Colon Cancer Cells Evade Drug Action by Enhancing Drug Metabolism

Abstract

Colorectal cancer (CRC) is the second most deadly cancer worldwide. One key reason is the failure of therapies that target RAS proteins, which represent approximately 40% of CRC cases. Despite the recent discovery of multiple alternative signalling pathways that contribute to resistance, durable therapies remain an unmet need. Here, we use liquid chromatography/mass spectrometry (LC/MS) analyses on Drosophila CRC tumour models to identify multiple metabolites in the glucuronidation pathway-a toxin clearance pathway-as upregulated in trametinib-resistant RAS/APC/P53 ("RAP") tumours compared to trametinib-sensitive RAS ^G12V tumours. Elevating glucuronidation was sufficient to direct trametinib resistance in RAS ^G12V animals while, conversely, inhibiting different steps along the glucuronidation pathway strongly reversed RAP resistance to trametinib. For example, blocking an initial HDAC1-mediated deacetylation step with the FDA-approved drug vorinostat strongly suppressed trametinib resistance in Drosophila RAP tumours. We provide functional evidence that pairing oncogenic RAS with hyperactive WNT activity strongly elevates PI3K/AKT/GLUT signalling, which in turn directs elevated glucose and subsequent glucuronidation. Finally, we show that this mechanism of trametinib resistance is conserved in an KRAS/APC/TP53 mouse CRC tumour organoid model. Our observations demonstrate a key mechanism by which oncogenic RAS/WNT activity promotes increased drug clearance in CRC. The majority of targeted therapies are glucuronidated, and our results provide a specific path towards abrogating this resistance in clinical trials.

Figure 1:. Glucuronidation pathway induces trametinib resistance in Drosophila.

(A, D, E, F, G and H) Percent survival of transgenic flies to adulthood relative to control flies was quantified in the present or absence of trametinib (1μM) or UDP-Glc as indicated. (A) Wild-type (WT), Ras^G12V and RAP; (D and E) RAP + GFP (control), RAP +Hex-C-RNAi, RAP +UGP-RNAi, RAP +Sgl-RNAi, RAP + GlcAT-P-RNAi; (F) Sgl-RNAi and GlcAT-P-RNAi; (G) WT; (H) Ras^G12V. (B) A heatmap of LC/MS showed top 50 metabolises. (C) An overview of the glucuronidation pathway. Transgene expression was induced in Drosophila hindguts by a byn-GAL4 driver. Drug concentrations indicate final food concentrations. Each data point represents a replicate.

Figure 2:. Pi3K/Akt signalling induces trametinib resistance by enhancing glucuronidation in Drosophila.

(A, C, F, H and K) Percent survival of transgenic flies to adulthood relative to control flies was quantified in the present or absence of trametinib (1μM), sucrose. (A) Ras^G12V; (C) Ras^G12V+GFP, Ras^G12V+Sgl-RNAi and Ras^G12V+GlcAT-P-RNAi; (F) RAP+GFP, RAP+Akt-RNAi; (H) RAP+AS160-RNAi; (K) Ras^G12V+Arm^CA were induced in Drosophila hindguts. (D and E) Western blot analysis of Drosophila hindguts pAkt and Akt levels in Ras^G12V, RAP, WT, Arm^CA, or Ras^G12V+Arm^CA with or without sucrose. (G, I and J) Released UDP analysis of RAP+GFP, RAP+Akt-RNAi, RAP+AS160-RNAi, Ras^G12V, Arm^CA or Ras^G12V+Arm^CA with trametinib in Drosophila hindguts. Transgene expression was induced in Drosophila hindguts by a byn-GAL4 driver. Increased dietary sugar led to increased glucuronidation and reduced trametinib activity, while targeting glucuronidation enzymes or Pi3K pathway activity strongly potentiated trametinib activity.

Figure 3:. Deacetylation, glucuronidation lead to trametinib resistance in mouse AKP organoids.

(A) Released UDP analysis of mouse AKP organoids in the present of trametinib. (B, C, E and F) Percent survival of AKP organoids relative to control was quantified in the present or absence of trametinib (5nM), fasentin (30μM), LY294002 (8μM), vorinostat (0.5μM) or phenacetin (100μM). (D) Representative images showing the impact of drugs on AKP organoids. Targeting glucuronidation led to increased effectiveness of trametinib.

Figure 4:. HDAC1 is required for glucuronidation of trametinib in Drosophila.

(A) Released UDP analysis of RAP+GFP or RAP+HDAC1-RNAi with trametinib in Drosophila hindguts. (B, C, D and E) Percent survival of adult tumour flies relative to control flies was quantified in the present or absence of trametinib (1μM), vorinostat (0.5μM) or phenacetin. (B) RAP +HDAC1-RNAi, (C, D and E) RAP. Reduced HDAC activity led to reduced trametinib-dependent UDP release. (F-J) Images of the digestive tract of third instar larvae in the present or absence of trametinib (1 μM), vorinostat (0.5 μM) which include the hindgut proliferation zone (HPZ). Nuclei are visualized with 4′,6-diamidino-2-phenylindole (DAPI) staining, hindgut is marked by GFP. Scale bar 1mm. (K) The average of hindgut proliferation zone (HPZ) size was measured by Fiji ImageJ and quantified as relative size to wild-type (WT) hindgut. Transgene expression was induced in Drosophila hindguts by a byn-GAL4 driver. Reducing deacetylation/glucuronidation with vorinostat increased trametinib’s ability to rescue hindgut size.

Figure 5:. Schematic summary

Trametinib (tram) is a potent MEK inhibitor with the demonstrated preclinical ability to block RAS pathway signalling and oncogenic transformation. Pairing activated RAS and WNT activities leads to activation of PI3K/AKT signalling, AS160, and GLUT1/4 to increase glucose flux into cells. The result is elevated glucuronidation and elimination of trametinib. Potential therapeutic targets include HDAC1: deacetylation is an obligatory pre-step required for glucuronidation of some drugs including trametinib.