α4-α5 Helices on Surface of KRAS Can Accommodate Small Compounds That Increase KRAS Signaling While Inducing CRC Cell Death

Abstract

KRAS is the most frequently mutated oncogene associated with the genesis and progress of pancreatic, lung and colorectal (CRC) tumors. KRAS has always been considered as a therapeutic target in cancer but until now only two compounds that inhibit one specific KRAS mutation have been approved for clinical use. In this work, by molecular dynamics and a docking process, we describe a new compound (P14B) that stably binds to a druggable pocket near the α4-α5 helices of the allosteric domain of KRAS. This region had previously been identified as the binding site for calmodulin (CaM). Using surface plasmon resonance and pulldown analyses, we prove that P14B binds directly to oncogenic KRAS thus competing with CaM. Interestingly, P14B favors oncogenic KRAS interaction with BRAF and phosphorylated C-RAF, and increases downstream Ras signaling in CRC cells expressing oncogenic KRAS. The viability of these cells, but not that of the normal cells, is impaired by P14B treatment. These data support the significance of the α4-α5 helices region of KRAS in the regulation of oncogenic KRAS signaling, and demonstrate that drugs interacting with this site may destine CRC cells to death by increasing oncogenic KRAS downstream signaling.

Keywords: AKT; ERK; KRAS; RAF; allosteric pocket; calmodulin; colorectal cancer; docking; molecular dynamics; small molecule inhibitors.

Figure 1

(A) KRAS binding site for calmodulin (CaM). Representation in blue surface of the binding site suggested both experimentally [37] and theoretically [36]. (B) Formula of the compound P14. (C,D) Spatial representation of the KRAS-P14 and KRAS-P14B complex at the end of 100ns of conventional molecular dynamics.

Figure 2

P14 and P14B treatment of DLD-1 cells increased endogenous downstream RAS signaling. (A) DLD-1-starved cells were incubated for different times with 100 μM P14, or for 30 min with 10% FBS. (B) DLD-1 starved cells were incubated for 3 h with 100 μM P14, P14A, P14B, P14C or P14D, or for 30 min with 10% FBS, or for 10 min with 50 ng/mL of EGF. (C) DLD-1 starved cells were incubated for different times with either 10% FBS or 100µM P14B. (A–C) The levels of activation and the total levels of RAF, MERK, ERK and AKT were analyzed by WB with specific antibodies against the active phosphorylated forms of these kinases or against the total forms of these proteins, respectively. In (C), tubulin was used as loading control. Tubulin 1 corresponds to P-AKT and P-MEK gel, and Tubulin 2 to P-ERK, P-RAF and RAF gel.

Figure 3

(A) Kinetic analysis (left) and sensograms (right) of the binding of P14B to GST-K-Ras-G12V (1–166) determined by surface plasmon resonance. RU, resonance units. The same code of colors indicated in the kinetic analysis are used in the sensogram to indicate each concentration of P14B (B) Competition between P14B and CaM for binding to oncogenic KRAS: CaM-sepharose pulldown assays were performed using GST-KRAS-G12V in the presence of P14B. Ca⁺² or EGTA containing buffers indicate specific or non-specific KRAS-G12V binding to CaM, respectively. Bound fractions were analyzed by WB with anti-KRAS specific antibodies.

Figure 4

P14B treatment increases oncogenic KRAS interaction with its effectors BRAF and C-RAF/P-C-RAF. (A) Serum starved DLD-1 cells stably expressing HA-KRAS-G12V (DLD-1-HA-KRAS G12V) were incubated for 3 h with 100 μM P14B, or for 30 min with 10% FBS. WB was performed with the indicated antibodies. (B) Co-immunoprecipitation of HA-KRAS G12V with BRAF, C-RAF/P-C-RAF, or ARAF was analyzed in starved DLD-1-KO cells stably expressing HA-KRAS G12V after being treated with P14B (100 μM) or EGF for 10 min. IP was performed with anti-HA antibodies and WB of the bound and input fractions with anti-BRAF, anti-C-RAF, anti-P-C-RAF(S338), and anti-ARAF-specific antibodies. (-) non-treated.

Figure 5

P14B decreases the viability of CRC cells expressing oncogenic KRAS. (A) Effect of P14B on the viability of DLD-1, DLD-1-KO and hTERT-RPE cells. Cells were treated with P14B at a dose ranging from 0 to 100 μM and incubated for 24 h, at which time cell viability was determined by MTS assay. The experiment was repeated at least three times. Differences were assessed using a one way ANOVA and Multiple Comparisons Test. **** means p < 0.0001 between DLD-1 and RPE or DLD-1-KO cells; #### means p < 0.0001 between different concentrations of P14B treatment and non-treated DLD-1 cells and considered significant when p ≤ 0.05. (B) 2.5 × 10⁴ DLD-1 cells were cultured on top of a thin basement membrane matrix (Matrigel) overlaid with a dilute solution of this matrix (3D on-top Matrigel assay). At the time of seeding or 24h later, cells were treated with P14B (10 μM, 40 μM or 100 μM), and 3 days after seeding the colonies formed were analyzed. Representative phase-contrast images are shown. All scale bars, 40 μm. (C) DLD-1 cells treated with P14B (100 μM) for 36 h and 48 h were lysed and the expression of the apoptosis marker cleaved caspase-3 was analyzed by WB. Cells treated with the apoptosis inducer palmitic acid (PA) were used as a positive control.

Figure 6

P14B treatment of CRC cells expressing oncogenic KRAS increases endogenous downstream RAS signaling. (A) DLD-1, DLD-1-KO and hTERT-RPE starved cells were incubated with 100 μM of P14B for 3 h and the levels of activation of ERK and AKT were analyzed by WB. Specific antibodies against the active phosphorylated and total proteins were used. (B) Graph showing quantification of four independent experiments. Differences were assessed using a one-way ANOVA and Multiple Comparisons Test, and considered significant when p ≤ 0.05. *: p-value < 0.05; **: p-value < 0.01; ****: p-value < 0.0001; and ns: non-significant.