• Adenosine-to-Inosine (A-to-I) editing is one of the most prevalent RNA modifications that occurs in metazoans and is mediated by the Adenosine Deaminase Acting on RNA (ADAR) family of enzymes.

• The enzyme ADAR/ADAR1 catalyzes the majority of A-to-I editing, where it has been demonstrated to effect coding sequence, miRNA function and silencing of Alu repetitive elements1.

1. Eisenberg et al, Nature Review Genetics, 2018 2. Ahmad et al, Cell, 20183. Chung et al, Cell, 20184. Crow et al, Nature Review Immunology, 20155. McDonald et al, Cell, 2017

Elevated Cancer-Intrinsic Type I Interferon Signaling Confers a Dependency on the RNA Editor ADAR1

Cindy Collins, Alexandra K. Gardino, Scott A. Ribich*, Robert A. CopelandAccent Therapeutics, 65 Hayden Ave., Lexington, MA

*Correspondence to: sribich@accenttx.com

mRNA (coding sequences)

ADAR1

Results: Nonsynonymous base changes (I read as G)

miRNAs

Results: Can alter miRNA function

mRNA (Intronic/UTR localized

Alu Elements)

Results: Prevents recognition by innate

immune system

AD

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Pro

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-log 1

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-log 1

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-log 1

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ADAR1-Dependent Cancer Cell Lines Have Elevated Interferon Stimulated Gene (ISG) Expression

The RNA Editor ADAR1: Background

A Subset of Cancer Cell Lines are Dependent on ADAR1

No Individual Gene Mutation Predicts Dependence on ADAR1 Dependence

Genes Elevated in ADAR1 Dependent Cells Include a Subset of Type I

Interferon Stimulated Genes (ISGs)

KEGG Pathway P-valueNOD-like receptor signaling pathway 2.45E-09Influenza A 2.91E-09Human papillomavirus infection 5.64E-09Herpes simplex infection 3.07E-07Hepatitis C 2.88E-06RIG-I-like receptor signaling pathway 4.15E-06TNF signaling pathway 6.57E-06NF-kappa B signaling pathway 9.69E-06Kaposi's sarcoma-associated herpesvirus 1.19E-05Measles 5.87E-05

GO Pathway P-valuetype I interferon signaling pathway 2.80E-29defense response to virus 7.11E-21response to type I interferon 8.39E-21response to biotic stimulus 1.33E-17interferon-gamma-mediated signaling pathway 5.38E-15epidermis development 6.96E-14negative regulation of viral genome replication 1.04E-12regulation of response to stress 1.87E-12positive regulation of NF-kappaB transcription factor activity 2.33E-10negative regulation of viral process 1.14E-09

KEGG Pathway P-valueInfluenza A 8.32E-05Measles 9.72E-04Amoebiasis 0.001498NOD-like receptor signaling pathway 0.002945Hepatitis C 0.004997Necroptosis 0.007552IL-17 signaling pathway 0.009242Legionellosis 0.012156Estrogen signaling pathway 0.012997Protein processing in endoplasmic reticulum 0.014772

GO Pathway P-valueregulation of viral life cycle 2.73E-07cuticle development 6.05E-07negative regulation of viral process 2.12E-06regulation of symbiosis, encompassing mutualism through parasitism 1.85E-05negative regulation of viral genome replication 3.17E-05modulation by virus of host morphology 1.91E-04multi-organism cellular process 2.31E-04NLRP1 inflammasome complex 4.15E-04response to interferon-alpha 4.49E-04type I interferon signaling pathway 5.64E-04

ADAR1

References

Model for ADAR1 Dependence in ISG-High Cancer

ADAR1 Knockout Isogenic Cells are Preferential ly Sensitive to Type I Interferon and dsRNA

• A critical function of ADAR1 is to edit double stranded RNA (dsRNA) structures that can activate the cytoplasmic nucleoside sensor MDA5 and induce an innate immune type I interferon (IFN) response2,3.

• Consistent with this, mutations in ADAR1 and other enzymes involved in nucleoside metabolism/sensing are found in Aicardi-Goutiéres Syndrome (AGS), an interferonopathy with spontaneous interferon production4.

Knockout of ADAR1 Leads to Rapid Cell Death In Cells with Elevated Basal ISG Expression

ADAR1 KO HAP-1 Isogenic Cell Line has no ADAR1 and Reduced A-to-I Editing

DMSO

Parental HAP-1 ADAR1 KO HAP-1IFNα 2A IFNβ IFNγIFNα 2B

ADAR1

Actin

PKR

pT451-PKR

p150

p110

0 0.01 1 100.10.001Poly I:C (mg/mL)

Parental HAP-1 ADAR1 KO HAP-1

p150

p110

DMSO IFNα 2A IFNβ IFNγIFNα 2B 0 0.01 1 100.10.001

Actin

Parental HAP-1 ADAR1 KO HAP-1

ADAR1p150

p110

20 6 24 20 6 24 Hours

Type I but not Type II Interferons Induce Cell Death in ADAR1 KO HAP-1s

The dsRNA Memetic Poly I:C Induces Rapid Cell Death in ADAR1 KO HAP-1s

APOBEC3D

AZIN1

MDM2

Mo

difi

ed/W

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NA

ISG15

High Expression of Example ISGs in ADAR1-Dependent Cell Lines

Top: RSA scores and CERES scores for ADAR were taken from Project Drive5 and Broad Avana6 pooled shRNA and CRISPR screens, respectively. Individual cell lines were colored by tumor type. Bottom Left: Graph of ADAR1 RSA and CERES scores for cell lines containing both shRNA and CRISPR data. Bottom Right: A volcano plot showing significance (as -log10 transformed p-values) against magnitude(log2(fold change)), with genes identified as having different levels between groups represented as red (up-regulated) or blue (down-regulated) dots. Demonstrates that ADAR1 knockdown and knockout are similar between both datasets.

• Tumor cells can have higher intrinsic type I Interferon signaling and dsRNA burden due to multiple factors, including chronic cytoplasmic DNA activation of STING, oncovirus infection, and other proinflammatory signals

• This chronic ISG expression and dsRNA burden creates a dependency on ADAR1 to prevent activation of MDA5 and PKR and suggests an ADAR1 inhibitor could be beneficial for the treatment of tumors with elevated ISG expression

• Similar results were recently reported by multiple groups in cell lines and tumor types9,10

dsRNA

ADAR1

MDA5(dsRNA sensor)

MAVS IFNa, IFNb, ISGs

Translational arrest

p-eIF2aPKR(dsRNA sensor)

OAS1(dsRNA sensor)

RNaseL RNA decay

ADAR1 Knockdown (shRNA) or Knockout (CRISPR) Kills a Select Number of Cancer Cell Lines in Project Drive5 and Project Achilles6 Pooled Screens

Good Correlation Between the shRNA and CRISPR Pooled Screening Datasets for ADAR1

Project Drive (shRNA) Broad Avana (CRISPR)

Cell lines (by CERES score)Cell lines (by RSA score) AD

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6. Meyers et al, Nature Genetics, 20177. Cancer Cell Line Encyclopedia Consortium, Nature 20158. Barretina et al, Nature, 20129. Liu et al, Nature Medicine, 201910. Gannon et al, Nature Communications, 2019

Top and Middle: Volcano plots for ADAR1 Project Drive shRNA data (left) and Broad Avana CRISPR data (right) showing significance (as -log10 transformed p-values) against magnitude (log2(fold change)) as a measurement of gene mutations7(top row) or gene expression8

(middle row) . Genes identified as having different mutations or expression between groups are represented as red (enriched or upregulated) or blue (not-enriched or down-regulated) dots. Middle Tables indicate KEGG or GO pathways associated with significantly enriched genes (P value <0.01). Bottom Row: Respective graphs of ADAR1 Broad Avana CRISPR data colored by gene expression for MX1 and IFIT1, the red bars are cell lines with expression greater than twice the standard deviation of the mean.

Cell lines (by CERES score)

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Left: Growth curves of two human esophageal cancer cell lines, OE21 and KYSE270, treated with CRISPR-Cas9 virus and ADAR1 guide sequences (2 sgRNAs/set) indicates that ADAR1 is essential for viability in these cell lines. This impact on viability is likely due to elevated basal interferon signaling (red boxes) in these cells as evidenced by the expression of ISGs which are further induced upon IFNb stimulation (right). No antiproliferative effects were seen in a cell line with low ISG expression (data not shown).

Top: Parental and ADAR1 KO HAP-1 cells are viable and respond to interferon as evidenced by the induction of the p150 isoform of ADAR1 in the HAP-1 parental cells. RESSq-PCR confirms a lack of A to I editing of ADAR1 substrates in the ADAR1 KO HAP-1 cells. Middle: 24 hour treatment with Type I and II interferons or the double stranded RNA mimetic Poly I:C results in ISG15 expression in both cell lines, but only in ADAR1 KO cells is a robust phospho-PKR signal observed. Bottom: In a separate experiment, effects of IFN and Poly I:C treatment on proliferation (Cell Titer Glo) were evaluated in parental and ADAR1 KO HAP-1 cells. Type I IFN’s and dose-dependent Poly(I:C) mediated cell death was observed only in ADAR KO HAP-1 cells. Type II IFN appears to have no effect on proliferation in either the ADAR KO or parental HAP-1 cells.