A PRMT5-ZNF326 axis mediates innate immune activation upon replication stress.

DNA replication stress (RS) is a widespread phenomenon in carcinogenesis, causing genomic instability and extensive chromatin alterations. DNA damage leads to activation of innate immune signaling, but little is known about transcriptional regulators mediating such signaling upon RS. Using a chemical screen, we identified protein arginine methyltransferase 5 (PRMT5) as a key mediator of RS-dependent induction of interferon-stimulated genes (ISGs). This response is also associated with reactivation of endogenous retroviruses (ERVs). Using quantitative mass spectrometry, we identify proteins with PRMT5-dependent symmetric dimethylarginine (SDMA) modification induced upon RS. Among these, we show that PRMT5 targets and modulates the activity of ZNF326, a zinc finger protein essential for ISG response. Our data demonstrate a role for PRMT5-mediated SDMA in the context of RS-induced transcriptional induction, affecting physiological homeostasis and cancer therapy.

Prdm15 acts upstream of Wnt4 signaling in anterior neural development of Xenopus laevis.

Mutations in PRDM15 lead to a syndromic form of holoprosencephaly (HPE) known as the Galloway-Mowat syndrome (GAMOS). While a connection between PRDM15, a zinc finger transcription factor, and WNT/PCP signaling has been established, there is a critical need to delve deeper into their contributions to early development and GAMOS pathogenesis. We used the South African clawed frog Xenopus laevis as the vertebrate model organism and observed that prdm15 was enriched in the tissues and organs affected in GAMOS. Furthermore, we generated a morpholino oligonucleotide-mediated prdm15 knockdown model showing that the depletion of Prdm15 leads to abnormal eye, head, and brain development, effectively recapitulating the anterior neural features in GAMOS. An analysis of the underlying molecular basis revealed a reduced expression of key genes associated with eye, head, and brain development. Notably, this reduction could be rescued by the introduction of wnt4 RNA, particularly during the induction of the respective tissues. Mechanistically, our data demonstrate that Prdm15 acts upstream of both canonical and non-canonical Wnt4 signaling during anterior neural development. Our findings describe severe ocular and anterior neural abnormalities upon Prdm15 depletion and elucidate the role of Prdm15 in canonical and non-canonical Wnt4 signaling.

SMARCAL1 is a dual regulator of innate immune signaling and PD-L1 expression that promotes tumor immune evasion.

Genomic instability can trigger cancer-intrinsic innate immune responses that promote tumor rejection. However, cancer cells often evade these responses by overexpressing immune checkpoint regulators, such as PD-L1. Here, we identify the SNF2-family DNA translocase SMARCAL1 as a factor that favors tumor immune evasion by a dual mechanism involving both the suppression of innate immune signaling and the induction of PD-L1-mediated immune checkpoint responses. Mechanistically, SMARCAL1 limits endogenous DNA damage, thereby suppressing cGAS-STING-dependent signaling during cancer cell growth. Simultaneously, it cooperates with the AP-1 family member JUN to maintain chromatin accessibility at a PD-L1 transcriptional regulatory element, thereby promoting PD-L1 expression in cancer cells. SMARCAL1 loss hinders the ability of tumor cells to induce PD-L1 in response to genomic instability, enhances anti-tumor immune responses and sensitizes tumors to immune checkpoint blockade in a mouse melanoma model. Collectively, these studies uncover SMARCAL1 as a promising target for cancer immunotherapy.

Anti-correlated feature selection prevents false discovery of subpopulations in scRNAseq.

While sub-clustering cell-populations has become popular in single cell-omics, negative controls for this process are lacking. Popular feature-selection/clustering algorithms fail the null-dataset problem, allowing erroneous subdivisions of homogenous clusters until nearly each cell is called its own cluster. Using real and synthetic datasets, we find that anti-correlated gene selection reduces or eliminates erroneous subdivisions, increases marker-gene selection efficacy, and efficiently scales to millions of cells.

MYOD-SKP2 axis boosts tumorigenesis in fusion negative rhabdomyosarcoma by preventing differentiation through p57Kip2 targeting.

Rhabdomyosarcomas (RMS) are pediatric mesenchymal-derived malignancies encompassing PAX3/7-FOXO1 Fusion Positive (FP)-RMS, and Fusion Negative (FN)-RMS with frequent RAS pathway mutations. RMS express the master myogenic transcription factor MYOD that, whilst essential for survival, cannot support differentiation. Here we discover SKP2, an oncogenic E3-ubiquitin ligase, as a critical pro-tumorigenic driver in FN-RMS. We show that SKP2 is overexpressed in RMS through the binding of MYOD to an intronic enhancer. SKP2 in FN-RMS promotes cell cycle progression and prevents differentiation by directly targeting p27Kip1 and p57Kip2, respectively. SKP2 depletion unlocks a partly MYOD-dependent myogenic transcriptional program and strongly affects stemness and tumorigenic features and prevents in vivo tumor growth. These effects are mirrored by the investigational NEDDylation inhibitor MLN4924. Results demonstrate a crucial crosstalk between transcriptional and post-translational mechanisms through the MYOD-SKP2 axis that contributes to tumorigenesis in FN-RMS. Finally, NEDDylation inhibition is identified as a potential therapeutic vulnerability in FN-RMS.

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.

RBFOX2 deregulation promotes pancreatic cancer progression and metastasis through alternative splicing.

RNA splicing is an important biological process associated with cancer initiation and progression. However, the contribution of alternative splicing to pancreatic cancer (PDAC) development is not well understood. Here, we identify an enrichment of RNA binding proteins (RBPs) involved in splicing regulation linked to PDAC progression from a forward genetic screen using Sleeping Beauty insertional mutagenesis in a mouse model of pancreatic cancer. We demonstrate downregulation of RBFOX2, an RBP of the FOX family, promotes pancreatic cancer progression and liver metastasis. Specifically, we show RBFOX2 regulates exon splicing events in transcripts encoding proteins involved in cytoskeletal remodeling programs. These exons are differentially spliced in PDAC patients, with enhanced exon skipping in the classical subtype for several RBFOX2 targets. RBFOX2 mediated splicing of ABI1, encoding the Abelson-interactor 1 adapter protein, controls the abundance and localization of ABI1 protein isoforms in pancreatic cancer cells and promotes the relocalization of ABI1 from the cytoplasm to the periphery of migrating cells. Using splice-switching antisense oligonucleotides (AONs) we demonstrate the ABI1 ∆Ex9 isoform enhances cell migration. Together, our data identify a role for RBFOX2 in promoting PDAC progression through alternative splicing regulation.

Nuclear RNA catabolism controls endogenous retroviruses, gene expression asymmetry, and dedifferentiation.

Endogenous retroviruses (ERVs) are remnants of ancient parasitic infections and comprise sizable portions of most genomes. Although epigenetic mechanisms silence most ERVs by generating a repressive environment that prevents their expression (heterochromatin), little is known about mechanisms silencing ERVs residing in open regions of the genome (euchromatin). This is particularly important during embryonic development, where induction and repression of distinct classes of ERVs occur in short temporal windows. Here, we demonstrate that transcription-associated RNA degradation by the nuclear RNA exosome and Integrator is a regulatory mechanism that controls the productive transcription of most genes and many ERVs involved in preimplantation development. Disrupting nuclear RNA catabolism promotes dedifferentiation to a totipotent-like state characterized by defects in RNAPII elongation and decreased expression of long genes (gene-length asymmetry). Our results indicate that RNA catabolism is a core regulatory module of gene networks that safeguards RNAPII activity, ERV expression, cell identity, and developmental potency.

Isoform-resolved transcriptome of the human preimplantation embryo.

Human preimplantation development involves extensive remodeling of RNA expression and splicing. However, its transcriptome has been compiled using short-read sequencing data, which fails to capture most full-length mRNAs. Here, we generate an isoform-resolved transcriptome of early human development by performing long- and short-read RNA sequencing on 73 embryos spanning the zygote to blastocyst stages. We identify 110,212 unannotated isoforms transcribed from known genes, including highly conserved protein-coding loci and key developmental regulators. We further identify 17,964 isoforms from 5,239 unannotated genes, which are largely non-coding, primate-specific, and highly associated with transposable elements. These isoforms are widely supported by the integration of published multi-omics datasets, including single-cell 8CLC and blastoid studies. Alternative splicing and gene co-expression network analyses further reveal that embryonic genome activation is associated with splicing disruption and transient upregulation of gene modules. Together, these findings show that the human embryo transcriptome is far more complex than currently known, and will act as a valuable resource to empower future studies exploring development.

WNTinib is a multi-kinase inhibitor with specificity against ß-catenin mutant hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is a leading cause of cancer-related deaths worldwide. ß-Catenin (CTNNB1)-mutated HCC represents 30% of cases of the disease with no precision therapeutics available. Using chemical libraries derived from clinical multi-kinase inhibitor (KI) scaffolds, we screened HCC organoids to identify WNTinib, a KI with exquisite selectivity in CTNNB1-mutated human and murine models, including patient samples. Multiomic and target engagement analyses, combined with rescue experiments and in vitro and in vivo efficacy studies, revealed that WNTinib is superior to clinical KIs and inhibits KIT/mitogen-activated protein kinase (MAPK) signaling at multiple nodes. Moreover, we demonstrate that reduced engagement on BRAF and p38α kinases by WNTinib relative to several multi-KIs is necessary to avoid compensatory feedback signaling-providing a durable and selective transcriptional repression of mutant ß-catenin/Wnt targets through nuclear translocation of the EZH2 transcriptional repressor. Our studies uncover a previously unknown mechanism to harness the KIT/MAPK/EZH2 pathway to potently and selectively antagonize CTNNB1-mutant HCC with an unprecedented wide therapeutic index.

Hepatic stellate cell stearoyl co-A desaturase activates leukotriene B4 receptor 2 - ß-catenin cascade to promote liver tumorigenesis.

Hepatocellular carcinoma (HCC) is the 3rd most deadly malignancy. Activated hepatic stellate cells (aHSC) give rise to cancer-associated fibroblasts in HCC and are considered a potential therapeutic target. Here we report that selective ablation of stearoyl CoA desaturase-2 (Scd2) in aHSC globally suppresses nuclear CTNNB1 and YAP1 in tumors and tumor microenvironment and prevents liver tumorigenesis in male mice. Tumor suppression is associated with reduced leukotriene B4 receptor 2 (LTB4R2) and its high affinity oxylipin ligand, 12-hydroxyheptadecatrienoic acid (12-HHTrE). Genetic or pharmacological inhibition of LTB4R2 recapitulates CTNNB1 and YAP1 inactivation and tumor suppression in culture and in vivo. Single cell RNA sequencing identifies a subset of tumor-associated aHSC expressing Cyp1b1 but no other 12-HHTrE biosynthetic genes. aHSC release 12-HHTrE in a manner dependent on SCD and CYP1B1 and their conditioned medium reproduces the LTB4R2-mediated tumor-promoting effects of 12-HHTrE in HCC cells. CYP1B1-expressing aHSC are detected in proximity of LTB4R2-positive HCC cells and the growth of patient HCC organoids is blunted by LTB4R2 antagonism or knockdown. Collectively, our findings suggest aHSC-initiated 12-HHTrE-LTB4R2-CTNNB1-YAP1 pathway as a potential HCC therapeutic target.

PBRM1-deficient PBAF complexes target aberrant genomic loci to activate the NF-κB pathway in clear cell renal cell carcinoma.

PBRM1 encodes an accessory subunit of the PBAF SWI/SNF chromatin remodeller, and the inactivation of PBRM1 is a frequent event in kidney cancer. However, the impact of PBRM1 loss on chromatin remodelling is not well examined. Here we show that, in VHL-deficient renal tumours, PBRM1 deficiency results in ectopic PBAF complexes that localize to de novo genomic loci, activating the pro-tumourigenic NF-κB pathway. PBRM1-deficient PBAF complexes retain the association between SMARCA4 and ARID2, but have loosely tethered BRD7. The PBAF complexes redistribute from promoter proximal regions to distal enhancers containing NF-κB motifs, heightening NF-κB activity in PBRM1-deficient models and clinical samples. The ATPase function of SMARCA4 maintains chromatin occupancy of pre-existing and newly acquired RELA specific to PBRM1 loss, activating downstream target gene expression. Proteasome inhibitor bortezomib abrogates RELA occupancy, suppresses NF-κB activation and delays growth of PBRM1-deficient tumours. In conclusion, PBRM1 safeguards the chromatin by repressing aberrant liberation of pro-tumourigenic NF-κB target genes by residual PBRM1-deficient PBAF complexes.

Identification of IGF2 as Genomic Driver and Actionable Therapeutic Target in Hepatoblastoma.

Management of hepatoblastoma (HB), the most frequent pediatric liver cancer, is based on surgical resection and perioperative chemotherapy regimens. In this study, we aimed to identify actionable targets in HB and assess the efficacy of molecular therapies in preclinical models of HB. Paired tumor and adjacent tissues from 31 HBs and a validation set of 50 HBs were analyzed using RNA-seq, SNP, and methylation arrays. IGF2 overexpression was identified as the top targetable HB driver, present in 71% of HBs (22/31). IGF2high tumors displayed progenitor cell features and shorter recurrence-free survival. IGF2 overexpression was associated in 91% of cases with fetal promoter hypomethylation, ICR1 deregulation, 11p15.5 loss of heterozygosity or miR483-5p overexpression. The antitumor effect of xentuzumab (a monoclonal antibody targeting IGF1/2) alone or in combination with the conventional therapeutic agent cisplatin was assessed in HB cell lines, in PDX-derived HB organoids and in a xenograft HB murine model. The combination of xentuzumab with cisplatin showed strong synergistic antitumor effects in organoids and in IGF2high cell lines. In mice (n = 55), the combination induced a significant decrease in tumor volume and improved survival compared with cisplatin alone. These results suggest that IGF2 is an HB actionable driver and that, in preclinical models of HB, the combination of IGF1/2 inhibition with cisplatin induces superior antitumor effects than cisplatin monotherapy. Overall, our study provides a rationale for testing IGF2 inhibitors in combination with cisplatin in HB patients with IGF2 overexpression.

G9a/GLP inhibition during ex vivo lymphocyte expansion increases in vivo cytotoxicity of engineered T cells against hepatocellular carcinoma.

Engineered T cells transiently expressing tumor-targeting receptors are an attractive form of engineered T cell therapy as they carry no risk of insertional mutagenesis or long-term adverse side-effects. However, multiple rounds of treatment are often required, increasing patient discomfort and cost. To mitigate this, we sought to improve the antitumor activity of transient engineered T cells by screening a panel of small molecules targeting epigenetic regulators for their effect on T cell cytotoxicity. Using a model for engineered T cells targetting hepatocellular carcinoma, we find that short-term inhibition of G9a/GLP increases T cell antitumor activity in in vitro models and an orthotopic mouse model. G9a/GLP inhibition increases granzyme expression without terminal T cell differentiation or exhaustion and results in specific changes in expression of genes and proteins involved in pro-inflammatory pathways, T cell activation and cytotoxicity.

Splice-switch oligonucleotide-based combinatorial platform prioritizes synthetic lethal targets CHK1 and BRD4 against MYC-driven hepatocellular carcinoma.

Deregulation of MYC is among the most frequent oncogenic drivers in hepatocellular carcinoma (HCC). Unfortunately, the clinical success of MYC-targeted therapies is limited. Synthetic lethality offers an alternative therapeutic strategy by leveraging on vulnerabilities in tumors with MYC deregulation. While several synthetic lethal targets of MYC have been identified in HCC, the need to prioritize targets with the greatest therapeutic potential has been unmet. Here, we demonstrate that by pairing splice-switch oligonucleotide (SSO) technologies with our phenotypic-analytical hybrid multidrug interrogation platform, quadratic phenotypic optimization platform (QPOP), we can disrupt the functional expression of these targets in specific combinatorial tests to rapidly determine target-target interactions and rank synthetic lethality targets. Our SSO-QPOP analyses revealed that simultaneous attenuation of CHK1 and BRD4 function is an effective combination specific in MYC-deregulated HCC, successfully suppressing HCC progression in vitro. Pharmacological inhibitors of CHK1 and BRD4 further demonstrated its translational value by exhibiting synergistic interactions in patient-derived xenograft organoid models of HCC harboring high levels of MYC deregulation. Collectively, our work demonstrates the capacity of SSO-QPOP as a target prioritization tool in the drug development pipeline, as well as the therapeutic potential of CHK1 and BRD4 in MYC-driven HCC.

Proof-of-Concept Method to Study Uncharacterized Methyltransferases Using PRDM15.

The PRDM family of methyltransferases has been implicated in cellular proliferation and differentiation and is deregulated in human diseases, most notably in cancer. PRDMs are related to the SET domain family of methyltransferases; however, from the 19 PRDMs only a few PRDMs with defined enzymatic activities are known. PRDM15 is an uncharacterized transcriptional regulator, with significant structural disorder and lack of defined small-molecule binding pockets. Many aspects of PRDM15 are yet unknown, including its structure, substrates, reaction mechanism, and its methylation profile. Here, we employ a series of computational approaches for an exploratory investigation of its potential substrates and reaction mechanism. Using the knowledge of PRDM9 and current knowledge of PRDM15 as basis, we tried to identify genuine substrates of PRDM15. We start from histone-based peptides and learn that the native substrates of PRDM15 may be non-histone proteins. In the future, a combination of sequence-based approaches and signature motif analysis may provide new leads. In summary, our results provide new information about the uncharacterized methyltransferase, PRDM15.

Novel microenvironment-based classification of intrahepatic cholangiocarcinoma with therapeutic implications.

OBJECTIVE: The diversity of the tumour microenvironment (TME) of intrahepatic cholangiocarcinoma (iCCA) has not been comprehensively assessed. We aimed to generate a novel molecular iCCA classifier that incorporates elements of the stroma, tumour and immune microenvironment ('STIM' classification). DESIGN: We applied virtual deconvolution to transcriptomic data from ~900 iCCAs, enabling us to devise a novel classification by selecting for the most relevant TME components. Murine models were generated through hydrodynamic tail vein injection and compared with the human disease. RESULTS: iCCA is composed of five robust STIM classes encompassing both inflamed (35%) and non-inflamed profiles (65%). The inflamed classes, named immune classical (~10%) and inflammatory stroma (~25%), differ in oncogenic pathways and extent of desmoplasia, with the inflammatory stroma showing T cell exhaustion, abundant stroma and KRAS mutations (p<0.001). Analysis of cell-cell interactions highlights cancer-associated fibroblast subtypes as potential mediators of immune evasion. Among the non-inflamed classes, the desert-like class (~20%) harbours the lowest immune infiltration with abundant regulatory T cells (p<0.001), whereas the hepatic stem-like class (~35%) is enriched in 'M2-like' macrophages, mutations in IDH1/2 and BAP1, and FGFR2 fusions. The remaining class (tumour classical: ~10%) is defined by cell cycle pathways and poor prognosis. Comparative analysis unveils high similarity between a KRAS/p19 murine model and the inflammatory stroma class (p=0.02). The KRAS-SOS inhibitor, BI3406, sensitises a KRAS-mutant iCCA murine model to anti-PD1 therapy. CONCLUSIONS: We describe a comprehensive TME-based stratification of iCCA. Cross-species analysis establishes murine models that align closely to human iCCA for the preclinical testing of combination strategies.

An interplay between BRD4 and G9a regulates skeletal myogenesis.

Histone acetylation and methylation are epigenetic modifications that are dynamically regulated by chromatin modifiers to precisely regulate gene expression. However, the interplay by which histone modifications are synchronized to coordinate cellular differentiation is not fully understood. In this study, we demonstrate a relationship between BRD4, a reader of acetylation marks, and G9a, a writer of methylation marks in the regulation of myogenic differentiation. Using loss- and gain-of-function studies, as well as a pharmacological inhibition of its activity, we examined the mechanism by which BRD4 regulates myogenesis. Transcriptomic analysis using RNA sequencing revealed that a number of myogenic differentiation genes are downregulated in Brd4-depleted cells. Interestingly, some of these genes were upregulated upon G9a knockdown, indicating that BRD4 and G9a play opposing roles in the control of myogenic gene expression. Remarkably, the differentiation defect caused by Brd4 knockdown was rescued by inhibition of G9a methyltransferase activity. These findings demonstrate that the absence of BRD4 results in the upregulation of G9a activity and consequently impaired myogenic differentiation. Collectively, our study identifies an interdependence between BRD4 and G9a for the precise control of transcriptional outputs to regulate myogenesis.

T cell immunity is key to the pandemic endgame: How to measure and monitor it.

As vaccine deployment improves the healthcare emergency status caused by the SARS-CoV-2 pandemic, we need reliable tools to evaluate the duration of protective immunity at a global scale. Seminal studies have demonstrated that while neutralizing antibodies can protect us from viral infection, T cell-mediated cellular immunity provides long-term protection from severe COVID-19, even in the case of emerging new variants of concern (VOC). Indeed, the emergence of VOCs, able to substantially escape antibodies generated by current vaccines, has made the analysis of correlates of humoral protection against infection obsolete. The focus should now shift towards immunological correlates of protection from disease based on quantification of cellular immunity. Despite this evidence, an assessment of T cell responses is still overlooked. This is largely due to technical challenges and lack of validated diagnostic tests. Here, we review the current state of the art of available tests to distinguish between SARS-CoV-2 antigen-specific Tcells and non-antigen specific T-cells. These assays range from the analysis of the T cell-receptor (TCR) diversity (i.e. Immunoseq and MHC tetramer staining) to the detection of functional T cell activation (i.e. ICS, AIM, Elispot, ELLA, dqTACT, etc.) either from purified Peripheral Blood Mononuclear Cells (PBMCs) or whole blood. We discuss advantages and disadvantages of each assay, proposing their ideal use for different scopes. Finally, we argue how it is paramount to deploy cheap, standardized, and scalable assays to measure T cell functionality to fill this critical diagnostic gap and manage these next years of the pandemic.

Dynamic plasticity of prostate cancer intermediate cells during androgen receptor-targeted therapy.

Treatment-emergent small cell neuroendocrine prostate cancer (t-SCNC) is associated with an epithelial lineage switch from an androgen receptor (AR)-positive to neuroendocrine (NE)-marker-positive status. Understanding the potential for reversibility of this aggressive disease state has been hampered by the paucity of models suitable for studying rate-limiting, transitional, or intermediate tumor cell subpopulations. We define a dual reporter model that measures acute transcriptional changes in response to castration or AR targeting agents. We identify steady-state transcriptional heterogeneity in AR and NE biomarkers, including intermediate subpopulations that are coordinately high for prostate-specific antigen (PSA) and neuron-specific enoclase (NSE) promoter activity. In the presence of castration or AR inhibitors, intermediate cells were necessary and sufficient for therapy-induced conversion of human PC cells to an NSE-high transcriptional status. Using hormone add-back studies, treatment-induced PSA-NSE transcriptional plasticity was reversible in PTEN-deficient PC cells but not in the presence of secondary genetic driver genes, including MYCN.