WNT Signalling Promotes NF-κB Activation and Drug Resistance in KRAS-Mutant Colorectal Cancer

Abstract

Approximately 40% of colorectal cancer (CRC) cases are characterized by KRAS mutations, rendering them insensitive to most CRC therapies. While the reasons for this resistance remain incompletely understood, one key aspect is genetic complexity: in CRC, oncogenic KRAS is most commonly paired with mutations that alter WNT and P53 activities ("RAP"). Here, we demonstrate that elevated WNT activity upregulates canonical (NF-κB) signalling in both Drosophila and human RAS mutant tumours. This upregulation required Toll-1 and Toll-9 and resulted in reduced efficacy of RAS pathway targeted drugs such as the MEK inhibitor trametinib. Inhibiting WNT activity pharmacologically significantly suppressed trametinib resistance in RAP tumours and more genetically complex RAP-containing 'patient avatar' models. WNT/MEK drug inhibitor combinations were further improved by targeting brm, shg, ago, rhoGAPp190 and upf1, highlighting these genes as candidate biomarkers for patients sensitive to this duel approach. These findings shed light on how genetic complexity impacts drug resistance and proposes a therapeutic strategy to reverse this resistance.

Figure 1:. WNT signalling induced trametinib resistance through enhancing canonical NF-κB signalling.

(A, C, D, E, F) Percent survival of transgenic flies to adulthood relative to control flies was quantified in the presence or absence of trametinib (1 μM). (A) Control, Ras^G12V, and RAP; (C) RAP +dif-RNAi and RAP +dl-RNAi; (D) dif-RNAi and dl-RNAi; (E) Ras^G12V+GFP (control) and Ras^G12V+cact-RNAi; (F) GFP and cact-RNAi. (B, G) Expression levels of drosomycin were quantified for each genotype by quantitative RT-PCR. (B) Control and RAP; (G) Ras^G12V, Arm^S10 and Ras^G12V +Arm^S10. (H-J) Images of the digestive tract of third instar larvae in the present of trametinib (1 μM), which include the hindgut proliferation zone (HPZ). Nuclei are visualized with 4′,6-diamidino-2-phenylindole (DAPI) staining, hindgut is visualized by GFP. Scale bar 100μm. (K) The average of hindgut proliferation zone (HPZ) size was measured by Fiji ImageJ and quantified as relative size to wild-type hindgut.

Figure 2:. Toll-1 and Toll-9 are required for upregulation of canonical NF-κB activity in RAP tumours.

(A-D) Control (A), RAP (B), RAP +Toll1-RNAi (C), RAP +Toll9-RNAi (D) were induced in hindguts and were stained with anti-dorsal antibody. DNA are visualized with Propidium iodide (PI). (E, F) Percent survival of transgenic flies to adulthood relative to control flies was quantified in the present or absence of trametinib (1 μM). € RAP +Toll1-RNAi and RAP +Toll9-RNAi; (F) Toll1-RNAi and Toll9-RNAi. (G) A model for WNT activity and p53 defect promoting drug resistance in CRC.

Figure 3:. PNU-74654 and LF3 suppressed trametinib resistance in RAP tumours.

(A) A summary of rescue rate of trametinib and WNT inhibitors drug combination in RAP hindgut tumours. (B-D) Percent survival of byn>RAP flies to adulthood relative to control flies was quantified in the present or absence of trametinib (1 μM), PNU-74654 (1 μM) or LF3 (10μM). (E) The average of hindgut proliferation zone (HPZ) size was measured by Fiji ImageJ and quantified as relative size to wild type (WT) hindgut. (F, G) Images of the digestive tract of third instar larvae in the present or absence of trametinib (1 μM) or PNU-74654 (1 μM).

Figure 4:. Combination of trametinib and PNU-74654 suppressed tumour progression in various genetically complex tumours.

(A-E) Percent survival of transgenic patient-specific avatar fly lines to adulthood relative to control flies was quantified in the present or absence of trametinib (1 μM) or PNU-74654. (A) CPCT006; (B) CPCT018; (C) CPCT045; (D) CPCT050; (E) CPCT029.

Figure 5:. Regulators of combination of trametinib and PNU-74654 in CRC tumours.

Survival of transgenic flies to adulthood relative to control flies was quantified in the present or absence of trametinib (1 μM) or PNU-74654 (1 μM except where noted). (A) RNAi-mediated knockdown of brm or ago improved survival of byn>RAP flies treated with trametinib. (B) rhoGAPp190 knockdown improved survival of RAP flies treated with PNU-74654. (C) Summary of five loci found to impact RAP response to trametinib and/or PNU-74654. (D) Knockdown of brm, shg, ago, rhoGAPp190, or upf1 improved survival to adulthood of RAP flies in the present of trametinib plus PNU-74654. These five loci did not significantly rescue RAP fly survival in the absence of drug (E), nor did they alter survival of control animals in the presence of drug (F). (G, H) Knockdown of brm, shg, ago, rhoGAPp190, or upf1 rescued more genetically complex avatar lines RAPp1 and RAPp2 when treated with trametinib plus PNU-74654 (5 μM); note shg rescue of RAPp1 did not rise to the level of statistical significance. (I) In the absence of knockdown, trametinib and/or PNU-74654 (5 μM) exhibited poor rescue of RAPp1 and RAPp2.

Figure 6:. Co-activation of WNT and KRAS was associated with an upregulation of canonical NF-κB activity in human CRC.

(A-H) Representative immunohistochemical (IHC) staining for IKKβ, IKKα in stage 2–3 colorectal cancer patient samples. (A, E) KRAS^WT (wild-type) plus low β-catenin expression CRC samples; (B, F) KRAS^MT (mutation in position G12/G13) plus low β-catenin expression CRC samples; (C, G) KRAS^WT plus high β-catenin expression CRC samples; (D, H) KRAS^MT plus high β-catenin expression CRC samples. (I, J) Graph shows the median of expression of IKKβ (I) or IKKα (J) in each different mutated human CRC, determined by IHC intensity values. Patients were grouped into 3 categories based on KRAS status and β-catenin expression.