Myotubularin-related proteins regulate KRAS function by controlling plasma membrane levels of polyphosphoinositides and phosphatidylserine

  1. Karen M. Henkels
  2. Taylor E. Miller
  3. Ali Naji
  4. Ransome van der Hoeven
  5. Hong Liang
  6. Yong Zhou
  7. Gerald R.V. Hammond
  8. John F. Hancock
  9. Kwang-jin Cho

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Abstract

KRAS is a small GTPase, ubiquitously expressed in mammalian cells, that functions as a molecular switch to regulate cell proliferation and differentiation. Oncogenic mutations that render KRAS constitutively active occur frequently in human cancers. KRAS must localize to the plasma membrane (PM) for biological activity. KRAS PM binding is mediated by interactions of the KRAS membrane anchor with phosphatidylserine (PtdSer), therefore, depleting PM PtdSer content abrogates KRAS PM binding and oncogenic function. From a genome-wide siRNA screen to search for genes that regulate KRAS PM localization, we identified a set of phosphatidylinositol (PI) 3-phosphatase family members: myotubularin-related (MTMR) proteins 2, 3, 4 and 7. Here we show that knockdown of MTMR 2/3/4/7 expression disrupts KRAS PM interactions. The molecular mechanism involves depletion of PM PI 4-phosphate (PI4P) levels, which in turn disrupts the subcellular localization and operation of oxysterol-binding protein related protein (ORP) 5, a PtdSer lipid transfer protein that maintains PM PtdSer content. Concomitantly, silencing MTMR 2/3/4/7 expression elevates PM levels of PI3P and reduces PM and total cellular levels of PtdSer. In summary we propose that the PI 3-phosphatase activity provided by MTMR proteins is required to generate PM PI for the synthesis of PM PI4P, which in turn, promotes the PM localization of PtdSer and KRAS.

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    Reply to the reviewers

    We thank the reviewers for their valuable comments, which definitely make our story stronger.

    2. Description of the planned revisions

    Reviewer 1

    Comments:

    No data are shown from the genome-wide screening approach, including the common regulators of KRAS and HRAS. Information about how imaging data were processed and analysed is missing. A final table of 8 selected factors with phosphatase activity is presented without providing further insight about the selection criteria and other factors.

    This information will be included in the revised manuscript. In the subsequent characterization via image-based quantification of GFP-KRAS membrane localization, a Manders´ coefficient was calculated. A respective chapter in the methods section on how this was done is missing.

    This information will be provided in the revised manuscript. I would be happy to see the following analyses to strengthen the dataset:

    • Reconstitution experiments and further validation to show that it is dependent on the enzymatic activity of MTMRs.

    MTMR3 knockdown (KD) cells will be rescued with wildtype (WT) MTMR3 or the phosphatase mutant MTMR3 (C413S, PMID: 11676921). MTMR4 KD cells will be rescued with WT MTMR4 or the phosphatase mutant MTMR4 (C407S, PMID: 20736309). In these cells, the PM localization of KRAS and PtdSer will be examined by confocal and electron microscopy.

    • Additive effect upon depletion of multiple MTMRs? Are they functionally co-operative?

    MTMR3 and 4 KD cells will be rescued with WT MTMR4 and 3, respectively, and the PM localization of KRAS and PtdSer will be examined by confocal and electron microscopy.

    • Signalling analysis is very limited (Fig. 5). Do the authors detect any defects in K-RAS driven downstream signaling in these cells upon depletion of MTMRs.

    Human pancreatic ductal adenocarcinoma (PDAC) cell lines that harbor oncogenic mutant KRAS and their growth is KRAS signaling-dependent (MiaPaCa2 and AsPC1), and PDAC cell line harboring WT KRAS and their growth is KRAS signaling-independent (BxPC3) will be infected with lentivirus expressing shRNAs against MTMR 2, 3, 4 or 7. Their growth (proliferation assay) and KRAS signaling (e.g. phosphorylated ERK and Akt by immunoblot) will be measure. Reviewer 2

    Major comments

    The unbiased siRNA screen used to identify proteins that impact KRAS membrane localization was a very nice approach to identify MTMR proteins. Although there is a clear phenotype of KRAS mislocalization associated with knockdown of the various MTMR proteins, the data provided does not prove a causational role for the MTMR proteins in maintaining PtdSer content, nor KRAS localization, at the PM. The current data does not provide a mechanism by which MTMR proteins are influencing this process, but rather speculates using existing literature that it is the loss in MTMR 3-phosphotase activity that leads to decreased PtdSer in the membrane. There is a series of conversions and exchanges that act upon PI3P (the substrate of MTMR proteins) and PI to generate PtdSer in the PM; thus, it is a dynamic process that is influenced by a variety of different proteins and transporters [3, 4, 5, 6]. To prove their single-protein-driven hypothesis, the authors should clone and express a mutant MTMR protein construct that contains an inactive phosphatase catalytic domain, to prove that it is indeed MTMR's generation of PI (which is further converted into PI4P) in the membrane that is responsible for maintaining PtdSer content and KRAS localization. Without this, there is not enough evidence to support this claim.

    MTMR3 knockdown (KD) cells will be rescued with wildtype (WT) MTMR3 or the phosphatase mutant MTMR3 (C413S, PMID: 11676921). MTMR4 KD cells will be rescued with WT MTMR4 or the phosphatase mutant MTMR4 (C407S, PMID: 20736309). In these cells, the PM localization of KRAS and PtdSer will be examined by confocal and electron microscopy. In addition, the authors speculate that ORP5 is a critical intermediate in this process, and that the loss in PI4P/ORP5 at the PM following MTMR knockdown is responsible for the decrease in PtdSer at the PM. The authors should knockdown ORP5 in MTMR-wildtype cells, since it is downstream of their proposed mechanism, and see whether this leads to comparable reductions in PtdSer levels and KRAS mislocalization at the PM. This would confirm ORP5 as having a major role in this setting and would support the initial mechanistic hypothesis. These experiments are imperative to forming an appropriate conclusion, especially since some of their current data contradicts their mechanistic hypothesis: the authors identify a decrease in whole cell PtdSer content, not just PM PtdSer content, when MTMR proteins are knocked down. Based on this result, one would predict that a secondary or supporting mechanism must exist that contributes to a reduction in whole cell PtdSer content, which likely contributes to its loss at the PM as well. The authors describe in line 360 how "previous work has shown that PM PI4P depletion indirectly blocks PtdSer synthase 1 and 2 activities," to explain this reduction in total cell levels of PtdSer. The authors should look at PtdSer synthase 1 and 2 activities in the presence of MTMR knockdown, as the loss in PtdSer at the PM may rely more heavily on synthase activity than ORP-dependent transfer of PtdSer.

    Investigating the PM localization of KRAS and PtdSer after silencing ORP5 in MTMR WT mammalian cell lines has been published (PMID: 31451509 and 34903667). In these studies, silencing ORP5 1) reduces the levels of PtdSer and KRAS from the plasma membrane (PM), 2) reduces KRAS signal output, 3) blocks the growth of KRAS-dependent PDAC in vitro and in vivo. These studies have been appropriately cited in our manuscript in lines 82 and 277. Although the c. Elegans model that was used to investigate downstream let-60 (RAS ortholog) activity through a multi-vulva phenotype is quite intriguing, it is more critical to assess downstream RAS pathway activation, especially in the human colorectal adenocarcinoma or the human mammary gland ductal carcinoma cell lines. Not only would this line of questioning provide a higher significance and increase the clinical applicability of these findings, but it is also crucial to support the author's claim that MTMR knockdown can influence mutant KRAS activity. Although small changes in KRAS localization to the PM can have significant effects on downstream signaling, these effects need to be measured and confirmed in this setting. The authors should perform western blots to assess the activation of both the PI3K and MAPK pathway in the MTMR knockdown cell lines.

    Human pancreatic ductal adenocarcinoma (PDAC) cell lines that harbor oncogenic mutant KRAS and their growth is KRAS signaling-dependent (MiaPaCa2 and AsPC1), and PDAC cell line harboring WT KRAS and their growth is KRAS signaling-independent (BxPC3) will be infected with lentivirus expressing shRNAs against MTMR 2, 3, 4 or 7. Their growth (proliferation assay) and KRAS signaling (e.g. phosphorylated ERK and Akt by immunoblot) will be measure. In addition to this, it might be important to know whether there are any changes in the levels of the KRAS protein itself, as recycling/transport pathways may be impacted by its lack of recruitment to the plasma membrane.

    Total KRAS protein expression will be measured in MTMR KD cell lines. Finally, the authors show that proliferation is inhibited by MTMR knockdown as a readout of RAS activity. The authors should also assess the levels of cell death, as the inhibition of mutant KRAS in cancer cells would likely lead to cell death. The authors do not describe why reducing any one of the MTMR proteins alone is sufficient to deplete the PM of PtdSer. This sort of discussion is important for understanding compensatory or regulatory mechanisms in place between the MTMR proteins, as this may influence PtdSer levels at the PM. For example, it has been shown that MTMR2 can stabilize MTMR13 on membranes. Do the levels, stability, or localization of the other MTMR proteins change when one specific MTMR is knocked down? Is this why we see an effect on PtdSer in any one of the knockdowns? The authors should at the very least provide western blots for each of the MTMR proteins discussed in the presence of each individual MTMR knockdown.

    MTMR3 knockdown (KD) cells will be rescued with WT MTMR3 or the phosphatase mutant MTMR3 (C413S, PMID: 11676921). MTMR4 KD cells will be rescued with WT MTMR4 or the phosphatase mutant MTMR4 (C407S, PMID: 20736309). In these cells, the PM localization of KRAS and PtdSer will be examined by confocal and electron microscopy. In addition, we will measure endogenous MTMR 2/3/4/7 proteins levels in the presence of each individual MTMR KD by immunoblotting. In addition to the above experiments, the MTMR hairpins should be expressed in a secondary or tertiary cell line to prove that these events are not specific to the current model used. Since their current human mammary gland ductal carcinoma cell line overexpresses a mutant KRAS-GFP construct, perhaps doing similar experiments in a cancer cell line that already expresses an endogenous mutant KRAS might provide a better model.

    Human pancreatic ductal adenocarcinoma (PDAC) cell lines that harbor oncogenic mutant KRAS and their growth is KRAS signaling-dependent (MiaPaCa2 and AsPC1), and PDAC cell line harboring WT KRAS and their growth is KRAS signaling-independent (BxPC3) will be infected with lentivirus expressing shRNAs against MTMR 2, 3, 4 or 7. Their growth (proliferation assay) and KRAS signaling (e.g. phosphorylated ERK and Akt by immunoblot) will be measure. Although this protein would not include a GFP-tag, other ways of visualizing its localization at the PM (such as immunofluorescent staining) could be used to confirm its localization there.

    The anti-KRAS antibody for IF has not been reported to my knowledge. In addition, the effects on downstream RAS signaling could be measured through western blot of PI3K and MAPK pathways.

    Human pancreatic ductal adenocarcinoma (PDAC) cell lines that harbor oncogenic mutant KRAS and their growth is KRAS signaling-dependent (MiaPaCa2 and AsPC1), and PDAC cell line harboring WT KRAS and their growth is KRAS signaling-independent (BxPC3) will be infected with lentivirus expressing shRNAs against MTMR 2, 3, 4 or 7. Their growth (proliferation assay) and KRAS signaling (e.g. phosphorylated ERK and Akt by immunoblot) will be measure. Supplemental Figure 4 is incorrectly referred to in the text as Supplemental Figure 3 (line 257-258). The text reads, "Confocal microscopy further demonstrates that HRASG12V cellular localization is not disrupted after silencing MTMR 2/3/4/7 (Fig. S3)" but Figure S3 is an EM image of PM basal sheets from T47D cells expressing GFP-KRASG12V. Supplemental Figure 4 shows that mutant HRAS is unaffected by the various MTMR knockdowns.

    They will be labeled correctly in the revised manuscript. Since the authors show decreased proliferation in mutant KRAS cells following MTMR knockdown, the authors should also investigate any changes to proliferation rates in mutant HRAS cell lines following MTMR knockdown. This data is necessary to prove that MTMR-driven changes in downstream RAS signaling are specific to mutant KRAS and not mutant HRAS.

    Cell proliferation assay will be performed using MTMR 2/3/4/7-silenced T47D cell lines stably expressing oncogenic mutant HRAS (HRASG12V) to address this questions. It may also be important for the authors to also show any effects on wildtype RAS localization to the PM when MTMR-2,-3,-4, and -7 are knocked down, to show whether this is a oncoprotein-specific event.

    Cells expressing the truncated mutant KRAS, which contains the minimal membrane anchor and does not have G-domain will be infected with lentivirus expressing shRNA against MTMR 2/3/4/7, and their localization will be examined. The representative images chosen for Figure 4 diminish the reliability of the data, as it is difficult to see a visible change in the PI3P probe between the control and MTMR knockdown cells in these images. Since the authors rely on the Mander's coefficient and the number of gold particles throughout much of the paper, having the same conclusion quantitatively but not qualitatively for these assays is confusing. Perhaps the authors should elaborate on whether MTMR knockdown has a stronger effect on PtSer and KRAS PM presence than PI3P PM presence.

    We will include the discussion in the revised manuscript. They should also describe their method for identifying early endosomes, since they switch back and forth between describing the content of the PM and of early endosomes, such as in Figure 1 and Figure 4.

    We will include the information in the revised manuscript. Minor comments:

    An additional experiment that may add another layer of clinical applicability would be the use of an MTMR inhibitor in this cell line, to see whether similar effects can be achieved pharmacologically [7]. This would provoke other researchers to investigate MTMR inhibitors in vitro and in vivo to assess the effect on mutant KRAS cancers.

    • This is an important point, but while vanadate, a general phospho-tyrosine phosphatase (PTP) inhibitor, has been reported to inhibit myotubulin, a family member of MTMR (PMID: 8995372 and 1943774), there are no commercially available MTMR-specific inhibitors. Using vanadate to inhibit MTMR proteins will produce non-specific effects by blocking other PTPs. The inclusion of cell lines that express KRAS proteins of different mutational statuses would be extremely interesting, as KRAS' orientation within the plasma membrane has been shown to be altered by these mutations. This fact should potentially be considered when choosing a secondary or tertiary cell line to do additional experiments in, but it is not necessary for the authors to elaborate on how MTMR proteins may impact different KRAS mutants for the scope of this project.

    For the aforementioned experiments using human KRAS-dependent and -independent PDAC cell lines, we will use MiaPaCa2 (KRASG12C) and AsPC1 (KRASG12D). Reviewer #3

    *Major comments: *

    One of the two main manuscript claims indicates that KRAS12V "function" is impaired upon MTMR knockdown. While this is an obvious phenotype expected by mislocalizing KRAS from the inner PM it is not sufficiently demonstrated in the current version of the manuscript. Western blots of at least MAPK and PI3K signalling following MTMR knockdown in KRAS-dependent cell lines should be included. In addition to the T47D cells used in the manuscript, it would be ideal to include a KRAS-mutant cell line from tumour types where KRAS mutations are more frequent that in breast.

    • Human pancreatic ductal adenocarcinoma (PDAC) cell lines that harbor oncogenic mutant KRAS and their growth is KRAS signaling-dependent (MiaPaCa2 and AsPC1), and PDAC cell line harboring WT KRAS and their growth is KRAS signaling-independent (BxPC3) will be infected with lentivirus expressing shRNAs against MTMR 2, 3, 4 or 7. Their growth (proliferation assay) and KRAS signaling (e.g. phosphorylated ERK and Akt by immunoblot) will be measure. Since the MTMR dependent phenotypes are mutant-KRAS specific it would be interesting to study the resulting phenotypes in HRAS-mutant cell line.

    Cell proliferation assay will be performed using MTMR 2/3/4/7-silenced T47D cell lines stably expressing oncogenic mutant HRAS (HRASG12V) to address these questions.

    **Referee cross-commenting**

    After reading the reviews of my colleagues I think there is a clear agreement on the need to further substantiate that KRAS membrane mis-localization is indeed affecting oncogenic output. The use of other KRAS addicted and non-addicted models would further enhance this analysis.

    Likewise, the other two reviewers request experimental evidences to validate the role of MTMR enzymatic activity in the process. This is a pertinent request that I failed to put forward. Suggestions include the use of reconstitution experiments catalytically dead mutants. Also, the use of MTMR small molecule inhibitors is proposed. If those exist with sufficient specificity this would indeed be appropriate to perform.

    Experiments addressing these comments have been described above.

    3. Description of the revisions that have already been incorporated in the transferred manuscript

    N/A

    4. Description of analyses that authors prefer not to carry out

    *Please include a point-by-point response explaining why some of the requested data or additional analyses might not be necessary or cannot be provided within the scope of a revision. *

    Reviewer 2

    R2 suggests to investigate PtdSer synthase 1 and 2 activities in presence of MTMR knockdown, as the loss in PtdSer at the PM may rely more heavily on synthase activity than ORP-dependent transfer of PtdSer.

    Although it is intriguing to examine the effect of MTMR loss on the activities of PtdSer synthase 1 and 2, our lab does not have resources/techniques to carry out the experiment.

    The results of this paper rely heavily on one experimental technique, which is calculating a Mander's coefficient and counting the co-localization of the probe of interest with the CellMask stain of the plasma membrane. How this coefficient is derived is explained in appropriate detail in the methods section of this manuscript; however, a secondary route of identifying these changes in membrane constituents would greatly enhance the paper's conclusions. This would eliminate any doubt surrounding the accuracy of the technique, since so much of the data relies on one experimental output.

    In addition to Manders' coefficient for examining the colocalization of KRAS and LactC2 (the PtdSer probe) to propose KRAS/PS redistribution to endomembranes after MTMR loss. To complement this, we also performed quantitative EM to demonstrate the PM depletion of KRAS and PtdSer from the inner PM leaflet. We believe these two techniques would appropriate to investigate KRAS/PtdSer PM depletion and cellular re-distribution.

    Reviewer 3

    To further support the conclusions, oncogenic signalling should be studied in the C.elegans model by immunofluorescence of immunohistochemistry. Furthermore, although not strictly required to support the author's claims, it would be interesting to elucidate whether the inhibition of the multivulva phenotype upon MTMR knockdown in vivo results as a consequence of cell death.

    Our collaborator for C. elegans study does not have resources to carry out the proposed IF and IHC experiment. Instead, we will measure KRAS signaling (e.g. phosphorylated ERK and Akt by immunoblot) and the growth of KRAS-dependent PDAC after MTMR loss. These experiments would be more clinically and physiologically relevant.

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    Referee #3

    Evidence, reproducibility and clarity

    In recent years, various genetic and pharmacological studies have clearly demonstrated a causal relationship between phosphatidylserine (PtdSer) distribution at the plasma membrane (PM) and KRAS clustering at the inner leaflet. In this manuscript, Henkels and colleagues have performed a high-content genome wide siRNA screen in search of hits that upon knockdown would result in membrane mislocalization of an exogenous KRAS12V-GFP fusion while not affecting HRASV12 membrane distribution. The results identified 4 of the 14 human members of the myotubularin-related (MTMR) protein family. Individual knockdown of MTMR2, 3, 4 and 7 resulted in specific relocalization of KRAS12V (and not of HRAS12V) from the cell membrane to endomembranes assessed both by confocal and electron microscopy. The MTMRs display enzymatic activity (3-phosphatase activity towards PI3P and PI(3,5)P2) controlling membrane trafficking. Given the known dependency of KRAS PM clustering on PtdSer, the authors showed that KRAS12V mislocalization upon MTMR depletion is due to an overall reduction of PtdSer accompanied by depletion of inner PM PtdSer. Using lipid-specific probes the authors went on to demonstrate that this phenotype occurs as a combined reduction of inner PM PI4P levels and concomitant elevation of PM PI3P. As expected, KRAS12V inner PM mislocalization affected oncogenic function. This is shown by a 50% reduction in cell proliferation of KRAS-transformed T47D (human mammary gland ductal carcinoma) cell line. More convincingly, si-RNA mediated depletion of the C.elegans MTMR3 and 7 orthologs potently reduces a KRAS-dependent multivulva phenotype.

    Overall, I find that the experimental part of the manuscript is satisfactory. Yet, the overall conclusion is that inactivation of a subset of MTMR phosphatases reduces KRAS PM localization and KRAS signalling. While changes of KRAS inner PM are well documented, there is not a single experiment demonstrating that this results in reduced oncogenic output. This needs to be further documented if a mention to KRAS function is included in the title.

    Major comments:

    1. One of the two main manuscript claims indicates that KRAS12V "function" is impaired upon MTMR knockdown. While this is an obvious phenotype expected by mislocalizing KRAS from the inner PM it is not sufficiently demonstrated in the current version of the manuscript. Western blots of at least MAPK and PI3K signalling following MTMR knockdown in KRAS-dependent cell lines should be included. In addition to the T47D cells used in the manuscript, it would be ideal to include a KRAS-mutant cell line from tumour types where KRAS mutations are more frequent that in breast.
    2. Since the MTMR dependent phenotypes are mutant-KRAS specific it would be interesting to study the resulting phenotypes in HRAS-mutant cell line.
    3. To further support the conclusions, oncogenic signalling should be studied in the C.elegans model by immunofluorescence of immunohistochemistry. Furthermore, although not strictly required to support the author's claims, it would be interesting to elucidate whether the inhibition of the multivulva phenotype upon MTMR knockdown in vivo results as a consequence of cell death.

    Referee cross-commenting

    After reading the reviews of my colleagues I think there is a clear agreement on the need to further substantiate that KRAS membrane mis-localization is indeed affecting oncogenic output. The use of other KRAS addicted and non-addicted models would further enhance this analysis. Likewise, the other two reviewers request experimental evidences to validate the role of MTMR enzymatic activity in the process. This is a pertinent request that I failed to put forward. Suggestions include the use of reconstitution experiments catalytically dead mutants. Also, the use of MTMR small molecule inhibitors is proposed. If those exist with sufficient specificity this would indeed be appropriate to perform.

    Significance

    In spite of its importance for oncogenic function, KRAS cellular trafficking remains one of the least studied processes. As such, reports like the current work are important to increase our biological knowledge. Furthermore, this increased biological understanding could identify vulnerabilities with future therapeutic potential.

    It is known, mainly from previous work of one of the co-authors (John Hancock), that a PtdSer interplay with oxysterol-binding protein related proteins ORP5 and 8 regulate KRAS membrane distribution. The current study describes a further layer of control depending on MTMR phosphatases. In my opinion the cellular phenotypes are properly addressed, but not the phenotypic consequences on KRAS-signalling.

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    Referee #2

    Evidence, reproducibility and clarity

    Summary:

    Henkels et al. propose the role of myotubularin-related proteins in promoting KRAS4B localization to the plasma membrane. Their data shows that shRNA-mediated knockdown of myotubularin-related proteins -2, -3, -4, or -7 led to measurable changes in RAS localization and in plasma membrane (PM) content. More specifically, knockdown of any one of these MTMR proteins led to a decrease in PI4P levels in the PM, an increase in PI3P content in the PM, a decrease in phosphatidyl serine (PtdSer) in the PM/whole cell, and a decrease in mutant KRAS localization to the PM. Their data also shows a decreased presence of ORP5 at the PM, a protein which is responsible for the exchange of PI4P in the plasma membrane for PtdSer in the endoplasmic reticulum. These results are somewhat predictable and are supported by the existing literature, as MTMR proteins are known to exhibit 3-phosphotase activity towards PI3P to generate PI (a precursor to PI4P), PI4P is known to recruit ORP5, and ORP5 is known to contribute to PtdSer content in the membrane [1, 2]. Regardless, the authors find that the individual knockdown of MTMR proteins is sufficient to cause measurable changes in PM content and mislocalization of mutant KRAS4B. Thus, despite the fact that many proteins are involved in regulating PM content, such as PI4KA, PtdSer synthase 1 and 2, Nir2/3, and PITPs, Henkels et al. speculate that MTMR proteins are the primary regulators of PtdSer PM levels [3, 4, 5, 6]. The authors propose that the loss of function in any one of these MTMR proteins alone is sufficient to cause significant changes in PM content through an ORP5-dependent process, and that this ultimately leads to a decrease in mutant KRAS signaling.

    Major comments:

    The unbiased siRNA screen used to identify proteins that impact KRAS membrane localization was a very nice approach to identify MTMR proteins. Although there is a clear phenotype of KRAS mislocalization associated with knockdown of the various MTMR proteins, the data provided does not prove a causational role for the MTMR proteins in maintaining PtdSer content, nor KRAS localization, at the PM. The current data does not provide a mechanism by which MTMR proteins are influencing this process, but rather speculates using existing literature that it is the loss in MTMR 3-phosphotase activity that leads to decreased PtdSer in the membrane. There is a series of conversions and exchanges that act upon PI3P (the substrate of MTMR proteins) and PI to generate PtdSer in the PM; thus, it is a dynamic process that is influenced by a variety of different proteins and transporters [3, 4, 5, 6]. To prove their single-protein-driven hypothesis, the authors should clone and express a mutant MTMR protein construct that contains an inactive phosphatase catalytic domain, to prove that it is indeed MTMR's generation of PI (which is further converted into PI4P) in the membrane that is responsible for maintaining PtdSer content and KRAS localization. Without this, there is not enough evidence to support this claim. In addition, the authors speculate that ORP5 is a critical intermediate in this process, and that the loss in PI4P/ORP5 at the PM following MTMR knockdown is responsible for the decrease in PtdSer at the PM. The authors should knockdown ORP5 in MTMR-wildtype cells, since it is downstream of their proposed mechanism, and see whether this leads to comparable reductions in PtdSer levels and KRAS mislocalization at the PM. This would confirm ORP5 as having a major role in this setting and would support the initial mechanistic hypothesis. These experiments are imperative to forming an appropriate conclusion, especially since some of their current data contradicts their mechanistic hypothesis: the authors identify a decrease in whole cell PtdSer content, not just PM PtdSer content, when MTMR proteins are knocked down. Based on this result, one would predict that a secondary or supporting mechanism must exist that contributes to a reduction in whole cell PtdSer content, which likely contributes to its loss at the PM as well. The authors describe in line 360 how "previous work has shown that PM PI4P depletion indirectly blocks PtdSer synthase 1 and 2 activities," to explain this reduction in total cell levels of PtdSer. The authors should look at PtdSer synthase 1 and 2 activities in the presence of MTMR knockdown, as the loss in PtdSer at the PM may rely more heavily on synthase activity than ORP-dependent transfer of PtdSer. Although the c. Elegans model that was used to investigate downstream let-60 (RAS ortholog) activity through a multi-vulva phenotype is quite intriguing, it is more critical to assess downstream RAS pathway activation, especially in the human colorectal adenocarcinoma or the human mammary gland ductal carcinoma cell lines. Not only would this line of questioning provide a higher significance and increase the clinical applicability of these findings, but it is also crucial to support the author's claim that MTMR knockdown can influence mutant KRAS activity. Although small changes in KRAS localization to the PM can have significant effects on downstream signaling, these effects need to be measured and confirmed in this setting. The authors should perform western blots to assess the activation of both the PI3K and MAPK pathway in the MTMR knockdown cell lines. In addition to this, it might be important to know whether there are any changes in the levels of the KRAS protein itself, as recycling/transport pathways may be impacted by its lack of recruitment to the plasma membrane. Finally, the authors show that proliferation is inhibited by MTMR knockdown as a readout of RAS activity. The authors should also assess the levels of cell death, as the inhibition of mutant KRAS in cancer cells would likely lead to cell death. The authors do not describe why reducing any one of the MTMR proteins alone is sufficient to deplete the PM of PtdSer. This sort of discussion is important for understanding compensatory or regulatory mechanisms in place between the MTMR proteins, as this may influence PtdSer levels at the PM. For example, it has been shown that MTMR2 can stabilize MTMR13 on membranes. Do the levels, stability, or localization of the other MTMR proteins change when one specific MTMR is knocked down? Is this why we see an effect on PtdSer in any one of the knockdowns? The authors should at the very least provide western blots for each of the MTMR proteins discussed in the presence of each individual MTMR knockdown.
    In addition to the above experiments, the MTMR hairpins should be expressed in a secondary or tertiary cell line to prove that these events are not specific to the current model used. Since their current human mammary gland ductal carcinoma cell line overexpresses a mutant KRAS-GFP construct, perhaps doing similar experiments in a cancer cell line that already expresses an endogenous mutant KRAS might provide a better model. Although this protein would not include a GFP-tag, other ways of visualizing its localization at the PM (such as immunofluorescent staining) could be used to confirm its localization there. In addition, the effects on downstream RAS signaling could be measured through western blot of PI3K and MAPK pathways. Supplemental Figure 4 is incorrectly referred to in the text as Supplemental Figure 3 (line 257-258). The text reads, "Confocal microscopy further demonstrates that HRASG12V cellular localization is not disrupted after silencing MTMR 2/3/4/7 (Fig. S3)" but Figure S3 is an EM image of PM basal sheets from T47D cells expressing GFP-KRASG12V. Supplemental Figure 4 shows that mutant HRAS is unaffected by the various MTMR knockdowns. Since the authors show decreased proliferation in mutant KRAS cells following MTMR knockdown, the authors should also investigate any changes to proliferation rates in mutant HRAS cell lines following MTMR knockdown. This data is necessary to prove that MTMR-driven changes in downstream RAS signaling are specific to mutant KRAS and not mutant HRAS. It may also be important for the authors to also show any effects on wildtype RAS localization to the PM when MTMR-2,-3,-4, and -7 are knocked down, to show whether this is a oncoprotein-specific event.
    The representative images chosen for Figure 4 diminish the reliability of the data, as it is difficult to see a visible change in the PI3P probe between the control and MTMR knockdown cells in these images. Since the authors rely on the Mander's coefficient and the number of gold particles throughout much of the paper, having the same conclusion quantitatively but not qualitatively for these assays is confusing. Perhaps the authors should elaborate on whether MTMR knockdown has a stronger effect on PtSer and KRAS PM presence than PI3P PM presence. They should also describe their method for identifying early endosomes, since they switch back and forth between describing the content of the PM and of early endosomes, such as in Figure 1 and Figure 4.

    Minor comments:

    An additional experiment that may add another layer of clinical applicability would be the use of an MTMR inhibitor in this cell line, to see whether similar effects can be achieved pharmacologically [7]. This would provoke other researchers to investigate MTMR inhibitors in vitro and in vivo to assess the effect on mutant KRAS cancers.

    The inclusion of cell lines that express KRAS proteins of different mutational statuses would be extremely interesting, as KRAS' orientation within the plasma membrane has been shown to be altered by these mutations. This fact should potentially be considered when choosing a secondary or tertiary cell line to do additional experiments in, but it is not necessary for the authors to elaborate on how MTMR proteins may impact different KRAS mutants for the scope of this project.

    The results of this paper rely heavily on one experimental technique, which is calculating a Mander's coefficient and counting the co-localization of the probe of interest with the CellMask stain of the plasma membrane. How this coefficient is derived is explained in appropriate detail in the methods section of this manuscript; however, a secondary route of identifying these changes in membrane constituents would greatly enhance the paper's conclusions. This would eliminate any doubt surrounding the accuracy of the technique, since so much of the data relies on one experimental output.

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    Significance

    The significance of this paper lies in providing the field with an additional regulator of KRAS localization at the PM, as this is localization is critical to KRAS function. Despite three decades worth of understanding and even successfully blocking KRAS membrane localization in vitro, no KRAS-membrane-localization inhibitors have been approved for the clinic. Thus, there is still room in the field for the development of a safe therapeutic target that can effectively block this process. There is a consensus in the literature that PtdSer is critical for KRAS anchoring to the membrane, and this paper describes how MTMR proteins may impact the supply of PtdSer to the PM. Since this work is done in a cancer background by utilizing a mutant KRAS construct (KRASG12V), this work would be interesting to many cancer researchers that are attempting to target mutant KRAS. This paper would also be interesting to researchers who investigate mechanisms of PM maintenance.

    Our lab studies RAS signaling in tumorigenesis. The authors are clear in their explanations of the mechanisms of PM maintenance and PM components relevant to this study.

  4. Review Commons

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

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    Referee #1

    Evidence, reproducibility and clarity

    In their manuscript with the title "Myotubularin-related proteins regulate KRAS function by controlling plasma membrane levels of polyphosphoinositides and phosphatidylserine", Henkels and colleagues describe the identification and characterization of factors modulating the plasma-membrane localization of KRASG12V, required for its activation. In an siRNA-based screening approach, they identify four members of the myotubularin-related (MTMR) protein family, namely MTMR 2, 3, 4 and 7, when downregulated result in impaired localization of KRAS to the plasma membrane.

    Validation was performed via confocal microscopy in cells overexpressing GFP-RASG12V, stained with the membrane marker CellMask and with gold labelling-Electron microscopy. Co-expression of GFP-LactC2, a well-established marker for PtdSer, and subsequent EM analysis revealed a reduction of PtdSer at the PM upon MTMR depletion. This observation was further validated by whole cell lipidomics, showing a significant reduction in total cellular PtdSer content in MTMR 2/3/4/7 KD conditions.Reduction of PI4P at the PM of PI4P and increase in overall (and PM) levels of PI3P - measured by overexpression of fluorescently tagged marker proteins for the respective phospholipid as well as EM.inally, to investigate the effect of MTMR knockdown on RAS signalling, the authors used transformed T47D cells as well as a C. elegans model system. In both systems, RAS signalling was found to be impaired upon MTMR depletion.

    Overall, the authors convincingly present MTMR proteins as regulators of KRAS plasma membrane localization. Upon depletion of MTMR 2,3,4 and 7, they see KRAS mis-localizing away from the PM and KRAS signalling being disrupted in cell culture and C. elegans model systems. The data are well presented and of high quality. Electron microscopy after immunogold labelling was used to provide quantitative data. The study can be further strengthened by uncovering the role of MTMR in KRAS driven pathobiology.

    Please find some minor comments below

    Comments:

    • no data are shown from the genome-wide screening approach, including the common regulators of KRAS and HRAS. Information about how imaging data were processed and analysed is missing. A final table of 8 selected factors with phosphatase activity is presented without providing further insight about the selection criteria and other factors.
    • in the subsequent characterization via image-based quantification of GFP-KRAS membrane localization, a Manders´ coefficient was calculated. A respective chapter in the methods section on how this was done is missing.

    I would be happy to see the following analyses to strengthen the dataset:

    • reconstitution experiments and further validation to show that it is dependent on the enzymatic activity of MTMRs
    • additive effect upon depletion of multiple MTMRs? Are they functionally co-operative?
    • signalling analysis is very limited (Fig. 5). Do the authors detect any defects in K-RAS driven downstream signaling in these cells upon depletion of MTMRs.

    Significance

    Very interesting and potentially important study. But needs further evidence on how MTMRs regulate pathobiology. Does MTMR depletion inhibit KRAS driven downs stream events needs to investigated here.

  5. Version published to 10.1101/2024.01.22.576612v1 on bioRxiv