Biology subjects

Stites, E. C.

Publications and source records attributed to Stites, E. C..

5 recordsLinked to original sources

bioRxiv2024

Analysis of the Modulation of RAF Signaling by 14-3-3 Proteins

The regulation of cellular biochemical signaling reactions includes the modulation of protein activity through a variety of processes. For example, signaling by the RAF kinases, which are key transmitters of extracellular growth signals downstream from the RAS GTPases, is modulated by dimerization, protein conformational changes, post-translational modifications, and protein-protein interactions. 14-3-3 proteins are known to play an important role in RAF signal regulation, and have the ability to stabilize both inactive (monomeric) and active (dimeric) states of RAF. It is poorly understood how these antagonistic roles ultimately modulate RAF signaling. To investigate, we develop a mathematical model of RAF activation with both roles of 14-3-3, perform algebraic and numeric analyses, and compare with available experimental data. We derive the conditions necessary to explain experimental observations that 14-3-3 overexpression activates RAF, and we show that strong binding of 14-3-3 to Raf dimers alone is not generally sufficient to explain this observation. Our integrated analysis also suggests that RAF-14-3-3 binding is relatively weak for the reasonable range of parameter values, and suggests the Raf dimer-14-3-3 interactions are stabilized primarily by avidity. Lastly we find that in the limit of paired weak/avidity driven interactions between RAF and 14-3-3, the paired binding interactions may be reasonably approximated with a strong, single, equilibrium reaction. Overall, our work presents a mathematical model that can serve as a foundational piece for future, extended, studies of signaling reactions involving regulated RAF kinase activity.

systems biology↗

bioRxiv2019

RAF autoinhibition and 14-3-3 proteins promote paradoxical activation

RAF kinase inhibitors can, in some conditions, increase RAF kinase signaling. This process, which is commonly referred to as "paradoxical activation" (PA), is incompletely understood. RAF kinases are regulated by autoinhibitory conformational changes, and the role of these conformational changes in PA is unclear. Our mathematical investigations reveal that a dynamic equilibrium between autoinhibited and non-autoinhibited forms of RAF, along with the RAF inhibitor stabilization of the non-autoinhibited form, can be sufficient to create PA. Using both computational and experimental methods we demonstrate that 14-3-3 proteins, which stabilize both RAF autoinhibition and RAF dimerization, potentiate PA. Our model led us to hypothesize that increased 14-3-3 expression would amplify PA for the third generation RAF inhibitors that normally display minimal to no PA. Our subsequent experiments find that 14-3-3 overexpression increases PA, increases RAF dimerization, and promotes resistance to these inhibitors, effectively "breaking" these "paradox breaker" and pan-RAF inhibitors. Overall, this work reveals a robust mechanism for PA based solely on equilibrium dynamics of canonical interactions in RAF signaling and identifies conditions which allow PA to occur.

cancer biology↗

bioRxiv2019

Inhibition of both mutant and wild-type RAS-GTP in KRAS G12C colorectal cancer through cotreatment with G12C and EGFR inhibitors

The combination of KRAS G12C inhibitors with EGFR inhibitors has reproducibly been shown to be beneficial. Here, we reveal a new benefit of this combination: it effectively inhibits both wild-type and mutant RAS. A role for WT RAS inhibition has not previously been reported for this important combination of targeted therapies. We believe that targeting both mutant and wild-type RAS helps explain why this combination of inhibitors is effective.

cancer biology↗

bioRxiv2018

A Systems Mechanism for KRAS Mutant Allele Specific Responses to Targeted Therapy

A well-established genotype to phenotype relationship in genomic medicine is that activating KRAS mutations indicate resistance to anti-EGFR agents. We used a computational model of Ras signaling to investigate a confusing exception to this relationship whereby colorectal cancers with one specific, constitutively-active, mutant, KRAS G13D, respond to anti-EGFR agents. Our computational simulations of the biochemical processes that regulate Ras suggest EGFR inhibition reduces wild-type Ras activation in KRAS G13D mutant cancer cells more than in other KRAS mutant cancer cells. The model also reveals a non-intuitive, mutant-specific, dependency of wild-type Ras activation on EGFR. This dependency is determined by the interaction strength between a KRAS mutant and tumor suppressor neurofibromin. Our prospective experiments confirm this mechanism that arises from the systems-level regulation of Ras pathway signaling. Overall, our work demonstrates how systems approaches enable mechanism-based inference in genomic medicine.

cancer biology↗

bioRxiv2017

A quantitative systems pharmacology analysis of KRAS G12C covalent inhibitors

The KRAS oncogene is the most common, activating, oncogenic mutation in human cancer. KRAS has proven difficult to target effectively. Two different strategies have recently been described for covalently targeting the most common activating KRAS mutant in lung cancer, KRAS G12C. Previously, we have developed a computational model of the processes that regulate Ras activation and this model has proven useful for understanding the complex behaviors of Ras signaling. Here, we use this model to perform a computational systems pharmacology analysis of KRAS G12C targeted covalent inhibitors. After updating our model to include Ras protein turnover, we verified the validity of our model for problems in this domain by comparing model behaviors with experimental behaviors. The model naturally reproduces previous experimental data, including several experimental observations that were interpreted as being contrary to conventional wisdom. Overall, this suggests that our model describes the Ras system well, including those areas where conventional wisdom struggles. We then used the model to investigate possible strategies to improve the ability of KRAS G12C inhibitors to inhibit Ras pathway signaling. We identify one, as of yet unexplored mechanism, that, if optimized, could improve the effectiveness of one class of KRAS inhibitor. We also simulated resistance to targeted therapies and found that resistance promoting mutations may reverse which class of KRAS G12C inhibitor inhibits the system better, suggesting that there may be value to pursuing both types of KRAS G12C inhibitors. Overall, this work demonstrates that systems biology approaches can provide insights that inform the drug development process.

systems biology↗