The Endosomal Protein CEMIP Links Wnt Signaling to MEK1-ERK1/2 Activation in Selumetinib-Resistant Intestinal Organoids

Author Manuscript Published OnlineFirst on June 18, 2018; DOI: 10.1158/0008-5472.CAN-17-3149 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

The endosomal protein CEMIP links Wnt signaling to MEK1-ERK1/2 activation in Selumetinib-

resistant intestinal organoids

Hong Quan Duong1,2,6,7, Ivan Nemazanyy8, Florian Rambow9, Seng Chuan Tang1,2, Sylvain Delaunay1,3,

Lars Tharun10, Alexandra Florin10, Reinhard Büttner10, Daniel Vandaele5, Pierre Close1,3, Jean-

Christophe Marine9, Kateryna Shostak1,2,£ and Alain Chariot1,2,4,£,*

1Interdisciplinary Cluster for Applied Genoproteomics (GIGA), 2Laboratory of Medical Chemistry,

3Laboratory of Cancer Signaling, GIGA-Molecular Biology of Diseases, 4Walloon Excellence in Life

Sciences and Biotechnology (WELBIO), 5Gastroenterology Department, University of Liege, CHU,

Sart-Tilman, 4000 Liège, Belgium, 6Institute of Research and Development, Duy Tan University, K7/25

Quang Trung, Danang, Vietnam, 7Department of Cancer Research, Vinmec Research Institute of Stem

Cell and Gene Technology, 458 Minh Khai, Hanoi 10000, Vietnam, 8Paris Descartes University,

Sorbonne Paris Cité, 75006 Paris, France, 9Laboratory for Molecular Cancer Biology, VIB Center for

Cancer Biology and KULeuven Department of Oncology, 3000 Leuven, Belgium, 10Institute for

Pathology, University Hospital Cologne, 50937 Cologne, Germany, £Equal contributions

*To whom correspondence should be addressed: Dr. Alain Chariot, Laboratory of Medical Chemistry,

GIGA Molecular Biology of Diseases, Tour GIGA, +2 B34, Sart-Tilman, University of Liège, 4000

Liège, Belgium. Tel: 32 (0) 4 366 24 72; FAX: 32 (0) 4 366 45 34; e-mail: [email protected].

Short title: CEMIP links Wnt and MEK1 signaling pathways

Keywords: CEMIP, colorectal cancer, resistance, inhibitors of RAS effectors, BRAF, MEK1, endosome.

The authors do not have any conflict of interest to declare. 1

Financial support: This study was supported by grants from the Belgian National Funds for Scientific

Research (FNRS), from the Concerted Research Action Program (Bio-Acet) and Special Research

Funds (FSR) at the University of Liege, the Belgian foundation against Cancer (FAF-F/2016/794), as well as from the Walloon Excellence in Life Sciences and Biotechnology (WELBIO-CR-2015A-02) and the Max Planck Society. We are also grateful to the “Fonds Leon Fredericq” and the “Centre

Anticancéreux” of the CHU Liege for their financial support. S. Delaunay, A. Chariot and P. Close are

Research Fellow, Research Director and Research Associates at the FNRS, respectively.

ABSTRACT

MAPK signaling pathways are constitutively active in colon cancer and also promote acquired resistance to MEK1 inhibition. Here we demonstrate that BRAFV600E-mutated colorectal cancers acquire resistance to MEK1 inhibition by inducing expression of the scaffold protein CEMIP through a E- catenin- and FRA-1-dependent pathway. CEMIP was found in endosomes and bound MEK1 to sustain

ERK1/2 activation in MEK1 inhibitor-resistant BRAFV600E-mutated colorectal cancers. The CEMIP- dependent pathway maintained c-Myc protein levels through ERK1/2 and provided metabolic advantage in resistant cells, potentially by sustaining amino acids synthesis. CEMIP silencing circumvented resistance to MEK1 inhibition, partly, through a decrease of both ERK1/2 signaling and c-Myc.

Together, our data identify a cross-talk between Wnt and MAPK signaling cascades, which involves

CEMIP. Activation of this pathway promotes survival by potentially regulating levels of specific amino acids via a Myc-associated cascade. Targeting this node may provide a promising avenue for treatment of colon cancers that have acquired resistance to targeted therapies.

PRECIS

MEK1 inhibitor-resistant colorectal cancer relies on the scaffold and endosomal protein CEMIP to maintain ERK1/2 signaling and Myc-driven transcription.

INTRODUCTION

Colorectal cancer (CRC) is the second leading cause of death from cancer in Western countries and arises from a variety of genetic alterations that result in the constitutive activation of both Wnt- and

ErbB-dependent oncogenic signaling pathways. Among the underlying genetic alterations are loss-of- function mutations of the adenomatous polyposis coli (APC) gene, which leads to E-catenin activation and constitutive Wnt signaling, followed by gain-of-function mutations in KRAS or BRAF proto- oncogenes (1). RAS signals though the RAF Ser/Thr kinase family and triggers the subsequent activation of the mitogen-activated protein/extracellular signal-regulated kinase 1 and 2 (MEK1/2) as well as the extracellular signal-regulated kinase 1 and 2 (ERK1/2). This signaling cascade gained significant attention due to the high frequency of KRAS and BRAF mutations found in human cancers

(2,3). Indeed, activating mutations of KRAS are found in 40% of advanced CRC (4). Additionally, the

BRAF valine 600 (BRAFV600E) mutation, which leads to constitutive activation of BRAF, is found in approximately 11% of CRCs and confers poor prognosis (5-7). As the pharmacological inhibition of

KRAS remains challenging, alternative approaches targeting downstream RAS effectors (RAF and

MEK1) have been proposed but were poorly effective in monotherapy for the treatment of CRC, largely because of a feedback reactivation of MAPK signaling (8,9). This reactivation occurs through the amplification of the driving oncogene KRAS or BRAF in colorectal cells treated with MEK1 inhibitors

(10,11). Other mechanisms involve the EGFR/HER1-dependent reactivation of MAPK in BRAFV600E- mutated colorectal cancer cells treated with a BRAF inhibitor (12,13). Similarly, MAPK reactivation in

KRAS-mutated colorectal cancer cells subjected to MEK1 inhibition also results from the induction of

HER3 (14). Clinical trials in which RAF and EGFR or RAF and MEK are co-targeted to suppress the feedback reactivation of MAPK signaling were carried out but patients showing initial benefit

nevertheless developed resistance and recurrence in disease progression (15). Here again, resistant colorectal cancer cells had KRAS or BRAF amplification as well as an activating MEK1 mutation (16).

The RAS-RAF-MEK1-ERK1/2 cascade is critically reliant on scaffold proteins which assemble pathway molecules to regulate signaling. Among them are Ras GTPase-activating-like protein

(IQGAP1) as well as kinase suppressor of RAS (KSR) (17-20). Another scaffold protein is KIAA1199, now referred to as CEMIP (“Cell Migration-inducing and hyaluronan-binding protein”), whose expression is enhanced in cervical, breast and colorectal cancer (21-25). CEMIP promotes cell survival and invasion, at least through EGFR-dependent MEK1 and ERK1/2 activation in cervical and breast cancer cells (23). It remains unclear which scaffold proteins, if any, are specifically involved in MAPK reactivation in colorectal cells showing intrinsic or acquired resistance to BRAF or MEK1 inhibitors.

It is intuitive that both Wnt- and MAPK-dependent signaling pathways are interconnected in promoting resistance to targeted therapies. Here we define CEMIP as a MEK1-binding protein induced by Wnt signaling. CEMIP promotes the acquired resistance to MEK1 inhibition in BRAFV600E-mutated colorectal cancer cells, at least through ERK1/2 signaling and Myc. This CEMIP-dependent cascade is essential for amino acid synthesis in resistant cells. Collectively, our data define CEMIP as a key driver of resistance to MEK1 inhibition in BRAFV600E-mutated colorectal cancer that acts upstream of ERK1/2 and Myc cascade.

MATERIALS AND METHODS

Cell culture and reagents

Colorectal cancer cell lines (HT-29, HCT116, SW480 and COLO-205) were purchased from American

Type Culture Collection (ATCC, Manassas, VA) in 2009. These cells were characterized by ATCC, using a comprehensive database of short tendem repeat (STR) DNA profiles. Frozen aliquots of freshly cultured cells were generated and experiments were done with resuscitated cells cultured for less than 6 months. All cell lines were mycoplasma tested. HT-29 and HCT116 cells were cultured in McCoy’s 5A supplemented with 10% heat-inactivated fetal bovine serum (HI-FBS; Gibco, Life Technologies,

Naerum, Denmark) and 100 units/ml penicillin/streptomycin. SW480 cells were cultured in DMEM supplemented with 10% HI-FBS, 1% glutamine and 100 units/ml penicillin/streptomycin. COLO-205 cells were cultured in RPMI-1640 supplemented with 10% HI-FBS, 1% glutamine and 100 units/ml penicillin/streptomycin. Selumetinib (AZD6244), Vemurafenib (PLX4032, RG7204), PD98059 and

PNU-74654 were from Selleck Chemicals (Houston, TX, USA).

Intestinal epithelial cell extraction and ex-vivo organoid cultures

Intestines and colons were extracted from C57BL/6 (Wnt OFF) or Apc+/Min (Wnt ON) mice. All our studies were approved by the Institutional Animal Care and Use Committee of the University of Liege.

Bowels were washed for 10 minutes at 37°C in a PBS-DTT (1 mM) buffer and then incubated for 15 minutes at 37°C in a HBSS-EDTA buffer (30 mM). Cells were harvested, washed twice in PBS and flashed frozen. For the generation of ex-vivo organoid cultures, small pieces of intestine were incubated in 2mM EDTA-PBS for 30 minutes at 4°C. Crypts were extracted, washed twice in PBS and cultured in

Matrigel (Biosciences, San Jose, CA, USA). DMEM/F12 supplemented with EGF (20ng/ml), Noggin

(100ng/ml) and R-Spondin (500ng/ml) was added every 2 days. Apc-mutated organoids were cultured in

DMEM/F12 supplemented with EGF (20ng/ml), Noggin (100ng/ml) without R-Spondin. The

enrichment of Lgr5+ stem cells in ex-vivo organoids generated with intestinal crypts from C57BL/6 mice was carried out by treating them with a combination of Valproïc acid (1 mM) and CHIR999021 (3

PM), a GSK3 inhibitor.

Generation of Selumetinib-resistant colorectal cancer cell lines (HT-29/SR, COLO-205/SR,

SW480/SR and HCT116/SR) and Selumetinib-resistant ex-vivo organoids

Four colorectal cancer cell lines (HT-29, COLO-205, SW480 and HCT116) were used as parental cell lines (HT-29/P, COLO-205/P, SW480/P and HCT116/P) from which were generated the Selumetinib- resistant cell lines (HT-29/SR, COLO-205/SR, SW480/SR and HCT116/SR). These cell lines were generated by repeated subculturing cells in the presence of incrementally increasing concentrations of

Selumetinib (from 0.05 to 1.5 μM for HT-29/P and SW480/P cells; from 0.05 to 2 μM for HCT116/P cells; from 0.005 to 0.3 μM for COLO-205/P) for six months. For the maintenance of Selumetinib- resistant colorectal cancer cell lines, the maximum concentration of Selumetinib, namely 1.5 μM (HT-

29/SR and SW480/SR cells), 2 μM (HCT116/SR cells), and 0.3 μM Selumetinib (COLO-205/SR cells) was added into the normal medium.

For the generation of Selumetinib-resistant ex-vivo organoids, organoids generated from Apc+/Min mice

were first cultured with 1 PM of Selumetinib for two weeks. The concentration was then increased by

0.5 PM every two weeks to reach a final concentration of 5 PM.

Lentiviral Cell Infection

Control shRNA, CEMIP, Myc, TAK1 and FRA-1 shRNA lentiviral pLKO1-puro plasmid constructs were purchased from Sigma (St. Louis, MO, USA). Control shRNA and CEMIP shRNA lentiviral pLKO1-puro-IPTG-inducible plasmid constructs were also purchased from Sigma. Lentiviral infections were carried out as described (23).

For lentiviral infections of ex-vivo organoids, they were manually disrupted, washed with PBS to eliminate debris and subsequently trypsinized for 30 minutes at 37°C. After washing with PBS, cells were washed via strainers (70 μM) with 20 ml of PBS and centrifuged (200 g) for 5 minutes at 4°C.

They were then diluted in 500 microliters of full media for organoid growth and 500 microliters of infectious supernatants were added, mixed and incubated in a CO2 incubator for 12 hours. Organoids were subsequently centrifuged for 5 minutes at 4°C, washed once with 1 ml of PBS and plated as usual.

24 hours later, full media contained 2 μg/ml of puromycin was added.

Quantitative real-time PCR

Total RNAs were extracted using the E.Z.N.A Total RNA kit (Promega). cDNAs were synthesized using the Revert aid H minus reverse transcriptase kit (Thermo Scientific) and Real-time PCR analyses were performed as described (23). mRNA levels in control organoids or cells were set to 1 and mRNA levels in other experimental conditions were relative to the control after normalization with E-Actin.

Data from at least two independent experiments performed in triplicates are shown.

MTS assay

Cells counted using the TC20TM Automated Cell Counter (Bio-Rad, Pleasanton, CA, USA) were plated in 96-well flat bottom plates at a density of 2000 cells per well in triplicate and then treated with various 8

concentrations of Selumetinib or Vemurafenib for 72 hours. Cell viability was determined using the

MTS assay reagent (CellTiter 96 AQueous One Solution Cell Proliferation Assay; Promega, Madison,

WI, USA) according to the manufacturer’s protocol. The absorbance was measured at 490 nm using a

Wallac Victor2 1420 Multilabel counter (Perkin Elmer, Wellesley, MA). Absorbance of untreated cells was designated as 100% and the number of viable cells in other experimental conditions were relative to the untreated cells.

Clonogenic assay

Cells were seeded in 60-cm dishes at a density of 3000 cells per dish in duplicate. 24 hours after plating, various concentrations of Selumetinib or Vemurafenib were added to each dish. After treatment for 24 hours, cells were washed with PBS and further incubated for 15 days. Cells were subsequently stained with 0.5% crystal violet in 25% methanol-containing PBS. Colonies were examined under a light microscope and counted after capturing images.

Western blot analysis

Cells were lysed in a buffer containing 20 mM Tris-HCl, 0.5 M NaCl, Triton X-100, 1 mM EDTA, 1 mM EGTA, 10 mM β-glycophosphate, 10 mM NaF, 300 μM Na3VO4, 1 mM Benzamidine, 2 μM

PMSF, and 1 mM DTT. Western blots were carried out as described, using antibodies listed in

Supplementary Table 1 (23).

Caspase-3/7 activity assay

Caspase-3/7 activity was quantified using the Caspase-3/7 Glo Assay (Promega). Cells were treated with

Selumetinib or Vemurafenib for the indicated periods of time and caspase-3/7 activity was quantified from cell lysates. Luminescence was measured at 490 nm using Wallac Victor2 1420 Multilabel counter

(Perkin Elmer). Luminescence values in vehicle-treated control samples were set to 1 and values obtained in other experimental conditions were relative to the vehicle control.

Extraction of cytoplasmic and nuclear proteins

Cells were incubated on ice for 10 minutes in cytoplasmic lysis buffer (10 mM HEPES pH 7.9, 10 mM

KCl, 0.1 mM EDTA, NP-40 0.3% and Protease inhibitor). After centrifugation at 3000 rpm for 5 minutes at 4oC, the supernatant fraction (cytoplasmic extract) was harvested and the pellet was resuspended in nuclear lysis buffer (20 mM HEPES pH 7.9, 0.4 M NaCl, 1 mM EDTA, Glycerol 25%, and Protease inhibitor), incubated on ice for 10 minutes and centrifuged at 14000 rpm for 5 minutes at

4oC and supernatant containing the nuclear fraction was retained.

Biochemical fractionation

Cells were resuspended and homogenized in a Dounce homogenizer with the lysis buffer (150 mM

NaCl, 5 mM DTT, 5 mM EDTA, 25 mM Tris HCl pH7.4, protease inhibitors) and centrifuged at 1000g for 10 minutes at 4°C. The supernatant was adjusted to 1% Triton X-100 and left on ice for 30 minutes.

4 vol of OptiPrep were added to 2 vol of supernatant. OptiPrep was diluted with the lysis buffer plus 1%

Triton X-100 to give 35, 30, 20 and 5% (w/v) iodixanol. 0.6 ml of each sample as well as the four gradient solutions were layered in tubes for the swinging-bucket rotor. Samples were centrifuged at

30000 rpm for 16 hours. Fractions of equal volume were collected for subsequent western blot analyses.

For the second fractionation experiment, the Optiprep Density Gradient centrifugation kit was purchased from Sigma Aldrich. Briefly, about 300 million HT-29 cells showing some acquired resistance to

Selumetinib were trypsinized, washed in PBS and centrifuged at 600 g for 5 minutes. Cells were then lysed with the extraction buffer, homogenized using the Dounce homogenizer and centrifuged at 1000 g for 10 minutes. The supernatant was collected and centriguged at 20000 g for 30 minutes. The pellet

(which includes ER, lysosomes, peroxisomes, mitochondria and endosomes) was diluted to a 19%

Optiprep Density gradient solution and centrifuged on an OptiPrep Density Gradient at 100000 g for 8 hours. Fractions of equal volume were collected for subsequent western blot analyses.

Immunoprecipitation

Anti-MEK1, -BRAF and -IgG (negative control) antibodies were first coupled non-covalently to a mixture of Protein A/G-Sepharose. The antibody-Protein A/G-Sepharose conjugates were then pelleted by centrifugation at 5,000 rpm for 2 minutes, the supernatant removed and the beads washed with 0.1 M sodium borate pH 9.3. This was repeated four times, after which the beads were resuspended in 20 mM dimethyl pimelimidate dihydrochloride (DMP) freshly made in 0.1 M sodium borate pH 9.3 and gently mixed on a rotating wheel for 30 minutes at room temperature. Following centrifugation at 5,000 rpm for 2 minutes, supernatant was removed and fresh 20 mM DMP/0.1 M sodium borate pH 9.3 solution was added to the beads, which were then gently mixed for a further 20 minutes. The beads were then spun down at 5,000 rpm for 2 minutes, the supernatant removed and four washes with 50 mM glycine pH 2.5 carried out to remove any antibody coupled non-covalently. Afterwards, the beads were washed twice with 0.2 M Tris-HCl pH 8.0 (neutralisation step) and then resuspended in the same solution and mixed gently on a rotating wheel at room temperature for 2 hours. The beads were then used immediately for immunoprecipitation analyses as described (23). 11

Kinase assay

Control or CEMIP-depleted HT29 cells showing acquired resistance to Selumetinib were subjected to anti-MEK1 or -IgG (negative control) immunoprecipitation. Selumetinib was added as control in some experimental conditions to inhibit MEK1 activity. The kinase assay was conducted at 30°C for 30 minutes with 1 μg of GST-ERK2 substrate (ThermoFisher Scientific), 10 μCi of [γ32P] ATP in 20 μl of kinase buffer (25 mM HEPES, pH 7.5, 10 mM MgCl2, 25 mM β-glycerophosphate, 1mM Na3VO4, and

1mM dithiothreitol). ERK2 phosphorylation was revealed by autoradiography.

Proximal ligation and ChIP assays

Parental and resistant HT-29 cells or control and CEMIP-depleted resistant HT-29 cells were plated in 8 chamber cell culture dishes and proximal ligation assays were performed as described (23).

Chromatin immunoprecipitation (ChIP) was performed using anti-TCF4 or IgG antibody as a negative control. A TCF4 binding site (site #1 located 839 bp upstream of the transcriptional start site on the

CEMIP promoter) was identified through in silico analysis (MatInspector, Genomatix). TCF binding sites #2, #3 and #4 located 26417, 75344 and 79348 bp downstream of the transcriptional start site within intron 1 respectively, were previously described (26). A negative binding site was randomly chosen in exon 1 of the CEMIP sequence. Primer sequences used are available upon request. Extracts from Selumetinib-resistant HT-29 cells, left untreated or treated with 3PM Selumetinib for 24 hours were precleared through an incubation step with protein A/BSA/Herring sperm DNA for 18 hours and immunoprecipitations were performed overnight at 4°C with the relevant antibody, followed by 1 hour incubation with protein A/BSA/Herring sperm DNA. Protein-DNA complexes were washed 3 times

with high salt buffer (1% Triton X-100, 0.1% SDS, 500 mM NaCl, 2 mM EDTA pH 8.0, 20 mM Tris

HCl pH 8.0, with protease inhibitors) and once with LiCl buffer (20 mM Tris HCl pH 8.0, 1 mM EDTA,

250 mM LiCl, 0.5% NP-40, 0.5% Na-deoxycholate, 0.5 mM PMSF and protease inhibitors). After elution, proteinase K treatment and reversal of crosslinks, DNA fragments were analysed by real time

PCR with SYBR Green detection. Values were calculated as ratios between ChIP signals obtained with the anti-TCF4 (specific) or IgG (non-specific) antibodies. Input DNA was analysed simultaneously and used for normalization purposes.

Immunofluorescence

Immunofluorescence on cells was carried out as described (23). For immunofluorescence on ex-vivo organoids, they were grown in 8 well chamber slides (Thermo Fisher Scientific, Lab-TekTM), fixed in

4% paraformaldehyde for 15 minutes and washed twice in PBS. They were then incubated in permeabilization solution (PBS with 0.5% Triton X-100) for 15 minutes, washed in PBS and incubated for 60 minutes in PBS containing 0.2% Triton X-100, 0.05% Tween and 1% bovine serum albumin

(BSA). Organoids were incubated overnight at 4°C with primary antibody in the same solution without

Triton. After incubation, organoids were washed 3 times in PBS and incubated for 40 minutes with secondary antibody, washed 3 times with PBS and incubated for 5 minutes with DAPI solution and mounted with the ProLong Gold Antifade Mountant from Invitrogen. The anti-Ki-67 mouse monoclonal antibody was from BD Biosciences (San Jose, CA, USA).

For immunofluorescence on cells expressing the SNAP-CEMIP construct, the CEMIP coding sequence was subcloned into the pSNAPf Vector (New England BioLabs, Ipswich, MA, USA) in which the SNAP tag is localized at the C-terminal end. HCT116 cells were transfected with controls (empty vector and

pSNAPf -Cox8A) and CEMIP-SNAP plasmids and labeled with SNAP-Cell TMR-Star (New England

BioLabs) 24 hours after the transfection. Cell were fixed with 4% PAF and co-stained with endosomal

or ER markers, as described (23). DAPI was used for nuclei staining.

FACS analysis

Organoids were manually disrupted, washed with PBS to eliminate debris and subsequently trypsinized

for 30 minutes at 37°C. After washing with PBS, cell were washed via strainers (70 μM) with 20 ml of

PBS and centrifuged (200 g) for 5 minutes at 4°C. They were then diluted in 500 microliters of PBS and

incubated for 40 minutes on ice with anti-mouse CD24-PE eBioscience and anti-mouse CD133-APC

eBioscience antibodies, washed once in PBS and analysed on the FACS CantoII. DAPI staining was

used for selection of live cells.

Tumor xenograft experiments

HT-29/SR cells were infected with an IPTG-inducible CEMIP shRNA (HT-29/SR-iCEMIP shRNA) or

control shRNA (HT-29/SR-iControl shRNA). HT-29/SR-iCEMIP shRNA or HT-29/SR-iControl

shRNA cells (1.5 x 106) mixed with matrigel at a ratio of 1:1 were injected subcutaneously into the right or left flank of 6- to 8-week old NOD/SCID male mice, respectively. Mice were monitored and tumors tracked via caliper measurements. Tumor volume was determined using the following formula: length x width x height x 0.5236 (n=4 mice/group). Mice were treated with a combination of 10 mM IPTG in their drinking water and 25 mM IPTG (200 μL) via intraperitoneal injection and either untreated or treated with Selumetinib (20 mg/kg) via intraperitoneal injection five days a week for two weeks. Mice

from all treatment groups were euthanized and tumors were excised and tissue archived for

immunofluorescence and real-time PCR analysis.

Establishment of metabolomic profiles by targeted metabolomics

For targeted metabolomics analysis of ex-vivo organoids, each sample was washed three times with cold

PBS, collected into an Eppendorf tube, frozen in liquid nitrogen and stored at -80°C until extraction. The extraction solution used was 50% methanol, 30% ACN, and 20% water. The volume of extraction solution added was calculated from the cell count (2 x 106 cells per ml). After addition of extraction

solution, samples were vortexed for 5 minutes at 4°C, and immediately centrifuged at 16,000 g for 15

minutes at 4°C. The supernatants were collected and analysed by liquid chromatography–mass

spectrometry using SeQuant ZIC-pHilic column (Merck) for the liquid chromatography separation.

Mobile phase A consisted of 20 mM ammonium carbonate plus 0.1% ammonia hydroxide in water.

Mobile phase B consisted of ACN. The flow rate was kept at 100 ml/minute, and the gradient was 0

minutes, 80% of B; 30 minutes, 20% of B; 31 minutes, 80% of B; and 45 minutes, 80% of B. The mass

spectrometer (QExactive Orbitrap, Thermo Fisher Scientific) was operated in a polarity switching mode

and metabolites were identified using TraceFinder Software (Thermo Fisher Scientific). To obtain a

robust statistical analysis, metabolomics data were normalized using the median normalization method

(27). The data were further pre-processed with a log transformation. MetaboAnalyst 3.0 software (28)

was used to conduct statistical analysis and heatmap generation, and an unpaired two-sample t-test was

chosen to perform the comparisons. The algorithm for heatmap clustering was based on the Pearson

distance measure for similarity and the Ward linkage method for biotype clustering. Metabolites with

similar abundance patterns were positioned closer together.

Statistical analysis

The two-tailed Student’s t-test was applied for statistical analysis when only 2 groups of interest were compared. Results were plotted as mean ± SD and were significant in all experiments at *** (p< 0.001),

** (p < 0.01) and * (p< 0.05).

RESULTS

Ex-vivo organoids with some acquired resistance to MEK1 inhibition show enhanced CEMIP expression

Cancer stem cells contribute to resistance to targeted therapies. To make the link between cancer stem cells and acquired resistance to targeted therapies, we subjected ex-vivo organoids showing constitutive Wnt signaling (i.e. generated with extracts of intestinal crypts from Apc+/Min mice) to increasing concentrations of Selumetinib to generate resistant organoids (Fig. 1A). The maintenance of these ex-vivo organoids relies on the self-renewal potential of cancer stem cells. Resistant organoids were larger in size but without changes in cell proliferation (as judged by the percentage of Ki-67+ cells) and were protected from caspase 3-dependent cell death, compared to parental organoids (Figs. 1A and

1B). The scaffold protein CEMIP may connect pro-tumorigenic Wnt- and MAPK signaling pathways as it is the most robust Wnt-induced gene candidate and is also required for MAPK activation upon activation of ErbB signaling (23,29). As such, CEMIP may actively contribute to acquired resistance to

Selumetinib as a signaling protein involved in MAPK reactivation. Ex-vivo organoids treated with a combination of valproic acid and CHIR999021, a GSK3 inhibitor, to induce Wnt signaling showed elevated mRNA levels of Wnt target genes such as Lgr5 and CEMIP while the level of Dclk1, a marker of differentiated Tuft cells, was downregulated (Fig. 1C). CEMIP induction by these drugs was also detected at the protein level (Fig. 1C). Immunofluorescence confirmed that treatment with valproic acid and CHIR999021, which enriches Lgr5+ cells in ex-vivo organoids, decreased the number of Dclk1+

Tuft cells (Fig. 1C). Therefore, CEMIP expression is transcriptionally induced by Wnt signaling.

Importantly, resistant organoids showed increased CEMIP, SOX9, HER3 and BRAF expression as well as enhanced activation of MEK1, ERK1/2 and mTOR (as judged by 4EBP1 phosphorylation) (Figs. 1D and 1E, respectively). CEMIP was actually increased at the mRNA level in resistant organoids (Fig. S1).

Moreover, Myc, which controls protein synthesis and organ size, was increased at the protein but not mRNA levels (Fig. 1D and S1, respectively). Of note, we did not detect any mutation on BRAF or on

MEK1 in these resistant organoids. Resistant organoids were enriched in CD24+/CD133+ cancer stem cells (Fig. 1F), which fits with the upregulation of CD133 in colon cancer cells showing hyperactivation of the RAS-RAF-MEK1 cascade (30). Therefore, Selumetinib-resistant organoids show all molecular features classically associated with the acquired resistance to MEK1 inhibition.

CEMIP is connected to ErbB/MEK1-, LEF1- and Myc-dependent pathways in colon adenocarcinoma

To explore whether CEMIP links Wnt-dependent gene transcription to MEK1 signaling, we depleted CEMIP in BRAFV600E-mutated HT-29 colorectal cancer cells and carried out RNA-Seq experiments combined with Gene Set enrichment analyses (GSEA) (Figs. 2A-C and S2A). In agreement with our previous observations (23), CEMIP expression was linked to ErbB/MEK1 signaling as a signature of genes induced through ErbB2, KRAS or MEK1 was lost upon CEMIP deficiency (Figs. 2C-

2D). Genes controlled by the transcription factor LEF1 were also identified to be regulated by CEMIP

(Figs. 2C and S2A). We next carried out an iRegulon analysis to identify all genes co-expressed with

CEMIP in colon adenocarcinoma and found 285 candidates (Fig. 2E). Interestingly, many of them are regulated by the Myc family of transcription factors (Fig. 2E). We also carried out an Ingenuity analysis

(31) on these 285 co-expressed genes and found a significant enrichment of genes controlled by p53,

Myc and E-catenin among others (Fig. S2B). Therefore, CEMIP expression is linked to ErbB-, Myc and

E-catenin-dependent pathways.

CEMIP promotes Myc expression through ERK1/2 activation

To explore how CEMIP and Myc are linked, we assessed the consequences of CEMIP deficiency in ex-vivo organoids. The depletion of CEMIP in parental or Selumetinib-resistant organoids severely impaired their maintenance (Fig. S3 and Fig. 3A, respectively). Consistently, the pool of CD24+/CD133+ cancer stem cells was impaired upon CEMIP deficiency (Fig. 3B). CEMIP deficiency downregulated

HER3, Cyclin D1, Cyclin D2, Myc, SOX9 and phosphorylated ERK1/2 levels (Fig. 3C). To explore whether the link between CEMIP and Myc was found in other experimental systems, we generated

BRAFV600E-mutated COLO205 cells with some acquired resistance to MEK1 inhibition by subjecting parental cells to increasing concentrations of Selumetinib (Fig. 3D). Selumetinib-resistant COLO205 cells showed elevated CEMIP mRNA and protein levels as well as increased levels of pMEK1/2, pERK1/2 and pRSK1 (Figs. 3E and 3F). Importantly, CEMIP deficiency in these cells also impaired

MEK1 and ERK1/2 activation and decreased protein levels of Myc (Fig. 3G). Of note, effects on MEK1 activation were largely due to decreased total levels of MEK1 in CEMIP-deficient cells, which was not the case in ex-vivo organoids (Figs. 3G and 3C, respectively). These Selumetinib-resistant COLO205 cells were also resistant to PD98059, another MEK1 inhibitor, as pERK1/2 levels barely decreased at high concentrations of this inhibitor (Fig. S4). Here also, CEMIP deficiency in these cells decreased pMEK1, pERK1/2 as well as Myc protein levels (Fig. S4). Conversely, the ectopic expression of

CEMIP alone in DLD-1 cells enhanced both pERK1/2 and Myc protein levels without impacting on

Myc mRNA levels and also protected from cell death triggered by Selumetinib (Figs. S5A and S5B, respectively). Therefore, CEMIP maintains Myc protein levels in multiple experimental models.

CEMIP expression is induced through BRAF, ERK1/2 and FRA-1 upon acquired resistance in

BRAFV600E- but not KRASG13D or G12A-mutated colorectal cancer cells 19

CEMIP expression is increased in ex-vivo organoids as well as in BRAFV600E-mutated COLO205 cells, both with resistance to MEK1 inhibition. To explore whether this also applies to other experimental models, we cultured parental HT-29 cells (HT-29/P) with increasing concentrations of

Selumetinib and generated highly resistant HT-29 cells (HT-29/SR) (Fig. 4A). These cells showed decreased E-cadherin levels, suggesting that they underwent epithelial-mesenchymal transition (EMT), a known feature of chemoresistance (Fig. S6A). Importantly, CEMIP mRNA and protein levels were strongly induced in resistant HT-29 cells (Fig. 4B). We next looked at the nuclear levels of transcription factors which drive CEMIP gene transcription, namely NF-NB and AP-1 family members (23,32). Both p65 and FRA-1 but not BCL-3 and c-JUN levels were increased in nuclear extracts from resistant HT-29 cells (Fig. 4C). Of note, cytoplasmic BRAF showed elevated levels in resistant cells (Fig. S6B), which reflects intrachromosomal amplification (10). These molecular changes persisted even in circumstances in which cells were constantly cultured with Selumetinib (Fig. S6C). Therefore, CEMIP expression is induced in resistant HT-29 cells in which mutated BRAFV600E, nuclear p65 and FRA-1 protein levels are increased. FRA-1 but not p65 was actually driving CEMIP expression in these cells as FRA-1 but not p65 deficiency impaired CEMIP expression at both mRNA and protein levels (Figs. S7A and S7B, respectively). FRA-1 controls the expression of several candidates such as WNT10, DKK-1 and DVL-1 acting in the canonical Wnt pathway in colon cancer cells (33). As CEMIP transcription is robustly induced upon Wnt activation (29), we hypothesized that FRA-1 indirectly controls CEMIP expression through Wnt signaling. FRA-1 deficiency indeed impaired nuclear E-catenin levels and both WNT10 and DKK-1, two E-catenin target genes downregulated upon FRA-1 deficiency in HT-29/SR cells (Figs.

S7C and S7D). FRA-1 deficiency also triggered cell death of HT-29/SR cells, as judged by clonogenic assays, at least due to Caspase-3/7 activation (Figs. S7E and S7F). As CEMIP expression is induced in

BRAFV600E-mutated cells, we reasoned that a BRAF inhibitor may decrease CEMIP expression. CEMIP

mRNA and protein levels were severely decreased in HT-29/SR cells subjected to Verumafenib (Figs.

S8A and S8B). As a consequence, Vemurafenib triggered some cell death and interfered with the

capacity of these cells to form colonies (Figs. S8C and S8D, respectively). Taken together, our results

define BRAFV600E, FRA-1 and E-catenin as upstream actors that drive CEMIP transcription in

BRAFV600E-mutated resistant colorectal cancer cells. As Selumetinib decreased CEMIP expression in

both Selumetinib-resistant organoids and HT-29 cells (Figs. 3C and 4D, respectively), we looked at

FRA-1 proteins levels upon MEK1 inhibition in HT-29/SR cells. FRA-1 but also p65, c-JUN, E-catenin

and TCF4 were downregulated upon MEK1 inhibition while the epithelial marker E-cadherin was

increased, (Fig. 4D). Moreover, TCF4 promotes CEMIP expression as CEMIP protein levels severely

decreased upon TCF4 deficiency in Selumetinib-resistant HT-29 cells (Fig. S9A). Consistently, the

treatment of two Selumetinib-resistant colon cancer cell lines with PNU-74654, which inhibits the

Wnt/E-catenin pathway by blocking the interaction between E-catenin and TCF4, decreased CEMIP

mRNA and protein levels (Fig. S9B). Both TCF4 and E-catenin were actually recruited at TCF binding

sites located on the CEMIP promoter as well as on intron 1 (Fig. 4E). Therefore, Selumetinib decreases

CEMIP expression, at least by negatively regulating protein levels of both FRA-1 and TCF4.

To explore whether KRASG13D or G12A-mutated colorectal cancer-derived cell lines showing some

acquired resistance to Selumetinib also show elevated levels of CEMIP, parental HCT116 and SW480

cells were also treated with increasing concentrations of Selumetinib to generate resistant HCT116 and

SW480 cells, respectively (HCT116/SR and SW480/SR) (Fig. S10A). Both resistant HCT116 and

SW480 cell lines did not upregulate CEMIP, in contrast to resistant HT-29 cells (Fig. S10A). Yet,

ERK1/2 reactivation was seen in all resistant cells (Fig. 4F). RSK1 activity was also specifically induced

in BRAFV600E- but not in KRASG13D or G12A-mutated resistant cells (Fig. 4F). Both p65 and FRA-1 were

not dramatically induced in both HCT116 and SW480 resistant cells (Fig. 4F and Fig. S10B). Levels of

another scaffold protein, IQGAP-1, remained unchanged in resistant HT-29 cells (Fig. 4F). BRAFV600E- mutated resistant HT-29 cells, and to a less extent KRASG13D-mutated HCT116 cells, also showed enhanced HER3 and MET reactivations (Fig. 4G). Finally, a somatic mutation in MEK1 exon 3

(H119R) was found in resistant HT-29 cells (Fig. 4H). The H119R mutation, among others in the same domain of MEK1, was demonstrated to underly resistance to the MEK1 inhibitor, PD184352 (34).

Taken together, our data indicate that BRAFV600E- but not KRASG13D or G12A-mutated colorectal cancer cells reactivate MAPK signaling and potently induce CEMIP gene transcription upon acquired resistance to MEK1 inhibition.

CEMIP deficiency sensitizes BRAFV600E-mutated resistant HT-29 cells to MEK1 inhibition

To explore whether CEMIP contributes to the resistance to Selumetinib in BRAFV600E-mutated colorectal cancer cells, we depleted CEMIP from HT-29/SR cells and subjected them to Selumetinib.

MEK1 inhibition decreased CEMIP mRNA levels (Fig. 5A). CEMIP deficiency enhanced DNA damage, as evidenced by increased pH2A.X levels (S139) as well as Caspase-3/7-dependent apoptotic cell death upon Selumetinib treatment (Figs. 5B and 5C, respectively). CEMIP-depleted HT-29/SR cells generated less colonies when subjected to Selumetinib (Fig. S11A). To assess whether this was also relevant in vivo, we carried out xenograft experiments in immunodeficient mice with control and

CEMIP-depleted HT-29/SR cells and subsequently treated mice with Selumetinib. CEMIP mRNA levels were expectedly decreased in cells infected with the inducible shRNA construct (Fig. S11B). Also,

Selumetinib failed to significantly trigger tumor regression in vivo in mice injected with HT-29/SR cells

(Fig. 5D). Importantly, CEMIP deficiency, combined with Selumetinib, caused significant tumor regression (Fig. 5D). This was due to DNA damage and apoptosis, as evidenced by anti-pH2A.X (S139) and cleaved caspase-3 immunofluorescence (Fig. S11C). CEMIP-deficient HT-29/SR cells showed 22

lower levels of HER3, even after treatment with Selumetinib and failed to maintain levels of phosphorylated ERK1/2 and RSK1 when treated with Selumetinib (Fig. 5E). Moreover, E-Cadherin levels increased upon CEMIP deficiency, indicating that CEMIP is required for EMT maintenance, a process linked to chemoresistance (Fig. 5E). CEMIP expression also decreased at the protein level in

Selumetinib-treated cells, similar to FRA-1 and also to BRAF but not KRAS levels, suggesting again that BRAF and FRA-1 controls CEMIP expression (Fig. 5E). Therefore, CEMIP contributes to the acquired resistance to Selumetinib, at least by promoting MEK1-ERK1/2 signaling.

CEMIP is an endosomal protein

We next carried out biochemical fractionation to identify cell compartments from which CEMIP contributes to MEK1 and ERK1/2 reactivation in resistant cells. CEMIP co-fractionated with EEA1 and

APPL1, two signaling endosome markers, and to a less extent with lysosomal markers (Rab7 and

LAMP2) and with Rab11, a recycling endosome marker (Fig. 6A). CEMIP also co-fractionated with

PDI, an endoplasmic reticulum marker, as previously described (24). Phosphorylated forms of MEK1 were also detected in CEMIP-positive fractions, suggesting that signaling endosomes are critical for

MEK1 reactivation (Fig. 6A). In contrast, CEMIP did not co-fractionate with Caveolin-1 and Flotillin-1, two lipid raft markers (Fig. 6A). To explore in which endosomes CEMIP is mainly located, we conducted a second fractionation experiment in which organelles of interest (ER, peroxisomes, mitochondria and endosomes) were enriched from cell extracts and separated on a gradient by ultracentrifugation. CEMIP mainly co-fractionated with EEA1+ endosomes and to a much less extent with Rab5/7+ or APPL1+ endosomes (Fig. 6B). A SNAP-CEMIP construct expressed in HCT116 cells also partially colocalized with EEA1+ endosomes and with the ER, as assessed by immunofluorescence

(Fig. 6C). CEMIP was the only endosomal protein to be upregulated in HT-29/SR cells, as both EEA1 23

and APPL1 levels remained unchanged (Fig. S12A). CEMIP associated with MEK1 in HT-29/SR cells, as evidenced by co-immunoprecipitation (Fig. 6D) and BRAF more weakly bound MEK1 in resistant versus parental HT-29 cells, more likely due to disengagement (Fig. 6E). Of note, pERK1/2 levels were totally abolished after 0.5 and 1 hour of treatment with Selumetinib in both parental and resistant HT-29 cells but pERK1/2 levels were again detectable after 24 hours of MEK1 inhibition (Fig. S12B). CEMIP actually contributes to MEK1 activity as an anti-MEK1 immunoprecipitate from CEMIP-depleted HT-

29/SR cells was less potent at phosphorylating ERK2 (Fig. S12C). CEMIP failed to bind mutated

BRAFV600E in resistant HT-29 cells (Fig. S13A). Although more BRAFV600E dimers were detected by the proximal ligation assay in resistant versus parental HT-29 cells, CEMIP was dispensable for BRAFV600E dimerization (Figs. S13B and S13C). Therefore, CEMIP is localized in several cell compartments and contributes to MEK1 reactivation from signaling endosomes in resistant BRAFV600E-mutated colorectal cancer cells as a MEK1-binding protein.

CEMIP promotes metabolic reprogramming potentially through Myc

To explore the biology downstream of CEMIP, we established the metabolomic signature of both parental and resistant organoids. Severe metabolic reprogramming was detected in resistant organoids as they showed elevated levels of TCA intermediates (Fumarate, Malate, Citrate and Succinate) (Fig. 7A).

Multiple nucleotides, whose synthesis relies on Myc (35), were increased in resistant organoids (Fig.

7A). Proline, whose degradation is inhibited by Myc (36), was detected at higher levels in resistant organoids (Fig. 7A). Moreover, levels of arachidonic acid which is regulated by Myc in lung cancer

(37), were also elevated in resistant organoids as were levels of other unsaturated fatty acids such as oleic acid, a candidate reported to be upregulated in colon cancer (Fig. 7A) (38). Finally, levels of

Cystathionine, which is generated by Cystathionine E-synthase (CBS), an enzyme downregulated in 24

gastrointestinal and hepatocellular malignancies (39,40), were decreased in resistant organoids (Fig.

7A). Therefore, metabolic reprogramming is seen in Selumetinib-resistant intestinal organoids.

Importantly, CEMIP expression contributes to this process as the production of lactate as well as levels of multiple amino acids was impaired upon CEMIP deficiency in these ex-vivo resistant organoids (Fig.

7B). To better define CEMIP as an upstream regulator of Myc, we reasoned that Myc deficiency would mimick CEMIP deficiency in Selumetinib-resistant ex-vivo organoids. Indeed, the depletion of Myc impaired ERK1/2 activation and also downregulated CEMIP protein but not mRNA levels, suggesting that CEMIP and Myc mutually post-transcriptionally control their expression (Fig. 7C and Fig. S14A).

Myc was also critical for the maintenance of Selumetinib-resistant ex-vivo organoids, similar to CEMIP

(Fig. S14B). Moreover, Myc depletion had a profound effect on the levels of multiple metabolites as levels of lactate and numerous amino acids were significantly downregulated in Myc-depleted cells (Fig.

S14C). A comparison of the metabolic signatures of Selumetinib-resistant ex-vivo organoids with depleted Myc or CEMIP confirmed that both proteins control the production of multiple metabolites such as amino acids (Methionine, Threonine, Tryptophane, Valine, Proline, Histidine, Asparagine,

Phenylalanine, IsoLeucine, Leucine, Glycine and L-Alanine) and lactate among other candidates (Fig.

7D). Therefore, CEMIP may promote the acquired resistance to MEK1 inhibition, in part by potentially regulating levels of specific metabolites via a Myc-associated signaling pathway.

DISCUSSION

We describe here the characterization of CEMIP as an endosomal protein that links Wnt-

dependent gene transcription to MEK1-ERK1/2 signaling to promote acquired resistance to MEK1

inhibition in BRAFV600E-mutated colorectal cancer cells. CEMIP expression is induced in resistant cells

through BRAFV600E, MEK1, RSK1 and FRA-1, which provides a mechanism by which these signaling proteins promote resistance to inhibitors of RAS effectors. In addition, CEMIP regulates levels of multiple amino acids seen in resistant cells, at least through Myc.

Multiple transcription factors govern CEMIP transcription, including the NF-NB proteins BCL-3

and p65 in cervical cancer cells (23). Functional NF-NB and AP-1 binding sites were also identified on

the CEMIP promoter in breast cancer cells (32). We define here FRA-1, one member of the AP-1 family

of transcription factors as well as TCF4 as key drivers of CEMIP expression in resistant BRAFV600E-

mutated colorectal cancer cells. The BRAF inhibitor, which indirectly turns off ERK1/2 activity, also

decreases FRA-1 protein levels. This observation fits with the fact that ERK1/2 signaling stabilizes

FRA-1 by preventing its proteasome-dependent degradation in colorectal cancer cells (41). Therefore,

interfering with ERK1/2 signaling downregulates CEMIP transcription, at least through the

destabilization of FRA-1 in resistant BRAFV600E-mutated colorectal cancer cells. This signaling cascade

critically drives CEMIP transcription to establish a positive loop as CEMIP physically binds MEK1 (but

not BRAF) to sustain MEK1 activity.

Our resistant BRAFV600E-mutated colorectal cancer cells have several features linked to acquired

resistance. They show enhanced phosphorylation of MET and HER3, elevated levels of BRAFV600E as

well as a MEK1 mutation, all events contributing to ERK1/2 reactivation. The upregulation of CEMIP

has been detected in all tested BRAFV600E- but not KRASG13D or G12A-mutated colorectal cancer cells

showing some acquired resistance to MEK1 inhibition. Yet, the induction of CEMIP upon acquired 26

resistance was more severe in resistant BRAFV600E-mutated colorectal cancer HT-29 cells in which the

MEK1 mutation within exon 3 (H119R) was found. Therefore, the combination of both BRAFV600E and

MEK1 mutations may be key genetic events to efficiently drive CEMIP expression upon acquired resistance.

CEMIP is found in signaling endosomes and is essential for ERK1/2 reactivation in BRAFV600E- but not KRASG13D or G12A-mutated cells, at least through binding to MEK1. Whether or not ERK1/2 activation downstream of tyrosine kinase receptors occurs from endosomes or from the cytoplasmic membrane has been the subject of an intense debate. While some studies support the notion that MAPK scaffold complexes found in endosomes are critical for signal transduction, other reports state that signaling from a tyrosine kinase receptor occurs from the cytoplasmic membrane (42,43). In support with this later hypothesis, EGFR endocytosis in endosomes helps to terminate Ras-dependent signaling to ERK1/2 as endogenous Ras is primarly located at the cytoplasmic membrane in low EGFR- expressing cells (44). BRAFV600E does not bind CEMIP, which fits with the hypothesis that CEMIP only binds signaling proteins such as EGFR or MEK1 found in endosomes but not candidates such as BRAF which is activated at the cytoplasmic membrane (23,45). The enhanced CEMIP expression that we specifically see in resistant BRAFV600E-mutated colorectal cancer cells may help to recycle HER3 and

MET at the cytoplasmic membrane to sustain ERK1/2 signaling and/or to favor the assembly of a specific endosomal signaling platform for ERK1/2 reactivation.

A previous study showed that a pool of CEMIP can be found in the ER (24). These results, combined with our study revealing CEMIP in endosomes, raises some questions on mechanisms by which a scaffold protein localized in two distinct cell compartments, promotes survival and chemoresistance. The answer may come from the existence of membrane contacts between endosomes and the ER, a process which contributes to EGFR-dependent signaling (46). These physical contacts

may help CEMIP to bring signaling proteins together to sustain ERK1/2 activation in resistant

BRAFV600E-mutated colorectal cancer cells.

One key mechanism through which CEMIP deficiency circumvents the acquired resistance to

MEK1 inhibition in BRAFV600E-mutated colorectal cancer cells may be through Myc, which is in agreement with the fact that the pharmacological inhibition of Myc circumvents the acquired resistance to c-Met inhibition (47). Our correlative metabolic data show that Myc is a key effector downstream of

CEMIP as CEMIP and Myc similarly control the production of multiple metabolites including lactate as well as amino acids such as Glycine, which has been defined, among others, as a driver of cancer pathogenesis (48). We demonstrated that Selumetinib-resistant ex-vivo organoids show high levels of multiple amino acids, which can be metabolized as a source of carbon and nitrogen for biosynthesis of fatty acids, lipids, nucleotides and proteins to support proliferation and survival (49). Essential amino acids such as Leucine, Tryptophane and Phenylalanine, whose levels are controlled by both CEMIP and

Myc in Selumetinib-resistant organoids, has been defined as signaling molecules for mTOR activation

(50). Therefore, CEMIP and its downstream effector Myc may indirectly control mTOR signaling through the production of specific essential amino acids to support acquired resistance to MEK1 inhibition.

In conclusion, our study defined the scaffold and endosomal protein CEMIP as an upstream regulator of Myc that links Wnt- and MEK1-dependent signaling pathways. As CEMIP is linked to Myc and to specific metabolic reprogramming seen in resistant cells, this oncogenic pathway may hold therapeutic interest.

ACKNOWLEDGEMENTS

We thank Ganna Panasyuk (INSERM, Paris) and Phillip Williams (GIGA-I3, ULiege, Belgium) for their critical reading of the manuscript and the GIGA Imaging and Flow Cytometry Facility.

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FIGURE LEGENDS

Figure 1. Selumetinib-resistant organoids show elevated levels of CEMIP and reactivation of the

MEK1/ERK1/2 pathway. A. Selumetinib-resistant organoids are larger in size. Ex-vivo organoids with intestinal crypts extracted from Apc+/Min mice were treated with increasing concentrations of

Selumetinib. Quantification of their size is illustrated. Statistical analysis was performed as described in the methods. B. Resistant organoids do not proliferate more but show less apoptotic cells. Ki67 and activated Caspase 3 staining were carried out to quantify the percentage of proliferative and apoptotic cells, respectively. Data from 20 organoids are illustrated. C. CEMIP expression is induced by Wnt signaling in ex-vivo organoids generated from intestinal crypts of C57BL/6 mice. Ex-vivo organoids were untreated or treated with Valproic acid (1 mM) and CHIR999021 (3 PM) to enrich for Lgr5+ stem cells. On top, mRNA levels of indicated candidates were quantified by real-time PCR analysis (see methods for the quantification). At the bottom, immunofluorescence (IF) analysis of the Tuft cell marker

Dclk1. Anti-CEMIP western blot (WB) analysis using extracts from untreated or Valproic acid and

CHIR999021-stimulated ex-vivo organoids is shown. D. and E. Selumetinib-resistant organoids show elevated levels of CEMIP, HER3, BRAF, SOX9 and c-Myc and enhanced activation of MEK1, ERK1/2 and mTOR. Protein extracts from parental and resistant ex-vivo organoids were subjected to WB analysis. F. Selumetinib-resistant organoids are enriched in CD24+/CD113+ cancer stem cells. FACS analysis was conducted to quantify the percentage of CD24+/CD113+ cells. Data from four experiments is illustrated.

Figure 2. CEMIP controls the expression of ErbB-, RAS-, MEK1- and LEF1-dependent signaling cascades. A. Generation of CEMIP-depleted colorectal cancer-derived HT-29 cells. Cells were

transduced with the indicated shRNA lentiviral constructs. Real-time PCR analysis was performed to assess mRNA expression of CEMIP. B. CEMIP controls the expression of gene candidates linked to

ErbB, RAS and LEF1 signaling. A Scatter plot of RNA-seq data obtained with RNA extracted from control versus CEMIP-deficient HT-29 cells is illustrated. C. Gene set enrichment analysis of RNA-Seq expression data obtained with total RNA from control and CEMIP-depleted HT-29 cells. Candidate genes up- or downregulated are illustrated in red and blue, respectively. D. Listing of the most robust up- or downregulated candidates (red and blue rectangles, respectively) upon CEMIP deficiency in HT-

29 cells. E. Identification of CEMIP co-expressed genes in colon adenocarcinoma through iRegulon analysis. The transcription factors (“TF”) known to regulate these co-expressed genes (N = 285) are listed.

Figure 3. CEMIP is required for the maintenance of Selumetinib-resistant ex-vivo organoid cultures and for Myc expression. A. CEMIP deficiency impairs the growth of Selumetinib-resistant ex-vivo organoids. Images of control and CEMIP-depleted ex-vivo organoids are illustrated. The number of organoids per dish was calculated in each experimental condition. B. CEMIP deficiency impairs the pool of CD24+/CD113+ cancer stem cells in Selumetinib-resistant ex-vivo organoids. FACS analysis was conducted to quantify the percentage of CD24+/CD113+ cells in control and CEMIP- depleted resistant organoids. Data from two experiments are illustrated. C. CEMIP deficiency impairs

HER3, Myc, SOX9, Cyclin D1/2 levels and ERK1/2 activation in Selumetinib-resistant ex-vivo organoids. Control or CEMIP-depleted resistant ex-vivo organoids were untreated or stimulated with

Selumetinib and cell extracts were subjected to WB analysis. D. Sensitivity of parental (COLO-205/P) and resistant (COLO-205/SR) COLO-205 cells to Selumetinib. COLO-205/P and COLO-205/SR cell lines were untreated or treated with Selumetinib at indicated concentrations for 72 hours. The percentage

of viable cells in untreated COLO-205/P or COLO-205/SR cells was set to 100% and the percentage of

viable cells upon treatment with Selumetinib in COLO-205/P or COLO-205/SR cells was relative to the

untreated cells. Data from two independent experiments performed in triplicate are shown. E. Resistant

COLO-205 cells show enhanced CEMIP expression. On top, mRNA levels of CEMIP in both COLO-

205/P and COLO-205/SR cells was assessed by Real-Time PCR. At the bottom, CEMIP protein levels

in both COLO-205/P and COLO-205/SR cells was assessed by WB analysis. F. Resistant COLO-205

cells show enhanced MEK1/2 and ERK1/2 activation. Extracts from parental and resistant COLO-205

cells were subjected to WB analysis. G. CEMIP deficiency in Selumetinib-resistent COLO205 cells

impairs MEK1 and ERK1/2 activation and Myc stability. Control or CEMIP-depleted COLO205SR

(0.3) cells were unstimulated or treated with Selumetinib for 24 hours at the indicated concentrations

and cell extracts were subjected to WB analysis.

Figure 4. Selumetinib-resistant colorectal cancer cells mutated for BRAFV600E but not for

KRASG13D or G12A show higher levels of CEMIP. A. Sensitivity of parental (HT-29/P) and resistant

(HT-29/SR) cells to Selumetinib. HT-29/P and HT-29/SR cell lines were untreated or treated with

Selumetinib at indicated concentrations for 72 hours. The percentage of viable cells in untreated HT-

29/P or HT-29/SR cells was set to 100% and values in other experimental conditions were relative to the untreated cells. Data from two independent experiments performed in triplicate are shown. B.

Acquisition of Selumetinib resistance in HT-29/SR cells induces CEMIP expression. HT-29/P or HT-

29/SR cell lines were growing without Selumetinib (1.5 μM) for 48 hours. Left, real-time PCR analysis was conducted on total RNA to assess CEMIP expression. Right, protein extracts were subjected to WB analysis. C. Acquisition of Selumetinib resistance in HT-29/SR cells induces nuclear expression of p65

and FRA-1. HT-29/P or HT-29/SR cell lines were growing without Selumetinib (1.5 μM) for 48 hours.

Cytoplasmic and nuclear protein extracts were subjected to WB analysis. D. Selumetinib decreases

CEMIP expression in Selumetinib-resistant and BRAFV600E-mutated colon cancer cells. HT-29/SR cells were untreated or stimulated with Selumetinib for 24 hours at indicated concentrations and both nuclear and cytoplasmic extracts were subjected to WB analysis. E. TCF4 is recruited at TCF binding sites on both the CEMIP promoter and intron 1 (see methods for details), as judged by ChIP assays using HT-

29/SR cells. Primers within exon 1 were randomly chosen and used as negative controls. Values for each primer pair were calculated as ratios between ChIP signals obtained with the anti-TCF4 (specific) and or

IgG (nonspecific) antibodies. F. Resistant colorectal cancer cell lines show elevated levels of pERK1/2 independent of the mutational status of BRAF and KRAS. Parental and Selumetinib-resistant

BRAFV600E-mutated (HT-29) and KRASG13D or G12A-mutated (HCT116 and SW480, respectively) cell lines were grown without Selumetinib for 48 hours. Cell extracts were subjected to WB analysis. G. Resistant

BRAFV600E-mutated cells show reactivation of HER3 and MET, as demonstrated by WBs. H. Resistant

HT-29 cells have a MEK1 somatic mutation. On top, identification of the MEK1 H119R mutation found in HT-29/SR cells. At the bottom, a representation of the MEK1 coding sequence with described somatic mutations of MEK1 in exons 2, 3 and 6.

Figure 5. CEMIP promotes acquired resistance to Selumetinib in BRAFV600E-mutated colorectal cancer cells. A. Selumetinib downregulates CEMIP expression in resistant HT-29 cells. Control or

CEMIP-depleted HT-29/SR cells were untreated or stimulated with Selumetinib at indicated concentrations for 12 hours and real-time PCR analysis was conducted on total RNA to assess CEMIP expression. B. CEMIP deficiency enhances Selumetinib-induced DNA damage in resistant HT-29 cells.

Control or CEMIP-depleted HT-19/SR cells were untreated or stimulated with Selumetinib at indicated concentrations for 24 hours. Protein extracts were subjected to WB analysis. C. CEMIP deficiency

induces apoptotic cell death by Selumetinib in resistant HT-29 cells. Control or CEMIP-depleted HT-

29/SR cells were untreated or treated with Selumetinib (3 μM) for 24 hours. Caspase-3/7 activity in control and unstimulated cells was set to 1 and caspase-3/7 activity in other experimental conditions were relative to the control. Data from three independent experiments performed in duplicate are shown.

D. CEMIP deficiency significantly enhances tumor regression induced by Selumetinib. A stable IPTG- inducible-control or CEMIP-depleted HT-19/SR cell line (2 x 106 cells) was injected subcutaneously into NOD/SCID male mice. Mice were treated with IPTG from day 3 post-injection and treated with

Selumetinib (20 mg/kg) from day 6 post-injection for two weeks, as described in the methods

(n=4/group). On top, representative images of tumors excised at day 20 post-injection. At the bottom, tumor weights were quantified. E. CEMIP deficiency increases E-Cadherin expression and impairs

Selumetinib-dependent HER3 expression in resistant HT-29 cells. Control or CEMIP-depleted HT-

29/SR cells were untreated or stimulated with Selumetinib at indicated concentrations for 24 hours.

Protein extracts were subjected to WB analysis.

Figure 6. CEMIP is an endosomal protein that binds MEK1 in resistant BRAFV600E-mutated HT-

29 cells. A. A pool of CEMIP is found in signaling endosomes. Protein extracts from HT-19/SR cells were biochemically fractionated on an OptiPrep gradient as described in methods and the resulting fractions were subjected to WB analysis. B. CEMIP mainly cofractionates with EEA1+ endosomes. A fractionation experiment in which some pellets enriched with organelles of interest were further separated into fractions by ultracentrifugation (see methods). Fractions were subjected to WB analysis.

C. CEMIP partially colocalizes with ER and endosomal markers. HCT116 cells were transfected with the SNAP-CEMIP construct and IF was conducted on resulting cells. Arrows indicate colocalization of

CEMIP with EEA1+ endosomes and with the ER using the PDI marker. D. CEMIP binds MEK1 in

resistant HT-29 cells. Protein extracts from parental and resistant HT-29 cells untreated or treated with

Selumetinib for the indicated periods of time were subjected to immunoprecipitation (IP) with anti-IgG

(negative control) or anti-MEK1 antibodies followed by WB analysis. Whole cell extracts (WCE) were also subjected to WB analysi. E. Enhanced MEK1 activation in resistant BRAFV600E-mutated HT-29 cells leads to disengagement from BRAF. Protein extracts from parental and resistant HT-29 cells treated or not with Selumetinib for the indicated periods of time were subjected to IP with anti-BRAF or anti-IgG antibodies followed by WB analysis carried out on the immunoprecipitates or on whole cell extracts.

Figure 7. Metabolomic reprograming regulated by CEMIP and Myc is linked to the acquired resistance to Selumetinib. A. Resistant organoids show metabolic reprogramming. The metabolomics profile was established in parental and resistant organoids and is presented as heatmap visualization and hierarchical clustering analysis of the top 50 compounds with p≤0.05, Student t-test. Rows: metabolites; columns: samples; color key indicates metabolite expression value (blue: lowest; red: highest). Data with triplicates is presented. B. CEMIP promotes the production of lactate and the synthesis of amino acids in Selumetinib-resistant ex-vivo organoids. The metabolomic profile was established in control and CEMIP-depleted resistant ex-vivo organoids and is presented as heatmap visualization and hierarchical clustering analysis of the top 50 compounds with p≤0.05, Student t-test. Rows: metabolites; columns: samples; color key indicates metabolite expression value (blue: lowest; red: highest).

Experiments were carried out in triplicates. C. Myc deficiency impairs CEMIP and ERK1/2 activation in

Selumetinib-resistant ex-vivo organoids. Extracts from control and Myc-depleted resistant ex-vivo organoids were subjected to WB analysis. D. CEMIP and Myc control the production of specific metabolites. The metabolic signatures established in control, Myc-depleted and CEMIP-depleted

Selumetinib-resistant ex-vivo organoids were compared. Control (CEMIP) and Control (Myc) experimental conditions represent Selumetinib-resistant ex-vivo organoids infected with control shRNA and used to compare CEMIP and Myc-depleted organoids, respectively. The data is presented as heatmap visualization and hierarchical clustering of the top 50 compounds with p≤0.05 Student t-test.

Rows: metabolites; columns: samples; color key indicates metabolite expression value (blue: lowest; red: highest).

A Parental B Parental C 4.5 Cleaved Caspase 3 Dapi *** 0.5 mm 4 Untreated Ki-67 3.5 Valproic acid / Valproic acid / kDa 3 CHIR999021 + CHIR999021 180 2.5 CEMIP 2 *** 130 1.5 *** 100 HSP90

1 12 0.5 Resistant 60 μm mRNA levels (fold induction) 0 0.5 mm Resistant Lgr5 CEMIP Dclk1 SOX9 Cleaved Caspase 3 Dapi Ki-67 Untreated organoids 60 μm 60 μm

14 *** DAPI DAPI 60 μm DCLK1 DCLK1 12 Organoids + inhibitors (6 days) 10 30 *** 60 μm 60 μm 8 25 Ki-67 6 Cleaved Caspase 3

4 20

2 15 DAPI DAPI

% of organoids > 0,5 mm 0 DCLK1 DCLK1

Parental Resistant 10 Percentage of cells of Percentage 5 F 70 *** D Organoids 60 Selumetinib (PM, 24 h) 0 50 Parental Resistant kDa 010,5 3 0 10,53 40 30 180 cells CD133+ CEMIP E 20 130 kDa PR

10 CD24+/ 55 of Percentage pMEK1 170 HER3 0 43 Parental Resistant 55 90 MEK1 Parental ResistantResistant BRAF 43 5 63,7 33,4 34,9 58,1 10 5 17 CD24+/CD133- CD24+/CD133+ 10 CD24+/CD133- CD24+/CD133+ 55 p4EBP1 pMEK1 4 4 45 10 10 10 55 3 17 4EBP1 10 3 MEK1 10 45 CD24PE-A 10 2 CD24PE-A 2 43 10 10 pERK1/2 100 HSP90 0 2,37 0,5 0 5,15 1,83 12 2 3 4 5 0 10 10 10 10 2 3 4 5 43 0 10 10 10 10 ERK1/2 CD133 APC-A CD133 APC-A 90 72 SOX9

72 Myc 55 100 HSP90

12 345 678

Parental Resistant

Figure 1 Downloaded from cancerres.aacrjournals.org on September 29, 2021. © 2018 American Association for Cancer Research. Author Manuscript Published OnlineFirst on June 18, 2018; DOI: 10.1158/0008-5472.CAN-17-3149 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. A B

no HT-29 isse

)noitcudni dloF( dloF( )noitcudni 1,2 rpxe ANR evitaleR ANR rpxe 1,0 0,8 0,6 0,4 *** 0,2

0 ShRNA CEMIP #2 Control C

Enrichment plot: KRAS.DF.V1_UP Enrichment plot: ERB2_UP.V1_UP

NES = 2.31 NES = 1.69 Enrichment score (ES)

Enrichment score (ES) Enrichment profile Hits Enrichment profile Hits shCTR shCEMIP HT-29 HT-29 shCTR shCEMIP HT-29 HT-29

Enrichment plot: LEF1_UP.V1_UP Enrichment plot: MEK1_UP.V1_UP

NES = 2.24 NES = 1.47 Enrichment score (ES) Enrichment score (ES) Enrichment profile Hits Enrichment profile Hits

shCTR shCEMIP shCTR shCEMIP HT-29 HT-29 HT-29 HT-29 D MIR1914 VTRNA2-1 SNORA21 SNORA21 RPL17-C8orf44 HIST1H4H HIST1H4E C8orf44-SGK3.SGK3 HIST1H2Al HIST1H2AK LOC285419 HIST1H4l TERC C19orf81 FGF8 RAB4B-EGLN2 STX16-NPEPL1 UBE2F-SCLY AKR1C4 RDH16 PLEKHF1 CDA VAV1 APOBEC3G SYS1-DBNDD2 SGK3 LOC100129216 SCARNA9 DIAPH3-AS1 URGCP-MRPS24 RPS10-NUDT3 TGIF2-C20orf24 IFITM1 MIR941-2.MIR941-3 SNORD128 SNORD88C SNORD119 SNORD888 MIR4648 SNORD19

TF NES (significance) #Targets #Motifs/Tracks MAX 6.564 199 9 MXI1 6.474 181 3 MYC 5.85 174 6 TFAP2C 5.124 130 18 Figure 2

A B Sh RNA Control Sh RNA CEMIP#2 Control ShRNAs CEMIP 5 5 9,0 76,0 10 27,0 - 49,8 10 CD24+/CD133- CD24+/CD133+ CD24+/CD133 CD24+/CD133+

4 4 10 10

200 μm 200 μm 3 3 10 10 Sh RNA CEMIP#3 Sh RNA CEMIP#5 CD24PE-A 2 CD24PE-A 2 10 10 0 5,85 9,1 0 15,7 7,49 2 3 4 5 0 102 103 104 105 0 10 10 10 10 CD133 APC-A CD133 APC-A 200 μm 200 μm

80 ** + Control *** ShRNAs *** + CEMIP 60 *** 60 180 CEMIP 50 130 40 100 40 HSP90 20

30 12

CD24+/CD133+ cells CD24+/CD133+ Percentage of Percentage 0 20 E ShRNAs Control CEMIP Number of organoids Number 10 4 CEMIP *** 0 COLO-205/P D 3 Control CEMIP#3 120 COLO-205/SR(0.3) ShRNAs CEMIP#2 CEMIP#5 ***

)lortnoc fo )lortnoc ** *** 2 C 100

80 Induction) (Fold 1 #1 *** ol laviv %( tr

ShRNAs mRNA Expression Relative ControlCEMIPCEMIP#2Con CEMIP#1CEMIP#2 60 0 *** P SR(0.3) Selumetinib (5 PM, 24 hours)

rus rus COLO-205 kDa +++ 40

lleC *** HER3 COL0-205 170 kDa 100 20 P SR(0.3) BRAF 180 CEMIP 55 pMEK1 0 130 40 000.01 .030.1 0.3 13 55 MEK1 Selumetinib (μM) 55 Į-Tubulin 40 12 pGSK3D COLO-205 43 FGkDa P SR(0.3) Selumetinib (PM, 24 hours) GSK3D/E 0,3 1 0,3 1 0,3 1 43 100 BRAF 0 0,5 0 0,5 0 0,5 55 pMEK1 pERK1/2 55 pMEK1/2 40 40 40 ERK1/2 55 55 MEK1 40 MEK1/2 40 72 40 Myc 55 pERK1/2 pERK1/2 40 40 SOX9 72 ERK1/2 40 ERK1/2 40 Cyclin D1 p-RSK1 34 70 72 34 Cyclin D2 Myc RSK1 55 70 180 180 40 CEMIP FRA-1 CEMIP 130 130 100 100 HSP90 55 Į-Tubulin HSP90 123456 12 123456789101112

Control CEMIP #2 ShRNAs Figure 3 CEMIP #1

)lortnoc fo %( lavivrus lleC lavivrus %( fo AB)lortnoc 140 C Cytoplasmic Nuclear HT-29/P CEMIP *** noiss HT-29 HT-29 120 HT-29/SR(1.5) 28 ** P SR(1.5) P SR(1.5) *** *** kDa 100 dloF )noitcudni 24 *** erpx HT-29 70 kDa p65 80 *** 20 P SR(1.5)

e ANR e 180 55 60 ** 16 CEMIP 130 55 BCL-3 12 40 evit 100 HSP90 40

20 8 40 c-JUN aleR ( ( 12 0 4 00.330.1 1 10 30 40 0 FRA-1 Selumetinib (PM) P SR(1.5) D 40 FRA-1 Selumetinib (PM, 24 hours) (long exposure) kDa 0 0,1 1 3 0 0,1 1 3 E 180 Site #1 Site #2 Site #3 Site #4 55 D-Tubulin CEMIP 130 100 NBS1 40 FRA-1 Exon 1 Exon 2 12 34 Promoter Intron 1 90 E-Catenin

100 IgG 70 p65 90 80 TCF4 0 hours Selumetinib 70 TCF4 70 TCF4 24 hours Selumetinib 60 50 40 c-JUN 40 30

130 enrichment Relative 20 E-Cadherin 10 0 70 Lamin B2 Site #1 Site #2 Site #3 Site #4 Negative Exon1 100 HSP90

12 34 56 78 G HT-29 HCT116 H HT-29/SR Cytoplasm Nucleus SR(1.5) SR(2) P P F HT-29 HCT116 SW480 kDa kDa P SR(1.5) P SR(2) P SR(1.5) pHER3 180 180 CEMIP 130 HER3 180 HT-29/P 180 IQGAP-1 180 pMET 100 BRAF 130

25 180 KRAS MET 130 15 55 H119R pMEK1/2 100 HSP90 40 55 40 MEK1/2 1234 Q56P K59del I99T I103N K104N C121S V211D 40 pERK1/2 I111N/C L115R/P E120D P124L/S G128D F129L F133L V2115P

40 ERK1/2

100 pRSK1 Helix C Activation loop 100 RSK1 Exon 2 Exon 3 Exon 6

70 pp65

70 p65

40 FRA-1

55 D-Tubulin 1234 5 6

Figure 4

ABKIAA1199 C CEMIP#2 8 ShRNA CEMIP#2 ShRNA Control Control *** Control CEMIP#2 ShRNA 1,4 *** *** )no 6

1,2 Selumetinib (μM) i

** kDa 0 0.1 1 3 00.11 3 t c

u 4 1,0 17 pH2A.X dni

0,8 dlo ** pDNA-PKcs

300 2 F 0,6 ( DNA PKcs 0,4 300 activity Caspase-3,7 (Fold induction) (Fold 0 0,2 55 α-Tubulin 03 12345678 Selumetinib (μM) Relative mRNA expression mRNA Relative 0 010.1 3 Selumetinib (μM) E HT-29/SR(1.5) D Control CEMIP#2 ShRNA Control Selumetinib (μM) kDa 00.11300.11 3 180 HER3 ShRNA

CEMIP Untreated 130 100 100 BRAF Control 25 KRAS 15 55

ShRNA pMEK1/2

CEMIP 40 Selumetinib 55 MEK1/2 40 2.0 pERK1/2 * ns 40 40 ERK1/2 1.5 *** * 100 pRSK1

1.0 100 RSK1 40 FRA-1 0.5

Tumor weight (g) 130 E-cadherin 180 0.0 CEMIP 130 Control Control ShRNA CEMIP CEMIP 100 HSP90 ++ 12345678

Selumetinib

Figure 5

Downloaded from cancerres.aacrjournals.org on September 29, 2021. © 2018 American Association for Cancer Research. Author Manuscript Published OnlineFirst on June 18, 2018; DOI: 10.1158/0008-5472.CAN-17-3149 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. AB12 34 567891011121314 Fractions kDa kDa 123456789101112 180 CEMIP 180 130 CEMIP 130 170 EEA1 170 EEA1 100 APPL1 72 26 Rab5 26 Rab5 26 Rab7 26 Rab7 100 APPL1 26 Rab11 72 72 90 LAMP2 55 PDI 26 Caveolin-1 55 C Flotillin-1 44 PDI SNAP-CEMIP SNAP-CEMIP DAPI 72 PDI PDI 55 20 μm 130 N-Cadherin 90

170 EGFR 20 μm 20 μm Merge 170 MET EEA1EEA1EEA1 SNAP-CEMIP SNAP-CEMIP DAPI 130 EEA1

72 SRC 55 20 μm 100 B-RAF 26 20 μm 20 μm K-RAS Merge 17 55 pMEK1/2 44 44 pERK1/2

D E B-RAF B-RAF IP IgG IgG MEK1 MEK1 IP IgG IgG Selumetinib (hours) Selumetinib (hours) kDa kDa 010 0102 2 00.5004002 .542 55 MEK1 180 CEMIP 44 130 IP IP 55 pMEK1/2 100 B-RAF 44 55 MEK1 100 44 B-RAF 55 55 pMEK1/2 pMEK1/2 44 44 55 55 MEK1/2 MEK1 44 44 WCE 44 pERK1/2 44 pERK1/2 WCE 44 ERK1/2 44 ERK1/2 180 CEMIP 170 130 CEMIP 130 100 HSP90 100 HSP90 12345678 123456789 10 HT-29 P HT-29 SR(1.5) HT-29 P HT-29 SR(1.5) Figure 6

A C

Class NAD Urate 1.5 kDa Oxypurinol ShRNAs Xanthine 180 Serine 1 CEMIP DKG Dodecanoyl-carnitine Class 130 Myristoyl-carnitine 0.5 Palmitoyl-carnitine 72 Thymidine P Cystathionine 0 Myc Acetyl-glutamine R 55 O-Phosphoethanolamine Guanine -0.5 CDP 43 pGSK3D/E GDP -1 AMP Adenosine UMP -1.5 CMP 43 GSK3D/E GMP ADP UDP-GlcNAc Coenzyme A Uridine diphosphate 43 pERK1/2 Aminoadipase Sedoheptulose-7-phosphate Fumarate Malate 43 PEP ERK1/2 Citrate GSH Fructose 1,6-diphosphate 100 Pantothenate HSP90 Proline Cystidine Lysine 12 34 Linolenic acid Top 50 T-test FAD 2 groups only Arachidonic acid Cis-aconitate Dihydroxyacetone p Glyceraldehyde 3-p Oleic acid Palmitoleic acid P – Parental S-Adenosyl-L-homoc Succinic acid R – Resistant Nicotinamide Ornithine GSSG P1 P2 P3 R1 R2 R3

B D CEMIP Control (CEMIP) ShRNAs CEMIPControl ShRNAs Myc#3y Myc#2y Control (y(Myc)

L-Sarcosine PEP Proline Butyryl-carnitine Aminoadipate Betaine Folate Ethanolamine Phosphate Glycerol 3-phosphate Eicosapentaenoic acid Lactate Arachidonic acid Oxypurinol 1.5 Aspartate Xanthine 1.0 Acetyl-carnitine Glutamine SuccinylCys 0.5 Myristoyl-carnitine Urate 0 Palmitoyl-carnitine L-Alanine Propionyl-carnitine -0.5 Citrulline Acetyl-carnitine Asparagine IsoLeucine -1.0 Carnitine Carnitine -1.5 Methionine Phenylalanine Threonine Leucine Tryptophane Acetyl-lysine Oxoadipate Oxoadipate Glycerol-3 phosphate Hypoxanthine Valine 2 Inosine Thymidine Proline Threonine Histidine 1 Argininosuccinate Asparagine Succinyladenosine Phenylalanine IMP IsoLeucine 0 Acetyl-glutamine Cystathionine Leucine aKG Glycine -1 UDP Acetyl-aspartate N CTP Acetyl-glutamine CDP Folate ADP L-Sarcosine -2 UTP ATP Aminoadipate Betaine Urate GTP Lactate NADP Thymidine N-carbamoyl-L-aspa Oxypurinol Pyruvate Xanthine PEP Citrulline NADH GSH Cystathionine Ethanolamine Phosphate Argininosuccinate O-Phosphoethanolamine SuccinylCys Creatine L-Alanine GDP Acetyl-lysine Aspartate Succinyladenosine Butyryl-carnitine Taurine Creatine GLN Serine Inosine IMP Arginine

Figure 7

The endosomal protein CEMIP links Wnt signaling to MEK1-ERK1/2 activation in Selumetinib-resistant intestinal organoids

Hong-Quan Duong, Ivan Nemazanyy, Florian Rambow, et al.

Cancer Res Published OnlineFirst June 18, 2018.

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