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. TCC-PAN2 G12R PATC50 PATC43 A AsPC-1 Hup-T3 G12R G12R A818.4 Pa04C G12D G12R PSN-1 G12R G12V G12R G12R PK-8 Supplementary Fig. S6 DMSO FITC-Dextran leii P-4 Pictilisib IPI-549 Alpelisib ; DAPI B C
E D Macropinocytotic index 10 20 30
Activity area 0 0 1 2 3 4 5 sC1a4 MIA AsPC-1Pa04C **** AZD8186 DMSO * PaCa-2 *** 884HuP- A818.4 T3 S- P- TCC- PK-8 PSN-1 G12R PAN2 PATC 43 PATC 50 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. The most sensitive cell line is shown on the left (N≥3 with SEMs; in triplicate). D, Response to selumetinib measured by the activity area. Activity area in 52 PDAC cell lines derived from 10-concentration dose response curves (N≥3 per cell line; in triplicate). Average Activity area (AA), as a measurement of maximum response and potency, aligned with
KRAS mutation information from COSMIC (if available). AA integrates potency (EC50 or IC50) and maximum response (Amax) at the highest drug concentration. E, Genetic mutations in 52 pancreatic cancer cell lines. The 250 most common cancer genes afflicted by somatic mutations were sequenced and genes with mutations found in at least two cell lines are shown Supplementary Fig. S7
A 6 G12D: G12R: 5 PF0282 PF0317 PF0195 hT2 4 PF0323 PF0301 PF0362 PF0049 3 hM1A PF0290
Area Area under the curve 2 G12D G12R B KRAS mutation
1.5 PF0195 1.5 PF0282 1.5 PF0362 1.5 PF0323 1.5 hM1A
IC50: 0.266 M 1.0 1.0 1.0 1.0 1.0 AUC: 3.47
0.5 0.5 0.5 0.5 0.5 IC : 1.65 M IC50: 2.92 M IC50: N/A IC50: 0.596 M 50 relative to DMSO to relative relative to DMSO to relative relative to DMSO to relative relative to DMSO to relative Fractional viability Fractional relative to DMSO to relative Fractional viability Fractional Fractional viability Fractional Fractional viability Fractional AUC: 4.37 viability Fractional AUC: 4.62 AUC: 3.81 AUC: 4.18 0.0 0.0 0.0 0.0 0.0 -3 -2 -1 0 1 -3 -2 -1 0 1 -3 -2 -1 0 1 -3 -2 -1 0 1 -3 -2 -1 0 1 Log [ERKi] (M) Log [ERKi] (M) Log [ERKi] (M) Log [ERKi] (M) Log [ERKi] (M) C
1.5 PF0049 1.5 PF0290 1.5 PF0301 1.5 PF0317 1.5 hT2 I C : 0.246 M 50 IC : 0.0839 M AUC: 3.41 50 1.0 1.0 AUC: 2.96 1.0 1.0 1.0
0.5 0.5 0.5 0.5 0.5 IC50:0.836 M IC50: 4 M IC50: 0.106 M relative to DMSO to relative relative to DMSO to relative relative to DMSO to relative Fractional viability Fractional relative to DMSO to relative relative to DMSO to relative Fractional viability Fractional viability Fractional Fractional viability Fractional Fractional viability Fractional AUC:3.92 AUC: 4.64 AUC: 3.39 0.0 0.0 0.0 0.0 0.0 -4 -3 -2 -1 0 1 -4 -3 -2 -1 0 1 -4 -3 -2 -1 0 1 -3 -2 -1 0 1 -4 -3 -2 -1 0 1 Log [ERKi] (M) Log [ERKi] (M) Log[ERKi] (M) Log [ERKi] (M) Log [ERKi] (M)
Figure S7. KRASG12R organoids are sensitive to ERK MAPK inhibition. A, Plot of the area under the curve for KRASG12D- and KRASG12R-mutant pancreas cancer organoids. AUC calculated from three independent experiments. The difference was not significant. B, Cell viability curves of KRASG12D-mutant organoids treated with ERKi for 10 days. Data are the average of three independent experiments. C, Cell viability curves of KRASG12R-mutant organoids treated with ERKi for 10 days. Data are the average of three independent experiments. Supplementary Fig. S8
sDSS with CTG for all inhibitors hT2(G12R) hM1A (G12D) B A 3 Mutation Mutation 100 AZ 3146 G12R 100 Niraparib 2 GSK−J4 Non−G12R MST−312 Selinexor SGC0946 1 UNC0638 AT 101 CUDC−907 Romidepsin 0 Bortezomib Carfilzomib 50 50 ONX−0914 −1 BCI Omacetaxine Auranofin % survival SH−4−54 −2 % survival Dactolisib Gedatolisib AZD8055 −3 NVP−BGT226 Ridaforolimus 0 0 Temsirolimus AVN944 PTC−209 PFI−1 -8 -7 -6 -5 -8 -7 -6 -5 I−BET151 JQ1 Atorvastatin PI3Ki (M) Simvastatin PI3Ki (M) SGC−CBP30 DMSO SGI−1776 DMSO PF−670462 Regorafenib LY3009120 GDC−0623 333 nm ERKi SCH772984 0 nM PI3Ki alone 37 nM ERKi PD0325901 Trametinib Ulixertinib 1000 nM ERKi Selumetinib 111 nM ERKi Binimetinib Cobimetinib AZD1208 MK−8745 Quisinostat Abexinostat Belinostat PI3Kγi PI3Kγi Panobinostat C Vorinostat Entinostat Mocetinostat DMSO (555 nM) DMSO (555 nM) Cilengitide PF−00562271 VS−4718 Dabrafenib Encorafenib PF−06463922 Idelalisib Amuvatinib Darapladib AT−406
PF−03758309 DMSO BX−912 DMSO Tipifarnib Mepacrine Chloroquine Palbociclib Ribociclib Pazopanib Tivozanib Bryostatin 1 Buparlisib Omipalisib ZSTK474 ERKi Copanlisib ERKi Pictilisib (111 nM) Amsacrine (111 nM) Valrubicin CEP−37440 Givinostat Birinapant Cladribine G12R G12D Resminostat hT2 (KRAS ) hM1A (KRAS ) Tacedinal in e Galiellalactone Indibulin BAY 87−2243 NMS−873 Crenolanib Bexarotene D E Tret in oin Cisplatin 15 Taselisib ASP3026 Danusertib **** Bleomycin Tivantinib Sabutoclax Verdinexor **** TAK−901 ENMD−2076 10 Fedratinib Dactinomycin KRAS Oxaliplatin 8−amino−adenosine Daunorubicin mutation Vector Idarubicin G12D G12V G12R AT9283 Clofarabine GSK−461364 5 FRAX486 KRAS Osimertinib Autophagic flux Alisertib (mCherry/eGFP) Toz as er tib Filanesib SB 743921 GAPDH Daporinad Thioguanine Etoposide 0 Gemcitabine Luminespib Onalespib EV G12D G12V G12R BIIB021 CUDC−305 Triciribine Clomifene Doramapimod 4−hydroxytamoxifen Fluorouracil Cytarabine 8−chloro−adenosine KRAS mutation Floxuridine Empty Prexasertib PF−00477736 UCN−01 F AZD7762 Vector G12D G12V G12R Nintedanib Mitomycin C Tas qu inimo d AZD−5363 Lucitanib AZ191 PHA 408 Ruboxistaurin Aldoxorubicin Doxorubicin VER 155008 Rocilinostat EGFP Filgotinib Mitoxantrone AT7519 SNS−032 Alvocidib Dinaciclib Pevonedistat Momelotinib Oprozomib Tosedostat GSK269962 Masitinib Merestinib
Ponatinib mCherry Methotrexate Plicamycin SN−38 Topot ecan AZD1152−HQPA Abemaciclib AZD−5438 Pacritinib Milciclib Silmitasertib BI 2536 Volasertib GSK923295 Merge TH588 Pemetrexed Raltitrexed Carboplatin Tal azo parib Rociletinib Docetaxel Rigosertib Vinblastine NVP−AEW541 BMS−754807 Linsitinib A−1210477 Cabazitaxel Paclitaxel ABT−751 Vincristine Vinorelbine BGB324 LY−2874455 Canertinib Dacomitinib Bosutinib Dasatinib Saracatinib Gandotinib Ralimetinib MDA−PA PK8 A818.4 TCCPAN2 PaCa−2 MIA HPAF−II Pa14C CFPAC−1 Capan−2 Pa18C Pa04C AsPC−1 HPAC MDA−PA Pa01C SW1990 −1 NC PA Pa03C Pa02C Pa16C PSN1 HuP−T3 MDA−PA T T T C43 C50 C53 Figure S8. KRASG12R PDAC cell lines are fundamentally different from nonKRASG12R cell lines. A, 525-drug sensitivity and resistance testing of multiple PDAC cell lines expressing KRASG12R and other KRAS mutations. Cell viability was measured using cell titer glo and response to each inhibitor was averaged and ranked according to its deviation from the average. Data are an average of three independent experiments. B, Cell viability of PDAC organoid cultures co-treated with ERKi (SCH772984) and PI3Kγi (IPI-549). Organoids were cultured for 10 days. Data are the average of two independent experiments. C, Bright field images from B. Right, hT2 (KRASG12R); left, hM1A (KRASG12D). D, Ectopic expression of KRASG12R in RIE-1 cells dos not suppress autophagy. RIE-1 cells were transduced to stably express KRAS and mCherry-EGFP-LC3B. Autophagic index indicates the ratio of the areas of mCherry+ punctae to EGFP+ punctae. Mean autophagic index is plotted, with each individual data point representing one field containing at least five analyzed cells. Data are representative of three 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 EV). Error bars, mean ± s.e.m. E, Immunoblot analysis of KRAS-expressing RIE-1 cells used in A. F, Representative confocal images of EGFP, MCHERRY and merged channels used to quantify the autophagic index, used in D. For D-F, EV and G12V were previously reported (1).
1. Bryant KL, Stalnecker CA, Zeitouni D, Klomp JE, Peng S, Tikunov AP, et al. Combination of ERK and autophagy inhibition as a treatment approach for pancreatic cancer. Nat Med 2019;25:628-40.