Fig 2: Loss of ICMT function obliterates the ability of established cancer cell lines carrying oncogenic mutant RAS to form tumors in vivo. (a) Schematic illustration of the predicted polypeptide sequences for each of the three alleles present in MiaPaca-2 cells expressing wild-type ICMT, as well as the truncated polypeptides predicted from each of the three mutant alleles in individual ICMT-null MiaPaCa-2 clones. The sequences of the mutated alleles are shown in Supplementary Table S2. The color scheme is the same as that in Figure 2b. (b) Growth of ICMT-wild-type MiaPaCa-2 xenografts. Tumor sizes were measured at 3–7 day intervals after implantation as shown; each trace denotes 1 tumor (n=3). (c) Measured sizes of ICMT-null MiaPaCa-2 implants from ICMT-null 1cr (n=3), ICMT-null 3cr (n=3), ICMT-null 4cr (n=3) cell lines. 3 × 106 cells were implanted for each MiaPaCa-2 xenograft, both ICMT wild-type and ICMT-null. (d) The mean final tumor size at the last measured time point for both ICMT wild-type and ICMT-null MiaPaCa-2 xenografts is shown. Two-tailed unpaired Welch’s t-test for unequal variances was performed; P=0.005 (**). (e) Photograph of a representative mouse showing the tumors at the time of killing. Red arrow: MiaPaCa-2-ICMT-wild-type tumor; blue arrow: remnant of the MiaPaCa-2-ICMT-null 4cr injection. (f) A similar schematic to (a) denoting the predicted polypeptide translated from each of the three alleles present in MDA-MB-231 expressing wild-type ICMT, as well as the truncated polypeptides translated from each of the three mutant alleles in individual MDA-MB-231 ICMT-null clones. The sequences of the mutated alleles are shown in Supplementary Table S2. Color scheme is the same as that in Figures 2b. (g) Growth of ICMT-wild-type MDA-MB-231 xenografts. Tumor sizes were measured every 3-7 days as indicated; each trace denotes 1 tumor (n=4). (h) Measured sizes of ICMT-null MDA-MB-231 xenografts resulting from the ICMT-null 1cr (n=4), ICMT-null 3cr (n=4), ICMT-null 4cr (n=4) lines. 1 × 107 cells were implanted for each MDA-MB-231 xenograft, both ICMT-wild-type or ICMT-null. (i) The mean final tumor size at the last measured time point for both ICMT-wild-type and ICMT-null MDA-MB-231 xenografts is shown. Two-tailed unpaired Welch’s t-test for unequal variances was performed; P<0.0001 (****). (j) Photograph of a representative mouse showing the tumors at the time of killing. Red arrow: MDA-MB-231-ICMT-wild-type tumor; blue arrow: MDA-MB-231-ICMT-null 4cr tumor.

Fig 3: ICMT function is critical for G12V-mutant RAS transformation both in vitro and in vivo. (a) Constitutively active (CA) KRAS4B-G12V and NRAS-G12V were introduced into HME1-shp53-ICMT-wild-type, ICMT-null 1cr and ICMT-null 4cr cells; GFP-tagged ICMT was also introduced into a subset of ICMT-null 1cr and 4cr cells to rescue ICMT activity. Cells were cultured in soft agar for 8 days, and subsequently stained using MTT. The image for each condition is a representative microscopic field from the five fields taken for each condition. (b) Cell lines from (a) were cultured for 48 h and lysates were used for immunoblot analysis. Lamin A antibody (sc-6214) was from Santa Cruz Biotechnology. Results presented in (a) and (b) are from a single experiment that is representative of three similarly performed studies. (c, g) 5 × 106 HME1-shp53-ICMT-wild-type cells expressing NRAS-G12V (c) or KRAS4A-G12V (g) were implanted into SCID mice (InVivos, Singapore) for subcutaneous xenografts following a previously established protocol.14 Tumor sizes were measured every 3-7 days as indicated until mice were killed. Each tracing on the plot represents 1 tumor (n=5 each for NRAS-G12V and KRAS4A-G12V groups). (d, h) 5 × 106 HME1-shp53-ICMT null cells expressing NRAS-G12V (d) or KRAS4A-G12V (h) were implanted on the contra-lateral side of the same mice as their ICMT-wild-type counterpart cells in (c) and (g), respectively. Sizes of the implants derived from HME1-shp53-ICMT-null cells were similarly monitored and plotted. n=5 for ICMT null NRAS-G12V and n=6 for KRAS4A-G12V groups, divided between 1cr and 4cr. (e, i) The mean final tumor sizes at the last measured time point for both ICMT-wild-type and ICMT-null HME-1-shp53 xenografts are shown; NRAS-G12V (e), KRAS4A-G12V (i). Two-tailed unpaired Welch’s t-test for unequal variances was performed; P=0.001 (**) for NRAS-G12V (e), P<0.001 (***) for KRAS4A-G12V (i). (f, j) Photograph of a representative mouse from NRAS-G12V (f) or KRAS4A-G12V (j) group, showing the tumors at the time of killing. The time of killing of each mouse was determined as that when a tumor reaches the size of 2 cm3 (all from ICMT-wild-type cells in this study), following the institutional IACUC guidelines. Red arrow: HME1-shp53-ICMT-wild-type tumor; blue arrow: remnant of the HME1-shp53-ICMT-null 1cr injection. All animal studies are performed in compliance with the protocol that has been approved by the Institutional Animal Care and Use Committee; randomization and blinding were not required for this study.

Fig 4: HME1-shp53 cells can be transformed by all four RAS-G12V mutant isoforms, but not by their wild-type counterparts. (a) Illustration of the scheme for creation of the HME1-shp53 cell line by sequential addition of SV40 small T antigen, shRNA against p53, and ER-tagged wild-type or G12V-mutant RAS isoforms into HME1-hTERT cells.26, 27 The parental HME1-hTERT (ATCC CRL-4010™) cells were obtained from American Type Cell Culture (ATCC) and mycoplasma-free. Stable expression of genes was achieved by transduction with retroviral expression vectors pBABE-hygro (for SV40 Small T Antigen), pRetroSuper (for shRNA against p53) or pBABE-puro (for all forms of RAS)28, 29 with viruses generated via calcium phosphate transfection,30 followed by stable selection using 20 µg/ml hygromycin for pBABE-hygro (Invitrogen, Carlsbad, CA, USA), 5 µg/ml blasticidin for pRetroSuper (Sigma Aldrich, St Louis, MO, USA), and 5 µg/ml puromycin (Sigma Aldrich) for pBABE-puro. Ras isoforms were cloned using primers listed in Supplementary Table S1. G12V mutations were introduced using QuikChange Site-Directed Mutagenesis Kit (Agilent Technologies) and the targeting oligonucleotides are listed in Supplementary Table S1. (b) HME1-shp53 cells expressing wild-type or ER-tagged G12V mutant of KRAS4A, KRAS4B, HRAS and NRAS as shown in (a) were cultured in soft agar in the presence or absence of 500 nM tamoxifen (Sigma Aldrich) for 14 days, and subsequently stained using methylthiazolyldiphenyl-tetrazolium bromide (MTT; Sigma Aldrich). This three-dimensional anchorage-independent soft agar culture was performed according to a previously published protocol25 supplemented with 0.5 µg/ml hydrocortisone (Sigma Aldrich), and 5 µg/ml insulin (I-DNA Biotech, Singapore). Using an Olympus SZX16 stereo microscope with 3.2 × zoom and 0.5 × objective, pictures of 4–5 random microscopy fields were taken per well after MTT staining; one representative field for each condition from one representative experiment out of three performed is presented. (c) Immunoblot study of cell lysates from HME1-shp53 control cells and those expressing RAS proteins. The cell lysates were prepared from the same cells used for the soft agar study in (b) and HME1-shp53 was used as the negative control. Immunoblots were done on cell lysates per standard protocol established in the laboratory.12 The antibodies for pan-RAS (Ras10, MA1-012) and GAPDH (14C10, #2118) were from Pierce Scientific (Waltham, MA, USA) and Cell Signaling Technology (Danver, MA, USA), respectively. Results presented in (b) and (c) are from a single experiment that is representative of three similarly performed studies.

Fig 5: PPI of CaM and Myc family membersWhole cell extracts of distinct cell lines and tissues were used in CaM-agarose (CaM-ag) pull-down experiments. A. For comparison, authentic CaM binding partners were analyzed first. Endogenous CaM-dependent protein kinase from whole mouse brain extracts was detected using monoclonal antibodies directed against a-CAMKII. Ras proteins were analyzed in extracts from the human colon adenocarcinoma cell line SW480 using monoclonal anti-pan-Ras antibodies. B. To analyze the binding of distinct Myc-family members to CaM, HA-tagged v-Myc, N-Myc, and L-Myc proteins were overexpressed in QT6 cells. Whole cell extracts were prepared and subjected to CaM-ag binding assays. Proteins eluted from the affinity matrix (upper panel) and aliquots of whole cell extracts (lower panel) were analyzed by immunoblotting using HA-specific antibodies. C. Endogenous c-Myc proteins were analyzed in CaM-ag binding assays using extracts from SW480 cells and monoclonal anti-c-Myc antibodies. For direct comparison, Ras proteins were analyzed from the same extracts using monoclonal anti-pan-Ras antibodies. GAPDH was used as a negative control, using monoclonal anti-GAPDH antibodies. Input: 0.25% (Ca2+), or 0.1% (EDTA). D. Analyses as in (C) of endogenous c-Myc proteins from the human embryonic kidney cell line HEK293. All binding assays were done in the presence of 2 mM EDTA, or 0.5 mM CaCl2 (Ca2+). E. Left panel: CaM-ag binding analysis of endogenous c-Myc proteins from HEK293 cell extracts containing 2 mM EDTA and supplemented either with no metal ions (no Me2+), or an excess (8 mM) of Ca2+ or Mg2+, respectively. Right panel: Analysis as before with variation of the excess Ca2+ concentration.