Targeting Eif4a Dependent Translation of KRAS Signaling Molecules

Author Manuscript Published OnlineFirst on February 25, 2021; DOI: 10.1158/0008-5472.CAN-20-2929 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

Targeting eIF4A Dependent Translation of KRAS Signaling Molecules

Kamini Singh1, Jianan Lin2, Nicolas Lecomte3, Prathibha Mohan1, Askan Gokce 3,

Viraj R. Sanghvi1, 4, Man Jiang1, Olivera Grbovic-Huezo1, Antonija Burčul5, Stefan

G. Stark5,6, Paul B. Romesser7, Qing Chang8, Jerry P. Melchor3, Rachel K.

Beyer9, Mark Duggan10, Yoshiyuki Fukase10, Guangli Yang11, Ouathek

Ouerfelli11, Agnes Viale12, Elisa de Stanchina8, Andrew W. Stamford10*, Peter T.

Meinke10, Gunnar Rätsch5,6, Steven D. Leach13, Zhengqing Ouyang14, and Hans-

Guido Wendel1**

1Cancer Biology and Genetics Program, Memorial Sloan-Kettering Cancer

Center, New York, NY 10065, USA, 2The Jackson Laboratory for Genomic

Medicine, Farmington, CT 06032 and Department of Biomedical Engineering,

University of Connecticut, Storrs, CT 06269, 3David M. Rubenstein Center for

Pancreatic Cancer Research, Memorial Sloan-Kettering Cancer Center, New

York, NY 10065, USA, 4Department of Molecular and Cellular Pharmacology,

Sylvester Comprehensive Cancer Center, Miller School of Medicine, University of

Miami, Miami, FL 33136, USA, 5Computational Biology Department, Memorial

Sloan-Kettering Cancer Center, New York, NY 10065, 6Biomedical Informatics,

Department of Computer Science, ETH, Zürich, 8092 Zürich,

Switzerland, 7Department of Radiation Oncology, Memorial Sloan-Kettering

Cancer Center, New York, NY 10065, USA, 8Molecular Pharmacology Program,

Memorial Sloan-Kettering Cancer Center, New York, NY 10065, USA, 9Cancer

Downloaded from cancerres.aacrjournals.org on October 2, 2021. © 2021 American Association for Cancer Research. Author Manuscript Published OnlineFirst on February 25, 2021; DOI: 10.1158/0008-5472.CAN-20-2929 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

Research Technology Program, Frederick National Laboratory for Cancer

Research, Frederick, MD, USA, 10Tri-Institutional Drug Development Initiative,

Memorial Sloan-Kettering Cancer Center, New York, NY 10065, USA, 11The

Organic Synthesis Core Facility, Memorial Sloan-Kettering Cancer Center, New

York, NY 10065, USA, 12Integrated Genomics Operation, Center for Molecular

Oncology, Memorial Sloan-Kettering Cancer Center, New York, NY 10065, USA,

13Molecular Systems Biology and Surgery, Geisel School of Medicine,

Dartmouth, Norris Cotton Cancer Center at Dartmouth-Hitchcock, Lebanon, NH

03756, USA, 14Department of Biostatistics and Epidemiology, School of Public

Health and Health Sciences, University of Massachusetts, Amherst, MA 01003.

*Present address, Bridge Medicines, 420 East 70th Street New York, NY 10021

**Correspondence and requests for materials should be addressed to:

Hans-Guido Wendel, Cancer Biology & Genetics Program, Sloan-Kettering

Institute, 1275 York Ave, New York, NY 10065, USA, Phone: 646-888-2526, Fax:

646-422-0197, Email: [email protected]

Conflict of Interest Statement PBR has received honorarium from Corning has served as a consultant for unrelated work for EMD Serono and AstraZeneca. The other authors declare no competing financial interests.

Abstract

Pancreatic adenocarcinoma (PDAC) epitomizes a deadly cancer driven by abnormal KRAS signalling. Here we show that the eIF4A RNA helicase is required for translation of key KRAS signaling molecules and that pharmacological inhibition of eIF4A has single-agent activity against murine and human PDAC models at safe dose levels. EIF4A was uniquely required for the translation of mRNAs with long and highly structured 5'UTRs including those with multiple G-quadruplex (GQ) elements. Computational analyses identified these features in mRNAs encoding KRAS and key downstream molecules.

Transcriptome-scale ribosome footprinting accurately identified eIF4A-dependent mRNAs in PDAC including critical KRAS signaling molecules such as PI3K,

RALA, RAC2, MET, MYC, and YAP1. These findings contrast with a recent study that relied on an older method, polysome fractionation, and implicated redox- related genes as eIF4A clients. Together, our findings highlight the power of ribosome footprinting in conjunction with deep RNA sequencing in accurately decoding translational control mechanisms and define the therapeutic mechanism of eIF4A inhibitors in PDAC.

Significance

Findings document the coordinate, eIF4A-dependent translation of RAS-related oncogenic signaling molecules and demonstrate therapeutic efficacy of eIF4A blockade in pancreatic adenocarcinoma.

Introduction

The translation of mRNAs into protein is tightly controlled at the level of the multi-subunit eIF4F initiation complex (1). The eIF4F complex assembles on the 5’cap structure and scans the 5’UTR for a translation start site. EIF4A

(DDX2) is the RNA helicase component of the eIF4F translation initiation complex and is especially required to initiate translation of mRNAs with long

5’UTRs that contain highly structured RNA sequences such as multiple G-

quadruplex (GQ) elements (2-4). To some extent this insight enables the predictive identification of eIF4A dependent mRNAs. Notably, the NRAS and

KRAS genes have predicted GQ forming sequences in their 5’UTRs, although the therapeutic impact of this prediction is not known (5,6). Importantly, the

natural compound silvestrol binds eIF4A with nanomolar affinity and disables its

RNA unwinding activity (7-10). Synthetic analogues of silvestrol (e.g. CR-1-31B)

have shown promise in models of leukaemia and lymphoma (2,11,12). In

principle, cancers driven by a GQ-controlled oncogene such as KRAS should be

susceptible to eIF4A blockade.

Genomic studies have catalogued the genetic drivers of PDAC and show

nearly ubiquitous activation of KRAS and loss of tumor suppressor genes p53,

p16/INK4A, and SMAD4 (13-17). Activation of mRNA translation is an important

biological consequence of KRAS activation. Accordingly, PDACs show increased

levels of mRNA translation and mediated through activation of MAPK, PI3K-AKT-

mTOR signals, NRF2, and MYC (18-20). mTORC1/S6K1 signaling activates

translation initiation through phosphorylating eIF4B, co-factor for eIF4A helicase and enhance translation of MYC in pancreatic cancer (21,22). There have been

significant advances in therapeutic development against PDAC (23). These

include, inhibitors of G12C KRAS mutation (24-26), and strategies to co-target

KRAS downstream signalling molecules like (e.g. MAPK, PI3K, mTOR, EGFR, c-

RAF) (27-30). While we have pharmacological inhibitors of the G12C KRAS

mutation, this specific mutation is present in only 3% of PDACs (24,25). A recent study reports effect of eIF4A inhibition on mouse PDAC (31). In this study, we explore exactly how eIF4A inhibition affects human PDACs and we measure effects on global and specific mRNA translation programs.

Materials and methods

Cell Culture and treatments

Pancreatic cancer cell lines were cultures as per specified by American Type

Culture Collection. PANC1 and MiaPaca2 cells were purchased from American

Type Culture Collection. PANC1 cells were cultured in DMEM supplemented with

10% fetal bovine serum and penicillin-streptomycin (100 U/ml; 100 ug/ml) (all Life

Technologies). MiaPaca-2 cells were cultured in DMEM supplemented with 10% fetal bovine serum, 2.5% horse serum penicillin-streptomycin (100 U/ml; 100 ug/ml) (all Life Technologies). All the cell lines used were regularly tested for

Mycoplasma contamination using PCR. The IC50 data on the panel of cancer cell lines was done in collaboration with Tri-Institutional Drug Initiative program at

MSKCC.

Drugs, Inhibitors and Plasmids

Silvestrol was purchased from ChemScene (CS-0543). []CR-1-31B and [-]CR-1-

31B were synthesised in house at organic chemistry core at MSKCC and Tri-

Institutional Drug Development Initiative at MSKCC, respectively. Each was suspended in DMSO for in vitro experiments and 5.2% Tween 80 5.2% PEG 400 for in vivo experiments. Cycloheximide (C7698) was purchased from Sigma. pLEX-HA-birA*-K-Ras(G12D)-IRES-Puro was a gift from Paul Khavari (Addgene plasmid #120562).

Ribosome Footprinting

Human pancreatic cancer PANC1 cells were treated with DMSO or []CR-1-31B

(25 nM; 45 minutes) followed by cycloheximide treatment for 10 minutes. Total

RNA and ribosome-protected fragments were isolated following published protocol (32). Deep sequencing libraries were generated from these fragments and sequenced on the HiSeq 2000 platform. Genome annotation was from the

human genome sequence GRCh37 downloaded from Ensembl public database:

http:// www.ensembl.org.

Sequence Alignment

First, Ribosome footprint (RF) reads were filtered based on the quality score,

which kept reads that have a minimum quality score of 25 for at least 75 percent

of the nucleotides. Second, the linker sequence (5’- CTGTAGGCACCATCAAT-

3’) was trimmed from the 3’ end of the reads. Next, we filtered out the reads

shorter than 15nt after the linker-trimming step. All these aforementioned steps

were done by using FASTX-Toolkit

(http://hannonlab.cshl.edu/fastx_toolkit/index.html). To remove ribosomal RNA, the footprint reads were then aligned to the ribosome RNA sequences of

GRCh37 downloaded from UCSC Table Browser (https://genome.ucsc.edu/cgi-

bin/hgTables). After removing the reads aligned to the ribosome RNAs, RF reads

were mapped to the human genome sequence GRCh37 downloaded from

Ensembl public database: http:// www.ensembl.org using HISAT2 with default

parameters. We only used the uniquely aligned reads for further analysis.

Total mRNA sequencing reads were aligned to the GRCh37 reference using

HISAT2. Similarly, as RF reads alignment, we performed the splice alignment for

the paired-end mRNA-seq datasets with the default parameters. We only kept

the uniquely aligned reads for the downstream analysis. The alignment

quantification for both RF and mRNA sequencing was done using featureCounts

with the annotations of the protein coding genes of GRCh37 as input. Only reads

aligned to the exonic regions of the protein coding genes were used for the

downstream analysis.

Footprint Profile Analysis using Ribo-diff

We used Ribo-diff to analyse the translation efficiency based on the ribosome

footprinting and mRNA sequencing data. Genes with at least 10 normalized read

counts as the sum of RF and RNA sequencing data were used as input, which

resulted in 10,861 genes in total. Genes with significantly changed translation efficiency were defined by the q-value cut-off equal to 0.001.

Motif analysis and G-quadruplex prediction

The longest transcript was selected to represent each corresponding gene. The

5’UTR sequences of the transcripts were collected for predicting motifs. Both the significant genes with increased or decreased TE and the corresponding background gene sets were used to predict motifs by DREME (33). The occurrences of the significant motifs (E < 0.05 and P < 110-8 from DREME) were called using FIMO (33) with default parameters for strand specific prediction of all the 5’UTR sequences.

We used RNAfold (version 2.1.6) to predict the G-quadruplex formation in the

RNA secondary structure with the –g option. The number of different types of G- quadruplex (GG4, GGG4, and GGGG4) was then calculated by counting the number of consecutive “G”s using customized python scripts.

KRAS 5’UTR reporter assays

Full-length 5’UTR of KRAS transcript variant b was cloned in MSCV-CMV- dsRed-IRES d1eGFP using Age I and Dra III restriction enzyme sites. This strategy replaced the IRES with the full length 5’UTR of KRAS. For mutant version, we changed the GG pattern in the full length 5’UTR of KRAS that disrupted the RNA G-quadruplex structure. The clones were validated by PCR sequencing using CMV forward primers and the presence of full length 5’UTR of

KRAS (wildtype and mutant, mentioned below) was confirmed downstream of

CMV promoter. Reporter plasmids were used for transient transfection in 293T cells. Destabilized d1eGFP has a half-life of 1 hr and the translation activity driven by respective 5’UTRs was measured by d1eGFP intensity using flow cytometry. The 5’UTR sequence for full-length wild type and mutant version with restriction enzyme sites is provided below.

AGE I KRAS 5UTR FOR CCGGTGGCCGCACCACCACACCCAGCAGCGCGCGCCGCAGTACCCGCAC CGAGCCTGCCACCGCCGCGCCCCGTGCTCCCGGCCCCCGCCGTTGCACA CTGACAGCGAGCGCACCGCAACCGCTGGAACCACCACCACACCCAGAACC TCAGCACCTCCCAACTGCGGGCGCGCGGCCTGCTGAGAATGCACGTT

DRAIII KRAS 5UTR REV

GTGCATTCTCAGCAGGCCGCGCGCCCGCAGTTGGGAGGTGCTGAGGTTCT GGGTGTGGTGGTGGTTCCAGCGGTTGCGGTGCGCTCGCTGTCAGTGTGCA ACGGCGGGGGCCGGGAGCACGGGGCGCGGCGGTGGCAGGCTCGGTGCG GGTACTGCGGCGCGCGCTGCTGGGTGTGGTGGTGCGGCCA

AGE I KRAS 5UTR Mutant FOR CCGGTGGCCGCGGCGGCGGAGGCAGCAGCGGCGGCGGCAGTGGCGGCG GCGAAGGTGGCGGCGGCTCGGCCAGTACTCCCGGCCCCCGCCATTTCGGA CTGGGAGCGAGCGCGGCGCAGGCACTGAAGGCGGCGGCGGGGCCAGAG GCTCAGCGGCTCCCAGGTGCGGGAGAGAGGCCTGCTGAAACACGTT

DRAIII KRAS 5UTR Mutant REV

GTGTTTCAGCAGGCCTCTCTCCCGCACCTGGGAGCCGCTGAGCCTCTGGC CCCGCCGCCGCCTTCAGTGCCTGCGCCGCGCTCGCTCCCAGTCCGAAATG GCGGGGGCCGGGAGTACTGGCCGAGCCGCCGCCACCTTCGCCGCCGCCA CTGCCGCCGCCGCTGCTGCCTCCGCCGCCGCGGCCA

Restriction enzyme sites are shown as underlined.

CMV Forward Primer- CGCAAATGGGCGGTAGGCGTG

CRISPR-cas9 mediated deletion of KRAS 5’UTR

To delete the RNA G-quadruplexes from KRAS 5’UTR we designed sgRNAs targeting the genomic regions on KRAS. Each sgRNA was cloned into the lenti-

CRISPRv2 (addgene cat no. 52961) using BsmB I restriction enzyme site.

PANC1 cells were co-transfected with sgRNA containing plasmids and then selected with Puromycin (2 ug/ml) for 24 hrs. Selected colonies were expanded for two weeks. The deletion was confirmed using PCR primers spanning the

KRAS 5’UTR region in genomic DNA. The sequence of sgRNA and PCR primers is provided below. sg-1

KRAS-5’UTR-5’sg1-F: CACCGCTAGGCGGCGGCCGCGGCGG KRAS-5’UTR-5sg1’-R: AAACCCGCCGCGGCCGCCGCCTAGC sg-2

KRAS-5’UTR-3’sg1-F: CACCGCCAGAGGCTCAGCGGCTCCC KRAS-5’UTR-3’sg1-R: AAACGGGAGCCGCTGAGCCTCTGGC

sg-3

KRAS-5’UTR-3’sg2-F: CACCGCTCAGCGGCTCCCAGGTGC KRAS-5’UTR-3’sg2-R: AAACGCACCTGGGAGCCGCTGAGC

PCR primers

KRAS 5’UTR F2: GACCGCCTCCAGCCTCA KRAS 5’UTR R2: AAGAAGAATCGAGCGCGGAA

Immunoblots

Lysates were made using TNN lysis buffer (50 mM Tris-Cl, 250 mM NaCl, 5 mM

EDTA, 0.5% NP-40 supplemented with protease inhibitor). 60ug of protein was

loaded onto SDS-PAGE gels then transferred onto Immobilon-FL Transfer

Membranes (Millipore IPFL00010). The antibodies used were KRAS2B

(Proteintech), MYC (Cell signalling Technology), HRAS (Abcam), ERK (BD

Pharmingen), p-ERK (p42/44), MET, YAP1, XPO1, DDX6, PARP1, RALBP1,

PI3KCA, RAC1/2, β-tubulin, GAPDH (Cell signalling Technology) and β-actin

(Sigma A5316). Quantification of western blot images were done by using Image

J software.

Annexin V staining assay

Human pancreatic cancer cell lines PANC1, were used for annexin V staining

using kit (Invitrogen) and following manufacturer’s instruction. Annexin V staining

were detected by FACS analysis.

Polysome profiling

We performed polysome profiling to evaluate the effect of []CR-1-31B on global

translation and translation of KRAS4B transcript. Briefly, we used polysome

lysate from PANC1 cells treated with DMSO or []CR-1-31B (50 nM) for 1 and 4

hours in duplicates and performed polysome fractionation using a published

protocol from Panda et. al. (34). The relative distribution of the % mRNA of KRAS

and B2M over the sucrose gradient was studied by RT-qPCR analysis of the

RNA in each of the 12 gradient fractions.

In vitro eIF4A duplex unwinding assay

To evaluate the eIF4A activity we have used a fluorescent duplex unwinding assay as reported in (35,36). Briefly, we designed oligo containing 12-mer CGG4

(GQ) motif and AG)7 repeats labelled with Cy3 on the positive strand and with

BHQ (Black Hole Quencher) on the negative strand. We purified human eIF4A1, eIF4H isoform 2, and eIF4GΔ using previously reported study (37). Purified proteins eIF4A and eIF4GΔ proteins are stored at −80 °C in storage buffer (20 mM Hepes, pH 7.5, 200 mM KCl, 1 mM DTT) supplemented with 10% glycerol.

To maintain stability, purified eIF4H is stored at −80 °C in storage buffer supplemented with 20% glycerol as reported in (35,36). We performed the duplex unwinding exactly as reported in Ozes et al using eIF4A1 (10 uM), eIF4H isoform

2 (10 uM), eIF4GΔ (5 uM) with 12 mer (CGG) and (AG)7 repeat oligos (50 nM) in

the presence of eIF4A inhibitor []CR-1-31B at (50 uM and 100 uM).

Oligo sequences are provided below.

1. 12 mer (CGG)-Cy3 REV- 5’-Cy3-CGGCGGCGGCGG-3’

2. 12 mer (CGG)-BHQ FOR-5’-GCCGCCGCCGCC-BHQ-3’

3. 12 mer (CGG)-Competitor DNA-5’-GCCGCCGCCGCC-3’

4. (AG)7 repeat-Cy3 REV-5’-Cy3-AGAGAGAGAGAG-3’

5. (AG)7 repeat-BHQ FOR-5’-CUCUCUCUCUCU-BHQ-3’

6. (AG)7 repeat-Competitor DNA-5’-CTCTCTCTCTCT-3’

Human Pancreatic cancer cell line xenografts and PDXs

Human pancreatic cancer MiaPaca-2 cells expressing stable GFP-Luciferase reporter were injected in subcutaneous flank in J:Nu mice (5 million cells per flank). IVIS imaging was performed weekly to monitor the tumor growth. When tumours were between 80-100 mm3, Silvestrol (0.5 m/kg) or []CR-1-31B (0.5 mg/kg) or [-]CR-1-31B (0.5 mg/kg) was injected in mice intra peritoneally twice a week until the control mice developed fully-grown tumors. P-values were calculated using 2-way repeated measures ANOVA. PDX tumors were established by the Antitumor Facility at MSK under an approved IRB protocol and were transplanted subcutaneously in nude mice. Once tumors reached 80-100 mm3, the mice were randomized into treatment groups as above. J:nu mice were purchased from Jackson Laboratories. All animal experiments were performed in accordance with regulations from Memorial Sloan-Kettering Cancer Center’s

Institutional Animal Care and Use Committee.

Orthotopic implantation studies

PDA cell line KPC4662 used for orthotopic implantation was obtained from RH

Vonderhide group and previously described (38) and stably transfected with

GFP-Luciferase reporter. C57BL/6 mice were obtained from The Charles River

Laboratories. For orthotopic implantation of PDEC, we followed previously described procedure with some modifications (39). In brief, mice were anesthetized using isoflurane and then the pancreas was exposed through an abdominal incision (laparotomy). PDAC (2 ×105 cells/mouse) were suspended in

Matrigel (Corning) diluted 1:1 with cold PBS (total volume of 25μl) and injected

into the tail region of the pancreas using a Hamilton Microliter Syringe. A successful injection was verified by the appearance of a fluid bubble without intraperitoneal leakage. The abdominal wall was closed with absorbable

Vicryl RAPIDE sutures (Ethicon) and the skin was closed with wound clips

(Roboz). Mice with luciferase imaging diagnosed tumors of volume 50 to 150 mm3 were enrolled and block randomized into treatment groups. Tumors were visualized and reconstructed for quantifying tumor volume using the integrated

Vevo 2100 Workstation software package. The Institutional Animal Care and Use

Committee at MSKCC approved all animal care and procedures.

In vivo therapy dosing

For the in vivo application in KPC mouse model of PDAC (PdxCre; KrasG12D,

TP53fl/fl), []CR-1-31B was diluted in 5.2% PEG400 (Sigma Aldrich), 5.2%

Tween 80 (Sigma Aldrich), 2% DMSO (Sigma Aldrich) and administered intraperitoneally at 0.5 mg/kg, twice a week, from d28-d83. For the in vivo application in PDA cell line KPC4662 derived orthotopic tumors in pancreas of

C57/Bl mice, []CR31B was diluted in 5.2% PEG400 (Sigma Aldrich), 5.2%

Tween 80 (Sigma Aldrich), 2% DMSO (Sigma Aldrich). Vehicle or []CR-1-31B was administered at 0.5mg/Kg dose via intra peritoneal (i.p.) injection on Mon-

Wed-Fri (three days a week). For the in vivo application in MiaPaca-2 cell line

derived xenografts nude mice, silvestrol was diluted in 5.2% PEG400 (Sigma

Aldrich), 5.2% Tween 80 (Sigma Aldrich), 2% DMSO (Sigma Aldrich). Vehicle or silvestrol was administered at 0.5 mg/kg dose via intra peritoneal (i.p.) injection

on Mon-Wed-Fri (three days a week). For the in vivo application in human PDAC derived tumors in nude mice, [-]CR-1-31B was diluted in 10% Captisol (Sigma

Aldrich) in sterile water. Vehicle or [-]CR31B was administered at 0.5 mg/kg dose via intra venous (i.v.) injection on Mon-Thur (e.g. twice a week). Toxicity in all the in vivo treatment experiments was monitored by weight loss and daily clinical observation until the end of the experiment. 24 hours after the last test article administration, 4-5 mice in each group were sacrificed and clinical chemistry, haematology and tissue specific histopathology were done at autopsy.

Patient-derived ex vivo PDAC organoid culture, treatment and read out

The study was conducted under Memorial Sloan-Kettering Cancer Center

Institutional Review Board approval (MSKCC IRB 15-149 or 06-107) and all

patients provided written informed consent prior to tissue acquisition. Tissue

resections and biopsies from pancreatic cancer patients were processed

according to protocols previously described by Pr Hans Clevers (40,41) slightly

modified to ensure maximum viable cell recovery and organoid formation

efficiency. Briefly, resected tissue or biopsy were minced in less than 1mm3

pieces and digested in organoid medium containing collagenase II (2.5mg/mL)

and Rho kinase inhibitor Y-27632 10.5μM (Selleck Chemicals). Tissue digestion

was performed up to 4 hours at 37°C. Red blood cells were removed by specific

lysis with ACK buffer (Gibco). After 3 successive wash in PBS and organoid

medium, dissociated cells were seeded in growth factor reduced Matrigel (BD

biosciences) and cultured in WNT-driven expansion medium containing: DMEM-

F12 Advanced (Gibco), Hepes 10mM (Gibco), antibiotics 500μg/mL (Gibco),

Glutamax 2mM (Gibco), A83-01 0.5μM (Tocris), human EGF 50ng/mL

(Peprotech), human FGF10 100 ng/mL (Peprotech), human Noggin 100ng/mL

(Peprotech), human Gastrin I 10 nM (Sigma), N-acetylcysteine 1.25 mM (Sigma),

Nicotinamide 10 nM (Sigma), B-27 supplement 1X (Gibco), Wnt3A conditioned media 50% (v/v, from Hans Clevers), R-spondin1 conditioned media 10 % (v/v, from Calvin Kuo). Organoid lines were considered established when sustained epithelial proliferation was maintained over 5 passages (about 3 to 5-week culture).

Evaluation of PDAC organoids sensitivity to CR31B was performed in 384-well plate format. Briefly 2,000 single organoid cells were plated into matrigel-coated well and precultured for 4 days. Organoids were then exposed to the drug over a

7-concentration range (from μM to sub-nM) or vehicle control for 6 days. Cell viability was assessed at day 10 using the Cell Titer-Glo 3D assay (Promega).

Clonogenic survival assay

Cells were seeded in 6-well plates (20 x 103 cells per well) and allowed to adhere overnight in regular growth media. DMSO or []CR-1-31B (10 nM) was added

and refreshed every 3 days until the end of the experiment (14 days). For each

independent experiment, the DMSO or []CR-1-31B treated cells were fixed in

4% formaldehyde for 15 minutes at RT and subsequently stained with 0.1%

crystal violet and digitalized on an image scanner. All experiments were

performed at least three times in triplicates and representative results are shown.

Histology and Immuno-histochemistry analysis

Tissue specimens were fixed in 4% buffered paraformaldehyde, dehydrated and embedded in paraffin wax. Formalin-fixed paraffin embedded sections of 3 um was stained with H&E, TUNNEL and Ki-67. Immuno-histochemical analysis was done on five samples in each vehicle and drug treated group by counting number of positive tumor cells at ten high power field (X400) and for ki67 staining one hot spot area at X400 was selected for each case.

Real Time PCR assay

Total RNA was extracted using AllPrep DNA/RNA/Protein Mini Kit (Qiagen

80004). cDNA was made using SuperScript III First-Strand (Invitrogen 18080-

400). Analysis was performed by ΔΔCt. Applied Biosystems Taqman

GeneExpression Assays: human KRAS Hs00364284_g1, Myc Hs00153408_m1,

MET Hs01565584_m1, YAP1 Hs00902712_g1, XPO1 Hs00185645_m1, DDX6

Hs00898915_m1 and B2M Hs00187842_m1.

Statistical analysis

All the results were analysed with two-tailed t-tests unless specified. The significance of motif enrichments was from DREME program based on the

Fisher’s Exact Test. Hypergeometric test was performed to test for the significance in the enrichment of the gene overlap in KEGG pathway.

Online Content Additional Methods, Supplementary display items and Source

Data are available in the online version of the paper; references unique to these sections appear only in the online paper.

The ribosome footprinting and total mRNA sequencing raw and processed data were deposited in the NCBI Gene Expression Omnibus database GSE120159 accession number available at following link. (To review GEO accession

GSE120159: Go to https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE120159 Enter token

yhkpamwsnpinfkt into the box).

Results

The KRAS protein is encoded by two variant transcripts (a, b) and two pseudogenes that do not encode a protein (Supplementary Fig. S1A). 5’UTR sequence of human and mouse KRAS transcripts is identical as shown by

ClustalX alignment (Supplementary Fig. S1A and S1B). The KRAS4B 5’UTR is

192 bp long and computational prediction using M-Fold indicates the presence of

three highly structured and potentially G-quadruplex (GQ) forming regions in the

5’UTR of both KRAS transcripts that are reminiscent of eIF4A dependent mRNAs

(Fig. 1 A and B) (2,3,5,6). The TSS can vary within different cell types and tissue

(32). We have compared the TSS in various PDAC cell lines (RNA seq data

reported in GSE dataset GSE160434) and observed that KRAS variant b

(KRAS4B) is the most expressed transcript across the nine PDAC cell lines

suggesting that the TSS for KRAS is very much stable in these PDAC cell lines.

We built a KRAS4B 5’UTR translation reporter assay to test the eIF4A requirement using the eIF4A inhibitor []CR-1-31B (11). Briefly, destabilized eGFP (d1eGFP, T1/2 = 1h) is expressed from a CMV promoter and its translation controlled by the KRAS4B 5’UTR or a mutant 5’UTR where predicted GQ

structures are disrupted without changing 5’UTR length or GC content (Fig. 1C).

[]CR-1-31B is a synthetic analogue of silvestrol synthesized by J. Porco’s group

at U. Boston and shows nanomolar eIF4A inhibition (2,11,12,31,42-44). We measured the basal expression of d1eGFP driven by KRAS4B 5’UTR or a mutant 5’UTR in 293T cells (Supplementary Fig. S1C). Next, we confirmed the

half-life of d1eGFP by adding a pan translation inhibitor cycloheximide. Both

KRAS4B 5’UTR or a mutant 5’UTR driven d1eGFP expression was reduced to

50% following 1h treatment with cycloheximide (Supplementary Fig. S1D and

S1E). In 293T cells transiently expressing the KRAS4B 5’UTR reporter constructs treatment with []CR-1-31B (50 nM) reduces translation of the d1eGFP reporter controlled by the wild type KRAS 5’UTR by 70% and has little

effect on the mutant 5’UTR (p<0.001) (Fig. 1D and Supplementary Fig. S1F).

Consistently, treatment of KRAS mutant PDAC cell lines (PANC1, in MiaPaca-2)

with different doses of []CR-1-31B (1-50nM, 72h) reduces the KRAS protein

levels consistent with reported KRAS protein half-life (45) (Fig. 1E-H). EIF4A inhibition resulted in slight reduction of phospho-ERK levels at 72h of []CR-1-

31B (1-50nM) treatment (Fig. 1E and 1F). KRAS mRNA expression was not

affected by []CR-1-31B (Supplementary Fig. S1G). Importantly, eIF4A

dependence of KRAS translation depends on an intact, endogenous KRAS

5’UTR. For example, using two pairs of CRISPR guideRNAs (sgRNAs) we

induce KRAS 5’UTR deletions sg1-sg2 and sg1-sg3, respectively, that abrogate

[]CR-1-31B sensitivity and eIF4A dependent KRAS translation in PANC1 cells

(Fig. 1I, J, Supplementary Fig. S1H and S1I). Deletion of KRAS 5’UTR did not

affect the protein expression of KRAS, phospho-ERK levels and mRNA of KRAS

in the PANC1 cells (Supplementary Fig. S1J and S1K). Hence, GQ elements in

the KRAS 5’UTR confer dependence on eIF4A in translation reporter assays and

in human PDAC cells.

Nanomolar concentrations (2-10nM) of []CR-1-31B inhibit cell growth in

KRAS mutant PDAC and other cancer cell lines indicated by IC50 analysis,

PARP1 cleavage, and block colony formation in human PDAC cell lines (PANC1,

MiaPaca-2) (Fig. 2A, B, Supplementary Fig. S2A, and B). Primary fibroblast

lines showed at least ~1000 fold higher IC50 for []CR-1-31B (Supplementary

Fig. S2A). Importantly, CRISPR-cas9 engineered KRAS 5’UTR deletions (sg1-

sg2 and sg1-sg3) are largely (> 80% colonies at 4 and 6 nM []CR-1-31B) able to

rescue colony formation by PANC1 cells under continued []CR-1-31B exposure

indicating that KRAS is a significant target of the drug’s action (Fig. 2C). Deletion

of KRAS 5’UTR in PANC1 cells increased the IC50 for []CR-1-31B to ~10 nM

compared with ~6 nM in Cas9 expressing PANC1 control cells (Supplementary

Fig. 2C and 2D). Next, overexpression of HA-BirA*-K-RAS4B (G12D) in PANC1

cells rescued the cell death induced by []CR-1-31B. We observed only 30%

inhibition in cell proliferation at the highest dose of []CR-1-31B (10 uM) in

PANC1 cells with ectopic expression of KRAS4B (G12D) (Supplementary Fig.

2E). While the []CR-1-31B treatment inhibited the endogenous KRAS4B protein it did not affect the ectopic expression of HA-BirA*-KRAS4B(G12D) as determined by the western blot analysis (Supplementary Fig. 2F). []CR-1-31B

treatment also equally inhibited the MYC protein levels similar to the wild type

PANC1 cells (Supplementary Fig. 2F). We observed higher levels of total ERK in PANC1 cells with ectopic expression of KRAS4B (G12D) compared to wild type PANC1 cells but the p-ERK levels were equally affected by the []CR-1-31B

treatment (Supplementary Fig. 2F).

Organoid culture more closely models the 3-dimensional tissue

organization of tumors that may also affect drug action (40). Here, []CR-1-31B

effectively blocked organoid formation from murine KRAS/p53 PDACs (Fig. 2D)

and similarly inhibited the growth of primary human PDAC organoids ([]CR-1-

31B IC50s: 0.4nM - 22nM; 72 hr) (Fig. 2E and F). Hence, the silvestrol analogue

[]CR-1-31B kills PDAC cells and the effects is reduced upon disruption of the

KRAS 5’UTR.

We performed ribosome footprinting (ribosome profiling) and deep

sequencing (32) in the presence and absence of the eIF4A inhibitor []CR-1-31B

to identify eIF4A dependent translation. Briefly, we normalized ribosome-

protected RNA fragments (RF reads) to the total RNA abundance to isolate

changes in translation efficiency (TE). We performed ribosome footprinting on

three control (DMSO) and three []CR-1-31B (25 nM) treated PANC1 samples

(Fig. 3A). We chose an early time point (45 min) to minimize secondary and

knock-on effects. Read mapping to ribosomal RNAs, non-coding RNAs, library

linkers, and incomplete alignments were removed from the analysis

(Supplementary Fig. S3A-F). Most of the remaining reads range from 25 to 35

nucleotides in length and map to protein coding genes (Supplementary Fig.

S3A-F). The total number of RF reads mapped to exons was 4.1 million in control

and 3.5 million in []CR-1-31B treated samples. This corresponds to 19,821 protein coding genes. Quality control analysis of replicates showed significant correlations among the replicates with a Pearson coefficient >0.97

(Supplementary Fig. S3G-H). We used the RiboDiff statistical framework to

isolate the effect on mRNA translation (46). With a very stringent statistical cut-off

at q < 0.001 (FDR <1%) we identified 614 mRNAs whose translation was

significantly repressed (TE down: n = 614; q < 0.001), and we also detect a set of

mRNAs showing a relative increase in ribosome occupancy (TE up: n = 456; q <

0.001) (Fig. 3B, Supplementary Table 1A, 1B; Complete dataset at GEO#

GSE120159). A full list of genes differentially affected on TE by eIF4A inhibitor

[]CR-1-31B in PANC1 cells is provided in Supplementary Table 2. Importantly,

we notice that eIF4A dependent (TE down) mRNAs included key oncogenic

drivers of PDAC, such as MYC, MET, YAP, TGF1/2, PI3K, and other proteins

involved in RAS signalling (Fig. 3B). KRAS translation efficiency was reduced by

35%, at q = 0.5 while MYC translation efficiency decreased by 40% at q < 0.001

(Supplementary Table 2).

These findings differ from results reported in a recent polysome fractionation study on PDAC cells treated with the same inhibitor (31). For example, we do not see the reported changes in the translation of redox and central carbon metabolism genes (31). A review of the published dataset (Suppl.

Table 2 in (31) shows that the polysome fractionation experiment identified exactly two genes (Lag3 and Tmcc2!) whose translation was significantly (q <

0.05) reduced upon eIF4A inhibition. Other genes implicated in the study as eIF4A targets are shown with significance values as high as q = 0.25 and in some instances q = 0.45 are chosen based on the manual analysis. Most likely this reflects poor inter-sample reproducibility of manual or instrumental separation of heavy and light polysome fractions using this older methodology.

We confirmed the effects on protein levels for KRAS and several targets across identified with as highly significant cell lines (PANC1, MiaPaca-2),

organoids, in PDACs arising in vivo (PdxCre; KrasG12D, TP53fl/fl) (47) (Fig. 3C-

F). []CR-1-31B did not affected the mRNA levels of these targets in PANC1

cells (Supplementary Fig. S3I). We also validated the effect of []CR-1-31B on

global and KRAS translation by polysome profiling. We observed a significant

increase of total mRNA in the light molecular weight (LMW) (p<0.05) and

corresponding reduction in the heavy molecular weight (HMW) polysome fraction

following []CR-1-31B treatment (50 nM for 1 and 4 hours) in PANC1 cells

(Supplementary Fig. S3J). KRAS mRNA was highly and significantly reduced in

the heavy molecular weight (HMW) polysome fraction (p<0.05) while B2M mRNA showed slight reduction in the HMW polysome fraction following []CR-1-31B treatment indicating that []CR-1-31B selectively target KRAS translation

(Supplementary Fig. S3K and S3L). Next, we evaluated the effect of []CR-1-

31B on the kinetics of KRAS protein degradation. Using cycloheximide treatment

(1 ng/ml) we observed ~40% degradation of KRAS protein between 4-24 hours in

PANC1 cells while []CR-1-31B treatment (50 nM) reduced KRAS protein

expression to similar extent (40-50%) in 4-24 hours as observed and quantified

by western blotting analysis (Supplementary Fig. S3M and S3N). Both

cycloheximide (1 ng/ ml) and []CR-1-31B (50 nM)) combined showed no

significant difference on KRAS protein degradation suggesting that []CR-1-31B

do not affect the half-life of KRAS protein in PANC1 cells (Supplementary Fig.

S3M and S3N). An unbiased gene ontology analysis of eIF4A dependent genes

(TE down) further supported the enrichment (p <1.18E-09) of RAS/MAPK

pathway genes including KRAS, RALA, RALBP1, MEK1/2, RAC2, and MYC (Fig.

3G and H). Together, these findings reveal coordinate translational downmodulation of key KRAS-MAPK-MYC signalling proteins following eIF4A inhibition.

Next, we explored these significant and confirmed eIF4A targets for common molecular features. We compared the []CR-1-31B sensitive (TE down)

group of mRNAs with annotated 5’UTRs (n=591), the []CR-1-31B independent

(TE up; n = 431) to each other and a background list of 623 equally expressed

and annotated mRNAs that showed no significant change in their translation

(Supplementary Table 3A-C). We noticed that []CR-1-31B sensitive mRNAs had significantly longer 5’UTRs (TE down vs Bkg p=4.0e-13; TE down vs TE up p=3.2e-24) (Fig. S4A). Next, we applied the MEME (Motif-Based sequence

analysis tool) (48) search tool to investigate sequence elements that were either

over- or underrepresented in any of the three groups. Comparing the TE down

group to TE up and background lists, we identify one significantly

overrepresented 12-mer sequence (CGGCGGCGGCGG) and two 9-mer motifs

(CGGCGGCGG and CCGCCGCCG) (p=3.8e-10; p=1.6e-14; and p=1.1e-8,

respectively) (Fig. 4A, Supplementary Table 4). We observed no significant

enrichment or depletion of sequences/motifs in the coding or 3’UTR sequences.

We also performed a separate search for differentially represented structural elements using RNAfold program. This search identified an enrichment of predicted RNA G-quadruplex (GQ) structures (sequence: GGX4) in the 5’UTRs of eIF4A dependent (TE down) genes compared to the background genes (p =

8.4e-7) and TE up genes (p = 3.8e-9); other potentially GQ forming sequences

(GGGX4 and GGGGX4), TOP motif, IRES, uORFs, Pyrimidine Rich Translation

Element (PRTE), and putative eIF4A binding sites (GAAG, (AG)3 repeats) were too infrequent to detect changes in their representation between groups

(Supplementary Fig. S4B and S4C). Next, the 12-mer sequence

(CGGCGGCGGCGG) and the 9-mer motif (CGGCGGCGG) significantly overlapped with GQ forming sequences while the 9-mer motif (CCGCCGCCG) did not coincide with the GQ sequences (Fig. 4B). 36% of the TE down

transcripts contain GQ sequences their 5’UTR in while only 19% and 23% of the

TE Up and background genes harbor GQ sequences, respectively (Fig. 4C).

Consistently, we found that 5’UTR GQ sequence elements were highly significantly (p<0.0001) associated with changes in translation efficiency across

the transcriptome upon eIF4A inhibition (Fig. 4D). A swimmer plot illustrates how

the number of GQ elements per 5’UTR corresponds to the degree of translational

repression []CR-1-31B treatment indicating a dosage effect (Fig. 4E). We also

mapped the relative position and number of GQ sequence elements in the 5’UTR

of key oncogenes using QGRS mapper and a stringent cut-off (QGRS score >

20), for example, the algorithm identifies three GQ sequence elements in the

KRAS 5’UTR, four in MYC, five in RALBP1, four in PI3K, four in HRAS, and

seven in YAP1 (Fig. 4F). Again, our findings are in stark contrast to Chan et al

(31) which report effects on short and unstructured 5’UTRs, instead we find that long and highly structured 5’UTRs are highly enriched among eIF4A dependent mRNAs.

We directly tested unwinding by the eIF4A helicase in the presence and absence of the inhibitor ([]CR-1-31B) in a sequence specific manner. Briefly, we used an in vitro duplex unwinding assay using the purified initiation factors

(eIF4A, eIF4H, eIF4G and ATP) to measure resolution of the 12-mer CGG4 (GQ)

motif and we used a ubiquitous (AG)7, that has been proposed as preferred

eIF4A binding sequence (35), as a control. The assay uses duplex probes

labelled with Cy3 on the positive strand and with BHQ (Black Hole Quencher) on

the negative strand such that unwinding results in an increased Cy3 signal (Fig.

4G). ATP addition results in activation of 12-mer CGG4 (GQ) motif unwinding that is blocked by the eIF4A inhibitor []CR-1-31B (pvehicle vs []CR-1-31B = 0.0001)

(Fig. 4H). On the other hand, []CR-1-31B led to increased unwinding of the

(AG)7 sequence (pvehicle vs []CR-1-31B < 0.001), potentially consistent with reports of

rocaglamide increasing eIF4A’s affinity ubiquitous and AG-rich sequences (7,49)

(Fig. 4I). Hence, []CR-1-31B inhibits the eIF4A helicase activity in a sequence specific manner on 12-mer CGG4 (GQ) sequence motif that are also highlighted in our ribosome profiling data.

Chan et al report activity of an eIF4A inhibitor against mouse PDAC (31) and we are pleased to confirm and expand the therapeutic benefit in the same

KPC model and also in human PDAC tumors. Briefly, in murine KPC tumors we observe an increase in median overall survival from 49 days (control) to 69 days upon treatment with []CR-1-31B (0.5 mg/kg, i.p., twice a week, d28-d83n = 7/5, p < 0.02 Log-rank Mantel-Cox test) (Fig. 5A). In orthotopically engrafted KPC

tumors engineered to express luciferase, responses were more varied and

[]CR-1-31B (0.5 mg/kg, i.p., twice a week, d10-d42) induced responses in 3 out

of 5 animals, while control animals uniformly succumbed to PDAC (n = 5, pvehicle

vs CR-1-31B < 0.01) (Fig. 5B, Supplementary Fig. S5A and S5B). Histology showed a cytostatic effect with loss of proliferation (Ki67 positive) (Fig. 5C, and

Supplementary Fig. S5C), animal weights, blood, and platelet counts were unchanged indicating tolerability in mice (Fig. 5D and Supplementary Fig. S5D-

F). Expression of KRAS, MYC, PI3K and YAP1 were significantly reduced in orthotopically engrafted KPC tumors treated with []CR-1-31B (0.5 mg/kg, i.p., twice a week, d10-d42) compared to vehicle treated tumors as observed by western blotting and quantification (p<0.05) (Fig. 5E and 5F).

We further find that the eIF4A inhibitor has single agent activity against human PDACs. Specifically, we purified the active [-] enantiomer of CR-1-31B.

We treated a primary, patient-derived PDX model of PDAC transplanted into the flanks of five nude mice once tumors had reached a volume of ~80mm3 (dosing

schedule: [-]CR-1-31B, 0.5mg/kg, i.v., twice a week from d22-d54). We observed

a significant delay in human PDX growth during the treatment period, although

tumors resumed growth when treatment was stopped, potentially indicating the

need for combination therapies (pvehicle vs [-]CR-1-31B <0.03, n=5) (Fig. 5G). When

tumors in either group reached a size of ~2000mm3 animals were euthanized

and we considered this as an endpoint for a survival analysis. Notably, [-]CR-1-

31B treatment of tumor-bearing animals significantly increased the median

overall survival from 49 days to 62 days (0.5 mg/kg i.v., twice a week, n=10 in

vehicle group, n=13 in treated group, Kaplan-Meier analysis: p < 0.0001 Log-rank

Mantel-Cox test) (Fig. 5H). Histology showed a striking reduction of proliferating

(Ki67 positive) adenocarcinoma cells (indicated by arrows) surrounded by tumor

stroma (Fig. 5I). We confirmed significant loss of KRAS, MYC, and phospho-

ERK (p<0.05) while total ERK remained unchanged as shown by immunoblots on

lysates of in vivo treated tumors collected from controls or 24h after the last [-

]CR-1-31B dosing (Fig. 5J). Image J quantification is shown where the KRAS,

MYC protein expression is normalized to -actin (Fig. 5K). p-ERK is normalized

to total ERK (Fig. 5K). As with the racemic mixture, we found [-]CR-1-31B was well tolerated without evidence of drug-related mortality or weight loss (Fig. 5L).

To enhance reproducibility of these important results, we also tested the

natural compound silvestrol and the analogue [-]CR-1-31B in more widely

available PDAC xenograft models. For example, silvestrol (0.5 mg/kg, i.p., twice

a week, from d10-d63) caused tumor growth arrest in MiaPaca-2 xenografts in

nude mice by luciferase imaging, and ex vivo tumor weights and volumes (n = 5,

p< 0.05) (Supplementary Fig. S5G-I). TUNEL stains (24h after last dosing)

indicated tumor cell apoptosis in silvestrol treated tumors (Supplementary Fig.

S5J and K). Silvestrol was well tolerated without weight loss or significant changes in blood counts (24h after last dosing) (Supplementary Fig. S5L-O). [-

]CR-1-31B showed similar tumor growth delay in this model (Supplementary

Fig. S5P), and endpoint analysis (tumor > 2000mm3) indicate a significant survival advantage in [-]CR-1-31B treated MiaPaca-2 bearing animals (0.5

mg/kg, i.p., twice a week, n=5/8, p<0.0012) (Supplementary Fig. S5Q).

Discussion

Our findings provide new insight into the therapeutic utility and the

mechanism of eIF4A inhibitor treatment. Using ribosome footprinting on human

PDAC cells, we find that genes with long- and highly structured 5’UTRs depend

on eIF4A for their translation. These include important KRAS signalling molecules such as RALA, RALGDS, RAF, RAC, PI3K, RALBP1, and MYC, whereas KRAS itself falls just outside our stringent statistical criteria and is still affected in protein studies. Consistent with our previous findings in T-cell leukemia we notice that many eIF4A dependent mRNAs harbor sequence

elements that are predicted to fold into secondary, non-covalent G-quadruplex

structures (2). Inserting GQ sequences confers eIF4A dependence in translation

reporter and helicase assays. Conversely, removing them from the endogenous

KRAS gene reduces the eIF4A requirement. However, it is not clear to what

extent these sequences exist in a stably folded state in cells. For example, one

RNA sequencing-based method suggested GQ elements are largely

unstructured in cells (50), another method (rG4-seq) indicates that they are

structured, and a third, antibody based method, also detects abundant GQ

structures in cells (51,52). Our study does not directly address this question,

although consistent and unbiased identification of GQ sequences in eIF4A

dependent RNAs and functional evidence consistently support a role for GQ

elements in eIF4A dependence. By contrast, ubiquitous AG-rich sequences that

act as accessible sites for eIF4A binding (49) do not confer eIF4A dependence

and may act to sequester eIF4A.

Ribosome profiling has emerged as the experimental standard to precisely

map and measure ribosome occupancy across the transcriptome and provides a

surrogate measure for mRNA translation into protein (53). The previous

polysome fractionation method relied on manual separation of heavy and light polysome fractions from sucrose gradients and suffered low reproducibility.

Highlighting these differences, our findings differ strongly from a recent polysome

study on PDAC cells treated with the same eIF4A inhibitor treated PDAC cells

(31). The other study ruled out MYC, KRAS, and instead implicated a number of redox enzymes and central carbon metabolism genes as a although using conventional statistical cut-offs (q < 0.05) none of named genes showed a

significant difference (31). Relying on this data the study reports preferential

eIF4A effects on genes with short and unstructured 5’UTRs which contradicts

much prior work on this helicase (54,55). On the other hand, we happily agree

that eIF4A inhibition has promising single agent activity against PDAC in vitro and in vivo (31). A prior study on a different rocaglamide (Infinity’s Compound 76) showed toxicity and low efficacy and these effects may be specific to that inhibitor (56). Our findings show in vivo efficacy at tolerable dose levels for the

CR-1-31B compound in mouse and human models of PDAC and indicate a

therapeutic window in treating this deadly cancer with this new and exciting class

of drugs.

Acknowledgements:

HGW is supported by NIH/NCI grants R01CA183876-03, R01 CA207217-04,

R01CA190384-05, P50 CA217694-02, P50 CA192937-04. HGW is supported by

Lymphoma Research Foundation; Mr. William H. Goodwin and Mrs. Alice

Goodwin and the Commonwealth Foundation for Cancer Research; the Center

for Experimental Therapeutics at MSKCC; the Starr Cancer Consortium;

the Geoffrey Beene Cancer Research Center; David Rubenstein Center for

Pancreatic Cancer; Druckenmiller Center for Lung Cancer Research; a Leukemia

and Lymphoma Society (LLS) SPORE grant; and the MSKCC Core Grant (P30

CA008748). H.G.W. is a scholar of the Leukemia Lymphoma Society. KS is

supported by the Pancreatic Cancer Action Network. GR, YZ were supported by

MSK Core funding. SDL is supported by NIH grant R01CA204228. EdS is

supported by NIH U54 OD020355-01. PBR is supported by a K12 CA184746,

GR is supported by the core funding of ETH Zürich. ZO is supported by NIH R35

GM124998. We acknowledge the use of the Integrated Genomics Operation

Core (funded by CCSG, P30 CA08748, Cycle for Survival, Marie-Josée and

Henry R. Kravis Center for Molecular Oncology, the mouse pharmacology and

Organic synthesis cores funded by the NCI CCSG, P30 CA08748. We thank G.

Sukenick, J. McCauley S. Kargman (Tri-I-TDI), T. Tammela (MSK) for assistance

with various part of the study. We thank Dr. Christopher S. Fraser for sharing the

human eIF4AI (406 amino acids), eIF4H isoform 2 (228 amino acids) and

eIF4GΔ (amino acids 682–1166 from eIF4GI) expression plasmids.

Author contributions KS designed, performed, analysed experiments, and co-

wrote the paper. JL, SS and AB performed the computational analysis. NL and

GHO performed the pancreatic cancer organoid treatment studies. PM, MJ, and

JPM provided technical assistance. AG analysed the pathology of human and

mouse PDAC tumor samples. VRS assisted with designing CRISPR donor DNA

oligos. QC performed the orthotopic implantations and PDX treatment studies.

EdS and PBR supervised generation of the PDX models and in vivo studies.

RKB provided various human pancreatic cancer cell lines. MD, AWS, and PTM provided [-]CR-1-31B and performed the PK and toxicity studies using this material. OO synthesised []CR-1-31B compound. LS and AV performed the

HiSeq-2000. SDL supervised the pancreatic cancer organoid and mouse model treatment studies. ZO and GR supervised the computational analyses. HGW designed the study and co-wrote the paper.

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Figure legends

Figure 1: The translation of KRAS mRNA depends on the eIF4A helicase.

A and B, the 5’UTRs of KRAS transcript variants “a” and “b” harbor three highly structured regions (red boxes; subsequent panels refer to variant “b” and

KRAS2B). C, Diagram of the KRAS 5’UTR translation reporter assay where wild type (structured) or mutant (unstructured, same length and GC content, sequence in methods) control d1eGFP expression in 293T cells. D,

Quantification of d1eGFP translation from wild type or mutant KRAS 5’UTR in

response to []CR-1-31B (50 nM; 24 hrs) or solvent (DMSO). E-H, KRAS, p-

ERK, ERK protein levels and β-actin in PANC1 and MiaPaca-2 cells following

[]CR-1-31B treatment at indicated doses and time points. I, Schematic diagram

showing CRISPR-Cas9 sgRNA pairs used to generate the two deletion mutants of the endogenous KRAS 5’UTR in PANC1 cells. J, In parental PANC1 cells the

KRAS protein decreases upon []CR-1-31B treatment (50 nM) (top panel), in cells with 5’UTR deletions corresponding to sgRNA pairs sg1-sg2 and sg1-sg3 the KRAS is not affected by []CR-1-31B. MYC protein is equally affected in the parental and KRAS 5’UTR deleted PANC1 cells. β-actin is used as loading control.

Figure 2: Activity of the eIF4A inhibitor CR-1-31B against PDAC cells and organoids.

A, IC50 analysis for []CR-1-31B in a panel of human PDAC cell lines. B and C,

Long term (14d) colony formation assay from MiaPaca-2 and PANC1 cells with different deletions of KRAS 5’UTR in []CR-1-31B (0-10 nM) or DMSO. D,

Primary murine PDAC (mKPC1/2) and normal pancreatic duct cell (mNormal1/2) organoids derived from the KPC (Pdx1-Cre; Kras+/LSL-G12D; Trp53+/LSL-

R172H) mouse model exposed to []CR-1-31B at indicated doses; p value for comparison of PDAC and normal pancreatic organoids. E, Six primary human

PDAC organoids exposed to indicated concentrations of []CR-1-31B and

assessed for viability. F, Representative images corresponding to human PDAC

organoids in (E) treated as indicated.

Figure 3: Ribosome profiling identifies the eIF4A dependent mRNAs in

PDAC cells.

A, Schematic of the ribosome profiling on PANC1 cells treated with DMSO or

[]CR-1-31B (25 nM; 45 minutes). Comparison of ribosome protected sequences and total mRNA isolates the translational efficiency for each mRNA (TE). B,

Frequency distribution of the change translation efficiency (TE) in untreated and

[]CR-1-31B treated PANC1 cells. Using the indicated statistical cut-offs we identify mRNAs with decreased (TE down, red) and increased (TE up, blue) and unchanged translation (background, grey); three biological replicates; the most significantly affected genes are indicated on each side. C-F, Immunoblot on lysates for PANC1 (C), MiaPaca-2 (D), KPC-4622 (E), and human PDAC organoid (F) treated with CR-1-31B and probed for the indicated proteins; β-

tubulin and GAPDH used as loading control; mRNA levels PANC1 in Fig. S3I. G,

GSEA KEGG pathway analysis of eIF4A dependent (TE down) genes. H, Key

proteins in the KRAS and MAPK, pathway are sensitive to eIF4A inhibition in

PDAC cells (marked with black star).

Figure 4: CR-1-31B impairs the translation of RNA G-quadruplex containing

mRNAs in PDAC.

A, An unbiased search for significantly enriched sequences (TE down versus

background and TE up; search across the entire coding sequence) identifies a

12-mer (CGG)4 and two 9-mer (CGG)3 and (CCG)3 sequences as significantly

enriched motifs that correspond to G quadruplex (GQ) forming sequences

(significance for GQ motifs: p12mer=3.8e-10, p9mer=1.6e-14, and p9mer=1.1e-8). B,

Analysis showing overlap between 12-mer (CGG)4 and two 9-mer (CGG)3 and

(CCG)3 sequences with predicted GQ structure forming sequences in TE down group of transcripts. C, Percentage of transcripts containing predicted GQ structure forming motifs in TE down, TE up and background. D, Frequency

distribution analysis across the TE range shows that transcripts with 5’UTR GQ

motifs are more sensitive to []CR-1-31B mediated inhibition compared to the transcripts without 5’UTR GQ sequences. E, Swimmer plot shows a reduction

(fold change) in TE upon []CR-1-31B treatment corresponds to the number of

5’UTR GQ sequences suggesting a dosage effect. F, Diagram of the 5’UTRs of key eIF4A dependent mRNAs indicating the presence of GQ sequences (red stars). G, Schematic of an eIF4A duplex unwinding assay that accounts for the sequence specific unwinding activity of eIF4A using a Cy3/BHQ labelled RNA duplex probe. H, eIF4A duplex unwinding assay using 12-mer (CGG)4 oligomers in RNA GQ folding conditions with DMSO or []CR-1-31B at the indicated doses and time points. I, eIF4A duplex unwinding assay with oligomers encoding the putative eIF4A binding site (AG)7 with DMSO or []CR-1-31B at the indicated

doses and time points.

Figure 5: In vivo safety and efficacy of eIF4A inhibitors in PDAC models.

A, Genetically engineered mouse model of PDAC. Kaplan-Meier survival

analysis comparing overall survival of KPC (PdxCre;KrasG12D,TP53fl/fl) mouse

treated with []CR-1-31B (0.5 mg/kg twice a week i.p.) (n=5) or vehicle (n=7)

starting at 4-5 weeks of age and followed for disease progression and death. B –

D, Orthotopically engrafted KPC mouse PDAC cells (KPC-4662) in the pancreas

of immune competent C57/B6 animals and treated with []CR-1-31B (0.5mg/kg,

i.p, twice a week, d1-d42) or vehicle. B, Luciferase imaging. C, Representative

H&E and Ki-67 stains on KPC tumors following []CR-1-31B treatment (d42). D,

Animal weights following []CR-1-31B (0.5mg/kg, i.p, twice a week, d1-d42) or vehicle treatment. E, KRAS, p-ERK, ERK protein levels and β-actin in

orthotopically engrafted KPC tumors treated with []CR-1-31B (0.5mg/kg, i.p,

twice a week, d1-d42) or vehicle at day42. F, Image J quantification of KRAS,

MYC, PI3K, and YAP1 protein levels normalized to β-actin (as in Fig. 5E). G-L

Human PDAC patient xenograft (PDX) implanted subcutaneously in NSG mice and treated with [-]CR-1-31B (0.5 mg/kg twice a week i.v.) or vehicle after tumors formed (~80mm3) from d22 until d54; G, Tumor volume curve, p value compares

[-]CR-1-31B and vehicle treated cohorts (n=5 per group). H, Kaplan-Meier survival analysis comparing overall survival of mice harbouring tumor volume

>2000 mm3 in vehicle treated (n=10) and [-]CR-1-31B treated animals (n=13). I,

Tumor histology (H&E and Ki-67 stains) on PDX tumors harvested after last treatment. J, Immunoblot on PDX lysates probed for KRAS, MYC, p-ERK, and

ERK 24h after last treatment (vehicle or [-]CR-1-31B). K and L, Image J quantification of KRAS and MYC protein levels normalized to β-actin, p-ERK is normalized to total ERK (as in Fig. 5J). M, Animal weights indicate tolerability of

[-]CR-1-31B treatment (as in G).

A 5’UTR-KRAS transcript variant b B 5’UTR-KRAS transcript variant a ENST00000311936 ENST00000256078

C D 150 KRAS WT KRAS 5’UTR Reporter *p<0.00003 KRAS Mutant Wild Type n.s. 100 d1eGFP or Mutant 50

d1eGFP % d1eGFP positive cells positive d1eGFP %

0 KRAS 5’UTR activity 0 25 (eGFP positive cells) [±]CR31B (nM) E F PANC1 MiaPaca-2 0 1 5 10 25 50 nM [±]CR31B (72hr) 0 1 5 10 25 50 nM [±]CR31B (72hr) KRAS4B KRAS4B

p-ERK (p42/44) p-ERK (p42/44)

ERK ERK

` -Actin ` -Actin G H PANC1 MiaPaca-2 0 24 48 72 hr [±]CR31B (50 nM) 0 24 48 72 hr [±]CR31B (50 nM) KRAS4B KRAS4B

`-Actin `-Actin

I J PANC1 0 24 48 72 hr [±]CR31B (50 nM) CRISPR-Cas9 targeting KRAS 5’UTR KRAS4B Full length MYC 5’UTR CDS 5’UTR [ `-Actin sg1 sg2 KRAS2B sg1-sg2 sg3 MYC Full length transcript 5’UTR del [ `-Actin

sg1-sg3 KRAS2B 5’UTR deleted transcript CDS 5’UTR del [ MYC `-Actin

Figure 2 A B C MiaPaca-2 PANC1 Full length sg1-sg2 sg1-sg3 5’UTR 5’UTR del 5’UTR del 8 DMSO DMSO

6 [±]CR31B [±]CR31B 2 nM 2 nM 4 [±]CR31B [±]CR31B 4 nM 4 nM 2 [±]CR31B [±]CR31B

[±]CR31B IC50 (nM) IC50 [±]CR31B 0 6 nM 6 nM [±]CR31B [±]CR31B PK-1 KP-4 KP-3 PL45 8 nM 8 nM PSN1 KLM-1 SUIT-2 PANC1 BxPC-3 HPAF-II SW1990

SNU-324 [±]CR31B SU.86.86

CAPAN-2 [±]CR31B MiaPaca2 10 nM 10 nM

D Mouse PDAC organoid E Human PDAC organoid mNormal 1 150 150 MSK-6 mNormal 2 MSK-8 mKPC 1 MSK-17 mKPC 2 MSK-3A 100 100 MSK-3B MSK-18 % viability %

% viability viability % 50 50 ] *p<0.0001 0 0 -2 -1 0 1 2 -1 0 1 2 3 4 [±]CR31B log (nM) [±]CR31B log10(nM) 10

F Control [±]CR31B (1 nM) [±]CR31B (10 nM) [±]CR31B (100 nM)

MSK-3B

600 +m 600 +m 600 +m 600 +m

MSK-17

600 +m 600 +m 600 +m 600 +m

Figure 3 A B 1000 Total RNA MYC Background [±]CR31B KRAS TE Down q<0.001 or DMSO MAP2K1 RALBP1 TE Up q<0.001 treatment RNA Seq eIF4A targets 800 Ribo Seq RALA n=10861 RAC2 += -0.07 RASA1 m=0.97 Ribosome- 600 MET PANC1 AKT1/2 protected RNA TGFB1/2 TK2 PI3K UCK1 FZD1/2/6 IMPDH2

Frequency 400 PLCG2 GMPS C PANC1 D MiaPaca-2 PAK1 CES1 RAC2 CES2 0 24 48 72 hr [±]CR31B (50 nM) 0 24 48 72 hr [±]CR31B (50 nM) 200 NFKB1 SEC61G SEC11A MYC MYC

RALBP1 RALBP1 0 -4 -2 0 2 4 PI3K PI3K TE log2 Fold change [±]CR31B +/- H-RAS H-RAS E KPC- 4622 mouse PDAC cell line RAC2 RAC2 (50 nM) 0 24 48 72 hr [±]CR31B (50 nM) MYC MET MET KRAS4B YAP1 YAP1 p-ERK ERK XPO1 XPO1 MET DDX6 DDX6 YAP1 `-Tubulin `-Tubulin XPO1 DDX6 GAPDH GAPDH `-Actin F G H RAS signaling Human PDAC Organoid Pancreatic cancer Cell Membrane Chronic myeloid leukemia 0 24 48 72 hr [±]CR31B (50 nM) KRAS PI3K AKT TGF-beta signaling pathway KRAS4B Wnt signaling pathway MET Cell cycle Neurotrophin signaling pathway RALGDS RAF TIAM1 YAP1 Ubiquitin mediated proteolysis MAPK signaling pathway RAL MEK1/2 RAC DDX6 Focal adhesion Pathways in cancer RALBP1 ERK1/2 PAK1 `-actin MAPK 0 10 20 30 40 TE down genes (% Enrichment KEGG pathways) Nucleus MYC

Indicate EIF4A target

p=3.8e-10 p=1.6e-14 p=1.1e-8

B C 40 TE Down F Motif and GQ RNA G-quadruplex motif in 5’UTR Only Motif TE Up 750 CDS 30 KRAS BKG (197 bp) 500 MYC 20 (1202 bp) Motif overlap 250 RALBP1 with GQ structure

% GQ motif GQ % (234 bp) 0 10 PI3K (323 bp) H-RAS 0 SVGCGGCGGCCGCCGCYG (188 bp) CGGCGGCGGCKG RAC2 (256 bp) MET D E (201 bp) 1.0 GQ genes 0 GQ YAP1 (388 bp) 0.8 [N=1015] 1-3 GQ XPO1 BKG genes (728 bp) 0.6 [N=4000] DDX6 4-7 GQ (361 bp) 0.4 TUBB > 7GQ (60 bp) 0.2 GAPDH Number of RNA GQ RNA of Number (79 bp) Cumulative Frequency 0.0 -1.5-2 0.50-1-0.5 1.0 1.5 -1.5 -1.0 -0.5 0.50 1.0

TE log Fold change [±]CR31B +/- TE log2 Fold change [±]CR31B +/- 2

G H I eIF4A duplex unwinding assay RNA Probe: (CGG)4 12 mer RNA Probe: (AG)7 repeat No drug No drug [±]CR31B (50 uM) [±]CR31B (50 uM) [±]CR31B (100 uM) RNA probe DNA competitor (10X) 160 [±]CR31B (100 uM) 150 5’ Cy3 5’ No Helicase 5’ BHQ + No Helicase 140 *p<0.001 Low Cy3 Signal *p=0.0001 100

eIF4A 120 +ATP RFU +eIF4H+eIF4G RFU 50 100 Cy3 5’ 5’ BHQ 5’ + 0 High Cy3 Signal 80 0 0 60 60 120 180 240 300 360 420 480 540 600 660 720 780 120 180 240 300 360 420 480 540 600660 720 780

Time (seconds) Time (seconds)

Targeting eIF4A Dependent Translation of KRAS Signaling Molecules

Kamini Singh, Jianan Lin, Nicolas Lecomte, et al.

Cancer Res Published OnlineFirst February 25, 2021.

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