FAK modulates glioblastoma stem cell energetics via regulation of glycolysis and glutamine oxidation

  1. Roza H. A. Masalmeh
  2. John C. Dawson
  3. Virginia Alvarez Garcia
  4. Morwenna T. Muir
  5. Roderick N. Carter
  6. Giles Hardingham
  7. Cameron Davies
  8. Rosina Graham
  9. Alex von Kriegsheim
  10. Jair Marques Junior
  11. Chinmayi Pednekar
  12. Steven M. Pollard
  13. Neil O. Carragher
  14. Valerie G. Brunton
  15. Margaret C. Frame

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Abstract

Glycolysis and the TCA cycle are reprogrammed in cancer cells to meet bioenergetic and biosynthetic demands, including by engagement with the extracellular matrix (ECM). We show that focal adhesion kinase (FAK), a mediator of integrin-ECM signalling, is driving cellular energetics in a stem cell model of glioblastoma (GBM). FAK gene deletion inhibits both glycolysis and glutamine oxidation, with increased mitochondrial fragmentation and elevated phosphorylation of the mitochondrial protein MTFR1L at S235. Simultaneously, FAK loss causes a mesenchymal to epithelial transition, enhanced acto-myosin contractility as shown by phospho-myosin light chain (p-MLC S19) and impaired cell migration/invasiveness. Rho-kinase (ROCK) inhibitors suppress p-MLC (S19) and restore glutamine oxidation and elongation of mitochondria. Thus, FAK is a key regulator of both glycolysis and glutamine oxidation mediated by acto-myosin contractility that controls both cell and mitochondrial morphology. Moreover, FAK-dependent cellular energetics are coincident properties with GBM stem cell migration, invasiveness and tumour growth in vivo.

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    Manuscript number: RC-2025-02946

    Corresponding author(s): Margaret, Frame

    Roza, Masalmeh

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    1. General Statements [optional]

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    We thank the reviewers for recognizing the significance of our work and for their constructive feedback and suggestions, most of which we have implemented in our revised manuscript.

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    Reviewer #1

    Evidence, reproducibility and clarity

    Review of Masalmeh et al. Title: "FAK modulates glioblastoma stem cell energetics..."

    Previous studies have implicated FAK and the related tyrosine kinase PYK2 in glioblastoma growth, cell migration, and invasion. Herein, using a murine stem cell model of glioblastoma, the authors used CRISPR to inactivate FAK, FAK-null cells selected and cloned, and lentiviral re-expression of murine FAK in the FAK-null cells (termed FAK Rx) was accomplished. FAK-/- cells were shown to possess epithelial characteristics whereas FAK Rx cells expressed mesenchymal markers and increased cell migration/invasion in vitro. Comparisons between FAK-/- and FAK Rx cells showed that FAK re-expressed increased mitochondrial respiration and amino acid uptake. This was associated with FAK Rx cells exhibiting filamentous mitochondrial morphology (potentially an OXPHOS phenotype) and decreased levels of MTFR1L S235 phosphorylation (implicated in mito morphology fragmentation). Mito and epithelial cell morphology of FAK-/- cells was reversed by treatment with Rho-kinase inhibitors that also increased mito metabolism and cell viability. Last, FAK-dependent glioblastoma tumor growth was shown by comparisons of FAK-/- and FAK Rx implantation studies.

    The studies by Masalmeh provide interesting findings associating FAK expression with changes in mitochondrial morphology, energy metabolism, and glutamate uptake. According to the authors model, FAK expression is supporting a glioblastoma stem cell like phenotype in vitro and tumor growth in vivo. What remains unclear is the mechanistic connection to cell changes and whether or not these are be dependent on intrinsic FAK activity or as the Frame group has previously published, potentially FAK nuclear localization. The associations with MTFR1L phosphorylation and effects by Rho kinase inhibition are likely indirect and remind this reviewer of long-ago studies with FAK-null fibroblasts that exhibit epithelial characteristics, still express PYK2, exhibited elevated RhoA GTPase activity. Some of these phenotypes were linked to changes in RhoGEF and RhoGAP signaling with FAK and/or Pyk2. At a minimum, it would be informative to know whether Pyk2 signaling is relevant for observed phenotypes and whether the authors can further support their associations with FAK-targeted or FAK-Pyk2-targeted inhibitors or PROTACs.

    Some questions that would enhance potential impact.

    1. Cell generation. Please describe the analysis of FAK-/- clones in more detail. The "low viability" phenotype needs further explanation with regard to clonal expansion and growth characteristics?

    Response:

    • We included a better description and a supplementary figure in our revised manuscript to indicate that we have examined several FAK -/- clones and confirmed that our observations were not due to clonal variation; multiple clones displayed similar morphological changes (Figure S1D). We also show that the elongated mesenchymal-like morphology was observed at 48 h after nucleofecting the cells with the FAK‑expressing vector, before beginning G418 selection to enrich for cells expressing FAK (Figure S1C). We also included experiments to acutely modulate FAK signalling (detaching and seeding cells on fibronectin) (Figure S2D, E, F and Figure S3) to exclude the possibility that the profound effects are due to protocols/selection we used for generating FAK-deleted cells.
    • Regarding the term "low viability", we have clarified in the text that there is no significant difference in cell number (Figure S1A) or 'cell viability' when it is assessed by trypan blue exclusion (a non-mitochondria-dependent read-out) (Figure S1B) between FAK-expressing FAK Rx and FAK-/- cells cultured for three days under normal conditions. Therefore, we agree the term 'cell viability' in this context could be confusing and have replace "cell viability" with "metabolic activity as measured by Alamar Blue." in Figure 1D and Figure 5B, and the corresponding text in the original manuscript. This wording more accurately reflects the data.

    Figure 1F: need further support of MET change upon FAK KO and EMT reversion.

    Response: We have added a heatmap (Figure S1E) illustrating the changes in protein expression of core-enriched EMT/MET genes products (by proteomics) after FAK gene deletion (EMT genes as defined in Howe et al., 2018) ; this strengthens the conclusion that the MET reversion morphological phenotype is accompanied by recognised MET protein changes.

    Fig. 2: Need further support if FAK effects impact glycolysis or oxidative phosphorylation in particular as implicated by the stem cell model.

    Response: We show that FAK impacts both glycolysis (Figure 2A, 2E, and 2F) and mitochondrial oxidative phosphorylation on the basis of the oxygen consumption rate (OCR) (Figure 2B, and 2D), showing both are contributing pathways to FAK-dependent energy production. We have clarified this in the text.

    Is there a combinatorial potential between FAKi and chemotherapies used for glioblastoma. Need to build upon past studies.

    Response: Yes, previous studies suggest that inhibiting FAK can sensitize GBM cells to chemotherapy (Golubovskaya et al., 2012; Ortiz-Rivera et al., 2023). We have included a paragraph in the discussion section to make sure this is clearer. Although it is not the subject of this study, we appreciate it is useful context.

    The notation of changes in glucose transporter expression should be followed up with regard to the potential that FAK-expressing cells may have different uptake of carbon sources and other amino acids. Altered uptake could be one potential explanation for increase glycolysis and glutamine flux.

    Response: We agree with the reviewer that glucose uptake could be contributing and we include data that 2 glucose transporters are indeed FAK-regulated namely Glucose transporter 1 (GLUT1, encoded by Slc2a1 gene) and Glucose transporter 3 (GLUT 3, encoded by Slc2a3 gene) (shown in Figure S2B and C).

    It would be helpful to support the confocal microscopy of mitos with EM.

    Response:

    We are concerned (and in our experience) that Electron microscopy (EM) may introduce artefacts during sample preparation. In contrast, immunofluorescence sample preparation is less susceptible to artefacts. The SORA system we used is not a conventional point-scanning confocal microscope, but is a super-resolution module based on a spinning disk confocal platform (CSU-W1; Yokogawa) using optical pixel reassignment with confocal detection. This method enhances resolution in all dimensions with resolution in our samples measured at 120nm. This has been instructive in defining a new level of changes in mitochondrial morphology upon FAK gene deletion.

    Lack of FAK expression with increased MTFR1 phosphorylation is difficult to interpret.

    Response: We do not directly show that this phosphorylation event is causal in our experiments; however, we think it important to document this change since it has been published that phosphorylation of MTFR1 has been causally linked to the mitochondrial morphology we observed in other systems (Tilokani et al., 2022).

    Need to have better support between loss of FAK and the increase in Rho signaling. Use of Rho kinase inhibitors is very limited and the context to FAK (and or Pyk2) remains unclear. Past studies have linked integrin adhesion to ECM as a linkage between FAK activation and the transient inhibition of RhoA GTP binding. Is integrin signaling and FAK involved in the cell and metabolism phenotypes in this new model?

    Response: To better support the antagonistic effect of FAK on Rho-kinase (ROCK) signalling, we included a new experiment in which the integrin-FAK signalling pathway has been disrupted by treating FAK WT cells with an agent that causes detachment from the substratum, Accutase, and growing the cells in suspension in laminin-free medium. We present ROCK activity data, as judged by phosphorylated MLC2 at serine 19 (pMLC2 S19), relating this to induced FAK phosphorylation at Y397 (a surrogate for FAK activity) that is supressed after integrin disengagement. These measurements have been compared with conditions whereby integrin-FAK signalling is activated by growing the cells on laminin coated surfaces. We observed a time-dependent decrease in pFAK(Y397) levels (normalised to total FAK) in suspended cells compared to those spread on laminin, while pMLC2(S19) levels increased in a reciprocal manner over time in detached cells relative to spread cells (S4A and B). There is therefore an inverse relationship between integrin-FAK signalling and ROCK-MLC2 activity, consistent with findings from FAK gene deletion experiments. In the former case, we do not rely on gene deletion cell clones.

    Significance

    The studies by Masalmeh provide interesting findings associating FAK expression with changes in mitochondrial morphology, energy metabolism, and glutamate uptake. According to the authors model, FAK expression is supporting a glioblastoma stem cell like phenotype in vitro and tumor growth in vivo. What remains unclear is the mechanistic connection to cell changes and whether or not these are be dependent on intrinsic FAK activity or as the Frame group has previously published, potentially FAK nuclear localization. The associations with MTFR1L phosphorylation and effects by Rho kinase inhibition are likely indirect and remind this reviewer of long-ago studies with FAK-null fibroblasts that exhibit epithelial characteristics, still express PYK2, exhibited elevated RhoA GTPase activity. Some of these phenotypes were linked to changes in RhoGEF and RhoGAP signaling with FAK and/or Pyk2. At a minimum, it would be informative to know whether Pyk2 signaling is relevant for observed phenotypes and whether the authors can further support their associations with FAK-targeted or FAK-Pyk2-targeted inhibitors or PROTACs.

    __Response: __

    Deleting the gene encoding FAK in mouse embryonic fibroblasts leads to elevated Pyk2 expression (Sieg, 2000). However, in the GBM stem cell model we used here, Pyk2 was not expressed (determined by both transcriptomics and proteomics). We have included Figure S1E to show that PYK2 expression was undetectable in FAK -/- and FAK Rx cells at the RNA level (Figure S1F). We conclude that there is no compensatory increase in Pyk2 upon FAK loss in these cells. In the transformed neural stem cell model of GBM, we do not consistently or robustly detect nuclear FAK.

    Review #2

    Masalmeh and colleagues employ a neural stem/progenitor cell-based glioma model (NPE cells) to investigate the role of Focal Adhesion Kinase (FAK) in GBM, with a focus on potential links between the regulation of morphological/adhesive and metabolic GBM cell properties. For this, the authors employ wt cells alongside newly generated FAK-KO and -reexpressing cells, as well as pharmacological interventions to probe the relevance of specific signaling pathways. The authors´ main claims are that FAK crucially modulates glioma cell morphology, cell-cell and cell-substrate interactions and motility, as well as their metabolism, and that these effects translate to changes to relevant in vivo properties such as invasion and tumor growth.

    My main issues are with the model chosen by the authors.

    As per the methods section, generation of FAK-KO and -"Rx" NPE cells entailed protracted selection/expansion processes, which may have resulted in inadvertent selection for cellular/molecular properties unrelated to the desired one (loss or gain of FAK expression) and which may have had cascading effects on NPE cells. The authors nonetheless repeatedly claim the parameters they quantify, such as mitochondrial or cytoskeletal properties or metabolic features, to have directly resulted from FAK loss or reintroduction. Examples of such causal inferences are to be found in lines 123, 134/135, 165, 181. Such causal claims are, in my view, unsupported.

    Acute perturbation of FAK expression/activity, genetically or pharmacologically, followed by a rapid assessment of the processes under investigation, would be needed to begin to assess causality, even if acute genetic perturbations may be technically challenging as sufficient gene expression reduction or restoration to physiologically relevant levels may be hard to achieve.

    Response:

    We would like to first comment on the model we used here, which we think will clarify the validity of our approach. The model is a transformed stem cell model of GBM that was published in (Gangoso et al., Cell, 2021) and is now used regularly in the GBM field. As mentioned in the response to Reviewer 1, we have added text (page 4 and 5 in the revised manuscript) and a new supplementary figure (Figure S1D) clarifying that the morphological changes we observed were consistent across multiple FAK -/- clones, showing this was not due to any inter-clonal variability. We also added images showing that the morphological changes were apparent at 48 h after nucleofecting FAK -/- cells with the FAK‑expressing vector specifically (not the empty vector), prior to starting G418 selection to enrich for FAK‑expressing cells (Figure S1C), addressing the worry that clonal variation and selection was the cause of the FAK-dependent phenotypes we observed. We believe that our model provides a type of well controlled, clean genetic cancer cell system of a type that is commonly used in cancer cell biology, allowing us to attribute phenotypes to individual proteins.

    We have also carried out a more acute treatment by using the FAK inhibitor VS4718 to perturb FAK kinase activity and assessed the effects on glycolysis and glutamine oxidation after 48h treatment (Figure S2D, E and F). We found that treating the transformed neural stem cells (parental population) with FAK inhibitor (300nM VS4718) decreases glucose incorporation into glycolysis intermediates and glutamine incorporation into TCA cycle intermediates, consistent with a role for FAK's kinase activity in maintaining glycolysis and glutamine oxidation.

    The employed pharmacological modulation of ROCK activity is the only approach that, given the presumably acute nature of the treatment, may have allowed the authors to probe the proposed functional links. The methods section of the manuscript does not however comprise details as to the duration of these treatments, which leaves open the possibility of long-term treatment having been carried out (data shown in Figure 5B refers to 72hr treatment).

    __Response: __

    We have added the duration of the treatment to the Methods section and Figure Legends, to clarify that cells were treated with ROCK inhibitors for 24h, before assessing the effects on mictochondria (Figure 4C, D, S4C and D) and glutamine oxidation (Figure 5A, and S5). For metabolic activity by AlamarBlue assay, cells were treated with ROCK inhibitors for 72h (Figure 5B).

    Even in the case of ROCK inhibitor experiments, it is however unclear if and how the effects on cell morphology and adhesion, mitochondrial organization and metabolic activity may be connected to each other and, if at all, to FAK expression.

    Given the above uncertainties due to the nature of the model and experimental approaches, it is hard to assess the reliability and thus the relevance of the findings.

    Response:

    FAK suppresses ROCK activity (as judged by pMLC2 S19, Figure 4A and B). Treating FAK -/- cells with two different ROCK inhibitors restored mesenchymal-like cell morphology, mitochondrial morphology and glutamine oxidation. As mentioned above, to strengthen our evidence for the antagonistic role of FAK in ROCK-MLC2 signalling, we have now introduced an experiment whereby integrin-FAK signalling was disrupted through treatment with a detachment agent (Accutase), and subsequently maintaining the cells in suspension in laminin-free medium. We assessed pMLC2 S19 levels (a measure of ROCK activity) relating this to FAK phosphorylation that is supressed after integrin disengagement. These results were evaluated relative to spread wild type cells growing on laminin* where Integrin-FAK signalling was active (Figure S4A and B). We observed an inverse relationship between Integrin-FAK signalling and ROCK-MLC2 activity in keeping with our conclusions (Figure 4A and B).*

    Experimental support for the ability of cell-substrate interaction modulation to concomitantly impact cellular metabolism and motility/invasion would be significant both in terms of advancing our understanding of glioma cell biology and of its translational potential, but the evidence being provided is at best compatible with the proposed model.

    Response: We carried out a new experiment to support the ability of cell-substrate interaction modulation to impact metabolism; specifically, we inhibited cell-substrate interactions by plating the cells on Poly-2-hydroxyethyl methacrylate (Poly 2-HEMA)-coated dishes. This suppressed FAK phosphorylation at Y397, as expected, with concomitant reduction in glutamine utilisation in the TCA cycle (Figure S3A, B and C).

    My background/expertise is in developmental and adult neurogenesis, in vivo modelling of gliomagenesis and cell fate control/reprogramming, with a focus on molecular mechanisms of differentiation and quantitative aspects of lineage dynamics; molecular details of the control of cellular metabolism, cell-cell adhesion and cytoskeletal dynamics are not core expertise of mine.

    We appreciate this reviewer's expertise are not necessarily in the cancer cell biology and genetic intervention aspects of our study. We hope that the explanations we have provided satisfy the reviewer that our conclusions are valid.

  2. Review Commons

    Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.

    Learn more at Review Commons

    Referee #2

    Evidence, reproducibility and clarity

    Masalmeh and colleagues employ a neural stem/progenitor cell-based glioma model (NPE cells) to investigate the role of Focal Adhesion Kinase (FAK) in GBM, with a focus on potential links between the regulation of morphological/adhesive and metabolic GBM cell properties. For this, the authors employ wt cells alongside newly generated FAK-KO and -reexpressing cells, as well as pharmacological interventions to probe the relevance of specific signaling pathways. The authors´ main claims are that FAK crucially modulates glioma cell morphology, cell-cell and cell-substrate interactions and motility, as well as their metabolism, and that these effects translate to changes to relevant in vivo properties such as invasion and tumor growth. My main issues are with the model chosen by the authors.

    As per the methods section, generation of FAK-KO and -"Rx" NPE cells entailed protracted selection/expansion processes, which may have resulted in inadvertent selection for cellular/molecular properties unrelated to the desired one (loss or gain of FAK expression) and which may have had cascading effects on NPE cells. The authors nonetheless repeatedly claim the parameters they quantify, such as mitochondrial or cytoskeletal properties or metabolic features, to have directly resulted from FAK loss or reintroduction. Examples of such causal inferences are to be found in lines 123, 134/135, 165, 181. Such causal claims are, in my view, unsupported. Acute perturbation of FAK expression/activity, genetically or pharmacologically, followed by a rapid assessment of the processes under investigation, would be needed to begin to assess causality, even if acute genetic perturbations may be technically challenging as sufficient gene expression reduction or restoration to physiologically relevant levels may be hard to achieve.

    The employed pharmacological modulation of ROCK activity is the only approach that, given the presumably acute nature of the treatment, may have allowed the authors to probe the proposed functional links. The methods section of the manuscript does not however comprise details as to the duration of these treatments, which leaves open the possibility of long-term treatment having been carried out (data shown in Figure 5B refers to 72hr treatment). Even in the case of ROCK inhibitor experiments, it is however unclear if and how the effects on cell morphology and adhesion, mitochondrial organization and metabolic activity may be connected to each other and, if at all, to FAK expression.

    Significance

    Given the above uncertainties due to the nature of the model and experimental approaches, it is hard to assess the reliability and thus the relevance of the findings.

    Experimental support for the ability of cell-substrate interaction modulation to concomitantly impact cellular metabolism and motility/invasion would be significant both in terms of advancing our understanding of glioma cell biology and of its translational potential, but the evidence being provided is at best compatible with the proposed model.

    My background/expertise is in developmental and adult neurogenesis, in vivo modelling of gliomagenesis and cell fate control/reprogramming, with a focus on molecular mechanisms of differentiation and quantitative aspects of lineage dynamics; molecular details of the control of cellular metabolism, cell-cell adhesion and cytoskeletal dynamics are not core expertise of mine.

  3. Review Commons

    Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.

    Learn more at Review Commons

    Referee #1

    Evidence, reproducibility and clarity

    Review of Masalmeh et al.

    Title: "FAK modulates glioblastoma stem cell energetics..."

    Previous studies have implicated FAK and the related tyrosine kinase PYK2 in glioblastoma growth, cell migration, and invasion. Herein, using a murine stem cell model of glioblastoma, the authors used CRISPR to inactivate FAK, FAK-null cells selected and cloned, and lentiviral re-expression of murine FAK in the FAK-null cells (termed FAK Rx) was accomplished. FAK-/- cells were shown to possess epithelial characteristics whereas FAK Rx cells expressed mesenchymal markers and increased cell migration/invasion in vitro. Comparisons between FAK-/- and FAK Rx cells showed that FAK re-expressed increased mitochondrial respiration and amino acid uptake. This was associated with FAK Rx cells exhibiting filamentous mitochondrial morphology (potentially an OXPHOS phenotype) and decreased levels of MTFR1L S235 phosphorylation (implicated in mito morphology fragmentation). Mito and epithelial cell morphology of FAK-/- cells was reversed by treatment with Rho-kinase inhibitors that also increased mito metabolism and cell viability. Last, FAK-dependent glioblastoma tumor growth was shown by comparisons of FAK-/- and FAK Rx implantation studies.

    The studies by Masalmeh provide interesting findings associating FAK expression with changes in mitochondrial morphology, energy metabolism, and glutamate uptake. According to the authors model, FAK expression is supporting a glioblastoma stem cell like phenotype in vitro and tumor growth in vivo. What remains unclear is the mechanistic connection to cell changes and whether or not these are be dependent on intrinsic FAK activity or as the Frame group has previously published, potentially FAK nuclear localization. The associations with MTFR1L phosphorylation and effects by Rho kinase inhibition are likely indirect and remind this reviewer of long-ago studies with FAK-null fibroblasts that exhibit epithelial characteristics, still express PYK2, exhibited elevated RhoA GTPase activity. Some of these phenotypes were linked to changes in RhoGEF and RhoGAP signaling with FAK and/or Pyk2. At a minimum, it would be informative to know whether Pyk2 signaling is relevant for observed phenotypes and whether the authors can further support their associations with FAK-targeted or FAK-Pyk2-targeted inhibitors or PROTACs.

    Some questions that would enhance potential impact.

    1. Cell generation. Please describe the analysis of FAK-/- clones in more detail. The "low viability" phenotype needs further explanation with regard to clonal expansion and growth characteristics?
    2. Figure 1F: need further support of MET change upon FAK KO and EMT reversion.
    3. Fig. 2: Need further support if FAK effects impact glycolysis or oxidative phosphorylation in particular as implicated by the stem cell model.
    4. Is there a combinatorial potential between FAKi and chemotherapies used for glioblastoma. Need to build upon past studies.
    5. The notation of changes in glucose transporter expression should be followed up with regard to the potential that FAK-expressing cells may have different uptake of carbon sources and other amino acids. Altered uptake could be one potential explanation for increase glycolysis and glutamine flux.
    6. It would be helpful to support the confocal microscopy of mitos with EM.
    7. Lack of FAK expression with increased MTFR1 phosphorylation is difficult to interpret.
    8. Need to have better support between loss of FAK and the increase in Rho signaling. Use of Rho kinase inhibitors is very limited and the context to FAK (and or Pyk2) remains unclear. Past studies have linked integrin adhesion to ECM as a linkage between FAK activation and the transient inhibition of RhoA GTP binding. Is integrin signaling and FAK involved in the cell and metabolism phenotypes in this new model?

    Significance

    The studies by Masalmeh provide interesting findings associating FAK expression with changes in mitochondrial morphology, energy metabolism, and glutamate uptake. According to the authors model, FAK expression is supporting a glioblastoma stem cell like phenotype in vitro and tumor growth in vivo. What remains unclear is the mechanistic connection to cell changes and whether or not these are be dependent on intrinsic FAK activity or as the Frame group has previously published, potentially FAK nuclear localization. The associations with MTFR1L phosphorylation and effects by Rho kinase inhibition are likely indirect and remind this reviewer of long-ago studies with FAK-null fibroblasts that exhibit epithelial characteristics, still express PYK2, exhibited elevated RhoA GTPase activity. Some of these phenotypes were linked to changes in RhoGEF and RhoGAP signaling with FAK and/or Pyk2. At a minimum, it would be informative to know whether Pyk2 signaling is relevant for observed phenotypes and whether the authors can further support their associations with FAK-targeted or FAK-Pyk2-targeted inhibitors or PROTACs.

  4. Review Commons

    Note: This response was posted by the corresponding author to Review Commons. The content has not been altered except for formatting.

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    Reply to the reviewers

    Manuscript number: RC-2025-02946

    Corresponding author(s): Margaret, Frame

    Roza, Masalmeh

    [Please use this template only if the submitted manuscript should be considered by the affiliate journal as a full revision in response to the points raised by the reviewers.

    If you wish to submit a preliminary revision with a revision plan, please use our "Revision Plan" template. It is important to use the appropriate template to clearly inform the editors of your intentions.]

    1. General Statements [optional]

    This section is optional. Insert here any general statements you wish to make about the goal of the study or about the reviews.

    We thank the reviewers for recognizing the significance of our work and for their constructive feedback and suggestions, most of which we have implemented in our revised manuscript.

    2. Point-by-point description of the revisions

    This section is mandatory. *Please insert a point-by-point reply describing the revisions that were already carried out and included in the transferred manuscript. *

    Reviewer #1

    Evidence, reproducibility and clarity

    Review of Masalmeh et al. Title: "FAK modulates glioblastoma stem cell energetics..."

    Previous studies have implicated FAK and the related tyrosine kinase PYK2 in glioblastoma growth, cell migration, and invasion. Herein, using a murine stem cell model of glioblastoma, the authors used CRISPR to inactivate FAK, FAK-null cells selected and cloned, and lentiviral re-expression of murine FAK in the FAK-null cells (termed FAK Rx) was accomplished. FAK-/- cells were shown to possess epithelial characteristics whereas FAK Rx cells expressed mesenchymal markers and increased cell migration/invasion in vitro. Comparisons between FAK-/- and FAK Rx cells showed that FAK re-expressed increased mitochondrial respiration and amino acid uptake. This was associated with FAK Rx cells exhibiting filamentous mitochondrial morphology (potentially an OXPHOS phenotype) and decreased levels of MTFR1L S235 phosphorylation (implicated in mito morphology fragmentation). Mito and epithelial cell morphology of FAK-/- cells was reversed by treatment with Rho-kinase inhibitors that also increased mito metabolism and cell viability. Last, FAK-dependent glioblastoma tumor growth was shown by comparisons of FAK-/- and FAK Rx implantation studies.

    The studies by Masalmeh provide interesting findings associating FAK expression with changes in mitochondrial morphology, energy metabolism, and glutamate uptake. According to the authors model, FAK expression is supporting a glioblastoma stem cell like phenotype in vitro and tumor growth in vivo. What remains unclear is the mechanistic connection to cell changes and whether or not these are be dependent on intrinsic FAK activity or as the Frame group has previously published, potentially FAK nuclear localization. The associations with MTFR1L phosphorylation and effects by Rho kinase inhibition are likely indirect and remind this reviewer of long-ago studies with FAK-null fibroblasts that exhibit epithelial characteristics, still express PYK2, exhibited elevated RhoA GTPase activity. Some of these phenotypes were linked to changes in RhoGEF and RhoGAP signaling with FAK and/or Pyk2. At a minimum, it would be informative to know whether Pyk2 signaling is relevant for observed phenotypes and whether the authors can further support their associations with FAK-targeted or FAK-Pyk2-targeted inhibitors or PROTACs.

    Some questions that would enhance potential impact.

    1. Cell generation. Please describe the analysis of FAK-/- clones in more detail. The "low viability" phenotype needs further explanation with regard to clonal expansion and growth characteristics?

    Response:

    • We included a better description and a supplementary figure in our revised manuscript to indicate that we have examined several FAK -/- clones and confirmed that our observations were not due to clonal variation; multiple clones displayed similar morphological changes (Figure S1D). We also show that the elongated mesenchymal-like morphology was observed at 48 h after nucleofecting the cells with the FAK‑expressing vector, before beginning G418 selection to enrich for cells expressing FAK (Figure S1C). We also included experiments to acutely modulate FAK signalling (detaching and seeding cells on fibronectin) (Figure S2D, E, F and Figure S3) to exclude the possibility that the profound effects are due to protocols/selection we used for generating FAK-deleted cells.
    • Regarding the term "low viability", we have clarified in the text that there is no significant difference in cell number (Figure S1A) or 'cell viability' when it is assessed by trypan blue exclusion (a non-mitochondria-dependent read-out) (Figure S1B) between FAK-expressing FAK Rx and FAK-/- cells cultured for three days under normal conditions. Therefore, we agree the term 'cell viability' in this context could be confusing and have replace "cell viability" with "metabolic activity as measured by Alamar Blue." in Figure 1D and Figure 5B, and the corresponding text in the original manuscript. This wording more accurately reflects the data.

    Figure 1F: need further support of MET change upon FAK KO and EMT reversion.

    Response: We have added a heatmap (Figure S1E) illustrating the changes in protein expression of core-enriched EMT/MET genes products (by proteomics) after FAK gene deletion (EMT genes as defined in Howe et al., 2018) ; this strengthens the conclusion that the MET reversion morphological phenotype is accompanied by recognised MET protein changes.

    Fig. 2: Need further support if FAK effects impact glycolysis or oxidative phosphorylation in particular as implicated by the stem cell model.

    Response: We show that FAK impacts both glycolysis (Figure 2A, 2E, and 2F) and mitochondrial oxidative phosphorylation on the basis of the oxygen consumption rate (OCR) (Figure 2B, and 2D), showing both are contributing pathways to FAK-dependent energy production. We have clarified this in the text.

    Is there a combinatorial potential between FAKi and chemotherapies used for glioblastoma. Need to build upon past studies.

    Response: Yes, previous studies suggest that inhibiting FAK can sensitize GBM cells to chemotherapy (Golubovskaya et al., 2012; Ortiz-Rivera et al., 2023). We have included a paragraph in the discussion section to make sure this is clearer. Although it is not the subject of this study, we appreciate it is useful context.

    The notation of changes in glucose transporter expression should be followed up with regard to the potential that FAK-expressing cells may have different uptake of carbon sources and other amino acids. Altered uptake could be one potential explanation for increase glycolysis and glutamine flux.

    Response: We agree with the reviewer that glucose uptake could be contributing and we include data that 2 glucose transporters are indeed FAK-regulated namely Glucose transporter 1 (GLUT1, encoded by Slc2a1 gene) and Glucose transporter 3 (GLUT 3, encoded by Slc2a3 gene) (shown in Figure S2B and C).

    It would be helpful to support the confocal microscopy of mitos with EM.

    Response:

    We are concerned (and in our experience) that Electron microscopy (EM) may introduce artefacts during sample preparation. In contrast, immunofluorescence sample preparation is less susceptible to artefacts. The SORA system we used is not a conventional point-scanning confocal microscope, but is a super-resolution module based on a spinning disk confocal platform (CSU-W1; Yokogawa) using optical pixel reassignment with confocal detection. This method enhances resolution in all dimensions with resolution in our samples measured at 120nm. This has been instructive in defining a new level of changes in mitochondrial morphology upon FAK gene deletion.

    Lack of FAK expression with increased MTFR1 phosphorylation is difficult to interpret.

    Response: We do not directly show that this phosphorylation event is causal in our experiments; however, we think it important to document this change since it has been published that phosphorylation of MTFR1 has been causally linked to the mitochondrial morphology we observed in other systems (Tilokani et al., 2022).

    Need to have better support between loss of FAK and the increase in Rho signaling. Use of Rho kinase inhibitors is very limited and the context to FAK (and or Pyk2) remains unclear. Past studies have linked integrin adhesion to ECM as a linkage between FAK activation and the transient inhibition of RhoA GTP binding. Is integrin signaling and FAK involved in the cell and metabolism phenotypes in this new model?

    Response: To better support the antagonistic effect of FAK on Rho-kinase (ROCK) signalling, we included a new experiment in which the integrin-FAK signalling pathway has been disrupted by treating FAK WT cells with an agent that causes detachment from the substratum, Accutase, and growing the cells in suspension in laminin-free medium. We present ROCK activity data, as judged by phosphorylated MLC2 at serine 19 (pMLC2 S19), relating this to induced FAK phosphorylation at Y397 (a surrogate for FAK activity) that is supressed after integrin disengagement. These measurements have been compared with conditions whereby integrin-FAK signalling is activated by growing the cells on laminin coated surfaces. We observed a time-dependent decrease in pFAK(Y397) levels (normalised to total FAK) in suspended cells compared to those spread on laminin, while pMLC2(S19) levels increased in a reciprocal manner over time in detached cells relative to spread cells (S4A and B). There is therefore an inverse relationship between integrin-FAK signalling and ROCK-MLC2 activity, consistent with findings from FAK gene deletion experiments. In the former case, we do not rely on gene deletion cell clones.

    Significance

    The studies by Masalmeh provide interesting findings associating FAK expression with changes in mitochondrial morphology, energy metabolism, and glutamate uptake. According to the authors model, FAK expression is supporting a glioblastoma stem cell like phenotype in vitro and tumor growth in vivo. What remains unclear is the mechanistic connection to cell changes and whether or not these are be dependent on intrinsic FAK activity or as the Frame group has previously published, potentially FAK nuclear localization. The associations with MTFR1L phosphorylation and effects by Rho kinase inhibition are likely indirect and remind this reviewer of long-ago studies with FAK-null fibroblasts that exhibit epithelial characteristics, still express PYK2, exhibited elevated RhoA GTPase activity. Some of these phenotypes were linked to changes in RhoGEF and RhoGAP signaling with FAK and/or Pyk2. At a minimum, it would be informative to know whether Pyk2 signaling is relevant for observed phenotypes and whether the authors can further support their associations with FAK-targeted or FAK-Pyk2-targeted inhibitors or PROTACs.

    __Response: __

    Deleting the gene encoding FAK in mouse embryonic fibroblasts leads to elevated Pyk2 expression (Sieg, 2000). However, in the GBM stem cell model we used here, Pyk2 was not expressed (determined by both transcriptomics and proteomics). We have included Figure S1E to show that PYK2 expression was undetectable in FAK -/- and FAK Rx cells at the RNA level (Figure S1F). We conclude that there is no compensatory increase in Pyk2 upon FAK loss in these cells. In the transformed neural stem cell model of GBM, we do not consistently or robustly detect nuclear FAK.

    Review #2

    Masalmeh and colleagues employ a neural stem/progenitor cell-based glioma model (NPE cells) to investigate the role of Focal Adhesion Kinase (FAK) in GBM, with a focus on potential links between the regulation of morphological/adhesive and metabolic GBM cell properties. For this, the authors employ wt cells alongside newly generated FAK-KO and -reexpressing cells, as well as pharmacological interventions to probe the relevance of specific signaling pathways. The authors´ main claims are that FAK crucially modulates glioma cell morphology, cell-cell and cell-substrate interactions and motility, as well as their metabolism, and that these effects translate to changes to relevant in vivo properties such as invasion and tumor growth.

    My main issues are with the model chosen by the authors.

    As per the methods section, generation of FAK-KO and -"Rx" NPE cells entailed protracted selection/expansion processes, which may have resulted in inadvertent selection for cellular/molecular properties unrelated to the desired one (loss or gain of FAK expression) and which may have had cascading effects on NPE cells. The authors nonetheless repeatedly claim the parameters they quantify, such as mitochondrial or cytoskeletal properties or metabolic features, to have directly resulted from FAK loss or reintroduction. Examples of such causal inferences are to be found in lines 123, 134/135, 165, 181. Such causal claims are, in my view, unsupported.

    Acute perturbation of FAK expression/activity, genetically or pharmacologically, followed by a rapid assessment of the processes under investigation, would be needed to begin to assess causality, even if acute genetic perturbations may be technically challenging as sufficient gene expression reduction or restoration to physiologically relevant levels may be hard to achieve.

    Response:

    We would like to first comment on the model we used here, which we think will clarify the validity of our approach. The model is a transformed stem cell model of GBM that was published in (Gangoso et al., Cell, 2021) and is now used regularly in the GBM field. As mentioned in the response to Reviewer 1, we have added text (page 4 and 5 in the revised manuscript) and a new supplementary figure (Figure S1D) clarifying that the morphological changes we observed were consistent across multiple FAK -/- clones, showing this was not due to any inter-clonal variability. We also added images showing that the morphological changes were apparent at 48 h after nucleofecting FAK -/- cells with the FAK‑expressing vector specifically (not the empty vector), prior to starting G418 selection to enrich for FAK‑expressing cells (Figure S1C), addressing the worry that clonal variation and selection was the cause of the FAK-dependent phenotypes we observed. We believe that our model provides a type of well controlled, clean genetic cancer cell system of a type that is commonly used in cancer cell biology, allowing us to attribute phenotypes to individual proteins.

    We have also carried out a more acute treatment by using the FAK inhibitor VS4718 to perturb FAK kinase activity and assessed the effects on glycolysis and glutamine oxidation after 48h treatment (Figure S2D, E and F). We found that treating the transformed neural stem cells (parental population) with FAK inhibitor (300nM VS4718) decreases glucose incorporation into glycolysis intermediates and glutamine incorporation into TCA cycle intermediates, consistent with a role for FAK's kinase activity in maintaining glycolysis and glutamine oxidation.

    The employed pharmacological modulation of ROCK activity is the only approach that, given the presumably acute nature of the treatment, may have allowed the authors to probe the proposed functional links. The methods section of the manuscript does not however comprise details as to the duration of these treatments, which leaves open the possibility of long-term treatment having been carried out (data shown in Figure 5B refers to 72hr treatment).

    __Response: __

    We have added the duration of the treatment to the Methods section and Figure Legends, to clarify that cells were treated with ROCK inhibitors for 24h, before assessing the effects on mictochondria (Figure 4C, D, S4C and D) and glutamine oxidation (Figure 5A, and S5). For metabolic activity by AlamarBlue assay, cells were treated with ROCK inhibitors for 72h (Figure 5B).

    Even in the case of ROCK inhibitor experiments, it is however unclear if and how the effects on cell morphology and adhesion, mitochondrial organization and metabolic activity may be connected to each other and, if at all, to FAK expression.

    Given the above uncertainties due to the nature of the model and experimental approaches, it is hard to assess the reliability and thus the relevance of the findings.

    Response:

    FAK suppresses ROCK activity (as judged by pMLC2 S19, Figure 4A and B). Treating FAK -/- cells with two different ROCK inhibitors restored mesenchymal-like cell morphology, mitochondrial morphology and glutamine oxidation. As mentioned above, to strengthen our evidence for the antagonistic role of FAK in ROCK-MLC2 signalling, we have now introduced an experiment whereby integrin-FAK signalling was disrupted through treatment with a detachment agent (Accutase), and subsequently maintaining the cells in suspension in laminin-free medium. We assessed pMLC2 S19 levels (a measure of ROCK activity) relating this to FAK phosphorylation that is supressed after integrin disengagement. These results were evaluated relative to spread wild type cells growing on laminin* where Integrin-FAK signalling was active (Figure S4A and B). We observed an inverse relationship between Integrin-FAK signalling and ROCK-MLC2 activity in keeping with our conclusions (Figure 4A and B).*

    Experimental support for the ability of cell-substrate interaction modulation to concomitantly impact cellular metabolism and motility/invasion would be significant both in terms of advancing our understanding of glioma cell biology and of its translational potential, but the evidence being provided is at best compatible with the proposed model.

    Response: We carried out a new experiment to support the ability of cell-substrate interaction modulation to impact metabolism; specifically, we inhibited cell-substrate interactions by plating the cells on Poly-2-hydroxyethyl methacrylate (Poly 2-HEMA)-coated dishes. This suppressed FAK phosphorylation at Y397, as expected, with concomitant reduction in glutamine utilisation in the TCA cycle (Figure S3A, B and C).

    My background/expertise is in developmental and adult neurogenesis, in vivo modelling of gliomagenesis and cell fate control/reprogramming, with a focus on molecular mechanisms of differentiation and quantitative aspects of lineage dynamics; molecular details of the control of cellular metabolism, cell-cell adhesion and cytoskeletal dynamics are not core expertise of mine.

    We appreciate this reviewer's expertise are not necessarily in the cancer cell biology and genetic intervention aspects of our study. We hope that the explanations we have provided satisfy the reviewer that our conclusions are valid.

  5. Review Commons

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    Referee #2

    Evidence, reproducibility and clarity

    Masalmeh and colleagues employ a neural stem/progenitor cell-based glioma model (NPE cells) to investigate the role of Focal Adhesion Kinase (FAK) in GBM, with a focus on potential links between the regulation of morphological/adhesive and metabolic GBM cell properties. For this, the authors employ wt cells alongside newly generated FAK-KO and -reexpressing cells, as well as pharmacological interventions to probe the relevance of specific signaling pathways. The authors´ main claims are that FAK crucially modulates glioma cell morphology, cell-cell and cell-substrate interactions and motility, as well as their metabolism, and that these effects translate to changes to relevant in vivo properties such as invasion and tumor growth. My main issues are with the model chosen by the authors.

    As per the methods section, generation of FAK-KO and -"Rx" NPE cells entailed protracted selection/expansion processes, which may have resulted in inadvertent selection for cellular/molecular properties unrelated to the desired one (loss or gain of FAK expression) and which may have had cascading effects on NPE cells. The authors nonetheless repeatedly claim the parameters they quantify, such as mitochondrial or cytoskeletal properties or metabolic features, to have directly resulted from FAK loss or reintroduction. Examples of such causal inferences are to be found in lines 123, 134/135, 165, 181. Such causal claims are, in my view, unsupported. Acute perturbation of FAK expression/activity, genetically or pharmacologically, followed by a rapid assessment of the processes under investigation, would be needed to begin to assess causality, even if acute genetic perturbations may be technically challenging as sufficient gene expression reduction or restoration to physiologically relevant levels may be hard to achieve.

    The employed pharmacological modulation of ROCK activity is the only approach that, given the presumably acute nature of the treatment, may have allowed the authors to probe the proposed functional links. The methods section of the manuscript does not however comprise details as to the duration of these treatments, which leaves open the possibility of long-term treatment having been carried out (data shown in Figure 5B refers to 72hr treatment). Even in the case of ROCK inhibitor experiments, it is however unclear if and how the effects on cell morphology and adhesion, mitochondrial organization and metabolic activity may be connected to each other and, if at all, to FAK expression.

    Significance

    Given the above uncertainties due to the nature of the model and experimental approaches, it is hard to assess the reliability and thus the relevance of the findings.

    Experimental support for the ability of cell-substrate interaction modulation to concomitantly impact cellular metabolism and motility/invasion would be significant both in terms of advancing our understanding of glioma cell biology and of its translational potential, but the evidence being provided is at best compatible with the proposed model.

    My background/expertise is in developmental and adult neurogenesis, in vivo modelling of gliomagenesis and cell fate control/reprogramming, with a focus on molecular mechanisms of differentiation and quantitative aspects of lineage dynamics; molecular details of the control of cellular metabolism, cell-cell adhesion and cytoskeletal dynamics are not core expertise of mine.

  6. Review Commons

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    Referee #1

    Evidence, reproducibility and clarity

    Review of Masalmeh et al.

    Title: "FAK modulates glioblastoma stem cell energetics..."

    Previous studies have implicated FAK and the related tyrosine kinase PYK2 in glioblastoma growth, cell migration, and invasion. Herein, using a murine stem cell model of glioblastoma, the authors used CRISPR to inactivate FAK, FAK-null cells selected and cloned, and lentiviral re-expression of murine FAK in the FAK-null cells (termed FAK Rx) was accomplished. FAK-/- cells were shown to possess epithelial characteristics whereas FAK Rx cells expressed mesenchymal markers and increased cell migration/invasion in vitro. Comparisons between FAK-/- and FAK Rx cells showed that FAK re-expressed increased mitochondrial respiration and amino acid uptake. This was associated with FAK Rx cells exhibiting filamentous mitochondrial morphology (potentially an OXPHOS phenotype) and decreased levels of MTFR1L S235 phosphorylation (implicated in mito morphology fragmentation). Mito and epithelial cell morphology of FAK-/- cells was reversed by treatment with Rho-kinase inhibitors that also increased mito metabolism and cell viability. Last, FAK-dependent glioblastoma tumor growth was shown by comparisons of FAK-/- and FAK Rx implantation studies.

    The studies by Masalmeh provide interesting findings associating FAK expression with changes in mitochondrial morphology, energy metabolism, and glutamate uptake. According to the authors model, FAK expression is supporting a glioblastoma stem cell like phenotype in vitro and tumor growth in vivo. What remains unclear is the mechanistic connection to cell changes and whether or not these are be dependent on intrinsic FAK activity or as the Frame group has previously published, potentially FAK nuclear localization. The associations with MTFR1L phosphorylation and effects by Rho kinase inhibition are likely indirect and remind this reviewer of long-ago studies with FAK-null fibroblasts that exhibit epithelial characteristics, still express PYK2, exhibited elevated RhoA GTPase activity. Some of these phenotypes were linked to changes in RhoGEF and RhoGAP signaling with FAK and/or Pyk2. At a minimum, it would be informative to know whether Pyk2 signaling is relevant for observed phenotypes and whether the authors can further support their associations with FAK-targeted or FAK-Pyk2-targeted inhibitors or PROTACs.

    Some questions that would enhance potential impact.

    1. Cell generation. Please describe the analysis of FAK-/- clones in more detail. The "low viability" phenotype needs further explanation with regard to clonal expansion and growth characteristics?
    2. Figure 1F: need further support of MET change upon FAK KO and EMT reversion.
    3. Fig. 2: Need further support if FAK effects impact glycolysis or oxidative phosphorylation in particular as implicated by the stem cell model.
    4. Is there a combinatorial potential between FAKi and chemotherapies used for glioblastoma. Need to build upon past studies.
    5. The notation of changes in glucose transporter expression should be followed up with regard to the potential that FAK-expressing cells may have different uptake of carbon sources and other amino acids. Altered uptake could be one potential explanation for increase glycolysis and glutamine flux.
    6. It would be helpful to support the confocal microscopy of mitos with EM.
    7. Lack of FAK expression with increased MTFR1 phosphorylation is difficult to interpret.
    8. Need to have better support between loss of FAK and the increase in Rho signaling. Use of Rho kinase inhibitors is very limited and the context to FAK (and or Pyk2) remains unclear. Past studies have linked integrin adhesion to ECM as a linkage between FAK activation and the transient inhibition of RhoA GTP binding. Is integrin signaling and FAK involved in the cell and metabolism phenotypes in this new model?

    Significance

    The studies by Masalmeh provide interesting findings associating FAK expression with changes in mitochondrial morphology, energy metabolism, and glutamate uptake. According to the authors model, FAK expression is supporting a glioblastoma stem cell like phenotype in vitro and tumor growth in vivo. What remains unclear is the mechanistic connection to cell changes and whether or not these are be dependent on intrinsic FAK activity or as the Frame group has previously published, potentially FAK nuclear localization. The associations with MTFR1L phosphorylation and effects by Rho kinase inhibition are likely indirect and remind this reviewer of long-ago studies with FAK-null fibroblasts that exhibit epithelial characteristics, still express PYK2, exhibited elevated RhoA GTPase activity. Some of these phenotypes were linked to changes in RhoGEF and RhoGAP signaling with FAK and/or Pyk2. At a minimum, it would be informative to know whether Pyk2 signaling is relevant for observed phenotypes and whether the authors can further support their associations with FAK-targeted or FAK-Pyk2-targeted inhibitors or PROTACs.

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    Reply to the reviewers

    Manuscript number: RC-2025-02946

    Corresponding author(s): Margaret, Frame

    Roza, Masalmeh

    [Please use this template only if the submitted manuscript should be considered by the affiliate journal as a full revision in response to the points raised by the reviewers.

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    1. General Statements [optional]

    This section is optional. Insert here any general statements you wish to make about the goal of the study or about the reviews.

    We thank the reviewers for recognizing the significance of our work and for their constructive feedback and suggestions, most of which we have implemented in our revised manuscript.

    2. Point-by-point description of the revisions

    This section is mandatory. *Please insert a point-by-point reply describing the revisions that were already carried out and included in the transferred manuscript. *

    Reviewer #1

    Evidence, reproducibility and clarity

    Review of Masalmeh et al. Title: "FAK modulates glioblastoma stem cell energetics..."

    Previous studies have implicated FAK and the related tyrosine kinase PYK2 in glioblastoma growth, cell migration, and invasion. Herein, using a murine stem cell model of glioblastoma, the authors used CRISPR to inactivate FAK, FAK-null cells selected and cloned, and lentiviral re-expression of murine FAK in the FAK-null cells (termed FAK Rx) was accomplished. FAK-/- cells were shown to possess epithelial characteristics whereas FAK Rx cells expressed mesenchymal markers and increased cell migration/invasion in vitro. Comparisons between FAK-/- and FAK Rx cells showed that FAK re-expressed increased mitochondrial respiration and amino acid uptake. This was associated with FAK Rx cells exhibiting filamentous mitochondrial morphology (potentially an OXPHOS phenotype) and decreased levels of MTFR1L S235 phosphorylation (implicated in mito morphology fragmentation). Mito and epithelial cell morphology of FAK-/- cells was reversed by treatment with Rho-kinase inhibitors that also increased mito metabolism and cell viability. Last, FAK-dependent glioblastoma tumor growth was shown by comparisons of FAK-/- and FAK Rx implantation studies.

    The studies by Masalmeh provide interesting findings associating FAK expression with changes in mitochondrial morphology, energy metabolism, and glutamate uptake. According to the authors model, FAK expression is supporting a glioblastoma stem cell like phenotype in vitro and tumor growth in vivo. What remains unclear is the mechanistic connection to cell changes and whether or not these are be dependent on intrinsic FAK activity or as the Frame group has previously published, potentially FAK nuclear localization. The associations with MTFR1L phosphorylation and effects by Rho kinase inhibition are likely indirect and remind this reviewer of long-ago studies with FAK-null fibroblasts that exhibit epithelial characteristics, still express PYK2, exhibited elevated RhoA GTPase activity. Some of these phenotypes were linked to changes in RhoGEF and RhoGAP signaling with FAK and/or Pyk2. At a minimum, it would be informative to know whether Pyk2 signaling is relevant for observed phenotypes and whether the authors can further support their associations with FAK-targeted or FAK-Pyk2-targeted inhibitors or PROTACs.

    Some questions that would enhance potential impact.

    1. Cell generation. Please describe the analysis of FAK-/- clones in more detail. The "low viability" phenotype needs further explanation with regard to clonal expansion and growth characteristics?

    Response:

    • We included a better description and a supplementary figure in our revised manuscript to indicate that we have examined several FAK -/- clones and confirmed that our observations were not due to clonal variation; multiple clones displayed similar morphological changes (Figure S1D). We also show that the elongated mesenchymal-like morphology was observed at 48 h after nucleofecting the cells with the FAK‑expressing vector, before beginning G418 selection to enrich for cells expressing FAK (Figure S1C). We also included experiments to acutely modulate FAK signalling (detaching and seeding cells on fibronectin) (Figure S2D, E, F and Figure S3) to exclude the possibility that the profound effects are due to protocols/selection we used for generating FAK-deleted cells.
    • Regarding the term “low viability”, we have clarified in the text that there is no significant difference in cell number (Figure S1A) or ‘cell viability’ when it is assessed by trypan blue exclusion (a non-mitochondria-dependent read-out) (Figure S1B) between FAK-expressing FAK Rx and FAK-/- cells cultured for three days under normal conditions. Therefore, we agree the term ‘cell viability’ in this context could be confusing and have replace "cell viability” with “metabolic activity as measured by Alamar Blue.” in Figure 1D and Figure 5B, and the corresponding text in the original manuscript. This wording more accurately reflects the data.

    Figure 1F: need further support of MET change upon FAK KO and EMT reversion.

    Response: We have added a heatmap (Figure S1E) illustrating the changes in protein expression of core-enriched EMT/MET genes products (by proteomics) after FAK gene deletion (EMT genes as defined in Howe et al., 2018) ; this strengthens the conclusion that the MET reversion morphological phenotype is accompanied by recognised MET protein changes.

    Fig. 2: Need further support if FAK effects impact glycolysis or oxidative phosphorylation in particular as implicated by the stem cell model.

    Response: We show that FAK impacts both glycolysis (Figure 2A, 2E, and 2F) and mitochondrial oxidative phosphorylation on the basis of the oxygen consumption rate (OCR) (Figure 2B, and 2D), showing both are contributing pathways to FAK-dependent energy production. We have clarified this in the text.

    Is there a combinatorial potential between FAKi and chemotherapies used for glioblastoma. Need to build upon past studies.

    Response: Yes, previous studies suggest that inhibiting FAK can sensitize GBM cells to chemotherapy (Golubovskaya et al., 2012; Ortiz-Rivera et al., 2023). We have included a paragraph in the discussion section to make sure this is clearer. Although it is not the subject of this study, we appreciate it is useful context.

    The notation of changes in glucose transporter expression should be followed up with regard to the potential that FAK-expressing cells may have different uptake of carbon sources and other amino acids. Altered uptake could be one potential explanation for increase glycolysis and glutamine flux.

    Response: We agree with the reviewer that glucose uptake could be contributing and we include data that 2 glucose transporters are indeed FAK-regulated namely Glucose transporter 1 (GLUT1, encoded by Slc2a1 gene) and Glucose transporter 3 (GLUT 3, encoded by Slc2a3 gene) (shown in Figure S2B and C).

    It would be helpful to support the confocal microscopy of mitos with EM.

    Response:

    We are concerned (and in our experience) that Electron microscopy (EM) may introduce artefacts during sample preparation. In contrast, immunofluorescence sample preparation is less susceptible to artefacts. The SORA system we used is not a conventional point-scanning confocal microscope, but is a super-resolution module based on a spinning disk confocal platform (CSU-W1; Yokogawa) using optical pixel reassignment with confocal detection. This method enhances resolution in all dimensions with resolution in our samples measured at 120nm. This has been instructive in defining a new level of changes in mitochondrial morphology upon FAK gene deletion.

    Lack of FAK expression with increased MTFR1 phosphorylation is difficult to interpret.

    Response: We do not directly show that this phosphorylation event is causal in our experiments; however, we think it important to document this change since it has been published that phosphorylation of MTFR1 has been causally linked to the mitochondrial morphology we observed in other systems (Tilokani et al., 2022).

    Need to have better support between loss of FAK and the increase in Rho signaling. Use of Rho kinase inhibitors is very limited and the context to FAK (and or Pyk2) remains unclear. Past studies have linked integrin adhesion to ECM as a linkage between FAK activation and the transient inhibition of RhoA GTP binding. Is integrin signaling and FAK involved in the cell and metabolism phenotypes in this new model?

    Response: To better support the antagonistic effect of FAK on Rho-kinase (ROCK) signalling, we included a new experiment in which the integrin-FAK signalling pathway has been disrupted by treating FAK WT cells with an agent that causes detachment from the substratum, Accutase, and growing the cells in suspension in laminin-free medium. We present ROCK activity data, as judged by phosphorylated MLC2 at serine 19 (pMLC2 S19), relating this to induced FAK phosphorylation at Y397 (a surrogate for FAK activity) that is supressed after integrin disengagement. These measurements have been compared with conditions whereby integrin-FAK signalling is activated by growing the cells on laminin coated surfaces. We observed a time-dependent decrease in pFAK(Y397) levels (normalised to total FAK) in suspended cells compared to those spread on laminin, while pMLC2(S19) levels increased in a reciprocal manner over time in detached cells relative to spread cells (S4A and B). There is therefore an inverse relationship between integrin-FAK signalling and ROCK-MLC2 activity, consistent with findings from FAK gene deletion experiments. In the former case, we do not rely on gene deletion cell clones.

    Significance

    The studies by Masalmeh provide interesting findings associating FAK expression with changes in mitochondrial morphology, energy metabolism, and glutamate uptake. According to the authors model, FAK expression is supporting a glioblastoma stem cell like phenotype in vitro and tumor growth in vivo. What remains unclear is the mechanistic connection to cell changes and whether or not these are be dependent on intrinsic FAK activity or as the Frame group has previously published, potentially FAK nuclear localization. The associations with MTFR1L phosphorylation and effects by Rho kinase inhibition are likely indirect and remind this reviewer of long-ago studies with FAK-null fibroblasts that exhibit epithelial characteristics, still express PYK2, exhibited elevated RhoA GTPase activity. Some of these phenotypes were linked to changes in RhoGEF and RhoGAP signaling with FAK and/or Pyk2. At a minimum, it would be informative to know whether Pyk2 signaling is relevant for observed phenotypes and whether the authors can further support their associations with FAK-targeted or FAK-Pyk2-targeted inhibitors or PROTACs.

    __Response: __

    Deleting the gene encoding FAK in mouse embryonic fibroblasts leads to elevated Pyk2 expression (Sieg, 2000). However, in the GBM stem cell model we used here, Pyk2 was not expressed (determined by both transcriptomics and proteomics). We have included Figure S1E to show that PYK2 expression was undetectable in FAK -/- and FAK Rx cells at the RNA level (Figure S1F). We conclude that there is no compensatory increase in Pyk2 upon FAK loss in these cells. In the transformed neural stem cell model of GBM, we do not consistently or robustly detect nuclear FAK.

    Review #2

    Masalmeh and colleagues employ a neural stem/progenitor cell-based glioma model (NPE cells) to investigate the role of Focal Adhesion Kinase (FAK) in GBM, with a focus on potential links between the regulation of morphological/adhesive and metabolic GBM cell properties. For this, the authors employ wt cells alongside newly generated FAK-KO and -reexpressing cells, as well as pharmacological interventions to probe the relevance of specific signaling pathways. The authors´ main claims are that FAK crucially modulates glioma cell morphology, cell-cell and cell-substrate interactions and motility, as well as their metabolism, and that these effects translate to changes to relevant in vivo properties such as invasion and tumor growth.

    My main issues are with the model chosen by the authors.

    As per the methods section, generation of FAK-KO and -"Rx" NPE cells entailed protracted selection/expansion processes, which may have resulted in inadvertent selection for cellular/molecular properties unrelated to the desired one (loss or gain of FAK expression) and which may have had cascading effects on NPE cells. The authors nonetheless repeatedly claim the parameters they quantify, such as mitochondrial or cytoskeletal properties or metabolic features, to have directly resulted from FAK loss or reintroduction. Examples of such causal inferences are to be found in lines 123, 134/135, 165, 181. Such causal claims are, in my view, unsupported.

    Acute perturbation of FAK expression/activity, genetically or pharmacologically, followed by a rapid assessment of the processes under investigation, would be needed to begin to assess causality, even if acute genetic perturbations may be technically challenging as sufficient gene expression reduction or restoration to physiologically relevant levels may be hard to achieve.

    Response:

    We would like to first comment on the model we used here, which we think will clarify the validity of our approach. The model is a transformed stem cell model of GBM that was published in (Gangoso et al., Cell, 2021) and is now used regularly in the GBM field. As mentioned in the response to Reviewer 1, we have added text (page 4 and 5 in the revised manuscript) and a new supplementary figure (Figure S1D) clarifying that the morphological changes we observed were consistent across multiple FAK -/- clones, showing this was not due to any inter-clonal variability. We also added images showing that the morphological changes were apparent at 48 h after nucleofecting FAK -/- cells with the FAK‑expressing vector specifically (not the empty vector), prior to starting G418 selection to enrich for FAK‑expressing cells (Figure S1C), addressing the worry that clonal variation and selection was the cause of the FAK-dependent phenotypes we observed. We believe that our model provides a type of well controlled, clean genetic cancer cell system of a type that is commonly used in cancer cell biology, allowing us to attribute phenotypes to individual proteins.

    We have also carried out a more acute treatment by using the FAK inhibitor VS4718 to perturb FAK kinase activity and assessed the effects on glycolysis and glutamine oxidation after 48h treatment (Figure S2D, E and F). We found that treating the transformed neural stem cells (parental population) with FAK inhibitor (300nM VS4718) decreases glucose incorporation into glycolysis intermediates and glutamine incorporation into TCA cycle intermediates, consistent with a role for FAK’s kinase activity in maintaining glycolysis and glutamine oxidation.

    The employed pharmacological modulation of ROCK activity is the only approach that, given the presumably acute nature of the treatment, may have allowed the authors to probe the proposed functional links. The methods section of the manuscript does not however comprise details as to the duration of these treatments, which leaves open the possibility of long-term treatment having been carried out (data shown in Figure 5B refers to 72hr treatment).

    __Response: __

    We have added the duration of the treatment to the Methods section and Figure Legends, to clarify that cells were treated with ROCK inhibitors for 24h, before assessing the effects on mictochondria (Figure 4C, D, S4C and D) and glutamine oxidation (Figure 5A, and S5). For metabolic activity by AlamarBlue assay, cells were treated with ROCK inhibitors for 72h (Figure 5B).

    Even in the case of ROCK inhibitor experiments, it is however unclear if and how the effects on cell morphology and adhesion, mitochondrial organization and metabolic activity may be connected to each other and, if at all, to FAK expression.

    Given the above uncertainties due to the nature of the model and experimental approaches, it is hard to assess the reliability and thus the relevance of the findings.

    Response:

    FAK suppresses ROCK activity (as judged by pMLC2 S19, Figure 4A and B). Treating FAK -/- cells with two different ROCK inhibitors restored mesenchymal-like cell morphology, mitochondrial morphology and glutamine oxidation. As mentioned above, to strengthen our evidence for the antagonistic role of FAK in ROCK-MLC2 signalling, we have now introduced an experiment whereby integrin-FAK signalling was disrupted through treatment with a detachment agent (Accutase), and subsequently maintaining the cells in suspension in laminin-free medium. We assessed pMLC2 S19 levels (a measure of ROCK activity) relating this to FAK phosphorylation that is supressed after integrin disengagement. These results were evaluated relative to spread wild type cells growing on laminin* where Integrin-FAK signalling was active (Figure S4A and B). We observed an inverse relationship between Integrin-FAK signalling and ROCK-MLC2 activity in keeping with our conclusions (Figure 4A and B).*

    Experimental support for the ability of cell-substrate interaction modulation to concomitantly impact cellular metabolism and motility/invasion would be significant both in terms of advancing our understanding of glioma cell biology and of its translational potential, but the evidence being provided is at best compatible with the proposed model.

    Response: We carried out a new experiment to support the ability of cell-substrate interaction modulation to impact metabolism; specifically, we inhibited cell-substrate interactions by plating the cells on Poly-2-hydroxyethyl methacrylate (Poly 2-HEMA)-coated dishes. This suppressed FAK phosphorylation at Y397, as expected, with concomitant reduction in glutamine utilisation in the TCA cycle (Figure S3A, B and C).

    My background/expertise is in developmental and adult neurogenesis, in vivo modelling of gliomagenesis and cell fate control/reprogramming, with a focus on molecular mechanisms of differentiation and quantitative aspects of lineage dynamics; molecular details of the control of cellular metabolism, cell-cell adhesion and cytoskeletal dynamics are not core expertise of mine.

    We appreciate this reviewer’s expertise are not necessarily in the cancer cell biology and genetic intervention aspects of our study. We hope that the explanations we have provided satisfy the reviewer that our conclusions are valid.

  8. Review Commons

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    Referee #2

    Evidence, reproducibility and clarity

    Masalmeh and colleagues employ a neural stem/progenitor cell-based glioma model (NPE cells) to investigate the role of Focal Adhesion Kinase (FAK) in GBM, with a focus on potential links between the regulation of morphological/adhesive and metabolic GBM cell properties. For this, the authors employ wt cells alongside newly generated FAK-KO and -reexpressing cells, as well as pharmacological interventions to probe the relevance of specific signaling pathways. The authors´ main claims are that FAK crucially modulates glioma cell morphology, cell-cell and cell-substrate interactions and motility, as well as their metabolism, and that these effects translate to changes to relevant in vivo properties such as invasion and tumor growth. My main issues are with the model chosen by the authors.

    As per the methods section, generation of FAK-KO and -"Rx" NPE cells entailed protracted selection/expansion processes, which may have resulted in inadvertent selection for cellular/molecular properties unrelated to the desired one (loss or gain of FAK expression) and which may have had cascading effects on NPE cells. The authors nonetheless repeatedly claim the parameters they quantify, such as mitochondrial or cytoskeletal properties or metabolic features, to have directly resulted from FAK loss or reintroduction. Examples of such causal inferences are to be found in lines 123, 134/135, 165, 181. Such causal claims are, in my view, unsupported. Acute perturbation of FAK expression/activity, genetically or pharmacologically, followed by a rapid assessment of the processes under investigation, would be needed to begin to assess causality, even if acute genetic perturbations may be technically challenging as sufficient gene expression reduction or restoration to physiologically relevant levels may be hard to achieve.

    The employed pharmacological modulation of ROCK activity is the only approach that, given the presumably acute nature of the treatment, may have allowed the authors to probe the proposed functional links. The methods section of the manuscript does not however comprise details as to the duration of these treatments, which leaves open the possibility of long-term treatment having been carried out (data shown in Figure 5B refers to 72hr treatment). Even in the case of ROCK inhibitor experiments, it is however unclear if and how the effects on cell morphology and adhesion, mitochondrial organization and metabolic activity may be connected to each other and, if at all, to FAK expression.

    Significance

    Given the above uncertainties due to the nature of the model and experimental approaches, it is hard to assess the reliability and thus the relevance of the findings.

    Experimental support for the ability of cell-substrate interaction modulation to concomitantly impact cellular metabolism and motility/invasion would be significant both in terms of advancing our understanding of glioma cell biology and of its translational potential, but the evidence being provided is at best compatible with the proposed model.

    My background/expertise is in developmental and adult neurogenesis, in vivo modelling of gliomagenesis and cell fate control/reprogramming, with a focus on molecular mechanisms of differentiation and quantitative aspects of lineage dynamics; molecular details of the control of cellular metabolism, cell-cell adhesion and cytoskeletal dynamics are not core expertise of mine.

  9. Review Commons

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    Referee #1

    Evidence, reproducibility and clarity

    Review of Masalmeh et al.

    Title: "FAK modulates glioblastoma stem cell energetics..."

    Previous studies have implicated FAK and the related tyrosine kinase PYK2 in glioblastoma growth, cell migration, and invasion. Herein, using a murine stem cell model of glioblastoma, the authors used CRISPR to inactivate FAK, FAK-null cells selected and cloned, and lentiviral re-expression of murine FAK in the FAK-null cells (termed FAK Rx) was accomplished. FAK-/- cells were shown to possess epithelial characteristics whereas FAK Rx cells expressed mesenchymal markers and increased cell migration/invasion in vitro. Comparisons between FAK-/- and FAK Rx cells showed that FAK re-expressed increased mitochondrial respiration and amino acid uptake. This was associated with FAK Rx cells exhibiting filamentous mitochondrial morphology (potentially an OXPHOS phenotype) and decreased levels of MTFR1L S235 phosphorylation (implicated in mito morphology fragmentation). Mito and epithelial cell morphology of FAK-/- cells was reversed by treatment with Rho-kinase inhibitors that also increased mito metabolism and cell viability. Last, FAK-dependent glioblastoma tumor growth was shown by comparisons of FAK-/- and FAK Rx implantation studies.

    The studies by Masalmeh provide interesting findings associating FAK expression with changes in mitochondrial morphology, energy metabolism, and glutamate uptake. According to the authors model, FAK expression is supporting a glioblastoma stem cell like phenotype in vitro and tumor growth in vivo. What remains unclear is the mechanistic connection to cell changes and whether or not these are be dependent on intrinsic FAK activity or as the Frame group has previously published, potentially FAK nuclear localization. The associations with MTFR1L phosphorylation and effects by Rho kinase inhibition are likely indirect and remind this reviewer of long-ago studies with FAK-null fibroblasts that exhibit epithelial characteristics, still express PYK2, exhibited elevated RhoA GTPase activity. Some of these phenotypes were linked to changes in RhoGEF and RhoGAP signaling with FAK and/or Pyk2. At a minimum, it would be informative to know whether Pyk2 signaling is relevant for observed phenotypes and whether the authors can further support their associations with FAK-targeted or FAK-Pyk2-targeted inhibitors or PROTACs.

    Some questions that would enhance potential impact.

    1. Cell generation. Please describe the analysis of FAK-/- clones in more detail. The "low viability" phenotype needs further explanation with regard to clonal expansion and growth characteristics?
    2. Figure 1F: need further support of MET change upon FAK KO and EMT reversion.
    3. Fig. 2: Need further support if FAK effects impact glycolysis or oxidative phosphorylation in particular as implicated by the stem cell model.
    4. Is there a combinatorial potential between FAKi and chemotherapies used for glioblastoma. Need to build upon past studies.
    5. The notation of changes in glucose transporter expression should be followed up with regard to the potential that FAK-expressing cells may have different uptake of carbon sources and other amino acids. Altered uptake could be one potential explanation for increase glycolysis and glutamine flux.
    6. It would be helpful to support the confocal microscopy of mitos with EM.
    7. Lack of FAK expression with increased MTFR1 phosphorylation is difficult to interpret.
    8. Need to have better support between loss of FAK and the increase in Rho signaling. Use of Rho kinase inhibitors is very limited and the context to FAK (and or Pyk2) remains unclear. Past studies have linked integrin adhesion to ECM as a linkage between FAK activation and the transient inhibition of RhoA GTP binding. Is integrin signaling and FAK involved in the cell and metabolism phenotypes in this new model?

    Significance

    The studies by Masalmeh provide interesting findings associating FAK expression with changes in mitochondrial morphology, energy metabolism, and glutamate uptake. According to the authors model, FAK expression is supporting a glioblastoma stem cell like phenotype in vitro and tumor growth in vivo. What remains unclear is the mechanistic connection to cell changes and whether or not these are be dependent on intrinsic FAK activity or as the Frame group has previously published, potentially FAK nuclear localization. The associations with MTFR1L phosphorylation and effects by Rho kinase inhibition are likely indirect and remind this reviewer of long-ago studies with FAK-null fibroblasts that exhibit epithelial characteristics, still express PYK2, exhibited elevated RhoA GTPase activity. Some of these phenotypes were linked to changes in RhoGEF and RhoGAP signaling with FAK and/or Pyk2. At a minimum, it would be informative to know whether Pyk2 signaling is relevant for observed phenotypes and whether the authors can further support their associations with FAK-targeted or FAK-Pyk2-targeted inhibitors or PROTACs.

  10. Version published to 10.1101/2024.12.11.627900 on bioRxiv