Logical design of synthetic cis-regulatory DNA for genetic tracing of cell identities and state changes.

Descriptive data are rapidly expanding in biomedical research. Instead, functional validation methods with sufficient complexity remain underdeveloped. Transcriptional reporters allow experimental characterization and manipulation of developmental and disease cell states, but their design lacks flexibility. Here, we report logical design of synthetic cis-regulatory DNA (LSD), a computational framework leveraging phenotypic biomarkers and trans-regulatory networks as input to design reporters marking the activity of selected cellular states and pathways. LSD uses bulk or single-cell biomarkers and a reference genome or custom cis-regulatory DNA datasets with user-defined boundary regions. By benchmarking validated reporters, we integrate LSD with a computational ranking of phenotypic specificity of putative cis-regulatory DNA. Experimentally, LSD-designed reporters targeting a wide range of cell states are functional without minimal promoters. Applied to broadly expressed genes from human and mouse tissues, LSD generates functional housekeeper-like sLCRs compatible with size constraints of AAV vectors for gene therapy applications. A mesenchymal glioblastoma reporter designed by LSD outperforms previously validated ones and canonical cell surface markers. In genome-scale CRISPRa screens, LSD facilitates the discovery of known and novel bona fide cell-state drivers. Thus, LSD captures core principles of cis-regulation and is broadly applicable to studying complex cell states and mechanisms of transcriptional regulation.

Pharmacological modulators of epithelial immunity uncovered by synthetic genetic tracing of SARS-CoV-2 infection responses.

Epithelial immune responses govern tissue homeostasis and offer drug targets against maladaptation. Here, we report a framework to generate drug discovery-ready reporters of cellular responses to viral infection. We reverse-engineered epithelial cell responses to SARS-CoV-2, the viral agent fueling the ongoing COVID-19 pandemic, and designed synthetic transcriptional reporters whose molecular logic comprises interferon-α/ß/γ and NF-κB pathways. Such regulatory potential reflected single-cell data from experimental models to severe COVID-19 patient epithelial cells infected by SARS-CoV-2. SARS-CoV-2, type I interferons, and RIG-I drive reporter activation. Live-cell image-based phenotypic drug screens identified JAK inhibitors and DNA damage inducers as antagonistic modulators of epithelial cell response to interferons, RIG-I stimulation, and SARS-CoV-2. Synergistic or antagonistic modulation of the reporter by drugs underscored their mechanism of action and convergence on endogenous transcriptional programs. Our study describes a tool for dissecting antiviral responses to infection and sterile cues and rapidly discovering rational drug combinations for emerging viruses of concern.

A Mutation-driven oncofetal regression fuels phenotypic plasticity in colorectal cancer.

Targeting cancer stem cells (CSCs) is crucial for effective cancer treatment 1 . However, the molecular mechanisms underlying resistance to LGR5 + CSCs depletion in colorectal cancer (CRC) 2,3 remain largely elusive. Here, we unveil the existence of a primitive cell state dubbed the oncofetal (OnF) state, which works in tandem with the LGR5 + stem cells (SCs) to fuel tumor evolution in CRC. OnF cells emerge early during intestinal tumorigenesis and exhibit features of lineage plasticity. Normally suppressed by the Retinoid X Receptor (RXR) in mature SCs, the OnF program is triggered by genetic deletion of the gatekeeper APC. We demonstrate that diminished RXR activity unlocks an epigenetic circuity governed by the cooperative action of YAP and AP1, leading to OnF reprogramming. This high-plasticity state is inherently resistant to conventional chemotherapies and its adoption by LGR5 + CSCs enables them to enter a drug-tolerant state. Furthermore, through phenotypic tracing and ablation experiments, we uncover a functional redundancy between the OnF and stem cell (SC) states and show that targeting both cellular states is essential for sustained tumor regression in vivo . Collectively, these findings establish a mechanistic foundation for developing effective combination therapies with enduring impact on CRC treatment.

Functional antagonism of chromatin modulators regulates epithelial-mesenchymal transition.

Epithelial-mesenchymal transition (EMT) is a developmental process hijacked by cancer cells to modulate proliferation, migration, and stress response. Whereas kinase signaling is believed to be an EMT driver, the molecular mechanisms underlying epithelial-mesenchymal interconversion are incompletely understood. Here, we show that the impact of chromatin regulators on EMT interconversion is broader than that of kinases. By combining pharmacological modulation of EMT, synthetic genetic tracing, and CRISPR interference screens, we uncovered a minority of kinases and several chromatin remodelers, writers, and readers governing homeostatic EMT in lung cancer cells. Loss of ARID1A, DOT1L, BRD2, and ZMYND8 had nondeterministic and sometimes opposite consequences on epithelial-mesenchymal interconversion. Together with RNAPII and AP-1, these antagonistic gatekeepers control chromatin of active enhancers, including pan-cancer-EMT signature genes enabling supraclassification of anatomically diverse tumors. Thus, our data uncover general principles underlying transcriptional control of cancer cell plasticity and offer a platform to systematically explore chromatin regulators in tumor-state-specific therapy.

Phenotypic Mapping of Pathologic Cross-Talk between Glioblastoma and Innate Immune Cells by Synthetic Genetic Tracing.

Glioblastoma is a lethal brain tumor that exhibits heterogeneity and resistance to therapy. Our understanding of tumor homeostasis is limited by a lack of genetic tools to selectively identify tumor states and fate transitions. Here, we use glioblastoma subtype signatures to construct synthetic genetic tracing cassettes and investigate tumor heterogeneity at cellular and molecular levels, in vitro and in vivo. Through synthetic locus control regions, we demonstrate that proneural glioblastoma is a hardwired identity, whereas mesenchymal glioblastoma is an adaptive and metastable cell state driven by proinflammatory and differentiation cues and DNA damage, but not hypoxia. Importantly, we discovered that innate immune cells divert glioblastoma cells to a proneural-to-mesenchymal transition that confers therapeutic resistance. Our synthetic genetic tracing methodology is simple, scalable, and widely applicable to study homeostasis in development and diseases. In glioblastoma, the method causally links distinct (micro)environmental, genetic, and pharmacologic perturbations and mesenchymal commitment. SIGNIFICANCE: Glioblastoma is heterogeneous and incurable. Here, we designed synthetic reporters to reflect the transcriptional output of tumor cell states and signaling pathways' activity. This method is generally applicable to study homeostasis in normal tissues and diseases. In glioblastoma, synthetic genetic tracing causally connects cellular and molecular heterogeneity to therapeutic responses.This article is highlighted in the In This Issue feature, p. 521.

NF1 regulates mesenchymal glioblastoma plasticity and aggressiveness through the AP-1 transcription factor FOSL1.

The molecular basis underlying glioblastoma (GBM) heterogeneity and plasticity is not fully understood. Using transcriptomic data of human patient-derived brain tumor stem cell lines (BTSCs), classified based on GBM-intrinsic signatures, we identify the AP-1 transcription factor FOSL1 as a key regulator of the mesenchymal (MES) subtype. We provide a mechanistic basis to the role of the neurofibromatosis type 1 gene (NF1), a negative regulator of the RAS/MAPK pathway, in GBM mesenchymal transformation through the modulation of FOSL1 expression. Depletion of FOSL1 in NF1-mutant human BTSCs and Kras-mutant mouse neural stem cells results in loss of the mesenchymal gene signature and reduction in stem cell properties and in vivo tumorigenic potential. Our data demonstrate that FOSL1 controls GBM plasticity and aggressiveness in response to NF1 alterations.