Novel Inhibition of Central Carbon Metabolism Pathways by Rac and CDC42 inhibitor MBQ167 and Paclitaxel

Abstract

Triple negative breast cancer (TNBC) represents a therapeutic challenge in which standard chemotherapy is limited to paclitaxel. MBQ167, a clinical stage small molecule inhibitor that targets Rac and Cdc42, inhibits tumor growth and metastasis in mouse models of TNBC. Herein, we investigated the efficacy of MBQ167 in combination with paclitaxel in TNBC preclinical models, as a prelude to safety trials of this combination in patients with advanced breast cancer. Individual MBQ167 or combination therapy with paclitaxel was more effective at reducing TNBC cell viability and increasing apoptosis compared with paclitaxel alone. In orthotopic mouse models of human TNBC (MDA-MB231 and MDA-MB468), individual MBQ167, paclitaxel, or the combination reduced mammary tumor growth with similar efficacy, with no apparent liver toxicity. However, paclitaxel single agent treatment significantly increased lung metastasis, whereas MBQ167, single or combined, reduced lung metastasis. In the syngeneic 4T1/BALB/c model, combined MBQ167 and paclitaxel decreased established lung metastases by ∼80%. To determine the molecular basis for the improved efficacy of the combined treatment on metastasis, 4T1 tumor extracts from BALB/c mice treated with MBQ167, paclitaxel, or the combination were subjected to transcriptomic analysis. Gene set enrichment identified specific downregulation of central carbon metabolic pathways by the combination of MBQ167 and paclitaxel but not individual compounds. Biochemical validation, by immunoblotting and metabolic Seahorse analysis, shows that combined MBQ167 and paclitaxel reduces glycolysis. This study provides a strong rationale for the clinical testing of MBQ167 in combination with paclitaxel as a potential therapeutic for TNBC and identifies a unique mechanism of action.

Figure 1.

MBQ-167 affects MDA-MB-231 TNBC cell viability, apoptosis, and tumor growth in combination with Paclitaxel. A, B, MDA-MB-231 cells treated with vehicle, 250 nM MBQ-167, 5 nM Paclitaxel, or the combination (250 nM MBQ-167 + 5 nM Paclitaxel), or, 500 nM MBQ-167, 10 nM Paclitaxel, or the combination (500 nMMBQ-167 + 10 nM Paclitaxel) for 96 hrs. A, Cell viability as determined using MTT assay. B, Caspase 3/7 assays for apoptosis. N=4 ± SEM in one-way ANOVA. ****=P<0.001. C, D, E, Effect of MBQ-167 and Paclitaxel treatments on MDA-MB-231 mammary fat pad tumor growth. SCID mice were inoculated with GFP-MDA-MB-231 cells at the mammary fat pad, when the tumors reached ~100 mm³ in diameter, mice were randomized (N=10), and treated with vehicle, 50 or 100 mg/kg BW 5X MBQ-167 5X/week per oral, 10 mg/kg BW paclitaxel (PTX) 1X week, or the combination by I.P. C, Relative tumor growth as a function of days. D, Relative tumor size at study termination. E, Plasma quantification of liver enzymes Asparagine transaminase (AST) and alanine transaminase (ALT).

Figure 2.

Effect of MBQ-167 and Paclitaxel treatments on MDA-MB-468 mammary fat pad tumor growth. SCID mice were inoculated with luciferase tagged MDA-MB-468 cells at the mammary fat pad, when the tumors reached ~100 mm³ in diameter, mice were randomized (N=10), and treated with vehicle, MBQ-167 at 50 or 100 mg/kg BW 5X week, Paclitaxel 10 mg/kg BW 1X week for 3 weeks, with 1 week rest in each cycle, or the combination. Mice were imaged 1X a week following administration of 1.5 mg luciferin. A, Tumor growth, as measured by total flux from chemiluminescence as a function of days. B, Representative whole-body images of mice under chemiluminescence on last day (day 136) and quantification of total chemiluminescence flux on day 136 relative to day 01 for each treatment; vehicle, MBQ-167 50 mg/kg (5X a week), MBQ-167 100 mg/kg (5X a week), Paclitaxel 10mg/kg (1X a week) ****=p<0.0001. C, Mouse weights (g)/week starting from day of tumor cell inoculation.

Figure 3.

Effect of MBQ-167, paclitaxel (PTX), or combination on lung metastases of mice bearing luciferase-tagged MDA-MB-468 mammary fat pad tumors. A, Total flux from luciferase tagged lung metastases. B, Representative H&E sections of lungs following 136 days of vehicle, MBQ-167 50 mg/kg 5X a week, Paclitaxel 10 mg/kg 1X a week or the combination. Arrows indicate metastatic foci of 10X images acquired from a Keyence microscope system. Scale bar, 10 μM. C, Number of metastatic foci/treatment; D, average area of lung metastases, as quantified from H&E stained lungs.

Figure 4.

MBQ-167 treatment with Paclitaxel abolishes established breast cancer metastasis. A-C, BALB/c mice were injected in the 4^th mammary fat pad with syngeneic 4T1 mammary carcinoma cells expressing firefly luciferase (150,000 cells). When the tumors reached ~150 mm³ (14d), tumors were surgically excised with tumor free margins, sutured and mice allowed to recover for 7 days. Mice were treated with 0, 25 or 50 mg/kg BW MBQ-167 5X/week or (and) 5 mg/kg Paclitaxel 1X a week (Scheme 03 in Materials & Methods). D, Lung metastases were imaged on day 21 and day 38. E, Representative excised lungs following bioluminescence imaging. F, Bioluminescence profile of Flux/ps of lung metastases. N=5.

Figure 5.

RNA-Seq analysis of vehicle or combination treatment of MBQ-167 (100mg/kg) and paclitaxel (5mg/kg) in a syngeneic mouse model (4T1 cells orthotopically injected in BALB/c mice). RNA-Seq was performed on d28 tumors (N=3 from each group) that were treated for 2 weeks (Suppl. Figure 2). A, Results of GSEA Hallmark analysis showing enriched gene sets. Bars in red indicate significant enrichment at FDR < 10%, bars in gray represent gene sets with FDR>10%. A positive Normalized Enrichment Score (NES) value indicates enrichment in the vehicle phenotype. B, Heat map of the top 20 genes of the most significant regulated pathway discovered with GSEA in the comparison of vehicle (left column) vs. combo treatment (right column). Expression values are represented as colors and range from red (high expression), pink (moderate), light blue (low) to dark blue (lowest expression). C, Enrichment plots for significant data sets enriched in GSEA Hallmark analysis, showing the profile of the running ES Score and positions of gene set members on the rank-ordered list. D, Overrepresentation (Enrichr) analysis for downregulated genes using Panther 2016. E, Top 20 differentially expressed genes of significantly enriched terms in the combination treatment phenotype, ordered by p-value. F, Enrichment map of the overrepresentation analysis using GO_Biological_Process f in EnrichR-KG.

Figure 6.

A, Representative immunoblots of genes differentially expressed in 4T1 tumors in Balb/c mice treated with vehicle or combined paclitaxel and MBQ-167 (combo) (as in Suppl. Fig. 2). B, Quantification of protein levels, normalized to loading control (Vinculin, n=3). C, Western blot of AMPKα activity (phospho) and expression (total) in vehicle versus combination treatment tumors. D, Quantification of protein levels, normalized to loading control (Vinculin, n=3). E, Relative ratio of p-AMPKα in combination versus vehicle treated tumors. F, Western blot analysis of the relative levels of the 5 OXPHOS complexes probed for a subunit that is labile when its complex is not assembled. G, Quantification of protein levels, normalized to loading control (Vinculin, n=3). H, Seahorse analysis of MDA-MB-468 cells treated with vehicle, MBQ-167 500 nM, paclitaxel 10 nM, or the combination for 24 hrs. Left, oxygen consumption rate (OCR); right, extracellular acidification rate (ECAR) relative to vehicle (1) as measured by an Agilent Seahorse XF analyzer.