Fig 2: Vitamin C impairs the Warburg effect in KRAS mutant cells through downregulation of GLUT-1 and PKM2A. in absence of vitamin C, PKM2 is phosphorylated at ser 37 and translocates to the cell nucleus. Then, p-PKM2 binds to β-catenin and TCF/LEF transcriptional complex promoting c-Myc transcription that, in turn enhances the expression of GLUT-1 and PTB that participates in the splicing of PKM2 mRNA. B. vitamin C enters into the cell via GLUT-1 and SVCT1 inducing RAS detachment from plasma membrane, blocking downstream phosphorylation of PKM2 phosphorylation at ser 37. Disruption of the transcriptional complex formed by p-PKM2, β-catenin and TCF/LEF leads to downregulation of c-Myc, GLUT-1 and PTB expression. Absence of PTB activity stalls the splicing of PKM2 mRNA.

Fig 3: Vitamin C selectively kills wild type and mutant KRAS colon cancer cells alone or in combination with cetuximabA. apoptosis-inducing activities of vitamin C for Normal human immortalized Colonocytes (HCEC) and SW480 and LoVo cancer cell lines were annalyzed by Annexin-PI assay. Each cell line (2,3 105 cells) was incubated with vitamin C (10 mM) and PBS for controls. for 20 hr, apoptosis in each cell line was measured by staining with FITC-conjugated Annexin-V and Propidium Iodide (PI) using a Sigma-Aldrich Apoptosis kit. The populations of cells (annexin-V positive/PI negative) and late apoptotic cells (PI positive) as a percent of total cells were evaluated. Vitamin C displayed a selective killing effect on SW480 and LoVo. One-way ANOVA followed by Dunnett's post-test for multiple comparisons. *p< 0.05, **p < 0.001, n = 3. B. vitamin C treatment at different concentrations were carried out with LoVo and SW480 cancer lines. SW480 and LoVo CRC lines were exposed to ascorbate at 2, 5 and 10mM, for 20 hr. Then, cells were tripsinized and fixed with trypan blue solution (Sigma-Aldrich). The effective concentration that decreased survival 50% (EC50) was determined. EC50 was <10 mM for both tumor cells tested. Cell counting was carried out using a TC20™ Automated Cell Counter (Biorad). One-way ANOVA followed by Dunnett's post-test for multiple comparisons. *p < 0.05, **p < 0.001, n = 3. C. SW480 and LoVo cells were treated with vitamin C (7mM) for 5, 12 and 36 hr. Then, cells were tripsinized and fixed with trypan blue solution (Sigma-Aldrich). Cell counting was carried out using a TC20™ Automated Cell Counter (Biorad). One-way ANOVA followed by Dunnett's post-test for multiple comparisons. *p < 0.05, **p < 0.001, n = 3. D. HT29 harboring wild type KRAS, and the KRAS mutants LoVo and SW480 were treated with cetuximab (calculated IC50=0,4 μM), vitamin C (5mM) alone and combination for 12 hr. Then, cells were tripsinized and fixed with trypan blue solution (Sigma-Aldrich). Cell counting was carried out using a TC20™ Automated Cell Counter (Biorad). One-way ANOVA followed by Dunnett's post-test for multiple comparisons. *p < 0.05, **p < 0.001, n = 3. Combination of both vitamin C and the anti-EGFR antibody cetuximab displayed a higher killing effect in the three lines tested.

Fig 4: Vitamin C impairs the Warburg effect in KRAS mutant cells through downregulation of GLUT-1 and PKM2A. immunofluorescence analysis of GLUT-1 expression in SW480 and LoVo cells after vitamin C treatment (8 mM) for 20 hr. Glucose receptor 1 (GLUT-1) is shown in green. Cell nucleus is depicted in blue after DAPI staining. B. western blot and Real Time Quantitative PCR analysis of GLUT-1 protein and mRNA expression in SW480 and LoVo cells after vitamin C treatment for 20 hr. C. immunofluorescence analysis of PKM2 and p-PKM2 expression in SW480 and cells after vitamin C treatment (5 mM) for 20 hr. PKM2 and p-PKM2 is shown in green. Cell nucleus is depicted in blue after DAPI staining. Western-Blot analysis of PKM2 and p-PKM2 expression in SW480 cells after vitamin C treatment for 20 hr. D. immunofluorescence analysis of PKM2 and p-PKM2 expression in LoVo cells after vitamin C treatment (8 mM) for 20 hr. PKM2 and p-PKM2 is shown in green. Cell nucleus is depicted in blue after DAPI staining. Western-Blot analysis of PKM2 and p-PKM2 expression in LoVo cells after vitamin C treatment for 20 hr.

Fig 5: K42 is a key residue regulating RAS SUMOylation(A) MiaPaCa-2 cells were transfected with Flag-KRAS and HA-SUMO3 expression constructs for 24 h and then treated with or without 2-D08 for 18 h. Cells were then lysed and equal amounts of the lysates were immunoprecipitated with the anti-Flag antibody. Flag precipitates, along with lysate inputs, were blotted with antibodies to HA and Flag, respectively. (B) HEK293T cells were co-transfected with plasmid constructs expressing Flag-KRAS, HA-SUMO3, and SENP isoforms as indicated. Equal amounts of protein lysates were immunoprecipitated with the anti-Flag antibody. Flag immunoprecipitates were immunoblotted with antibodies to Flag or HA. Protein lysates of various treatments were also blotted with antibodies to HA, Flag, β-actin, SENP1, SENP2, or SENP6. (C) Alignment of RAS isoform amino acid sequences with a predicted SUMOylation site (in red). The residue for optimal sumoylation was predicted with SUMOplot (http://www.abgent.com/sumoplot). (D) HEK293T cells were co-transfected with constructs expressing Flag-tagged HRAS (WT) or HRAS42R, HA-UBC9, and HA-SUMO3. Equal amounts of protein lysates from various treatments were immunoprecipitated with the anti-Flag antibody. Flag immunoprecipitates, along with lysate inputs, were blotted with the anti-Flag antibody or with the anti-HA antibody. (E) HEK293T cells were co-transfected with HA-SUMO3 and WT KRAS or with HA-SUMO3 and various forms of KRAS mutants as indicated for 24 h. Equal amounts of cell lysates were immunoprecipitated with the anti-Flag antibody. Flag immunoprecipitates, along with cell lysate inputs, were blotted with antibodies to HA and Flag, respectively.