KRAS Genotype Correlates with Proteasome Inhibitor Ixazomib Activity in Preclinical In Vivo Models of Colon and Non-Small Cell Lung Cancer: Potential Role of Tumor Metabolism

Abstract

In non-clinical studies, the proteasome inhibitor ixazomib inhibits cell growth in a broad panel of solid tumor cell lines in vitro. In contrast, antitumor activity in xenograft tumors is model-dependent, with some solid tumors showing no response to ixazomib. In this study we examined factors responsible for ixazomib sensitivity or resistance using mouse xenograft models. A survey of 14 non-small cell lung cancer (NSCLC) and 6 colon xenografts showed a striking relationship between ixazomib activity and KRAS genotype; tumors with wild-type (WT) KRAS were more sensitive to ixazomib than tumors harboring KRAS activating mutations. To confirm the association between KRAS genotype and ixazomib sensitivity, we used SW48 isogenic colon cancer cell lines. Either KRAS-G13D or KRAS-G12V mutations were introduced into KRAS-WT SW48 cells to generate cells that stably express activated KRAS. SW48 KRAS WT tumors, but neither SW48-KRAS-G13D tumors nor SW48-KRAS-G12V tumors, were sensitive to ixazomib in vivo. Since activated KRAS is known to be associated with metabolic reprogramming, we compared metabolite profiling of SW48-WT and SW48-KRAS-G13D tumors treated with or without ixazomib. Prior to treatment there were significant metabolic differences between SW48 WT and SW48-KRAS-G13D tumors, reflecting higher oxidative stress and glucose utilization in the KRAS-G13D tumors. Ixazomib treatment resulted in significant metabolic regulation, and some of these changes were specific to KRAS WT tumors. Depletion of free amino acid pools and activation of GCN2-eIF2α-pathways were observed both in tumor types. However, changes in lipid beta oxidation were observed in only the KRAS WT tumors. The non-clinical data presented here show a correlation between KRAS genotype and ixazomib sensitivity in NSCLC and colon xenografts and provide new evidence of regulation of key metabolic pathways by proteasome inhibition.

Fig 1. Ixazomib activity in tumor xenografts.

(A) Antitumor activity of ixazomib in 14 non small cell lung (NSCLC) and 6 colon xenograft models. The panel included both cell line-derived and primary human xenografts harboring either WT or mutant KRAS. Tumor-bearing animals (n = 8–10 per group) were treated with either vehicle or MTD dose of ixazomib (between 11 and 14 mg/kg, IV, BIW) for approximately three weeks. T/C was calculated by dividing average tumor volume of drug treated animals by that of vehicle treated animals on day 19–21. Tumor concentration of ixazomib (B) and 20S proteasome inhibition (β5 site) (C) in various NSCLC and colon xenografts at different time points following a single IV administration of MTD dose of ixazomib. % 20S β5-inhibition was calculated by considering the average (n = 3 for treatment group or 4 for vehicle group) tumor 20S activity of vehicle treated animals as 100%. Each bar represents the average concentration of ixazomib or 20S inhibition in the tumor from 3–4 different animals +/- SD.

Fig 2. In vitro and in vivo activity of ixazomib in SW48 isogenic cells and xenografts.

(A) Summary table of EC₅₀, IC₅₀ and T/C values for SW48, SW48-G13D and SW48-G12V cells and xenografts from in vitro cell viability, in vitro colony formation and in vivo antitumor activity assays. EC₅₀ represents the mean concentration of ixazomib (+/-SD) required for 50% cell killing in three different experiments in cell viability assay. In the colony formation assay (CFA) the mean concentration of the drug to inhibit 50% colony formation was calculated in three different experiments and the number is mean +/-SD. 13mg/kg, IV, BIW dose was used in xenograft studies and T/C was calculated as described before. (B) Antitumor activity of ixazomib in SW48, SW48-G13D and SW48-G12V xenografts. The tumor growth over time is presented for vehicle and ixazomib treated animals bearing SW48, SW48-G13D and SW48-G12V xenografts. The treatment started when average tumor volume reached approximately 200mm³ and there were n = 10 animals per arm. The T/C was calculated at day 19–21. C and D: Tumor concentration of ixazomib (C) and 20S proteasome inhibition (D) at different time points following an acute IV administration of ixazomib at 13mg/kg dose in SW48 and SW48-G13D tumors. Each data point represents tumors from three different animals +/- SD

Fig 3. Expression of GLUT1 and GLUT4 receptors in xenograft tumors with either WT or mutant KRAS.

Proteins were extracted from untreated tumors and 10μg protein was loaded in each lane. Levels of GLUT1 and GLUT4 proteins were determined by western blot and tubulin was used as a loading control. n = 3 for each tumor.

Fig 4. Baseline metabolic differences between SW48 KRAS WT and G13D mutant tumors.

A-C: Glutathione metabolism pathways (A), glycogen metabolism (B) and relative expression of pathway metabolites in KRAS WT vs KRAS mutant tumors treated with vehicle for 1 and 8 hrs. The numbers indicate the fold change of each metabolite in KRAS mutant tumors compared to KRAS WT tumors and are the average of metabolite levels from 5 different tumors. C: Relative expression of free long chain fatty acids, acetyl coA, acetylcarnitine and citrate in KRAS mutant vs KRAS WT tumors. Green boxes indicates a ratio <1, with dark green boxes as highly significant (p ≤0.05) and light green boxes as less significant (0.051, with dark red boxes as highly significant (p ≤0.05) and light red boxes as less significant (0.05

Fig 5. Ixazomib effect on amino acid metabolism.

(A) Relative level of amino acids at 1 hr and 8hrs following ixazomib treatment of SW48 KRAS WT and G13D mutant tumors. The numbers are expressed as relative level of each amino acid following ixazomib treatment compared to vehicle treatment at that time point. Each data point is an average of tumors from five different animals. Color coding is similar as described in Fig 4. (B) Expression of pGCN2 (T899), GCN2, peIF2α (Ser52) and eIF2α in tumors from vehicle or ixazomib treatment. The vehicle samples were collected at 4hrs after dosing and the ixazomib treated samples were collected at different time points (as indicated in the figure) after the drug treatment. Tubulin was used as a loading control. Note: for pGCN2/GCN2 western blot, only 2 samples were evaluated at 24hr timepoint and 3 samples were evaluated for all other markers and time points in this figure (14 samples used in GCN2/pGCN2 blots and 15 samples in the other blots).

Fig 6. Induction of β-oxidation by ixazomib treatment in SW48 WT tumors.

metabolites involved in fatty acid beta oxidation pathway. (A) Table summarizing free fatty acids in tumor samples. The numbers indicate the fold change in drug treated tumors over vehicle treated tumors at 1 and 8hrs time points. Each data point represents an average of tumors from five different animals. Table summarizing the levels of acetyl Co-A and 3 Hydroxy butyrate at 1hr after ixazomib treatment. The numbers indicate fold change in ixazomib treated tumors over vehicle treated tumors, n = 5 per data point. Changes in CPT-1 protein level following ixazomib treatment in SW48 and SW48-G13D tumors. The vehicle samples were collected at 4hrs after dosing and the ixazomib treated samples were collected at different time points (as indicated in the figure) after the drug treatment. The color coding is as described in Fig 4.

Fig 7. Ixazomib effect on key enzymes of lipid metabolism.

(A) Enzymes and metabolites in lipid synthesis and β-oxidation pathways. (B) Regulation of FASN, pACC-1(S79) and ACC-1 proteins following vehicle and ixazomib treatment. The vehicle samples were collected at 4hrs after dosing and the ixazomib treated samples were collected at different time points (as indicated in the figure) after the drug treatment. (Note: For SW48 ACC1 blot there were two samples in vehicle group and thus there were only 14 samples for that blot)