide variants, but large-scale screening in primary human T cells is limited due to low editing efficiency, among other challenges1. Here, we developed a high-throughput approach for high-efficiency and massively parallel adenine and cytosine base-editor screening in primary human T cells. We performed multiple large-scale screens editing 102 genes with central functions in T cells and full-length tiling mutagenesis of selected genes, and read out variant effects on hallmarks of T cell anti-tumor immunity, including activation, proliferation, and cytokine production. We discovered a broad landscape of gain- and loss-of-function mutations, including in PIK3CD and its regulatory subunit encoded by PIK3R1, LCK, AKT1, CTLA-4 and JAK1. We identified variants that affected several (e.g., PIK3CD C416R) or only selected (e.g. LCK Y505C) hallmarks of T cell activity, and functionally validated several hits by probing downstream signaling nodes and testing their impact on T cell polyfunctionality and proliferation. Using primary human T cells in which we engineered a T cell receptor (TCR) specific to a commonly presented tumor testis antigen as a model for cellular immunotherapy, we demonstrate that base edits identified in our screens can tune specific or broad T cell functions and ultimately improve tumor elimination while exerting minimal off-target activity. In summary, we present the first large-scale base editing screen in primary human T cells and provide a framework for scalable and targeted base editing at high efficiency. Coupled with multi-modal phenotypic mapping, we accurately nominate variants that produce a desirable T cell state and leverage these synthetic proteins to improve models of cellular cancer immunotherapies.

immunology↗

bioRxiv2023

GET: a foundation model of transcription across human cell types

Transcriptional regulation, involving the complex interplay between regulatory sequences and proteins, directs all biological processes. Computational models of transcription lack generalizability to accurately extrapolate in unseen cell types and conditions. Here, we introduce GET, an interpretable foundation model designed to uncover regulatory grammars across 213 human fetal and adult cell types. Relying exclusively on chromatin accessibility data and sequence information, GET achieves experimental-level accuracy in predicting gene expression even in previously unseen cell types. GET showcases remarkable adaptability across new sequencing platforms and assays, enabling regulatory inference across a broad range of cell types and conditions, and uncovering universal and cell type specific transcription factor interaction networks. We evaluated its performance on prediction of regulatory activity, inference of regulatory elements and regulators, and identification of physical interactions between transcription factors. Specifically, we show GET outperforms current models in predicting lentivirus-based massive parallel reporter assay readout with reduced input data. In fetal erythroblasts, we identify distal (>1Mbp) regulatory regions that were missed by previous models. In B cells, we identified a lymphocyte-specific transcription factor-transcription factor interaction that explains the functional significance of a leukemia-risk predisposing germline mutation. In sum, we provide a generalizable and accurate model for transcription together with catalogs of gene regulation and transcription factor interactions, all with cell type specificity.

bioinformatics↗

bioRxiv2023

Extrachromosomal Telomeres Derived from Excessive Strand Displacements

Alternative Lengthening of Telomeres (ALT) is a telomere maintenance mechanism mediated by break-induced replication (BIR), evident in approximately 15% of human cancers. A characteristic feature of ALT cancers is the presence of C-circles, circular single-stranded telomeric DNAs composed of C-rich sequences. Despite the fact that extrachromosomal C-rich single-stranded DNAs (ssDNAs), unique to ALT cells, are considered potential precursors of C-circles, their generation process remains undefined. Here, we introduce a highly sensitive method to detect single stranded telomeric DNA, called 4SET (Strand-Specific Southern-blot for Single-stranded Extrachromosomal Telomeres) assay. Utilizing 4SET, we are able to capture C-rich single stranded DNAs that are near 200 to 1500 nucleotides in size. Both linear C-rich ssDNAs and C-circles are abundant in the fractions of cytoplasm and nucleoplasm, which supports the idea that linear C-rich ssDNA accumulation may indeed precede C-circle formation. We also found that C-rich ssDNAs originate during Okazaki fragment processing during lagging strand DNA synthesis. The generation of C-rich ssDNA requires CST-PP (CTC1/STN1/TEN1-PRIMASE-Polymerase alpha) complex-mediated priming of the C-strand DNA synthesis and subsequent excessive strand displacement of the C-rich strand mediated by the DNA Polymerase delta and the BLM helicase. Our work proposes a new model for the generation of C-rich ssDNAs and C-circles during ALT-mediated telomere elongation.

biochemistry↗

bioRxiv2023

Mechanism of DNA unwinding by hexameric MCM8-9 in complex with HROB

The human MCM8-9 helicase functions in concert with HROB in the context of homologous recombination, but its precise function is unknown. To gain insights into how HROB regulates MCM8-9, we first used molecular modeling and biochemistry to define their interaction interface. We show that HROB makes important contacts with both MCM8 and MCM9 subunits, which directly promotes its DNA-dependent ATPase and helicase activities. MCM8-9-HROB preferentially binds and unwinds branched DNA structures, and single-molecule experiments reveal a low DNA unwinding processivity. MCM8-9 unwinds DNA as a hexameric complex that assembles from dimers on DNA in the presence of ATP, which is prerequisite for its helicase function. The hexamer formation thus involves two repeating protein-protein interfaces forming between the alternating MCM8 and MCM9 subunits. One of these interfaces is rather stable and forms an obligate heterodimer, while the other interface is labile and mediates the assembly of the hexamer on DNA, independently of HROB. The ATPase site composed of the subunits forming the labile interface disproportionally contributes to DNA unwinding. HROB does not affect the MCM8-9 ring formation, but promotes DNA unwinding downstream by possibly coordinating ATP hydrolysis with structural transitions accompanying translocation of MCM8-9 on DNA.

biochemistry↗

bioRxiv2023

Arginine shortage induces replication stress and confers genotoxic resistance by inhibiting histone H4 translation and promoting PCNA polyubiquitination

The unique arginine dependencies of cancer cell proliferation and survival creates metabolic vulnerability. Here, we investigate the impact of extracellular arginine availability on DNA replication and genotoxic resistance. Using DNA combing assays, we find that when extracellular arginine is limited, cancer cells are arrested at S-phase and DNA replication forks slow or stall instantly until arginine is re-supplied. The translation of new histone H4 is arginine-dependent and impacts DNA replication and the expression of newly synthesized histone H4 is reduced in the avascular nutrient-poor breast cancer xenograft tumor cores. Furthermore, we demonstrate that increased PCNA occupancy and HLTF-catalyzed PCNA K63-linked polyubiquitination protects arginine-starved cells from hydroxyurea-induced, DNA2-catalyzed nascent strand degradation. Finally, arginine-deprived cancer cells are tolerant to genotoxic insults in a PCNA K63-linked polyubiquitination-dependent manner. Together, these findings reveal that extracellular arginine is the "linchpin" for nutrient-regulated DNA replication. Such information could be leveraged to expand current modalities or design new drug targets against cancer.

cancer biology↗

bioRxiv2019

Identification of MCM8IP, an interactor of MCM8-9 and RPA1 that promotes homologous recombination and DNA synthesis in response to DNA damage

Homologous recombination (HR) mediates the error-free repair of DNA double-strand breaks to maintain genomic stability. HR is carried out by a complex network of DNA repair factors. Here we identify C17orf53/MCM8IP, an OB-fold containing protein that binds ssDNA, as a novel DNA repair factor involved in HR. MCM8IP-deficient cells exhibit HR defects, especially in long-tract gene conversion, occurring downstream of RAD51 loading, consistent with a role for MCM8IP in HR-dependent DNA synthesis. Moreover, loss of MCM8IP confers cellular sensitivity to crosslinking agents and PARP inhibition. Importantly, we identify a direct interaction with MCM8-9, a putative helicase complex mutated in Primary Ovarian Insufficiency, that is crucial for MCM8IPs ability to promote resistance to DNA damaging agents. In addition to its association with MCM8-9, MCM8IP also binds directly to RPA1. We show that the interactions of MCM8IP with both MCM8-9 and RPA are required to maintain replication fork progression in response to treatment with crosslinking agents. Collectively, our work identifies MCM8IP as a key regulator of DNA damage-associated DNA synthesis during DNA recombination and replication.

genetics↗