RAS internal tandem duplication disrupts GTPase-activating protein (GAP) binding to activate oncogenic signaling

Abstract

The oncogene RAS is one of the most widely studied proteins in cancer biology, and mutant active RAS is a driver in many types of solid tumors and hematological malignancies. Yet the biological effects of different RAS mutations and the tissue-specific clinical implications are complex and nuanced. Here, we identified an internal tandem duplication (ITD) in the switch II domain of NRAS from a patient with extremely aggressive colorectal carcinoma. Results of whole-exome DNA sequencing of primary and metastatic tumors indicated that this mutation was present in all analyzed metastases and excluded the presence of any other clear oncogenic driver mutations. Biochemical analysis revealed increased interaction of the RAS ITD with Raf proto-oncogene Ser/Thr kinase (RAF), leading to increased phosphorylation of downstream MAPK/ERK kinase (MEK)/extracellular signal-regulated kinase (ERK). The ITD prevented interaction with neurofibromin 1 (NF1)-GTPase-activating protein (GAP), providing a mechanism for sustained activity of the RAS ITD protein. We present the first crystal structures of NRAS and KRAS ITD at 1.65-1.75 Å resolution, respectively, providing insight into the physical interactions of this class of RAS variants with its regulatory and effector proteins. Our in-depth bedside-to-bench analysis uncovers the molecular mechanism underlying a case of highly aggressive colorectal cancer and illustrates the importance of robust biochemical and biophysical approaches in the implementation of individualized medicine.

Keywords: RAS protein; bedside-to-bench analysis; colon cancer; exome sequencing; oncogene; personalized medicine; protein structure; structural biology; switch II domain; tandem duplication.

Figure 1.

Whole-exome sequencing of a clinically aggressive CRC suggests the NRAS ITD is the primary oncogenic driver mutation. A, overview of filtered variants prioritized for molecular pathology review in whole-exome NGS of the patient's primary tumor, lung, and CNS metastases; asterisk indicates variants unique to the metastases versus the primary tumor. B, molecular pathologist interpretation of clinically relevant variants, including tumor site(s) at which each variant was identified. C, overall survival of patients with NRAS or CSMD1 mutations (red, n = 48; median overall survival, 38.9 months) was significantly (p = 0.002) shorter than patients without mutations in these genes (blue, n = 162) in the CRC TCGA cohort.

Figure 2.

Switch II internal tandem duplication of both NRAS and KRAS proteins increases the affinity for RAF effector. NanoBRET saturation curves of transiently transfected RAF1 NanoLuc donor constructs (constant) and titrated, transiently transfected Halotag-RAS acceptor constructs. BRET_max and BRET₅₀ values represent the maximum number of protein–protein interactions and protein affinity, respectively. A, top, Halotag-NRAS ITD fusion proteins show increased interaction with effector RAF1 compared with the WT protein when overexpressed in Caco-2 cells. *Interactions with NRAS WT to be significantly different to both NRAS Q61R and NRAS ITD (p < 0.0005 from 0.0625–4 µg DNA; p < 0.02 at 8 µg DNA). A, bottom, both KRAS ITD and NRAS ITD mutants display increased RAF1 interaction compared with KRAS WT in HEK293T cells. *NRAS ITD mBRET values are significantly different to KRAS WT (p < 0.0001 from 0.0094–0.3 µg DNA; p < 0.008 at 0.6 µg DNA). **KRAS ITD mBRET values are significantly different from KRAS WT (p < 0.0001 from 0.0094–0.075 µg DNA; p < 0.03 at 0.15 µg DNA). B, nanoBRET saturation curves of NRAS ITD and NRAS WT in HEK293T cells. *RAF1 interactions are significantly different (p < 0.0005) between 0.00195 and 0.00781 µg DNA. C, nanoBRET saturation curves of KRAS ITD and KRAS WT. *RAF1 interactions are significantly different (p < 0.0001) between 0.00195 and 0.25 µg DNA. Nonlinear regressions were performed in GraphPad Prism (see Tables S2–S5 for curve fit parameters). Error bars represent n = 3 technical replicates. Statistical significance of the differences was determined by 2-way ANOVA in GraphPad Prism.

Figure 3.

Expression of ITD mutant RAS leads to MEK and ERK activation. A and B, Halotag-NRAS ITD fusion proteins overactivate the MAP kinase cascade when transiently overexpressed in titrated amounts in (A) Caco-2 cells and (B) HEK293T cells. Pathway induction, as measured by phospho-ERK and phospho-MEK using Western blotting, is comparable with oncogenic NRAS Q61R constructs, and less than NRAS WT. C and D, KRAS ITD constructs are similarly able to induce pathway overactivation when overexpressed in HEK293T cells (C) at a greater level than KRAS4b WT and (D) at similar levels to KRAS G12D.

Figure 4.

The ITD mutation in RAS does not affect RAS–RAF1 interaction but blocks RAS-RasGAP binding. A and B, isothermal titration calorimetry experiments to measure the dissociation constant for GMPPNP-bound WT NRAS and NRAS ITD with (A) RasGAP NF1 (GRD) and (B) effector RAF1 (RBD). Differential power (DP) is a measure of energy required to maintain isothermal conditions between the reference cell and the sample cell. C, mBRET saturation values assessing the ability of NF1 GAP co-expression to squelch RAF–RAS interaction for WT and ITD mutant KRAS and NRAS. The error bars represent the standard deviation of three technical replicates.

Figure 5.

Crystal structure of GDP-bound NRAS ITD and KRAS ITD provide insights into the effect of ITD on RAS interaction with GAP and effector proteins. A, the tertiary structure of GDP-bound NRAS ITD. B, the tertiary structure of GDP-bound KRAS ITD. C and D, structural superposition of GDP-bound NRAS ITD with (C) GDP-bound WT KRAS and (D) GMPPNP-bound WT NRAS. E–G, models of ITD mutants of NRAS and KRAS in complex with (E) NF1-GRD (PDB ID: 6OB2), (F) PI3Kγ (PDB ID: 1HE8), and (G) RAF1-RBD (PDB ID: 4G0N) generated using the structural superposition of NRAS ITD on K/HRAS present in KRAS-NF1, HRAS-PI3Kγ, and HRAS-RAF1 (RBD) complexes. These models suggest that the ITD (shown in red) in RAS would sterically clash with NF1 GAP and PI3Kγ and not with RAF1.