Journal: Cancer research communications

Article Title: Inhibitor of the Nuclear Transport Protein XPO1 Enhances the Anticancer Efficacy of KRAS G12C Inhibitors in Preclinical Models of KRAS G12C–Mutant Cancers

doi: 10.1158/2767-9764.crc-21-0176

Figure Lengend Snippet: (A) KRAS G12C mutant MiaPaCa-2 cells exposed to incremental doses of AMG510 and MRTX1257 in long term cell culture, eventually developed drug-resistance as shown by their unresponsiveness to drug treatment in MTT assay and several fold increase in the drug IC50 values compared to parental cells. (B) AMG510- and MRTX1257-resistant MiaPaCa-2 cell lines show sensitivity toward selinexor induced growth inhibition. Parental as well as resistant cells were treated with selinexor for 72h and MTT assay was performed as described in Methods. All results are expressed as percentage of control ± S.E.M of six replicates.

Article Snippet: MRTX1257 (ChemieTek, Indianapolis, IN, USA), AMG510, MRTX849 (Selleck Chemical LLC, Houston, TX) and selinexor (Karyopharm Therapeutics, Newton, MA, USA) were dissolved in DMSO to make 10 mM stock solutions.

Techniques: Mutagenesis, Cell Culture, MTT Assay, Inhibition, Control

Journal: Cancer research communications

Article Title: Inhibitor of the Nuclear Transport Protein XPO1 Enhances the Anticancer Efficacy of KRAS G12C Inhibitors in Preclinical Models of KRAS G12C–Mutant Cancers

doi: 10.1158/2767-9764.crc-21-0176

Figure Lengend Snippet: NCI-H2122 (A), NCI-H358 (B) and MiaPaCa-2 (C) cells were exposed to the indicated concentrations of either selinexor, MRTX1257, or a combination of both for 72 h, and cell proliferation was evaluated by MTT assay as described in Methods. CalcuSyn software was employed to generate isobolograms and determine CI values from the resulting data. CI < 1 indicates synergistic effect of the drug combination at the corresponding doses. All results are expressed as percentage of control ± S.E.M of six replicates.

Article Snippet: MRTX1257 (ChemieTek, Indianapolis, IN, USA), AMG510, MRTX849 (Selleck Chemical LLC, Houston, TX) and selinexor (Karyopharm Therapeutics, Newton, MA, USA) were dissolved in DMSO to make 10 mM stock solutions.

Techniques: MTT Assay, Software, Control

Journal: Cancer research communications

Article Title: Inhibitor of the Nuclear Transport Protein XPO1 Enhances the Anticancer Efficacy of KRAS G12C Inhibitors in Preclinical Models of KRAS G12C–Mutant Cancers

doi: 10.1158/2767-9764.crc-21-0176

Figure Lengend Snippet: MiaPaCa-2 cells were plated in 6-well plates (500 cells per well) and treated with combinations of selinexor with MRTX1257 (A), MRTX849 (B) and AMG510 (C) at the indicated concentrations for 72 h and colony formation assay was performed as described in Methods. Images of crystal violet-stained colonies were captured and NIH ImageJ 1.5Oi software was used to measure the number and size of colonies. Data is representative of three independent experiments.

Article Snippet: MRTX1257 (ChemieTek, Indianapolis, IN, USA), AMG510, MRTX849 (Selleck Chemical LLC, Houston, TX) and selinexor (Karyopharm Therapeutics, Newton, MA, USA) were dissolved in DMSO to make 10 mM stock solutions.

Techniques: Colony Assay, Staining, Software

Journal: Cancer research communications

Article Title: Inhibitor of the Nuclear Transport Protein XPO1 Enhances the Anticancer Efficacy of KRAS G12C Inhibitors in Preclinical Models of KRAS G12C–Mutant Cancers

doi: 10.1158/2767-9764.crc-21-0176

Figure Lengend Snippet: (A) Immunoblots showing suppression of P70 S6 Kinase and Erk activation in NCI-H358 and MiaPaCa-2 cells treated with MRTX1257, AMG510 and MRTX849 as single agents (300 nM) and in combination with selinexor (100 nM) for 24h. (B) Immunoblots showing the inhibition of cyclin B1 and CDK4 in NCI-H358 and MiaPaCa-2 cells treated with single agent KRAS G12C inhibitors (300 nM) or their combinations with selinexor (100 nM) for 24h. (C) Immunoblot showing the expression of Rb in nuclear and cytosolic fractions of MiaPaCa2 cells treated with AMG510 alone (300 nM) or in combination with selinexor (100 nM) for 24h. Lamin B1 and GAPDH were used as loading controls for the nuclear and cytosolic fractions, respectively. (D) Immunoblot showing reduced expression of KRAS and NF-κB in MiaPaCa-2 cells treated with selinexor (300 nM) and MRTX1257 (90 nM) or AMG510 (120 nM) combinations for 12h. (E) Schematic of the proposed mechanisms by which the combination of KRAS G12C inhibitor and selinexor induces antineoplastic activity. This drug combination can block multiple nodes (represented with red arrows) of the cell proliferation and survival machinery. The quantitative analysis of mean pixel density of the blots was performed using NIH ImageJ 1.5Oi software.

Article Snippet: MRTX1257 (ChemieTek, Indianapolis, IN, USA), AMG510, MRTX849 (Selleck Chemical LLC, Houston, TX) and selinexor (Karyopharm Therapeutics, Newton, MA, USA) were dissolved in DMSO to make 10 mM stock solutions.

Techniques: Western Blot, Activation Assay, Inhibition, Expressing, Activity Assay, Blocking Assay, Software

Journal: Science (New York, N.Y.)

Article Title: Defining the KRAS- and ERK-dependent transcriptome in KRAS-mutant cancers

doi: 10.1126/science.adk0775

Figure Lengend Snippet: (A) KRAS-dependent gene expression changes upon acute (24 hours) KRAS suppression in eight KRAS-mutant PDAC cell lines transiently transfected with KRAS or control non-specific (NS) siRNA. Enrichment of Hallmark KRAS signaling gene sets is shown below. The 677 KRAS-dependent (UP) and 1,051 KRAS-suppressed (DN) genes (log2 fold-change/FC > 0.5, adj. p-val. < 0.05) are indicated by blue and red shaded circles, respectively. The top 200 KRAS-dependent (UP) and KRAS-inhibited (DN) genes comprising the PDAC KRAS UP/DN signatures are indicated by the dotted outlines. (B) Venn diagram indicates the overlap of differentially expressed genes (with unique Entrez gene IDs) upon KRAS siRNA treatment (refer to blue/red shading in (A)) compared to Hallmark KRAS signaling genes. N = 8 (cell lines as biological replicates) for each treatment and control. (C) GSEA for 50 Hallmark gene sets within DE genes following KRAS siRNA treatment. Detection is based on presence of a gene in all 8 PDAC cell lines at >5 reads. NES, normalized enrichment score. (D) KRAS-G12Ci (MRTX1257, 20 nM) induced expression changes summarized over 24 hours and three cell lines (PDAC, CRC, and NSCLC). Genes from siKRAS experiment shown as blue/red boxes in (A) are highlighted with blue/red and indicated by barcode plot below. Enrichment scores (ES) for top 200 UP/DN genes are indicated. (E) GSEA for 50 Hallmark gene sets within RNA-seq data from the indicated human cancer cell line-derived (CDX) or patient-derived (PDX) xenograft tumor samples. CDX mice were treated orally (24 hours) with either KRASG12C or KRASG12D selective inhibitors (G12Ci adagrasib or G12Di MRTX1133, respectively) or vehicle control. PDX mice were treated (21 days) with G12Ci sotorasib alone or together with the EGFRi panitumumab. The top 200 genes from the PDAC KRAS UP/DN gene sets (panel A) were also evaluated. N = 3 for each cell line/treatment combination except HPAC (control) and LS-180 (G12Di), where n = 6.

Article Snippet: The KRAS G12C -selective inhibitor MRTX1257 and the KRAS G12D -selective inhibitor MRTX1133 were synthesized at WuXi AppTec (Wuhan, China) or purchased from Selleckchem (E1051).

Techniques: Gene Expression, Mutagenesis, Transfection, Control, Expressing, RNA Sequencing, Derivative Assay

Journal: Science (New York, N.Y.)

Article Title: Defining the KRAS- and ERK-dependent transcriptome in KRAS-mutant cancers

doi: 10.1126/science.adk0775

Figure Lengend Snippet: (A) Immunoblot analysis of ERK activity in PDAC cell lines stably infected with control vector (Luc) or constitutively activated MEK1 (MEK1-DD), treated with vehicle (DMSO) or indicated inhibitors of each level of the RAF-MEK-ERK cascade (G12Ci/G12Di, MRTX1257/MRTX1133 (20 nM); RAFi, LY3009120 (600 nM); MEKi, trametinib (6 nM); ERKi, SCH772984 (600 nM). Images are representative of 2-3 biological replicates. (B) Growth of PDAC cells stably infected with control vector (Luc) or activated MEK1-DD and treated with the indicated inhibitors. Error bars indicate SE of the mean with 3-4 biological replicates, each with three technical replicates. (C) Immunoblot analysis of ERK and AKT phosphorylation in PDAC cells stably infected with control vector (Luc) or constitutively activated AKT (myr-AKT), treated with G12Ci/G12Di (MRTX1257/MRTX1133, 20 nM) or ERKi (SCH772984, 600 nM). Representative of 2-3 biological replicates. (D) Growth of PDAC cells expressing activated AKT or control vector (Luc) and treated with KRAS G12C/D inhibitors. Error bars indicate SE of the mean with 3-4 biological replicates, each with 3 technical replicates. (E) Differential gene expression analysis for seven PDAC cell lines subjected to ERKi (SCH772984, 1000 nM) treatment for 24 hours versus paired untreated control cells. (F) Top 200 UP/DN genes from 24 hours KRAS siRNA and ERKi RNA-seq experiments are shown as filled blue (UP) or red (DN) circles, with a positive predictive value (agreement/total) for logFC = 0.84. Genes with strongest logFC values (n = 40) are labeled. (G) Evaluation of DE genes following 100 nM treatment of Pa16C PDAC cells with KRASi-G12Di (MRTX1133) or ERKi (SCH772984) for 24 hours (each condition, n = 2). Colored points are DE genes in either treatment (FDR < 0.05); red, log2FC > 0 with either treatment; blue, log2FC < 0 with either treatment; tan, log2FC opposite directions between treatments. Coordinate labels indicate points outside of plotting range. Barcode plots below and left represent log2FC for “median rank KRAS-ERK” signature genes within each experiment and ssGSEA enrichment statistics. (H) GSEA for PDAC KRAS UP/DN, PDAC KRAS-ERK UP/DN, and KRAS-ERK UP/DN based on median rank, along with Hallmark KRAS Signaling signatures in CDX and PDX models treated with G12Di, G12Ci or G12Ci+EGFRi with replicates as described in Fig. 1E. Inhibitors used in (H): G12Ci (CDX), adagrasib; G12Di, MRTX1133; G12Ci (PDX), sotorasib; EGFRi, panitumumab.

Article Snippet: The KRAS G12C -selective inhibitor MRTX1257 and the KRAS G12D -selective inhibitor MRTX1133 were synthesized at WuXi AppTec (Wuhan, China) or purchased from Selleckchem (E1051).

Techniques: Western Blot, Activity Assay, Stable Transfection, Infection, Control, Plasmid Preparation, Phospho-proteomics, Expressing, Gene Expression, RNA Sequencing, Labeling