Theoretical analysis reveals a role for RAF conformational autoinhibition in paradoxical activation

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1. 

   Version published to 10.7554/elife.82739 on eLife

   Oct 12, 2023
2. 

   eLife

   Jul 19, 2023

   Author Response

   Reviewer #1 (Public Review):

   The authors investigate the mechanistic underpinning of paradoxical activation (PA) of RAF by small molecule kinase inhibitors using mathematical modeling. The main novelty of the study is the consideration of RAF conformational autoinhibition by its N-terminal regulatory domains as a new determinant of PA. This mechanism has not been explicitly considered in previous theoretical studies, which are based on two other mechanisms: drug-induced RAF oligomerization into active dimers (dimer potentiation DP) and negative cooperativity (NC) of inhibitor binding by a second monomer in the inhibitor-induced RAF kinase dimerization. An important discovery of this study is that conformational autoinhibition is a critical determinant of PA and that in some cases, it can contribute to PA in the …

   Author Response

   Reviewer #1 (Public Review):

   The authors investigate the mechanistic underpinning of paradoxical activation (PA) of RAF by small molecule kinase inhibitors using mathematical modeling. The main novelty of the study is the consideration of RAF conformational autoinhibition by its N-terminal regulatory domains as a new determinant of PA. This mechanism has not been explicitly considered in previous theoretical studies, which are based on two other mechanisms: drug-induced RAF oligomerization into active dimers (dimer potentiation DP) and negative cooperativity (NC) of inhibitor binding by a second monomer in the inhibitor-induced RAF kinase dimerization. An important discovery of this study is that conformational autoinhibition is a critical determinant of PA and that in some cases, it can contribute to PA in the absence of DP and NC. Another novelty is the consideration of RAF interaction with 14-3-3 proteins, as a determinant of PA. The 14-3-3 dimeric scaffolds play an important role in the regulation of both autoinhibited and active states of RAF and thus understanding how their interaction with RAF influences PA by RAF inhibitors is important. Using mathematical modeling the authors show that 14-3-3 binding does indeed enhance PA in response to a spectrum of RAF inhibitors.

   We thank Reviewer #1 for reviewing our manuscript, and we agree with this summary.

   Strengths

   The overall strength of this study is that it increases the mechanistic understanding of how PA of RAF originates in response to its inhibitors. Consideration of the effect that the inhibitors play in breaking the autoinhibited conformation has been overlooked by previous mathematical analyses of PA, and this study bridges this gap. By doing so, the authors discover that breaking that autoinhibited state is in fact the biggest contribution to PAB by RAF inhibitors. In my opinion, this is the most impactful finding of this study, which additionally speaks to how important are the autoinhibitory mechanisms for constraining basal RAF signaling in cells. The presented analysis also shows that consideration of conformational autoinhibition can explain PA by all different types of RAF inhibitors (1, 1.5, and 2), which until now has been difficult to reconcile.

   Another important contribution of this study is the investigation of how the 14-3-3 scaffold proteins can further contribute to PA. This is exciting, especially in light of recent elegant structural studies that unveiled complex regulation of RAF by 14-3-3 (which are both important for RAF inhibition and stabilization of the active dimers). The authors dissect these opposing roles of 14-3-3 in their model and show the autoinhibitory interaction with 14-3-3, but not the activating one, significantly increases the PA response. Their findings that an increase in the 143-3 levels amplifies PA is very interesting and somewhat provocative as it is unclear how much 14-3-3 levels in cells can oscillate. To this end, the authors show that elevated 14-3-3 levels are observed with increased time of RAF inhibitor treatment, which might point to a new mechanism of resistance to RAF inhibitors.

   We thank reviewer #1 for the enthusiastic review and for highlighting the value of bringing conformational autoinhibition into the study and understanding of paradoxical activation. We also appreciate the positive consideration of the 14-3-3 section of the manuscript and the helpful suggestions later in the review. In this revision, we have taken the offered option of removing all of the 14-3-3 theoretical and experimental work. We plan to expand the 14-3-3 work in our ongoing work, in accordance with the thoughtful input from reviewers #1, #2, and #3 on this topic. Thank you.

   Weaknesses

   The main weakness of the study is the limited experimental analysis conducted to test the predictions that arise from the mathematical models. While some of these predictions might be challenging to test, the one which is tested is not tested rigorously. The experiments focus on 14-3-3-based regulation and are conducted in cells by observing the effect of 14-3-3 overexpression on the inhibition of RAF signaling by its different kinase inhibitors. While the authors acknowledge that too, 14-3-3 overexpression will have a multifaceted effect on signaling as these scaffold proteins participate in the regulation of almost all signaling events. Thus, the proposed experiments are not sufficient to conclude that the observed effects are in fact a result of 14-3-3/RAF interaction.

   The authors consider conformational autoinhibition and 14-3-3 stabilization of autoinhibited RAF as two different mechanisms. While it is not a weakness, I am curious how accurate is the consideration of the autoinhibited state of RAF in the absence of 14-3-3. Is it known how the proportion of RAF in cells in its inactive state exists while not bound to 14-3-3?

   We thank Reviewer #1 for this input on how we can significantly improve the 14-3-3 section of the manuscript. We have removed the 14-3-3 sections due to the consensus input of all three reviewers and the presented option of focusing on the theoretical results of how conformational autoinhibition influences PA. We do plan to continue this research program on beyond this manuscript, and we therefore very much appreciate these insights into which aspects should be supported with additional experiments and the challenges that follow from the pleiotropic activities of 14-3-3 proteins. The suggestion of quantifying the ratio of autoinhibited to non-autoinhibited forms of RAF when 14-3-3 proteins are present and absent is an experiment we plan to pursue in our future work. It will require us to learn new methods and/or to form new collaborations, and we therefore appreciate the consensus opinion that this would be outside of our current expertise and outside of the scope of the focused manuscript on modeling the impact of conformational autoinhibition on PA.

   Reviewer #2 (Public Review):

   In this study, the authors set out to investigate factors that have been neglected in existing mathematical models for the paradoxical activation (PA) of RAF by pharmacological inhibitors. The PA phenomenon is well known and is thought to be an important factor in limiting the effectiveness of RAF inhibitors. The authors primarily use mathematical models, first to examine the importance of conformational autoinhibition of RAF monomers, and later to investigate the potential role played by binding of 14-3-3 proteins to either autoinhibited monomers or active dimers. The authors develop several model variants containing different candidate mechanisms and generate analytical solutions that demonstrate under which parameter conditions PA may occur within these models. The use of analytical solutions is a strong point of the paper, as it allows evaluation of the models independently of specific parameter values. This analysis suggests that conformational autoinhibition is a very strong contributor to paradoxical activation, as models that include this mechanism show substantially larger concentration ranges under which RAF is activated by inhibitors. Fitting the parameters of the model to a published dataset on multiple inhibitors further suggests that conformational activation is important, as models containing this mechanism can fit the dataset with significantly lower error. Another interesting observation is that the different types of RAF inhibitors (1, 1.5, 2) fit the data with parameter values that are reasonably similar within each type. A moderate weakness in this analysis is that all of these observations provide indirect evidence for the importance of conformational autoinhibition. A direct test of whether PA is reduced when conformational autoinhibition is removed would be more compelling, but such a test could be difficult to set up experimentally.

   We thank Reviewer #2 for reviewing our manuscript, and we agree with this summary. We agree that an experimental test where conformational autoinhibition is removed from the system would a very compelling experiment, but that it would be difficult to set up experimentally. We appreciate the option to focus on the theoretical advance in our revision, and we will be working toward such an experiment.

   The authors then perform an analysis of how 14-3-3 binding to either autoinhibited monomers or active dimers might enhance PA. A new model is constructed that contains these binding events in the context of conformational activation, but without negative cooperativity or dimer potentiation included, for the sake of limiting complexity. These models implicate monomer binding, but not dimer binding as a contributor to PA. They follow up on this model result by overexpressing 14-3-3 proteins in two RAS-mutant cell lines, which leads to both higher baseline ERK phosphorylation and to a wider range of inhibitor-induced PA, as predicted by the model. A cell-based RAF dimerization assay also shows higher dimerization effects when 14-3-3 plasmids are transfected as well. This experimental evidence provides strong support for the model, although one drawback, which is noted by the authors in the discussion, is that 14-3-3 overexpression could potentially exert effects on RAF activity through pleiotropic effects other than the binding actions included in the model.

   We thank Reviewer #2 for the input on the 14-3-3 section of the manuscript. Although it has been removed from the revision, all of the comments from the review will be helpful for our ongoing work.

   Overall, this study makes a strong contribution to understanding the paradoxical effects of RAF inhibitors on the RAS/ERK signaling pathway, which remains a significant problem in the use of targeted inhibitors for cancer. Demonstrating that both conformational activation and 14-3-3 binding strongly contribute to the PA effect is an important step forward, as it establishes that these mechanisms should not be overlooked when designing strategies to use Raf inhibitors.

   We appreciate the thoughtful review and helpful comments to improve the manuscript.

   Reviewer #3 (Public Review):

   The authors describe a mathematical and computational modeling study of RAF paradoxical activation (PA), a phenomenon in which RAF inhibitors exhibit a bell-shaped dose-response curve of Erk phosphorylation - activating signaling through wild-type RAF at low drug concentrations before inhibiting it at higher concentrations. They explore three distinct mechanisms that may contribute to PA - conformational autoinhibition, negative cooperativity, and drug-induced dimerization - and conclude that all three are required to best fit published data that show the PA phenomenon. They explore the effect of 14-3-3 binding to RAF both computationally and experimentally and reach the conclusion that 14-3-3 can potentiate the PA phenomenon via stabilization of the autoinhibited conformation.

   We thank Reviewer #3 for reviewing our manuscript, and for the helpful comments in the review.

   Strengths:

   One key finding will be quite valuable to the field - that paradoxical activation can arise in the absence of negative cooperativity and without any effect of the inhibitor on the propensity of RAF to dimerize, provided that there exists a "conformationally autoinhibited" state that cannot dimerize and cannot bind inhibitor. This finding is important because negative cooperativity and dimer-induction have been a major focus - arguably the main focus - of prior studies of the phenomenon and also a source of considerable confusion. Inhibitors with very different chemical structures and binding properties - type 1.5 inhibitors that are dimer-breakers (and may or may not exhibit negative cooperativity) and type I and II inhibitors that can promote dimers (and almost certainly do not exhibit negative cooperativity) can nevertheless both exhibit PA. Thus the authors' modeling provides a unifying explanation - it is not dimerinduction or negative cooperativity that is at the root of PA, rather it is that there exists an autoinhibited state that can neither bind inhibitor nor dimerize. The authors further show that negative cooperativity and dimer-induction can act in concert with "conformational autoinhibition" to modify the PA response in a drug-specific manner.

   We thank Reviewer #3 for highlighting these strengths and their value to the field. In the focused paper, we have updated our discussion of the fits and of the model to highlight these points better.

   Weaknesses:

   Unfortunately, the authors don't really explain in a straightforward manner what is going on with the conformational autoinhibition model (Figure 2A). One has to read carefully and all the way to section 3 of appendix 1 to piece it together. In short, what the math shows is that at least for certain ranges of parameter values, the presence of an inhibitor can increase the concentration of dimers, even when it does not change the equilibrium constant for dimer formation, and some of those dimers will have an active, drug-free protomer. This is because the inhibitor effectively traps open monomers, which can then capture drug-free open monomers to form active dimers (active in one subunit, inactive and drug-bound in the other). As inhibitor concentration increases, the pool of autoinhibited RAF is diminished, and eventually, it is shifted completely to fully inhibited dimers. But at low concentrations of inhibitor, there is a net increase in dimerized (active) but drug-free protomers (see figure on page 27 of the appendix). Voila, paradoxical activation, with no need to invoke negative cooperativity.

   We apologize for the confusion, and agree that the description/walk through in the appendix should be featured more prominently in the manuscript. To this end, we have added a section to the main manuscript (titled “Paradoxical activation reflects a shifting balance of signaling complexes”) that includes the content that was previously in the appendix, and we have added a supplementary figure (Figure 2 – figure supplement 2) which includes the figures from the appendix. Thank you for your thorough review and working through the appendix, and we appreciate this suggestion.

   Considering the potential for confusion around what is meant by "drug-induced dimerization" as an effect distinct from the effect of the drug in promoting RAF dimerization in their conformational autoinhibition model, it would have been helpful for the authors to explicitly address the distinction (drug-induced dimerization alters the equilibrium constant for dimerization; this is not a feature of the conformational autoinhibition model).

   Thank you for this suggestion. We have clarified our text by rewriting it to read: … some RAF inhibitors have been shown to result in an increased level of RAF dimerization (Hatzivassiliou et al., 2010; Jin et al, 2017; Karoulia et al., 2016; Lavoie et al, 2013). This druginduced dimer potentiation is commonly thought of as manifesting in a higher affinity between RAF protomers when one (or both) are bound to a RAF inhibitor (Kholodenko, 2015).

   Also, I am confused by Figure 3C. The figure shows, and the authors state in the text, that for type II inhibitors an f > ~1 indicates a propensity to break dimers. But type 1.5 inhibitors should break dimers, and Type I and II inhibitors should promote dimers (at least some Type I and II drugs have been shown to promote kinase dimers). Seems that the predictions of the model are inconsistent with experimental data, at least for some inhibitors.

   We agree that discussing the fits, relating them to experimental data and current thinking in the field, is important. We have therefore significantly extended our discussion of the fits in Figure 3C in the Discussion of the text. The new text reads:

   It has previously been difficult to reconcile PA for Type I.5 inhibitors, which are sometimes thought of as dimer breakers because they position the alpha-C helix in the “out” position (in contrast to Type I and Type II inhibitors). Studies with recombinant protein and analytic ultracentrifugation clearly found type I.5 inhibitors to predominantly be in the monomeric form (Lavoie et al., 2013). Within-cell assays have similarly found type I.5 inhibitors to promote dimerization less than other Type I and Type II RAF inhibitors (Hatzivassiliou et al., 2010; Peng et al., 2015; Thevakumaran et al, 2015), however, RAF inhibitors still appeared to promote some dimerization in those in-cell assays. 14-3-3 binding proteins, which can help stabilize RAF dimers, may help explain this discrepancy (Kondo et al., 2019; Liau et al, 2020; Park et al., 2019). For example, by promoting the non-autoinhibited form, a type I.5 inhibitorbound RAF monomer is more dimerization capable than an autoinhibited (and non-inhibitor bound) RAF monomer, and even if the affinity is reduced compared to a non-autoinhibited and non-inhibitor bound RAF monomer, 14-3-3 proteins may be able to bind and overcome the effect. As our model does not explicitly include 14-3-3 proteins, this effect may contribute to our parameter estimation process finding an elevated binding affinity for type I.5 bound RAF monomers.

   Although negative cooperativity has been difficult to precisely measure experimentally, it has widely been assumed to be present to help explain the paradoxical activation caused by Type I.5 inhibitors that do not promote dimerization as strongly as other RAF inhibitors. Our best fit parameters did tend to have g values that were larger than 1, indicating that the model fit best when there was some negative cooperativity. This could suggest that negative cooperativity is more abundant than widely believed. Alternatively, the model without negative cooperativity was able to fit the data nearly as well as the full model that included negative cooperativity (i.e., Figure 3D). This may suggest that other processes not included in the model may be modulating paradoxical activation and the g parameter, as the only other term the model, is contributing to the models ability to account for these otherwise not included effects.

   We found parameter sets that reproduced available, published, data in order to test our model and investigate the potential for it to help illuminate aspects of PA. The best fit parameter sets further support a role for conformational autoinhibition and its modulation by RAF inhibitors in PA. However, it is also important not to read too deeply into the fits. For example, the data for the type II inhibitors AZ-628, LY3009120, and TAK-632 had small total fold-change PA magnitudes, and our fits for them have even less PA. We anticipate that the model-fitting approach would converge to increasingly accurate estimates for the parameters as the set of data being fit to expands. Additionally, quantitative experimental measurements of the parameters being fit should also cascade to impact other parameters and result in better estimates (Gutenkunst et al, 2007).

   A large part of the paper deals with the effect of 14-3-3 binding. In my view, this part of the manuscript is not particularly helpful. There is no evidence (that I am aware of) that 14-3-3 concentrations vary significantly, or that their variation affects RAF activity/signaling. Considering their abundance relative to RAF, and relatively high affinity for RAF, it is likely that both autoinhibited and active RAF are saturated with 14-3-3. (RAF that is not 14-3-3-bound is likely mostly bound to chaperones and not active). That said, the authors' conclusion (based on modeling) that 14-3-3 can increase the extent of paradoxical activation by stabilizing the autoinhibited state seems sensible, but hard to reconcile with their experimental result where they find increased basal signaling with 14-3-3 over-expression. It is also difficult to understand how increased 14-3-3 binding to RAF could lead to active RAF dimers that are not inhibited at 10-100 uM concentrations of potent RAF dimer inhibitors like LY3009120 (Fig. 5C). It seems more likely that 14-3-3 overexpression is promoting Erk phosphorylation in a manner that is (at least partially) Raf-independent. To their credit, the authors acknowledge this concern.

   We thank Reviewer #3 for the helpful critique of the section on 14-3-3. Although we have cut this section as part of the consensus review and suggestions for how to proceed with the revision, these points are very helpful for us as we consider how to interpret the modeling and experimental results of this section, how it fits into what is known, and what we should investigate next. Thank you.

   Finally, one comment regarding the presentation. The authors discuss conformational inhibition and 14-3-3 binding as if they are promoting and/or inducing paradoxical activation. This is pervasive in the paper, including in the title, and is distracting and potentially will mislead some readers. Obviously, it is RAF inhibitor that induces or promotes paradoxical activation. Conformational autoinhibition - mediated by 14-3-3 - is a feature of the system that makes paradoxical activation possible.

   We completely agree. We have rephrased to avoid this interpretation and we apologize for not recognizing it previously. Thank you for catching this and noting it for us to fix. As examples of the revisions to address this point, the last sentence of our abstract now reads:

   Overall, this work establishes conformational autoinhibition as a robust mechanism for RAFinhibitor driven PA based solely on equilibrium dynamics of canonical interactions that comprise RAF signaling and inhibition.

   And as another example, the third to last sentence in our Introduction now reads:

   Our modeling reveals that, under certain conditions, RAF autoinhibitory conformational changes and their modulation by RAF inhibitor binding can be sufficient to drive PA.

   Lastly, we have a last paragraph in the discussion that summarizes and hypothesizes to generalization:

   \Our analysis was motivated by RAF inhibitors and PA in RAS mutant cells treated with a RAF inhibitor. Our model, however, is generalizable to other systems that share the modeled features. We anticipate that PA will be observed for other proteins (a) that have a dynamic-equilibrium of conformations, (b) where not all conformations can dimerize, and (c) where drug binding the protein stabilizes one or more of the conformations that can dimerize. As dimerization and conformational autoinhibition are both common features for kinase regulation (Huse & Kuriyan, 2002; Lavoie et al, 2014), it seems reasonably to hypothesize PA will be observed for more kinases through modulation of the conformation and dimerization dynamic-equilibrium. Thank you for suggesting these changes.

   Read the original source
3. 

   eLife

   Jul 16, 2023

   eLife assessment

   This important study uses mathematical modelling to demonstrate that conformational autoinhibition of the RAF kinase is an important feature of its paradoxical activation by pharmacological inhibitors. This part of the theoretical analysis is highly compelling but its extension to the investigation of how the binding of 14-3-3 adaptors additionally contributes to the paradoxical activation phenomenon is incomplete and would benefit from more rigorous experimental validation. With the experimental part addressing 14-3-3-dependent regulation strengthened or the 14-3-3 part completely removed, this paper would be of considerable interest to cell biologists and cancer biologists, ultimately paving the way for improved RAF therapeutics.

   Read the original source
4. 

   eLife

   Jul 16, 2023

   Reviewer #1 (Public Review):

   The authors investigate the mechanistic underpinning of paradoxical activation (PA) of RAF by small molecule kinase inhibitors using mathematical modeling. The main novelty of the study is the consideration of RAF conformational autoinhibition by its N-terminal regulatory domains as a new determinant of PA. This mechanism has not been explicitly considered in previous theoretical studies, which are based on two other mechanisms: drug-induced RAF oligomerization into active dimers (dimer potentiation DP) and negative cooperativity (NC) of inhibitor binding by a second monomer in the inhibitor-induced RAF kinase dimerization. An important discovery of this study is that conformational autoinhibition is a critical determinant of PA and that in some cases, it can contribute to PA in the absence of DP and NC. …

   Reviewer #1 (Public Review):

   The authors investigate the mechanistic underpinning of paradoxical activation (PA) of RAF by small molecule kinase inhibitors using mathematical modeling. The main novelty of the study is the consideration of RAF conformational autoinhibition by its N-terminal regulatory domains as a new determinant of PA. This mechanism has not been explicitly considered in previous theoretical studies, which are based on two other mechanisms: drug-induced RAF oligomerization into active dimers (dimer potentiation DP) and negative cooperativity (NC) of inhibitor binding by a second monomer in the inhibitor-induced RAF kinase dimerization. An important discovery of this study is that conformational autoinhibition is a critical determinant of PA and that in some cases, it can contribute to PA in the absence of DP and NC. Another novelty is the consideration of RAF interaction with 14-3-3 proteins, as a determinant of PA. The 14-3-3 dimeric scaffolds play an important role in the regulation of both autoinhibited and active states of RAF and thus understanding how their interaction with RAF influences PA by RAF inhibitors is important. Using mathematical modeling the authors show that 14-3-3 binding does indeed enhance PA in response to a spectrum of RAF inhibitors.

   Strengths
   The overall strength of this study is that it increases the mechanistic understanding of how PA of RAF originates in response to its inhibitors. Consideration of the effect that the inhibitors play in breaking the autoinhibited conformation has been overlooked by previous mathematical analyses of PA, and this study bridges this gap. By doing so, the authors discover that breaking that autoinhibited state is in fact the biggest contribution to PAB by RAF inhibitors. In my opinion, this is the most impactful finding of this study, which additionally speaks to how important are the autoinhibitory mechanisms for constraining basal RAF signaling in cells. The presented analysis also shows that consideration of conformational autoinhibition can explain PA by all different types of RAF inhibitors (1, 1.5, and 2), which until now has been difficult to reconcile.

   Another important contribution of this study is the investigation of how the 14-3-3 scaffold proteins can further contribute to PA. This is exciting, especially in light of recent elegant structural studies that unveiled complex regulation of RAF by 14-3-3 (which are both important for RAF inhibition and stabilization of the active dimers). The authors dissect these opposing roles of 14-3-3 in their model and show the autoinhibitory interaction with 14-3-3, but not the activating one, significantly increases the PA response. Their findings that an increase in the 14-3-3 levels amplifies PA is very interesting and somewhat provocative as it is unclear how much 14-3-3 levels in cells can oscillate. To this end, the authors show that elevated 14-3-3 levels are observed with increased time of RAF inhibitor treatment, which might point to a new mechanism of resistance to RAF inhibitors.

   Weaknesses
   The main weakness of the study is the limited experimental analysis conducted to test the predictions that arise from the mathematical models. While some of these predictions might be challenging to test, the one which is tested is not tested rigorously. The experiments focus on 14-3-3-based regulation and are conducted in cells by observing the effect of 14-3-3 overexpression on the inhibition of RAF signaling by its different kinase inhibitors. While the authors acknowledge that too, 14-3-3 overexpression will have a multifaceted effect on signaling as these scaffold proteins participate in the regulation of almost all signaling events. Thus, the proposed experiments are not sufficient to conclude that the observed effects are in fact a result of 14-3-3/RAF interaction.

   The authors consider conformational autoinhibition and 14-3-3 stabilization of autoinhibited RAF as two different mechanisms. While it is not a weakness, I am curious how accurate is the consideration of the autoinhibited state of RAF in the absence of 14-3-3. Is it known how the proportion of RAF in cells in its inactive state exists while not bound to 14-3-3?

   Read the original source
5. 

   eLife

   Jul 16, 2023

   Reviewer #2 (Public Review):

   In this study, the authors set out to investigate factors that have been neglected in existing mathematical models for the paradoxical activation (PA) of RAF by pharmacological inhibitors. The PA phenomenon is well known and is thought to be an important factor in limiting the effectiveness of RAF inhibitors. The authors primarily use mathematical models, first to examine the importance of conformational autoinhibition of RAF monomers, and later to investigate the potential role played by binding of 14-3-3 proteins to either autoinhibited monomers or active dimers. The authors develop several model variants containing different candidate mechanisms and generate analytical solutions that demonstrate under which parameter conditions PA may occur within these models. The use of analytical solutions is a strong …

   Reviewer #2 (Public Review):

   In this study, the authors set out to investigate factors that have been neglected in existing mathematical models for the paradoxical activation (PA) of RAF by pharmacological inhibitors. The PA phenomenon is well known and is thought to be an important factor in limiting the effectiveness of RAF inhibitors. The authors primarily use mathematical models, first to examine the importance of conformational autoinhibition of RAF monomers, and later to investigate the potential role played by binding of 14-3-3 proteins to either autoinhibited monomers or active dimers. The authors develop several model variants containing different candidate mechanisms and generate analytical solutions that demonstrate under which parameter conditions PA may occur within these models. The use of analytical solutions is a strong point of the paper, as it allows evaluation of the models independently of specific parameter values. This analysis suggests that conformational autoinhibition is a very strong contributor to paradoxical activation, as models that include this mechanism show substantially larger concentration ranges under which RAF is activated by inhibitors. Fitting the parameters of the model to a published dataset on multiple inhibitors further suggests that conformational activation is important, as models containing this mechanism can fit the dataset with significantly lower error. Another interesting observation is that the different types of RAF inhibitors (1, 1.5, 2) fit the data with parameter values that are reasonably similar within each type. A moderate weakness in this analysis is that all of these observations provide indirect evidence for the importance of conformational autoinhibition. A direct test of whether PA is reduced when conformational autoinhibition is removed would be more compelling, but such a test could be difficult to set up experimentally.

   The authors then perform an analysis of how 14-3-3 binding to either autoinhibited monomers or active dimers might enhance PA. A new model is constructed that contains these binding events in the context of conformational activation, but without negative cooperativity or dimer potentiation included, for the sake of limiting complexity. These models implicate monomer binding, but not dimer binding as a contributor to PA. They follow up on this model result by overexpressing 14-3-3 proteins in two RAS-mutant cell lines, which leads to both higher baseline ERK phosphorylation and to a wider range of inhibitor-induced PA, as predicted by the model. A cell-based RAF dimerization assay also shows higher dimerization effects when 14-3-3 plasmids are transfected as well. This experimental evidence provides strong support for the model, although one drawback, which is noted by the authors in the discussion, is that 14-3-3 overexpression could potentially exert effects on RAF activity through pleiotropic effects other than the binding actions included in the model.

   Overall, this study makes a strong contribution to understanding the paradoxical effects of RAF inhibitors on the RAS/ERK signaling pathway, which remains a significant problem in the use of targeted inhibitors for cancer. Demonstrating that both conformational activation and 14-3-3 binding strongly contribute to the PA effect is an important step forward, as it establishes that these mechanisms should not be overlooked when designing strategies to use Raf inhibitors.

   Read the original source
6. 

   eLife

   Jul 16, 2023

   Reviewer #3 (Public Review):

   The authors describe a mathematical and computational modeling study of RAF paradoxical activation (PA), a phenomenon in which RAF inhibitors exhibit a bell-shaped dose-response curve of Erk phosphorylation - activating signaling through wild-type RAF at low drug concentrations before inhibiting it at higher concentrations. They explore three distinct mechanisms that may contribute to PA - conformational autoinhibition, negative cooperativity, and drug-induced dimerization - and conclude that all three are required to best fit published data that show the PA phenomenon. They explore the effect of 14-3-3 binding to RAF both computationally and experimentally and reach the conclusion that 14-3-3 can potentiate the PA phenomenon via stabilization of the autoinhibited conformation.

   Strengths:

   One key finding …

   Reviewer #3 (Public Review):

   The authors describe a mathematical and computational modeling study of RAF paradoxical activation (PA), a phenomenon in which RAF inhibitors exhibit a bell-shaped dose-response curve of Erk phosphorylation - activating signaling through wild-type RAF at low drug concentrations before inhibiting it at higher concentrations. They explore three distinct mechanisms that may contribute to PA - conformational autoinhibition, negative cooperativity, and drug-induced dimerization - and conclude that all three are required to best fit published data that show the PA phenomenon. They explore the effect of 14-3-3 binding to RAF both computationally and experimentally and reach the conclusion that 14-3-3 can potentiate the PA phenomenon via stabilization of the autoinhibited conformation.

   Strengths:

   One key finding will be quite valuable to the field - that paradoxical activation can arise in the absence of negative cooperativity and without any effect of the inhibitor on the propensity of RAF to dimerize, provided that there exists a "conformationally autoinhibited" state that cannot dimerize and cannot bind inhibitor. This finding is important because negative cooperativity and dimer-induction have been a major focus - arguably the main focus - of prior studies of the phenomenon and also a source of considerable confusion. Inhibitors with very different chemical structures and binding properties - type 1.5 inhibitors that are dimer-breakers (and may or may not exhibit negative cooperativity) and type I and II inhibitors that can promote dimers (and almost certainly do not exhibit negative cooperativity) can nevertheless both exhibit PA. Thus the authors' modeling provides a unifying explanation - it is not dimer-induction or negative cooperativity that is at the root of PA, rather it is that there exists an autoinhibited state that can neither bind inhibitor nor dimerize. The authors further show that negative cooperativity and dimer-induction can act in concert with "conformational autoinhibition" to modify the PA response in a drug-specific manner.

   Weaknesses:

   Unfortunately, the authors don't really explain in a straightforward manner what is going on with the conformational autoinhibition model (Figure 2A). One has to read carefully and all the way to section 3 of appendix 1 to piece it together. In short, what the math shows is that at least for certain ranges of parameter values, the presence of an inhibitor can increase the concentration of dimers, even when it does not change the equilibrium constant for dimer formation, and some of those dimers will have an active, drug-free protomer. This is because the inhibitor effectively traps open monomers, which can then capture drug-free open monomers to form active dimers (active in one subunit, inactive and drug-bound in the other). As inhibitor concentration increases, the pool of autoinhibited RAF is diminished, and eventually, it is shifted completely to fully inhibited dimers. But at low concentrations of inhibitor, there is a net increase in dimerized (active) but drug-free protomers (see figure on page 27 of the appendix). Voila, paradoxical activation, with no need to invoke negative cooperativity.

   Considering the potential for confusion around what is meant by "drug-induced dimerization" as an effect distinct from the effect of the drug in promoting RAF dimerization in their conformational autoinhibition model, it would have been helpful for the authors to explicitly address the distinction (drug-induced dimerization alters the equilibrium constant for dimerization; this is not a feature of the conformational autoinhibition model).

   Also, I am confused by Figure 3C. The figure shows, and the authors state in the text, that for type II inhibitors an f > ~1 indicates a propensity to break dimers. But type 1.5 inhibitors should break dimers, and Type I and II inhibitors should promote dimers (at least some Type I and II drugs have been shown to promote kinase dimers). Seems that the predictions of the model are inconsistent with experimental data, at least for some inhibitors.

   A large part of the paper deals with the effect of 14-3-3 binding. In my view, this part of the manuscript is not particularly helpful. There is no evidence (that I am aware of) that 14-3-3 concentrations vary significantly, or that their variation affects RAF activity/signaling. Considering their abundance relative to RAF, and relatively high affinity for RAF, it is likely that both autoinhibited and active RAF are saturated with 14-3-3. (RAF that is not 14-3-3-bound is likely mostly bound to chaperones and not active). That said, the authors' conclusion (based on modeling) that 14-3-3 can increase the extent of paradoxical activation by stabilizing the autoinhibited state seems sensible, but hard to reconcile with their experimental result where they find increased basal signaling with 14-3-3 over-expression. It is also difficult to understand how increased 14-3-3 binding to RAF could lead to active RAF dimers that are not inhibited at 10-100 uM concentrations of potent RAF dimer inhibitors like LY3009120 (Fig. 5C). It seems more likely that 14-3-3 overexpression is promoting Erk phosphorylation in a manner that is (at least partially) Raf-independent. To their credit, the authors acknowledge this concern.

   Finally, one comment regarding the presentation. The authors discuss conformational inhibition and 14-3-3 binding as if they are promoting and/or inducing paradoxical activation. This is pervasive in the paper, including in the title, and is distracting and potentially will mislead some readers. Obviously, it is RAF inhibitor that induces or promotes paradoxical activation. Conformational autoinhibition - mediated by 14-3-3 - is a feature of the system that makes paradoxical activation possible.

   Read the original source
7. 

   Version published to 10.1101/849489v2 on bioRxiv

   Oct 29, 2021
8. 

   Version published to 10.1101/849489v1 on bioRxiv

   Nov 20, 2019