Supplementary Fig. S1 A All cancers (3.4%) PDAC (17.2%) CRC (1.1%) LAC (2.0%) G12 G12 G12 G12 G13 G13 G13 G13 Q61 Q61 Q61 Q61 84% N117 93% N117 68% N117 89% N117 A146 A146 A146 A146 G12S G12S G12S G12S G12R 17% G12R G12R 21% G12R 31% 28% G12C 35% G12C G12C G12C G12D G12D G12D G12D 42% G12A 43% G12A 44% G12A 13% 52% G12A G12V G12V G12V G12V G0/G1 B C S G2M 50 100 NS si K1 40 75 30 50 20 % cell cycle 25 % apoptotic cells apoptotic % 10 0 0 NS K1 NS K1 NS K1 NS K1 NS K1 NS K1 NS K1 NS K1 NS K1 NS K1 1 C 2 4 1 8 3 0 4 - . C- 8 -T3 K- C 4 3 2 3 0 a0 Ca P SN- P AN2 -1 4 A . -T -1 -8 N 4 5 sP 81 P C a0 I 18 P N K A C TC A P Pa A Hu P ATC4ATC5 P P M 2 8 u S P P AT A P P As a- A H P - P P A CC- CC MI T PaC T G12R Figure S1. KRASG12R is the third most prevalent mutation in PDAC. A, KRAS mutation frequency in PDAC, CRC and LAC. The top row provides the mutational frequency by amino acid position, and the bottom row provides the prevalence of G12 mutations in each cancer. B, Apoptosis assays of siRNA-transfected PDAC cells measured by Annexin V- FITC staining. Data are representative of two independent experiments. C, Cell cycle analyses of siRNA-transfected PDAC cells determined by staining with propidium iodide. At least 104 cells were collected for apoptosis and cell cycle assays, and data are representative of at least two independent experiments. Error bars, mean ± s.e.m. Supplementary Fig. S2 0.5 h 2 h 6 h 24 h A B * 200 ** Vector EV G12D RIE-1 G12V 150 G12R KRASG12D DAPI ; 100 ** KRASG12V * 50 FITC-Dextran *** Macropinocytotic index Macropinocytotic **** * ** KRASG12R 0 0.5 h 2 h 6 h 24 h RIE-1 CF I EV EV G12V G12V G12D G12R G12D G12R RIE-1 HA-KRAS HA-KRAS Vinculin Vinculin NIH 3T3 HPNE-DT Vector D Vector KRASG12D G Vector KRASG12D DAPI DAPI ; ; KRASG12D FITC-Dextran FITC-Dextran KRASG12V KRASG12V KRASG12R KRASG12V KRASG12R E H ** **** 20 30 **** *** 20 10 KRASG12R index 10 index 0 0 Macropinocytotic V V R Macropinocytotic E D 2 2 EV D 2V 2R NIH 3T312 1 1 HPNE-DT 12 1 1 G G G G G G Figure S2. Ectopic expression of KRASG12D and KRASG12V mutants elevate macropinocytosis in model systems. A, Time-dependent measurement of FITC-dextran labeled macropinosomes in KRAS- transformed RIE-1 cells. Macropinosomes, green; nuclei, blue, for the indicatedtime.Assayused 1 ml of 1:20 FITC-dextran/DMEM. B, Quantification of macropinocytosis index. Quantification performed with at least 50 cells per condition, and data are representative of two independent experiments. C, Immunoblot analysis of HA epitope-tagged KRAS expression levels in NIH/3T3 cells. D, FITC-dextran labeled macropinosomes in KRAS-transformed NIH/3T3 cells. E, Quantification of macropinocytotic index. Quantification of macropinocytosis index with at least 50 cells per condition, and data are representative of two independent experiments. F, Immunoblot analysis of HA epitope-tagged KRAS expression levels in HPNE-DT cells. G, FITC- dextran labeled macropinosomes in KRAS-transformed HPNE-DT cells. H, Quantification of macropinocytotic index. Quantification of macropinocytosis index with at least 50 cells per condition, and data are representative of three independent experiments. I, Brightfield microscopy images of RIE-1 cells transfected with KRAS mutants. Scale bar, 100 µm. For all data; ****P<0.0001, ***P<0.0002, **P<0.0021, *P<0.032, p values from Dunnett’s multiple comparison test after one-way ANOVA, comparing all lanes to G12R at each time point. Scale bar is 20 μm, unless otherwise noted. Error bars, mean ± s.e.m. Supplementary Fig. S3 A KRASG12R B HRASG12V HRASG12R (GMPPNP/Mg) SI SI SII T35 SII G12R G12V G12R E62 Q61 C D TM KRASG12R 100 WT: 52.25 C HRASG12R G12D: 57.25 C 80 G12V: 57.75 C G12R: 57.75 C 60 SII SI 40 Normalized G12R mGDP fluorescence 20 0 30 40 50 60 70 80 Temperature (C) Figure S3. KRASG12R structure is unique among G12 mutations. A, Overlay of the ribbon diagrams of KRASG12R (green) and HRASG12V (pink; PDB 3OIW). SI, light blue; SII, red. Stick models of G12V and G12R are shown. GMPPNP is shownwith phosphates colored orange. B, A ribbon diagram of the HRASG12R crystal structure with selected side chains represented as sticks. Carbons in Q61 and E62 are colored red, nitrogen is blue and oxygen light red. T35 carbons colored in light blue. Hydrogen bonds are shown as gray dashed lines. C, Overlay of the ribbon diagrams of HRASG12R (teal) with KRASG12R (green). All other coloring is the same as previous. D, Thermal denaturation of KRAS proteins as measured by mGDP binding. Supplementary Fig. S4 A B D KRAS KRAS E37G RAF RALGEF PI3K Vector G12D G12D/T35S G12D/E37G G12D/Y40C G12V G12V/T35S G12V/E37G G12V/Y40C C HA-KRAS KRAS 35 pERK 30 ERK 20 pAKT AKT 10 Vinculin 0 Macropinocytotic index Macropinocytotic RIE-1 S G C V EV D 7G 0C 37 40 35S 4 T35 T 3 G12 E Y G12 / /E /Y D/ D/ D/ V V V 12 E G12 G12 G12 G12 G G12 KRAS PDK1 mTORC2 PIP3 PI3Kα P P T308 AKT S473 mTORC1 PIP2 P P P P PRAS40 T246MDM2 S166FKHR T24 S6K T421 T389 F KRAS Effector WT G12D G12R CRAF-RBD 61.0 ± 0.53 nM 481 ± 10 nM 499 ± 23 nM RGL2-RA 3.52 ± 0.12 μM 5.28 ± 0.15 μM 4.47 ± 0.11 μM PLCε-RA 0.73 ± 0.04 μM 2.15 ± 0.20 μM 1.96 ± 0.19 μM PI3K 1.85 ± 0.07 μM 1.91 ± 0.20 μM No binding G12R G12R G12V G12V G12D G12D -FBS +FBS Figure S4. KRASG12R does not interact with p110α/p85 in vitro and in RIE-1 cells. A, Immunoblot analysis of RIE-1 cells transfected with HA epitope-tagged KRAS effector-specific mutants. Vinculin was used a total lysate control, and phospho-specific and total AKT and ERK levels were probed. B, A diagram of the expected signaling preference for the KRAS effector-specific mutants. C, Quantification of macropinocytosis in KRAS effector-specific mutants with at least 50 cells per condition, and data are representative of two independent experiments. Error bars, mean ± s.e.m. D, Heatmap of the relative RPPA intensity of 162 proteins in RIE-1 cells ectopically expressing KRAS mutants cultured in the absence or presence of serum. E, KRAS-PI3K effector signaling diagram displaying the expected phosphorylation sites downstream of PI3K activation in cells. F, Table of KRAS WT and mutant protein binding affinities to select effector RBD/RA domains and full length p110α/p85. Values were determined using the inhibition of nucleotide dissociation assay as described. All experiments were performed in triplicate or greater, and the results are the average binding affinity. Supplementary Fig. S5 A B **** NS **** 4.5 M1 si 3.0 M2 1.5 ** ** ** ** ** 1.0 ** ** Normalized 0.5 0.0 2D colony formation colony 2D AsPC-1 Pa04C A818.4 HuP-T3 PSN-1 PK-8 C AsPC-1 Pa04C A818.4 HuP-T3 PSN-1 PK-8 NS DAPI ; M1 siRNA FITC-Dextran M2 G12D G12V G12R Figure S5. KRASG12R cell lines are distinct from nonKRASG12R cell lines. A, Heatmap comparing RPPA protein phosphorylation/expression in KRASG12D and KRASG12R pancreas cancer cell lines under basal conditions. RPPA analysis of over 160 proteins cell lines with a native KRASG12R (6 cell lines, magenta) or non-KRASG12R (7 cell lines, cyan) mutation. B, 2D colony formation of MYC- silenced G12R-mutant PDAC. All cells were siRNA transfected for 48 h and allowed to proliferate for 10 days. Data are an average of four independent experiments. (***P<0.0002, **P<0.0021, *P<0.032, p values from Dunnett’s multiple comparison test after one-way ANOVA, comparing all lanes to NS for each treatment). Error bars, mean ± s.e.m. C,Imagesof FITC-dextran labeled macropinosomes with and without MYC silencing by siRNA. 70-kDa FITC-dextran, green; nuclei, blue. Scale bar, 20 μm. Supplementary Fig. S6 A DMSO Alpelisib IPI-549 Pictilisib B DMSO AsPC-1 30 AZD8186 G12D **** *** 20 * Pa04C G12V 10 0 A818.4 index Macropinocytotic AsPC-1Pa04C MIA A818.4 HuP- PSN-1 PK-8 TCC- PATC PATC G12R PaCa-2 T3 PAN2 43 50 G12R C Hup-T3 G12R PSN-1 G12R PK-8 G12R 5 TCC-PAN2 G12R D 4 3 PATC43 G12R 2 PATC50 Activity area 1 G12R 0 FITC-Dextran; DAPI E Figure S6. KRASG12R-mutant PDAC rely on p110γ for macropinocytosis and are sensitive to MEK/ERK inhibition. A, Images of FITC-dextran labeled macropinosomes of PDAC cell lines treated for 24 h with DMSO, alpelisib (p110α-selective inhibitor, 100 nM), IPI-549 (p110γ-selective inhibitor, 300 nM) or pictilisib (pan-p110 inhibitor, 1.5 µM) for 18 hrs. Images are representative from four independent experiments. B, Quantification of FITC-dextran labeled macropinosomes in PDAC cell lines treated for 18 h with DMSO or AZD8186 alpelisib (p110α/β/δ-selective inhibitor, 1.0 μM). C, Response to selumetinib (AZD6244) measured by IC50 values (in nM) in a pancreas cancer cell line panel.