Supplementary Table S2 from FGFR1-Activated Translation of WNT Pathway Components with Structured 5′ UTRs Is Vulnerable to Inhibition of EIF4A-Dependent Translation Initiation
Supplementary Table S2 provides lists of TE UP genes that are WNT pathway components, TE UP genes... more Supplementary Table S2 provides lists of TE UP genes that are WNT pathway components, TE UP genes and TE UP WNT pathway components with the (CGG)4 motif, and TE UP WNT pathway components with the 6-nt polypurine sequences at the 5' UTR.
Supplementary Table S1 from FGFR1-Activated Translation of WNT Pathway Components with Structured 5′ UTRs Is Vulnerable to Inhibition of EIF4A-Dependent Translation Initiation
Supplementary Table S1 provides expression data of RNA, ribosome occupancy and protein used for m... more Supplementary Table S1 provides expression data of RNA, ribosome occupancy and protein used for multi-omics correlation analysis as well as complete lists of TE UP, TE DOWN and background genes.
Supplementary Methods and Figure Legends from FGFR1-Activated Translation of WNT Pathway Components with Structured 5′ UTRs Is Vulnerable to Inhibition of EIF4A-Dependent Translation Initiation
Supplementary Methods and Figure Legends provide further details of the study methods and legends... more Supplementary Methods and Figure Legends provide further details of the study methods and legends for supplementary figures and tables.
Cooperativity between WNT and FGF signaling is well documented in embryonic development and cance... more Cooperativity between WNT and FGF signaling is well documented in embryonic development and cancer progression, but the molecular mechanisms underlying this cross-talk remain elusive. In this study, we interrogated the dynamics of RNA levels, ribosome occupancy, and protein expression as a function of inducible FGF signaling in mouse mammary glands with constitutive WNT hyperactivation. Multiomics correlation analysis revealed a substantial discrepancy between RNA and ribosome occupancy levels versus protein levels. However, this discrepancy decreased as cells became premalignant and dynamically responded to FGF signaling, implicating the importance of stringent gene regulation in nontransformed cells. Analysis of individual genes demonstrated that acute FGF hyperactivation increased translation of many stem cell self-renewal regulators, including WNT signaling components, and decreased translation of genes regulating cellular senescence. WNT pathway components translationally upregulated by FGF signaling had long and structured 5 0 UTRs with a high frequency of polypurine sequences, several of which harbored (CGG) 4 motifs that can fold into either stable G-quadruplexes or other stable secondary structures. The FGF-mediated increase in translation of WNT pathway components was compromised by silvestrol, an inhibitor of EIF4A that clamps EIF4A to polypurine sequences to block 43S scanning and inhibits its RNA-unwinding activity important for translation initiation. Moreover, silvestrol treatment significantly delayed FGF-WNT-driven tumorigenesis. Taken together, these results suggest that FGF signaling selectively enhances translation of structured mRNAs, particularly WNT signaling components, and highlight their vulnerability to inhibitors that target the RNA helicase EIF4A. Significance: The RNA helicase EIF4A may serve as a therapeutic target for breast cancers that require FGF and WNT signaling. Cancer Res; 78(15); 4229-40. Ó2018 AACR.
Supplementary Figure S5 from FGFR1-Activated Translation of WNT Pathway Components with Structured 5′ UTRs Is Vulnerable to Inhibition of EIF4A-Dependent Translation Initiation
Supplementary Figure S5 demonstrates that silvestrol treatment does not affect mouse body weight ... more Supplementary Figure S5 demonstrates that silvestrol treatment does not affect mouse body weight and proliferation rate of tumor cells, but selectively inhibits translation of WNT signaling components, and also shows that EIFA1 levels predominate EIF4A2 expression in the WNT/iR1 model, whose tumors are enriched with basal-like and Her2 signatures.
Supplementary Figure S1 from FGFR1-Activated Translation of WNT Pathway Components with Structured 5′ UTRs Is Vulnerable to Inhibition of EIF4A-Dependent Translation Initiation
Supplementary Figure S1 describes the iFGFR1 system, mating scheme for generation of WNT/iR1 mice... more Supplementary Figure S1 describes the iFGFR1 system, mating scheme for generation of WNT/iR1 mice and alterations in the mammary glands induced by iFGFR1 activation.
Supplementary Figure S3 from FGFR1-Activated Translation of WNT Pathway Components with Structured 5′ UTRs Is Vulnerable to Inhibition of EIF4A-Dependent Translation Initiation
Supplementary Figure S3 shows comprehensive multi-omics correlation analysis and assessment of th... more Supplementary Figure S3 shows comprehensive multi-omics correlation analysis and assessment of the magnitude of improvement in inter-omic correlations as the cells switched from a steady state to a dynamic state induced by iFGFR1 activation.
ABSTRACTProteolysis Targeting Chimeras (PROTACs), a class of heterobifunctional molecules that re... more ABSTRACTProteolysis Targeting Chimeras (PROTACs), a class of heterobifunctional molecules that recruit target proteins to E3 ligases, have gained traction for targeted protein degradation. However, pomalidomide, a widely used E3 ligase recruiter in PROTACs, can independently degrade other targets, such as zinc-finger (ZF) proteins, that hold key functions in normal development and disease progression. This off-target degradation of pomalidomide-based PROTACs raises concerns about their therapeutic applicability and long-term side effects. Therefore, there is a crucial need to develop rules for PROTAC design that minimize off-target degradation. In this study, we developed a high-throughput platform that interrogates the off-target degradation of ZF domains and discovered, using this platform, that PROTACs with the current design paradigm induce degradation of several ZF proteins. To identify new rules for PROTAC design, we generated a library of pomalidomide analogs that allowed sys...
Cancer-induced immune responses affect tumor progression and therapeutic response. In multiple mu... more Cancer-induced immune responses affect tumor progression and therapeutic response. In multiple murine models and clinical datasets, we identified large variations of neutrophils and macrophages, which define "immune subtypes" of triple negative breast cancer (TNBC) including neutrophil-enriched (NES) and macrophage-enriched subtypes (MES). Different tumor-intrinsic pathways and mutual regulation between macrophages/monocytes and neutrophils contribute to the development of dichotomous myeloid compartment. MES contains predominantly macrophages that are CCR2-dependent and exhibit variable responses to immune checkpoint blockade (ICB). NES exhibits systemic and local accumulation of immunosuppressive neutrophils (or granulocytic myeloid-derived suppressor cells (gMDSCs), is resistant to ICB, and contains a minority of macrophages that appear to be unaffected by CCR2 knockout. A MES-toNES conversion mediated acquired ICB resistance of initially sensitive MES models. Our results demonstrate diverse myeloid cell frequencies, functionality, and potential roles in immunotherapies, and highlight the need to better understand the inter-patient heterogeneity of the myeloid compartment.
Myeloid-derived suppressor cells (MDSCs) play critical roles in primary and metastatic cancer pro... more Myeloid-derived suppressor cells (MDSCs) play critical roles in primary and metastatic cancer progression. MDSC regulation is widely variable even among patients harbouring the same type of malignancy, and the mechanisms governing such heterogeneity are largely unknown. Here, integrating human tumour genomics and syngeneic mammary tumour models, we demonstrate that mTOR signalling in cancer cells dictates a mammary tumour's ability to stimulate MDSC accumulation through regulating G-CSF. Inhibiting this pathway or its activators (for example, FGFR) impairs tumour progression, which is partially rescued by restoring MDSCs or G-CSF. Tumour-initiating cells (TICs) exhibit elevated G-CSF. MDSCs reciprocally increase TIC frequency through activating Notch in tumour cells, forming a feedforward loop. Analyses of primary breast cancers and patient-derived xenografts corroborate these mechanisms in patients. These findings establish a non-canonical oncogenic role of mTOR signalling in r...
In response to DNA double strand breaks, the histone variant H2AX at the break site is phosphoryl... more In response to DNA double strand breaks, the histone variant H2AX at the break site is phosphorylated at serine 139 by DNA damage sensor kinases such as ataxia telangiectasia-mutated, forming ␥-H2AX. This phosphorylation event is critical for sustained recruitment of other proteins to repair the break. After repair, restoration of the cell to a prestress state is associated with ␥-H2AX dephosphorylation and dissolution of ␥-H2AXassociated damage foci. The phosphatases PP2A and PP4 have previously been shown to dephosphorylate ␥-H2AX. Here, we demonstrate that the wild-type p53-induced phosphatase 1 (WIP1) also dephosphorylates ␥-H2AX at serine 139 in vitro and in vivo. Overexpression of WIP1 reduces formation of ␥-H2AX foci in response to ionizing and ultraviolet radiation and blocks recruitment of MDC1 (mediator of DNA damage checkpoint 1) and 53BP1 (p53 binding protein 1) to DNA damage foci. Finally, these inhibitory effects of WIP1 on ␥-H2AX are accompanied by WIP1 suppression of DNA double strand break repair. Thus, WIP1 has a homeostatic role in reversing the effects of ataxia telangiectasia-mutated phosphorylation of H2AX.
The novel coronavirus, 2019-nCov (named as SARS-CoV-2 by ICTV Coronaviridae Study Group on Februa... more The novel coronavirus, 2019-nCov (named as SARS-CoV-2 by ICTV Coronaviridae Study Group on February 12, 2020), causes severe respiratory illness 1 and has been spreading around the world rapidly. 2 As of February 14, 2020, there are over 64,000 confirmed cases with 1,384 deaths. This raises an urgent need for an effective treatment of the deadly disease. However, current antiviral drugs have limited effects on 2019-nCov (SARS-CoV-2). Although Gilead's NUC (nucleoside) inhibitor, which previously failed to treat Ebola, seemed to benefit a 2019-nCov (SARS-CoV-2) patient in Washington, USA, it remains unknown whether the drug will be effective against the virus in other patients, who may have been infected by different variants of the virus. Our analysis of 2019-nCov (SARS-CoV-2) RNA genome from 19 patients in China, USA and Australia reveals that these viruses have differences in sequence (Fig. 1a). These differences are mostly single nucleotide variations. Fig. 1b shows an example of single nucleotide variations that result in changes in amino acids 62 and 84 of ORF8 of 2019-nCov (SARS-CoV-2), a polypeptide implicated in driving coronavirus transition from bat to human. 3 The evidence from patient samples suggests that 2019-nCov (SARS-CoV-2) is actively acquiring new mutations that may enable it to escape antiviral drugs. This raises a serious challenge to the development of conventional drugs and of vaccines. The same limitations apply to other deadly RNA viruses such as SARS or MERS. Our group has implemented a flexible and efficient approach for targeting RNA in the laboratory using CRISPR/Cas13d technology (under review), and here we propose that this system can be used to specifically chew up 2019-nCov (SARS-CoV-2) RNA genome, hence limiting its ability to reproduce. To functionally disrupt the virus, we will specifically use guide RNAs (gRNAs) that concomitantly target ORF1ab and S, which represent the replicase-transcriptase (ORF1ab) and the spike (S) of the virus. The Gilead's NUC inhibitor, remdesivir, having a similar chemical structure to HIV reverse-transcriptase inhibitors, is currently being tested in clinical trials for 2019-nCov (SARS-CoV-2), while a drug targeting the spike glycoprotein has also been tested in phase I trials for the treatment of HIV and SARS-CoV. 4 CRISPR/Cas13d is an RNA-guided, RNA-targeting CRISPR system. 5 To cleave the 2019-nCov (SARS-CoV-2) RNA genome, a Cas13d protein and guide RNAs-containing spacer sequences specifically complementary to the virus RNA genome are used (Fig. 1c). One advantage of the CRISPR/Cas13d system is its flexibility with respect to designing guide RNAs, because the RNAtargeting cleavage activity of Cas13d does not depend on the presence of specific adjacent sequences such as the NGG motif for the DNA-editing effector, Cas9. 5 This unique feature of the system meets the requirement for rapid development of guide RNAs to target different virus variants that evolve and may escape traditional drugs. In total, we have designed 10,333 guide RNAs
Transposable elements (TEs) comprise a large proportion of long non-coding RNAs (lncRNAs). Here, ... more Transposable elements (TEs) comprise a large proportion of long non-coding RNAs (lncRNAs). Here, we employed CRISPR to delete a short interspersed nuclear element (SINE) in Malat1, a cancerassociated lncRNA, to investigate its significance in cellular physiology. We show that Malat1 with a SINE deletion forms diffuse nuclear speckles and is frequently translocated to the cytoplasm. SINE-deleted cells exhibit an activated unfolded protein response and PKR and markedly increased DNA damage and apoptosis caused by dysregulation of TDP-43 localization and formation of cytotoxic inclusions. TDP-43 binds stronger to Malat1 without the SINE and is likely 'hijacked' by cytoplasmic Malat1 to the cytoplasm, resulting in the depletion of nuclear TDP-43 and redistribution of TDP-43 binding to repetitive element transcripts and mRNAs encoding mitotic and nuclear-cytoplasmic regulators. The SINE promotes Malat1 nuclear retention by facilitating Malat1 binding to HNRNPK, a protein that drives RNA nuclear retention, potentially through direct interactions of the SINE with KHDRBS1 and TRA2A, which bind to HN-RNPK. Losing these RNA-protein interactions due to the SINE deletion likely creates more available TDP-43 binding sites on Malat1 and subsequent TDP-43 aggregation. These results highlight the significance of lncRNA TEs in TDP-43 proteostasis with potential implications in both cancer and neurodegenerative diseases.
Cooperativity between WNT and FGF signaling is well documented in embryonic development and cance... more Cooperativity between WNT and FGF signaling is well documented in embryonic development and cancer progression, but the molecular mechanisms underlying this crosstalk remain elusive. In this study, we interrogated the dynamics of RNA levels, ribosome occupancy, and protein expression as a function of inducible FGF signaling in mouse mammary glands with constitutive WNT hyperactivation. Multi-omics correlation analysis revealed a substantial discrepancy between RNA and ribosome occupancy levels versus protein levels. However, this discrepancy decreased as cells became pre-malignant and dynamically responded to FGF signaling, implicating the importance of stringent gene regulation in non-transformed cells. Analysis of individual genes demonstrated that acute FGF hyperactivation increased translation of many stem cell self-renewal regulators, including WNT signaling components, and decreased translation of genes regulating cellular senescence. WNT pathway components translationally upr...
Myeloid lncRNA LOUP Mediates Opposing Regulatory Effects of RUNX1 and RUNX1-ETO in t(8;21) AML
ABSTRACTThe mechanism underlying cell type-specific gene induction conferred by ubiquitous transc... more ABSTRACTThe mechanism underlying cell type-specific gene induction conferred by ubiquitous transcription factors as well as disruptions caused by their chimeric derivatives in leukemia is not well understood. Here we investigate whether RNAs coordinate with transcription factors to drive myeloid gene transcription. In an integrated genome-wide approach surveying for gene loci exhibiting concurrent RNA- and DNA-interactions with the broadly expressed transcription factor RUNX1, we identified the long noncoding RNA LOUP. This myeloid-specific and polyadenylated lncRNA induces myeloid differentiation and inhibits cell growth, acting as a transcriptional inducer of the myeloid master regulator PU.1. Mechanistically, LOUP recruits RUNX1 to both the PU.1 enhancer and the promoter, leading to the formation of an active chromatin loop. In t(8;21) acute myeloid leukemia, wherein RUNX1 is fused to ETO, the resulting oncogenic fusion protein RUNX1-ETO limits chromatin accessibility at the LOUP...
New twists on long noncoding RNAs: from mobile elements to motile cancer cells
RNA Biology
ABSTRACT The purpose of this review is to highlight several areas of lncRNA biology and cancer th... more ABSTRACT The purpose of this review is to highlight several areas of lncRNA biology and cancer that we hope will provide some new insights for future research. These include the relationship of lncRNAs and the epithelial to mesenchymal transition (EMT) with a focus on transcriptional and alternative splicing mechanisms and mRNA stability through miRNAs. In addition, we highlight the potential role of enhancer e-lncRNAs, the importance of transposable elements in lncRNA biology, and finally the emerging area of using antisense oligonucleotides (ASOs) and small molecules to target lncRNAs and their therapeutic implications.
45 The mechanism underlying cell type-specific gene induction conferred by ubiquitous transcripti... more 45 The mechanism underlying cell type-specific gene induction conferred by ubiquitous transcription 46 factors as well as disruptions caused by their chimeric derivatives in leukemia is not well understood. 47 Here we investigate whether RNAs coordinate with transcription factors to drive myeloid gene 48 transcription. In an integrated genome-wide approach surveying for gene loci exhibiting concurrent 49 RNAand DNA-interactions with the broadly expressed transcription factor RUNX1, we identified the 50 long noncoding RNA LOUP. This myeloid-specific and polyadenylated lncRNA induces myeloid 51 differentiation and inhibits cell growth, acting as a transcriptional inducer of the myeloid master 52 regulator PU.1. Mechanistically, LOUP recruits RUNX1 to both the PU.1 enhancer and the promoter, 53 leading to the formation of an active chromatin loop. In t(8;21) acute myeloid leukemia, wherein RUNX1 54 is fused to ETO, the resulting oncogenic fusion protein RUNX1-ETO limits chromatin acces...
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