Immunohistochemistry

Immunohistochemistry of AmotL2 in human tissues was performed in collaboration with http://www.proteinatlas.org. The following primary antibody was used: LDS-AmotL2 (polyclonal antibodies reactive to human AmotL2 C-terminal peptide and detecting both p60 and p100 isoforms [17]).

Cell culture

Madin-Darby Canine Kidney (MDCK; product number PTA-6500, ATCC), A549 (product number CCL-185, ATCC) and SW480 (product number 87092801, ECACC) cells were cultured in Dulbecco's Modified Eagle Medium (DMEM; product number D6429, Sigma‒Aldrich) supplemented with 10% fetal bovine serum (FBS, product number 10270106, Thermo Fisher Scientific) and penicillin/streptomycin (product number 15070063, Thermo Fisher Scientific).

MDCK cells stably expressing doxycycline-inducible p60AmotL2 were produced with the Gateway™ system (Thermo Fisher) as previously described [13]. To induce p60AmotL2 expression, doxycycline (Dox; product number D3447, Sigma‒Aldrich) was added to the culture at a final concentration of 10 ng/mL. SW480 p60AmotL2 shRNA cells were generated using lentiviral vectors as previously described [14]. A549 cells constitutively expressing p60AmotL2 and respective controls were established by lentiviral infection for 3 days followed by puromycin selection (2 µg/mL, product number A1113803, Thermo Fisher Scientific). Lentiviral vectors constitutively expressing p60AmotL2 were designed by VectorBuilder using vectors coding for BC011454 (mRNA) or AAH11454.1 (protein). The exact sequence used for p60AmotL2 expression was optimized by VectorBuilder (Vector ID VB210610-1214uwv) and can be provided upon request.

RNA-seq analysis

For RNA-sequencing, samples from the MDCK WT control (− Dox) group, MDCK WT control (+ Dox) group, MDCK p60AmotL2 control (− Dox) group and MDCK p60AmotL2 (+ Dox) group were collected. Each group consisted of quadruplicate samples to ensure data accuracy. The cells were seeded on 6-well plates and treated with or without doxycycline (10 ng/mL) for 24 h or 48 h and harvested using a Qiagen RNA extraction kit (Qiagen). All samples were processed using an RNA-seq pipeline by Novogene Co., Ltd. (Beijing, China), and RNA integrity was assessed using the RNA Nano 6000 Assay Kit on a Bioanalyzer 2100 system (Agilent Technologies, CA, USA). Subsequently, the resulting count files were employed as inputs for the DESeq2 package within the R environment. This package utilized a negative binomial distribution model to analyse differentially-expressed genes (DEGs). To enhance precision, P values were adjusted using the Benjamini and Hochberg approach. DEGs were identified based on an adjusted P value of less than 0.05 (q-value) and a log fold change greater than 0.5. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses were conducted using the clusterProfiler (v3.16.1) R package. Visual representations including volcano plots and bar plots were generated using the ggplot2 and the Enhanced Volcano R package.

Proteomic analysis

MDCK cells were lysed with 4% SDS lysis buffer and prepared for mass spectrometry analysis using a modified version of the SP3 protein cleanup and digestion protocol [21]. Peptides were labelled with TMT10-plex reagent according to the manufacturer’s protocol (Thermo Scientific) and separated by immobilized pH gradient—isoelectric focusing (IPG-IEF) on 3–10 strips as described previously [22]. Extracted peptide fractions from the IPG-IEF were separated using an online 3000 RSLCnano system coupled to a Thermo Scientific Q Exactive-HF. MSGF + and Percolator in the Galaxy platform were used to match MS spectra to the Ensembl_92 Homo sapiens protein database [23].

Western blotting (WB)

To prepare whole-cell lysates, cells were treated with a lysis buffer that contained 50 mM HEPES buffer, 150 mM NaCl, 1.5 mM MgCl2, 1 mM EGTA, 10% glycerol, and 1% Triton X-100 (product number X100, Sigma‒Aldrich), along with a freshly added 1 × protease inhibitor (product number 04693159001, Roche). This process was performed on ice. Next, the mixture was subjected to centrifugation at 15,000 rpm for a span of 3 min and the supernatant was collected. Lysates were then combined with SDS sample buffer (4X, product number 1225644, Novex) that was enhanced with a 10% sample reducing agent (product number 1176192, Novex). The proteins present were fractionated using a Bis–Tris precast polyacrylamide gel with a gradient of 4–12% (product number NP0322BOX, Novex). These fractionated proteins were then transferred onto a nitrocellulose membrane (product number 10401396, Whatman). To block any non-specific binding, the membrane was treated with a solution of 5% nonfat dry milk and 0.1% Tween 20 in phosphate-buffered saline (PBS) for an hour at room temperature. Following this, the membrane was incubated overnight at 4 °C with a primary antibody and was then subjected to treatment with a secondary antibody for an additional hour at room temperature. Finally, proteins that were labelled with antibodies were identified using a chemiluminescent substrate (ECL; product number RPN2232, Amersham) on an iBright imaging system (Thermo Fisher Scientific, USA). For primary antibodies we used LDS-AmotL2 (in-house polyclonal antibodies reactive to human AmotL2 C-terminal peptide and detecting both p60 and p100AmotL2 isoforms), β-actin (product number ab3280, Abcam) and Vinculin (product number 13901, Cell Signaling Technology). For secondary antibodies we used ECL anti-mouse IgG horseradish peroxidase (product number NA931V, Cytiva) and ECL anti-rabbit IgG horseradish peroxidase (product number NA934V, Cytiva).

Drug sensitivity and resistance testing

Cell lines were seeded using a Multidrop dispenser (Thermo Fisher Scientific) into pre-spotted 384-well plates (product number 6007668, PerkinElmer) containing 528 oncology compounds in five concentrations (FIMM oncology collection, FIMM High-Throughput Biomedicine Unit) or a custom drug library made with an Echo 550 (Beckman Coulter) [24]. MDCK cells were added at a final concentration of 2000 cells/well. Doxycycline was added to the cell culture media prior to dispensing. The drug plates were incubated for 72 h at 37 °C, after which cell viability was determined by adding CellTiterGlo (CTG; product number G7573, Promega) and measuring the luminescence signal with an EnSight plate reader (PerkinElmer). Dose response curves and drug sensitivity scores (DSS) were calculated with an in-house analysis pipeline termed Breeze [25]. Briefly, dimethyl sulfoxide (DMSO) and benzethonium chloride (BzCl) were used as negative and positive controls, respectively, to calculate the dynamic range of the assay for each plate, which was then used to calculate DSS values for each compound [26]. An initial primary screen with the 528 compounds tested over 5 different doses was performed. Selective drug sensitivity scores (sDSS) were used to quantify the selective response of p60AmotL2-expressing cells relative to non-expressing control cells. This was done by subtracting the DSS value for control cells from the DSS value of p60AmotL2-expressing cells for each compound [26]. The top 60 compounds in terms of sDSS values were selected for a second round of screening, where 9 doses per compound were used to make the dose‒response profiles instead of the initial 5. For these studies, custom plates were created from the Nordic Oncology Set by the Compound Center at Chemical Biology Consortium Sweden (CBCS), and the same in-house analysis pipeline was used to format the data for analysis in Breeze [25]. New DSS and sDSS values were calculated based on these results, and the compounds from the two top classes identified across both the primary screen and the validation screen were used for further validation.

Fluorescence staining of nuclei in 2D

Briefly, MDCK cells were seeded on 96-well plates (product number SW96G-EC-HTS, Cell Guidance Systems) at a density of 10 000 cells per well. Once cells reached approximately 60 to 70 percent confluency (approximately 2 days), compounds (BETi and RTKi) were added at different concentrations. After 72 h treatment, the cells were washed two times with 1 × PBS and fixed with 4% paraformaldehyde (PFA; product number sc-281692, Santa Cruz Biotechnology) for 10 min at room temperature. After two more rounds of washing with 1 × PBS, cells were incubated for 30 min at room temperature with NucBlue™ Live ReadyProbes™ Reagent (product number R37605, Thermo Fisher Scientific), diluted in 1 × PBS according to the manufacturer’s instructions, to stain the nuclei. Plates were imaged using a Leica Thunder Wide Field Fluorescence Microscope (Leica, Germany).

Validation of hit compounds in 2D and 3D using CellTiterGlo

For 2D cell viability testing, cells were seeded in 384-well plates (product number 6007668, PerkinElmer) at a density of 1000 cells per well in 20 µL growth media. Compounds were added the following day in 10 µL growth media and incubated for 72 h. DMSO and BzCl were used as negative and positive controls, respectively. Cell viability was determined by adding 30 µL of CellTiterGlo (CTG; product number G7573, Promega) and incubating for 15 min while protected from light. 3D viability testing was performed in parallel from the same cell stocks. We seeded approximately 500 cells per well in a 20 µL Geltrex (product number A1413202, Thermo Fisher Scientific) plus 10 µL growth media mix. Organoids were allowed to grow for 3 to 4 days before adding compounds in 10uL growth media for 72 h. The exception was SW480 cells, since these were grown in a collagen matrix instead of Geltrex, using the same seeding protocol as the one used for MDCK cells detailed in the method section below pertaining to the Immunofluorescence Staining of 3D Collagen Gels. We then added 40 µL of CellTiter-Glo® 3D Cell Viability Assay (product number G9681, Promega) and incubated for 30 min with gentle agitation, protected from light. The luminescence signals were measured with a Varioskan LUX microplate reader (Thermo Fisher Scientific, USA). The dose‒response curves and relative IC50 values were generated by using a dose‒response nonlinear regression model from GraphPad Prism (Dotmatics, USA).

Immunofluorescence staining of 3D collagen gels

Cells were seeded on 8-well chamber slides (product number 154534, Thermo Fisher Scientific) in a collagen matrix (PureCol type I collagen; product number 5005, Advanced Biomatrix) at a density of 500 cells per well. Compounds were added at different concentrations after five to seven days in culture. After 48 h of treatment, collagen gels underwent two washes in 1 × PBS and were then fixed in 0.5 mL of 4% paraformaldehyde (PFA; product number sc-281692, Santa Cruz Biotechnology) for 30 min. Afterwards, the gels were washed three times with 1 × PBS and permeabilized using 0.1% Triton X-100 (product number X100, Sigma‒Aldrich) for a duration of 15 min. After an additional two washes in 1 × PBS, the primary antibody, diluted in 5% normal horse serum, was added and allowed to incubate at 4 °C overnight. The gels were subsequently washed three more times in 1 × PBS and then incubated with the secondary antibody for 1.5 h at room temperature. Following another three washes in 1 × PBS, the gels were prepared for imaging by removing the chambers from the slides and using Fluoroshield™ with DAPI (product number F6057, Sigma‒Aldrich) to carefully mount the gels between the slides and coverslips. Cleaved Caspase-3 (product number 9661, Cell Signaling Technology) was used as the primary antibody and Alexa Fluor 488 anti-rabbit (product number A21441, Thermo Fisher Scientific) was used as the secondary antibody. Gels were also stained with Phalloidin-Atto 647 N (product number 65906, Sigma‒Aldrich) to visualize actin filaments. Confocal images were captured using an LSM 700 microscope (Zeiss, Germany) and all image processing was carried out using ImageJ software.

Patient-derived colon organoid culturesIsolation and culture of colon organoids

Fresh samples from surgically resected colon tumor specimens and paired healthy colon tissues were obtained from the Department of Clinical Pathology and Cancer Diagnostics at Karolinska University Hospital, Stockholm, Sweden. Experimental procedures and protocols were approved by the regional ethics review board (Etikprövningsnämnden) in Stockholm. Clinical information for each patient specimen involved in this study is listed in Supplemental data 6. The tissue samples were washed with ice-cold PBS containing 1% penicillin–streptomycin (Gibco) repeatedly until the solution became clear. The tissues were then minced into small fragments (1–2 mm) and incubated with Gentle Cell Dissociation Reagent (product number 100-0485, Stemcell Technologies) on a shaker (~ 20 rpm) for 15 min followed by centrifugation. After carefully removing the supernatant, the remaining tissues in the bottom were resuspended in cold PBS with 1% BSA and filtered through a 70 μm cell strainer to remove undigested tissue debris. The processed suspension was centrifuged, and the resulting pellet was mixed with Geltrex (product number A1413202, Thermo Fisher Scientific) at a 1:1 ratio. 50 µl of Geltrex-organoid mixture was then plated in pre-warmed 24-well plates and allowed to solidify at 37 °C. IntestiCult™ Organoid Growth Medium (Human) (product number 06010, Stemcell Technologies) with Y-27632 (product number 72302, Stemcell Technologies) was gently added to the wells. The organoids were cultured in a humidified incubator at 37 °C with 5% CO2 and the medium was changed every 3 days.

Lentiviral infection and generation of stable PDO models

A third-generation lentiviral transduction system was used to establish stable p60AmotL2-expressing organoids. p60AmotL2 and the respective control plasmids were co-transfected with plasmids expressing virus coat and assembly proteins (REV, RRE, and VSVG) into 70% -80% confluent HEK293T cells using Lipofectamine 3000 (product number L3000001, Invitrogen). Conditioned media were collected after 24, 48 and 72 h, filtered through 0.45 μm low protein binding membranes (Sarstedt) and later concentrated at 3200 xg for 15 min. The supernatant media containing virus particles were then concentrated using a PEG Virus Precipitation Kit (product number ab102538, Abcam) and stored at \(-\) 80 °C for future use. Colon organoids were dissociated into small clusters using Gentle Cell Dissociation Reagent (product number 100-0485, Stemcell Technologies) and resuspended in culture medium with concentrated lentiviral particles. The organoid suspension was then mixed with Geltrex (product number A1413202, Thermo Fisher Scientific) and plated in a 24-well plate. The growth media was replaced every other day. Protein expression was examined by western blotting and fluorescence microscopy. The selected organoids were expanded by passaging and maintained in culture for further experimentation.

Drug screening using stable PDO models and cell lines in 3D

Stable PDO models were plated in 384-well plates (product number 6007668, PerkinElmer) at approximately 500 cells per well in a 20 µL Geltrex (product number A1413202, Thermo Fisher Scientific) plus 10 µL growth media mix. Organoids were allowed to grow for 3 to 4 days before adding compounds in 10 µL growth media for 72 h. We then added 40 µL of CellTiter-Glo® 3D Cell Viability Assay (product number G9681, Promega) and incubated for 30 min with gentle agitation, protected from light. The luminescence signals were measured with a Varioskan LUX microplate reader (Thermo Fisher Scientific, USA). The dose‒response curves and relative IC50 values were generated by using a dose‒response nonlinear regression model from GraphPad Prism (Dotmatics, USA).

Nascent RNA sequencing using Global Run-on sequencing (GRO-seq)Pulse labelling of RNA

MDCK cells were seeded in 10 cm dishes at a confluency of 500 000 cells per dish. After 48 h, Dox was added overnight to the culture at a final concentration of 10 ng/mL to induce p60AmotL2 expression. Cells were then treated with 5 μM iBET151 or DMSO vehicle control for 3 h, after which 0.5 mM (final concentration) 5’-ethynyl uridine (EU; Click-it Nascent RNA Capture kit, product number C10365, Invitrogen) was added for 30 min to the cells to label newly synthesized (nascent) RNA. Cells were then washed briefly for three times with 1 × PBS and, after detachment with trypsin, pelleted for 5 min at 500 xG and lysed with Cell Disruption Buffer (product number AM1921, Invitrogen) for 5 min on ice. Total RNA was extracted using PARIS kit (product number AM1921, Invitrogen) according to the manufacturer’s instructions.

Streptavidin pulldown of labelled RNA

The Click-it reaction to biotinylate the labelled RNA was performed according to manufacturer’s instructions with minor modifications (Click-it Nascent RNA Capture kit (product number C10365, Invitrogen). Briefly, 1000 ng of EU-labelled RNA was added to a buffer containing CuSO4 and biotin azide for biotinylation of the ethynyl group. The reaction started with addition of Additive 1 from the kit and took place for 3 min before being ceased by adding Additive 2. After 30 min of incubation in a rotator at RT, biotinylated RNA was precipitated overnight in a solution of ethanol and LiCl2 in a -80 °C ultrafreezer. After isolation (20 min centrifugation at 14,000 RPM, followed by two washes in 70% ethanol), biotinylated RNA was purified using streptavidin beads provided by the kit. Incubation of 60 min at RT was followed by ten washes as instructed in the user’s guide, and the captured RNA adhered to the beads was used for library preparation. As a control, click-it reaction and pull down was performed on unlabelled RNA isolated from MDCK cells with no EU treatment.

Library generation and sequencing

Lexogen’s QuantSeq 3’ mRNA library preparation kit FWD for Illumina (product number 015, Lexogen) with i7 indices was used for library preparation following the manufacturer’s protocol for low input RNA samples, with minor modifications. Briefly, the 5 μl of resuspended beads with 1 µl of diluted (1/10000) ERCC ExFold RNA Spike-In Mix (product number 4456740, Thermo Fisher Scientific) was used as starting material for the first strand synthesis reaction, followed by RNA removal and second strand synthesis reaction according to the manufacturer’s instruction. The converted dsDNA library was carefully removed from the beads by using a magnetic rack. These libraries were further purified using purification beads provided with the kit, followed by library amplification with i7 indices and repurification. The quality and fragmentation of purified libraries was assessed on a Bioanalyzer (Agilent Technologies, USA). Sequencing was performed on Novogene’s (Cambridge, United Kingdom) sequencing platform Novaseq 6000 (Flowcell S4, software version V1.7, reagent V1.5) using a Pair-end 150 sequencing strategy (50 M read depth).

Data processing

Data analysis was performed by Lexogen’s proprietary Data Analysis Pipeline (DAP) with default parameters for QuantSeq (https://www.lexogen.com/quantseq-data-analysis/). In brief, adapters and low-quality reads were filtered by Cutadapt (1.18) (https://cutadapt.readthedocs.io/en/stable/). Differential expression analysis on gene counts based on full gene coordinates and exon coordinates was performed using DESeq2 (1.18.1) [27] and Canis lupus familiaris Ensembl 107 (https://ftp.ensembl.org/pub/release-107/fasta/canis_lupus_familiaris/, https://ftp.ensembl.org/pub/release-107/gtf/canis_lupus_familiaris/) as a reference genome.

For this, clean reads were aligned to the reference genome and ERCC RNA spike-in fasta (Thermo Fisher Scientific) with STAR aligner (2.6.1a) (https://github.com/alexdobin/STAR) [28] using custom parameters summarized in the table as follows:

Parameter

Value

outFilterMismatchNoverLmax

0.6

alignSJoverhangMin

8

alignEndsType

Local

outFilterMultimapNmax

200

alignSJDBoverhangMin

1

outFilterMismatchNmax

999

alignIntronMin

20

alignIntronMax

1,000,000

alignMatesGapMax

1,000,000

limitOutSJcollapsed

5,000,000

Gene counts from uniquely aligned reads were obtained using subread’s featureCounts (1.6.4) (https://subread.sourceforge.net/). Significant genes identified based on exon feature unique counts from differential gene analysis were proceeded for Gene ontology (GO) enrichment analysis. Assessment for all relations such as Molecular Function, Cellular Component, Biological Process (< 0.05 p-adj, BH) was performed using ClusterProfiler R package (v4.10.0) [29], universe baseMean > 1. For the Gene Set Enrichment Analysis (GSEA) and the pathway analysis msigdbr (7.5.1) and ReactomePA (1.46.0) R packages were used, respectively.