Oncogenic RAS isoforms show a hierarchical requirement for the guanine nucleotide exchange factor SOS2 to mediate cell transformation

Abstract

About a third of tumors have activating mutations in HRAS, NRAS, or KRAS, genes encoding guanosine triphosphatases (GTPases) of the RAS family. In these tumors, wild-type RAS cooperates with mutant RAS to promote downstream effector activation and cell proliferation and transformation, suggesting that upstream activators of wild-type RAS are important modulators of mutant RAS-driven oncogenesis. The guanine nucleotide exchange factor (GEF) SOS1 mediates KRAS-driven proliferation, but little is understood about the role of SOS2. We found that RAS family members have a hierarchical requirement for the expression and activity of SOS2 to drive cellular transformation. In mouse embryonic fibroblasts (MEFs), SOS2 critically mediated mutant KRAS-driven, but not HRAS-driven, transformation. Sos2 deletion reduced epidermal growth factor (EGF)-dependent activation of wild-type HRAS and phosphorylation of the kinase AKT in cells expressing mutant RAS isoforms. Assays using pharmacological inhibitors revealed a hierarchical requirement for signaling by phosphoinositide 3-kinase (PI3K) in promoting RAS-driven cellular transformation that mirrored the requirement for SOS2. KRAS-driven transformation required the GEF activity of SOS2 and was restored in Sos2^-/- MEFs by expression of constitutively activated PI3K. Finally, CRISPR/Cas9-mediated deletion of SOS2 reduced EGF-stimulated AKT phosphorylation and synergized with MEK inhibition to revert the transformed phenotype of human KRAS mutant pancreatic and lung tumor cells. These results indicate that SOS2-dependent PI3K signaling mediates mutant KRAS-driven transformation, revealing therapeutic targets in KRAS-driven cancers. Our data also reveal the importance of three-dimensional culture systems in investigating the mediators of mutant KRAS.

Fig. 1.. Oncogenic mutant RAS isoforms show a hierarchical requirement for SOS2 to drive transformation in MEFs.

(A) Sos2^+/+ and Sos2^−/− MEFs were transduced with lentiviruses expressing either empty vector (V) or the indicated HA-tagged mutant RAS isoform (H, N, and K: HRAS^G12V, NRAS^G12V, and KRAS^G12V, respectively). Whole cell lysates (WCLs) were analyzed by Western blotting with antibodies specific for EGFR, SOS1, SOS2, HA (for RAS^G12V), total RAS, or β-actin to assess total protein. GST-RAS binding domain pulldowns were analyzed by Western blotting with an antibody specific for the HA epitope to assess activation of mutant HA-RAS^G12V. Blots are representative of three independent experiments. (B-D) Sos2^+/+ and Sos2^−/− MEFs expressing the indicated mutant RAS isoform were assessed for (B) proliferation in 2D culture plates, (C) colony growth in soft agar to assess anchorage-independent growth, and (D) loss of contact inhibition as assessed by a focus forming assay. Data are mean ± SD from three independent experiments. ** P<0.01 by ANOVA using the Tukey’s method to correct for multiple comparisons. (E) 10× images of post-confluent MEFs from D. Images are representative from three independent experiments. See also fig. S1 for an overlay of the proliferation curves in B.

Fig. 2.. Sos2 is required for mutant KRAS driven transformation in MEFs.

(A) Sos2^+/+ (+) and Sos2^−/− (–) MEFs were transduced with lentiviruses expressing either empty vector (V), wild-type KRAS, or the indicated HA-tagged mutant KRAS constructs. Whole cell lysates (WCLs) were analyzed by Western blotting with antibodies specific for SOS2, HA (KRAS), or β-actin. Blots are representative of three independent experiments. (B-C) Sos2^+/+ and Sos2^−/− MEFs expressing the indicated KRAS constructs were assessed for (B) colony growth in soft agar to assess anchorage independent growth, and (C) loss of contact inhibition as assessed by focus forming assay. Data are mean ± SD from three independent experiments. Statistical significance was determined by ANOVA using the Tukey’s method to correct for multiple comparisons. ** P<0.01; *** P<0.001.

Fig. 3.. Sos2 RASGEF activity contributes to KRAS4B–driven transformation.

(A) Schematic showing potential routes of SOS2-dependent wild-type RAS activation in the presence of mutant KRAS. SOS2 point mutants block either RASGEF activity (F927A) or putative allosteric SOS2 activation by KRAS (W727E). (B) Sos2^−/− MEFs expressing KRAS^G12C, KRAS^G12V, or KRAS^Q61R were transduced with lentiviruses expressing either empty vector (V), wild-type SOS2, RASGEF-dead (F927A) SOS2, or feedback-defective (W727E) SOS2. Whole cell lysates (WCLs) were analyzed by Western blotting with antibodies specific for SOS2 or β-actin. Blots are representative of three independent experiments. (C-D) Sos2^+/+ MEFs, Sos2^−/− MEFs, or Sos2^−/− MEFs expressing the indicated SOS2 constructs along with either KRAS^G12C (closed), KRAS^G12V (hashed), or KRAS^Q61R (open) were assessed for (C) colony growth in soft agar to assess anchorage independent growth, and (D) loss of contact inhibition by focus forming assay. Data are mean ± SD from three independent experiments. Statistical significance was determined by ANOVA using the Tukey’s method to correct for multiple comparisons. For C, ** P<0.01 versus Sos2^+/+ and WT. (E) 10 × images of post-confluent MEFs from D. Images are representative of 3 independent experiments. (F-G) Western blotting for activated V5-HRAS from GST-RBD pulldowns (middle, quantified above) or for total V5-HRAS from WCLs (below) from (F) Sos2^+/+ or Sos2^−/− MEFs expressing HA-KRAS^G12C and V5-wild-type HRAS in either actively cycling cells (left) or cells serum starved overnight and then lysed or stimulated with 100 μg/mL EGF for 5 minutes (right) or (G) Sos2^−/− MEFs expressing HA-KRAS^G12C, V5-wild-type HRAS, and the indicated SOS2 construct. Data are mean ± SD from three independent experiments. Blots are representative of three independent experiments. Statistical significance was determined by ANOVA using the Tukey’s method to correct for multiple comparisons. For F, * P<0.05; ** P<0.01. For G, **P<0.01 versus vector and SOS2^F927A.

Fig. 4:. SOS2 is required for optimal RTK−dependent AKT phosphorylation in cells expressing mutant RAS.

Sos2^+/+ and Sos2^−/− MEFs expressing the indicated mutant RAS isoforms were placed in serum-free media overnight, and then stimulated with 100 μg/mL EGF for the indicated times. (A) WCLs were analyzed by multiplex Western blotting for pERK1/2, ERK1/2, pAKT (Ser⁴⁷³), pAKT (Thr³⁰⁸), AKT, and β-actin on a LI-COR Odyssey machine. Blots are representative of three independent experiments. (B) Quantification of pERK1/2, pAKT (Ser⁴⁷³), and pAKT (Thr³⁰⁸) levels versus a weighted average of total proteins (ERK1/2, AKT, and β-actin). Data are mean ± SD from three independent experiments. Statistical significance was determined by ANOVA using the Tukey’s method to correct for multiple comparisons. * P<0.05; ** P<0.01; *** P<0.001; **** P<0.0001.

Fig. 5:. Mutant RAS isoforms show a hierarchical requirement for PI3K signaling to drive transformation in MEFs.

Sos2^+/+ MEFs expressing the indicated mutant RAS isoforms were seeded onto either tissue-culture treated 96-well plates to assess anchorage-dependent growth or low-attachment 96-well plates to assess anchorage-independent growth. Cells were treated with the indicated concentrations of (A) the PI3K inhibitor LY294002, (B) the AKT inhibitor AZD5363, or (C) the MEK1/2 inhibitor trametinib for four days, and cell number was assessed. Data are expressed relative to vehicle-treated controls and are expressed as mean ± SD four independent experiments. IC₅₀ values and AUC measurements are shown. Statistical significance was determined by ANOVA using the Tukey’s method to correct for multiple comparisons. ** P<0.01 versus HRAS^G12V. HRAS^G12V (salmon triangles), NRAS^G12V (purple inverted triangles), KRAS^G12V (blue diamonds). (D) Sos2^+/+ MEFs expressing the indicated mutant RAS isoforms were seeded in low-attachment 96-well round-bottomed plates and treated with the indicated concentrations of the PI3K inhibitor LY294002 to assess the effects of PI3K inhibition on RAS-induced cancer spheroid formation (left). Sos2^−/− MEFs expressing the indicated mutant RAS isoforms were seeded in parallel and left untreated for comparison (right). Images of spheroids were taken 16 hours after plating (Day 0) and again seven days later. Data are representative of three independent experiments. The outlined image for each cell line represents the LY294002 concentration where cancer spheroid size did not increase relative to Day 0. Images are representative of three independent experiments. See fig. S4 for inhibition of downstream protein phosphorylation by specific inhibitors, and fig. S5 for quantification of spheroid growth between Sos2^+/+ and Sos2^−/− MEFs expressing mutant RAS.

Fig. 6:. Activated PI3K (p110α) cooperates with mutant KRAS4B to transform Sos2^−/− MEFs.

(A) Sos2^+/+ and Sos2^−/− MEFs were transduced with lentiviruses expressing KRAS^G12V +/− p110α^H1047R. Whole cell lysates (WCLs) were analyzed by Western blotting with antibodies specific for SOS2, p110α, HA (KRAS^G12V), or β-actin. Blots are representative of three independent experiments. (B) Sos2^+/+ and Sos2^−/− MEFs expressing KRAS^G12V +/− p110α^H1047R were assessed for loss of contact inhibition by focus forming assay (stained dishes below, quantified above). Images are representative from three independent experiments. See fig. S6 for 10 × images of cells from B.

Fig. 7:. SOS2 is required for transformation of KRAS mutated tumor cells.

(A) YAPC pancreatic cancer cells (harboring a KRAS^G12V mutation) were transduced with lentiviruses expressing Cas9 and either a non-targeting sgRNA (NT), an sgRNA targeting KRAS, or one of three different sgRNAs targeting SOS2. WCLs were analyzed by Western blotting with antibodies specific for KRAS, SOS2, SOS1, or tubulin (left). The SOS2 protein abundance relative to the NT sgRNA control in the SOS2 CRISPR samples is given. Cells were assessed for colony growth in soft agar 21 days after plating to assess anchorage independent growth (right), and 10× images showing transformed colonies growing in soft agar were taken (bottom). Data are mean ± SD from three independent experiments. Blots and images are representative of 3 independent experiments. Statistical significance was determined by ANOVA using the Tukey’s method to correct for multiple comparisons. * P<0.05; *** P<0.001; **** P<0.0001. (B) H358 NSCLC cells (harboring a KRAS^G12C mutation) were transduced with lentiviruses expressing Cas9 and either a non-targeting sgRNA (NT), an sgRNA targeting KRAS, or one of two different sgRNAs targeting SOS2. WCLs were analyzed by Western blotting with antibodies specific for KRAS, SOS2, SOS1, or tubulin (left). The SOS2 protein abundance relative to the NT sgRNA control in the SOS2 CRISPR samples is given. Cells were assessed for anchorage independent growth by cancer spheroid assay (right), and cancer spheroid growth 16 hours after plating (day 0) or 14 days later (10× images below). Data are mean ± SD from three independent experiments. Blots and images are representative of 3 independent experiments. Statistical significance was determined by ANOVA using the Tukey’s method to correct for multiple comparisons. * P<0.05; *** P<0.001; **** P<0.0001. (C-D) YAPC cells from A were either lysed actively cycling (C) or starved overnight and then stimulated with EGF 100 ng/mL for 5 minutes (D) prior to lysis. Multiplex Western blotting for pERK1/2, ERK1/2, pAKT (Ser⁴⁷³), AKT, and β-actin was performed on a LI-COR Odyssey machine. Quantification of pERK1/2 and pAKT (Ser⁴⁷³) levels versus a weighted average of total proteins (ERK1/2, AKT, and β-actin) are shown above. Data are mean ± SD from three independent experiments. Blots and images are representative of 3 independent experiments. Statistical significance was determined by ANOVA using the Tukey’s method to correct for multiple comparisons. * P<0.05; *** P<0.001; **** P<0.0001.

Fig. 8:. SOS2 deletion synergizes with MEK inhibition to inhibit transformation of KRAS mutant tumor cells.

(A-D) KRAS mutant YAPC pancreatic cancer cells (A, C) or H358 NSCLC cells (B, D) transduced with lentiviruses expressing Cas9 and either a non-targeting sgRNA (NT), an sgRNA targeting KRAS, or an sgRNAs targeting SOS2 were seeded onto either tissue-culture treated 96-well plates to assess anchorage-dependent growth (left) or low-attachment 96-well plates to assess anchorage-independent growth (right). Cells were treated with the indicated concentrations of the PI3K inhibitor buparlisib (A-B) or the MEK1/2 inhibitor trametinib (C-D) for five days, and cell number was assessed. For C-D, NT cells were either treated with the indicated concentration of trametinib alone or in the presence of 100 ng/mL buparlisib. Data are expressed relative to vehicle-treated controls and are expressed as mean ± SD for three independent experiments. IC₅₀ values and AUC measurements are shown. Statistical significance was determined by ANOVA using the Tukey’s method to correct for multiple comparisons. * P<0.05 versus NT. # P<0.05 versus KRAS deletion. NT (grey squares), KRAS deleted (black circles), SOS2 deleted (blue triangles), NT + 100 ng/mL buparlisib (red diamonds).