It Takes Two To Target: A Study in KRAS Dimerization - Biochemistry

Publication Date (Web): April 12, 2018. Copyright © 2018 American Chemical Society. *E-mail: ... Selective Synergism Created by Interactive Nacre Fra...
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It Takes Two To Target: A Study in KRAS Dimerization Behnam Nabet*,†,‡ and Nathanael S. Gray*,†,‡ †

Department of Cancer Biology, Dana-Farber Cancer Institute, Boston, Massachusetts 02215, United States Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts 02115, United States



biochemical evaluation of KRASWT in lipid bilayers indicates that KRASWT is a monomer that lacks intrinsic dimerization ability.5 Furthermore, past studies have left open the question of whether KRASWT or KRASMUT dimerization has significant biological consequences. Ambrogio and co-workers hypothesized that KRAS dimerization may underlie the lack of sensitivity to MEK inhibitors and that KRAS dimerization may be important for KRAS activity. Using KRAS crystal structures, the authors identified D154 and R161 as candidate residues that could form a salt bridge and promote interactions between KRAS monomers. Fluorescence resonance energy transfer (FRET)-based proximity assays were employed to assess whether mutations in the putative dimerization residues (D154Q or R161E) could disrupt KRASWT or KRASMUT dimerization. While KRASD154Q did not impair the ability of KRAS to exchange GDP−GTP or to bind KRAS partners such as CRAF, D154Q effectively impaired homodimerization of KRASWT, KRASG12C, and KRASG12D. Importantly, impaired heterodimerization of KRASWT and KRASMUT had profound biological consequences in lung cancer model systems. KRASWT suppressed cell proliferation and diminished sensitivity to MEK inhibitors in xenograft models, potentially through reactivation of CRAF. However, the dimerization defective mutant failed to impair cell proliferation while also retaining sensitivity to MEK inhibitors in vivo. This provides support for a novel mechanism by which KRASWT expression and dimerization may determine responsiveness of KRAS mutant cancers to MEK inhibitors. Intriguingly, KRASWT status in KRAS-driven cancers may serve as a biomarker to predict patient populations in which MEK inhibition may have an improved response. As dimerization was critical for the activity of KRASWT, the authors next evaluated whether dimerization was required for the oncogenic ability of KRASMUT. Remarkably, cells expressing dimerization defective double mutants of KRASG12C/D154Q, KRASG12V/D154Q, and KRASG12D/D154Q, while biochemically behaving like their point mutant counterparts, were significantly impaired in their ability to proliferate and form tumors in mice, due to a collapse in the KRAS-driven signaling network. Although biochemical evidence indicates that KRAS is a monomer in lipid bilayers,5 these cell-based studies suggest that dimerization is crucial for the oncogenic activity of KRASMUT. Therefore, these two recent studies open a new set of questions for RAS investigators to explore. Further studies will be necessary to identify the molecular events that drive dimerization and the potential role of downstream signaling

RAS proteins (KRAS, HRAS, and NRAS) are exquisitely controlled GTPases that coordinate complex signaling networks to drive a cell toward a biological response. Common activating mutations in RAS genes, particularly KRAS, drive oncogenesis, and numerous strategies are under investigation for treatment of RAS-driven cancers. Advances toward the covalent inhibition of KRASG12C, a prevalent mutation in lung adenocarcinoma, have spurred excitement and wide-ranging research interests, which recently culminated in the disclosure of the first KRASG12C selective probe with in vivo efficacy in preclinical models.1 While there is optimism that covalent KRASG12C inhibitors will have clinical efficacy, it is essential to develop strategies to directly target other common KRAS mutants, including KRASG12D, KRASG12V, KRASQ61L, and KRASQ61R. Currently, mutant selective inhibitors targeting these oncoproteins remain far from clinical translation.2 An alternative targeting strategy for RAS-driven cancers includes inhibiting key signaling nodes downstream of RAS, particularly MEK or ERK. However, pharmacological inhibition of MEK or ERK has failed to show clinical efficacy in the context of KRAS mutations due to toxicity, relief of feedback control, and resistance.3 Nevertheless, it remains imperative not only to identify novel vulnerabilities of oncogenic KRAS but also to investigate whether there are specific contexts where MEK or ERK inhibition may be effective. In a recent issue of Cell, Ambrogio, Westover, Jänne, and colleagues combined genetic approaches, structural biology, and mouse models to identify dimerization as a general and potentially targetable feature of wild-type (KRASWT) and mutant KRAS (KRASMUT) function.4 They also present new insights into why MEK inhibitors fail to exhibit clinical efficacy in KRAS-driven lung adenocarcinomas. In these studies, the authors first take advantage of a system in which KRASWT or KRASMUT alleles, as well as combinations thereof, are introduced into engineered cells deficient in KRas, HRas, and NRas expression. Expression of oncogenic KRAS mutants (KRASG12C, KRASG12V, or KRASG12D) in cells lacking Ras expression increased cell growth, while the concomitant presence of KRASWT severely blunted cell growth, consistent with the described tumor-suppressive activity of KRASWT.3 Strikingly, KRASMUT-transformed cells were more sensitive to clinically relevant MEK inhibitors in the absence of KRASWT, indicating that KRASWT contributes to the diminished sensitivity of KRAS-transformed cells to MEK inhibitors. In order for active RAS to exert influence over signaling networks, RAS must be loaded with GTP and localized to the plasma membrane. In recent years, investigators have provided evidence that RAS dimerization may also be an important requirement for proper RAS activation of signaling pathways.3 However, discrepancies with these observations exist, as © XXXX American Chemical Society

Received: April 2, 2018

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DOI: 10.1021/acs.biochem.8b00376 Biochemistry XXXX, XXX, XXX−XXX

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Biochemistry

recent work. Future studies will ultimately reveal the generalizability of RAS dimerization and the exciting potential of exploiting RAS dimerization as a therapeutic strategy.

effectors, upstream kinases, or other factors in this process. It also remains unclear how KRASWT heterodimerization with KRASMUT impacts the oncogenic activity of KRASMUT, whether KRAS dimerization is a generalizable feature in other KRASdriven cancers with very limited therapeutic opportunities, such as pancreatic adenocarcinoma, and whether dimerization is a key feature of HRAS, NRAS, and other common activating KRAS lesions. The elegant study by Ambrogio and co-workers lends significant merit to the hypothesis that therapeutic strategies blocking the interaction between KRASWT and KRASMUT may improve sensitivity to MEK inhibition (Figure 1A,B) and that



AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. *E-mail: [email protected]. ORCID

Behnam Nabet: 0000-0002-6738-4200 Nathanael S. Gray: 0000-0001-5354-7403 Funding

This work was supported by an American Cancer Society Postdoctoral Fellowship (PF-17-010-01-CDD) (B.N.) and generous philanthropic gifts from the Hale Center for Pancreatic Cancer Research and the Katherine L. and Steven C. Pinard Research Fund. Notes

The authors declare the following competing financial interest(s): N.S.G. is a Scientific Founder and member of the Scientific Advisory Board of Syros Pharmaceuticals, C4 Therapeutics, and Petra Pharmaceuticals and is the inventor on IP licensed to these entities.

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ACKNOWLEDGMENTS We thank M. Kostic and S. Nabet for comments and helpful discussions. REFERENCES

(1) Janes, M. R., Zhang, J., Li, L. S., Hansen, R., Peters, U., Guo, X., Chen, Y., Babbar, A., Firdaus, S. J., Darjania, L., Feng, J., Chen, J. H., Li, S., Li, S., Long, Y. O., Thach, C., Liu, Y., Zarieh, A., Ely, T., Kucharski, J. M., Kessler, L. V., Wu, T., Yu, K., Wang, Y., Yao, Y., Deng, X., Zarrinkar, P. P., Brehmer, D., Dhanak, D., Lorenzi, M. V., Hu-Lowe, D., Patricelli, M. P., Ren, P., and Liu, Y. (2018) Targeting KRAS Mutant Cancers with a Covalent G12C-Specific Inhibitor. Cell 172, 578−589. (2) Ostrem, J. M., and Shokat, K. M. (2016) Direct small-molecule inhibitors of KRAS: from structural insights to mechanism-based design. Nat. Rev. Drug Discovery 15, 771−785. (3) Simanshu, D. K., Nissley, D. V., and McCormick, F. (2017) RAS Proteins and Their Regulators in Human Disease. Cell 170, 17−33. (4) Ambrogio, C., Kohler, J., Zhou, Z. W., Wang, H., Paranal, R., Li, J., Capelletti, M., Caffarra, C., Li, S., Lv, Q., Gondi, S., Hunter, J. C., Lu, J., Chiarle, R., Santamaria, D., Westover, K. D., and Janne, P. A. (2018) KRAS Dimerization Impacts MEK Inhibitor Sensitivity and Oncogenic Activity of Mutant KRAS. Cell 172, 857−868. (5) Chung, J. K., Lee, Y. K., Denson, J. P., Gillette, W. K., Alvarez, S., Stephen, A. G., and Groves, J. T. (2018) K-Ras4B Remains Monomeric on Membranes over a Wide Range of Surface Densities and Lipid Compositions. Biophys. J. 114, 137−145.

Figure 1. Disruption of KRAS dimerization improves sensitivity to MEK inhibitors and abrogates the oncogenic potential of mutant KRAS. (A) Despite the tumor-suppressive effects of wild-type KRAS (KRAS WT ), heterodimerization of KRAS WT with KRAS G12C , KRASG12V, or KRASG12D promotes insensitivity to MEK inhibitors (MEKi: trametinib and selumetinib). (B) Development of strategies to disrupt KRASWT heterodimerization with KRASG12C, KRASG12V, or KRAS G12D may enhance sensitivity to MEK inhibition. (C) Homodimerization of KRASG12C, KRASG12V, or KRASG12D promotes aberrant signaling and oncogenic proliferation. (D) Development of strategies to disrupt KRASG12C, KRASG12V, or KRASG12D homodimerization may diminish the aberrant biological effects of oncogenic KRAS.

forcing KRASMUT to function as a monomer may serve as a new strategy for targeting KRAS-driven cancers (Figure 1C,D). Tractable strategies to disrupt dimerization include development of synthetic monobodies or stapled peptides, which have been previously developed to alter RAS dimerization or interactions with key regulators.2,3 It will be important to use such tools to determine whether disruption of dimerization between endogenous KRAS proteins will have consequences similar to those of the engineered model systems used in the B

DOI: 10.1021/acs.biochem.8b00376 Biochemistry XXXX, XXX, XXX−XXX