Analysis of RAS protein interactions in living cells reveals a mechanism for pan-RAS depletion by membrane-targeted RAS binders

Li, Yao Cheng; Lytle, Nikki K.; Gammon, Seth T.; Wang, Luke; Hayes, Tikvah K.; Sutton, Margie N.; Bast, Robert C.; Der, Channing J.; Piwnica‐Worms, David; McCormick, Frank; Wahl, Geoffrey M.

doi:10.1073/pnas.2000848117

Cited by 19 publications

(24 citation statements)

References 54 publications

(66 reference statements)

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“…The varied chemistry of the HVRs, thus membrane preferences, favors distinct isoform nanoclusters. However, recently KRas4B nanoclusters were shown to be co-inhibited also by other isoforms and notably, by a subset of prenylated small GTPase family members, confirming the importance of dimer/co-cluster formation on cell membranes (Li et al 2020b), in line with an earlier hypothesis making such prediction (Nussinov et al 2020a).…”

Section: Earlier Discussion On Ras Isoformssupporting

confidence: 74%

Ras isoform-specific expression, chromatin accessibility, and signaling

et al. 2021

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The anchorage of Ras isoforms in the membrane and their nanocluster formations have been studied extensively, including their detailed interactions, sizes, preferred membrane environments, chemistry, and geometry. However, the staggering challenge of their epigenetics and chromatin accessibility in distinct cell states and types, which we propose is a major factor determining their specific expression, still awaits unraveling. Ras isoforms are distinguished by their C-terminal hypervariable region (HVR) which acts in intracellular transport, regulation, and membrane anchorage. Here, we review some isoform-specific activities at the plasma membrane from a structural dynamic standpoint. Inspired by physics and chemistry, we recognize that understanding functional specificity requires insight into how biomolecules can organize themselves in different cellular environments. Within this framework, we suggest that isoform-specific expression may largely be controlled by the chromatin density and physical compaction, which allow (or curb) access to “chromatinized DNA.” Genes are preferentially expressed in tissues: proteins expressed in pancreatic cells may not be equally expressed in lung cells. It is the rule—not an exception, and it can be at least partly understood in terms of chromatin organization and accessibility state. Genes are expressed when they can be sufficiently exposed to the transcription machinery, and they are less so when they are persistently buried in dense chromatin. Notably, chromatin accessibility can similarly determine expression of drug resistance genes.

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Section: Earlier Discussion On Ras Isoformssupporting

confidence: 74%

Ras isoform-specific expression, chromatin accessibility, and signaling

et al. 2021

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“…Multiple lines of evidence, including oncogenic KRAS copy number gain, and loss of the wild-type KRAS allele in human tumors, as well as functional studies in mouse models, suggest that wild-type KRAS is tumor-suppressive (also called “RAS allelic imbalance”), although the exact role of wild-type RAS in lung cancer is still controversial (3,33,35,45,46). Recent data have also suggested that interactions among H-, N- and KRAS proteins exist, thus raising the question of the roles of wild-type HRAS and NRAS in oncogenic KRAS-driven cancer (14,15,43).…”

Section: Discussionmentioning

confidence: 99%

“…Much of our understanding of RAS signaling has stemmed from diverse cellular and cellfree systems [12][13][14] . Thus, while recent studies have mapped KRAS protein-protein interaction networks and identified synthetic lethal interactions with oncogenic KRAS in human cell lines 10,11,15,16 , it remains difficult to assess the relevance of these biochemical and genetic interactions to cancer growth in vivo. Genetically engineered mouse models of oncogenic KRAS-driven cancer uniquely recapitulate autochthonous tumor growth and have contributed to our understanding of KRAS signaling 17 .…”

Section: Introductionmentioning

confidence: 99%

Multiplexed identification of RAS paralog imbalance as a driver of lung cancer growth

Tang

Shuldiner

Kelly

et al. 2021

Preprint

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Oncogenic KRAS mutations occur in approximately 30% of human lung adenocarcinoma. Despite tremendous effort over the past several decades, oncogenic KRAS-driven lung cancer remains difficult to treat, and our understanding of the positive and negative regulators of RAS signaling is incomplete. To uncover and corroborate the functional impact of diverse KRAS-interacting proteins on lung cancer growth in vivo, we integrate somatic CRISPR/Cas9-based genome editing in genetically engineered mouse models with tumor barcoding and high-throughput barcode sequencing (Tuba-seq). Through a series of in vivo CRISPR/Cas9 screens, we identified HRAS and NRAS as key suppressors of KRASG12D-driven lung tumor growth in vivo and confirmed these effects in oncogenic KRAS-driven human lung cancer cell lines. Mechanistically, we find that these RAS paralogs interact with oncogenic KRASG12D, suppress KRASG12D-KRASG12D interaction, and reduce downstream ERK signaling. Patient-derived mutations HRAST50M and HRASR123C partially abolished this effect. Comparison of the tumor-suppressive effects of HRAS and NRAS in KRASG12D- and BRAFV600E-driven lung cancer models confirmed that these RAS paralogs are specific suppressors of oncogenic KRAS-driven lung cancer in vivo. Our study outlines a technological avenue to specifically uncover positive and negative regulators of oncogenic KRAS-driven cancer in a multiplexed manner and highlights the role of RAS paralog imbalance in oncogenic KRAS-driven cancers.

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“…Work using recombinant proteins with model membranes indicated NRAS and KRAS did not colocalize but formed separate nanoclusters [ 41 ]. However, the expression of endogenous levels of fusions of split luciferase with all RAS isoforms indicates that all isoform pairs could cluster [ 49 ]. Indeed, the expression of each RAS isoform’s HVR was sufficient to reconstitute this behavior.…”

Section: Membrane Organization Ras Dynamics and Ras Clusteringmentioning

confidence: 99%

RAS Nanoclusters: Dynamic Signaling Platforms Amenable to Therapeutic Intervention

et al. 2021

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RAS proteins are mutated in approximately 20% of all cancers and are generally associated with poor clinical outcomes. RAS proteins are localized to the plasma membrane and function as molecular switches, turned on by partners that receive extracellular mitogenic signals. In the on-state, they activate intracellular signal transduction cascades. Membrane-bound RAS molecules segregate into multimers, known as nanoclusters. These nanoclusters, held together through weak protein–protein and protein–lipid associations, are highly dynamic and respond to cellular input signals and fluctuations in the local lipid environment. Disruption of RAS nanoclusters results in downregulation of RAS-mediated mitogenic signaling. In this review, we discuss the propensity of RAS proteins to display clustering behavior and the interfaces that are associated with these assemblies. Strategies to therapeutically disrupt nanocluster formation or the stabilization of signaling incompetent RAS complexes are discussed.

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Analysis of RAS protein interactions in living cells reveals a mechanism for pan-RAS depletion by membrane-targeted RAS binders

Cited by 19 publications

References 54 publications

Ras isoform-specific expression, chromatin accessibility, and signaling

Ras isoform-specific expression, chromatin accessibility, and signaling

Multiplexed identification of RAS paralog imbalance as a driver of lung cancer growth

RAS Nanoclusters: Dynamic Signaling Platforms Amenable to Therapeutic Intervention

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