Integrated signaling and transcriptome analysis reveals Src family kinase individualities and novel pathways controlled by their constitutive activity

  1. Nikolaos Koutras
  2. Vasileios Morfos
  3. Kyriakos Konnaris
  4. Adamantia Kouvela
  5. Athanasios-Nasir Shaukat
  6. Constantinos Stathopoulos
  7. Vassiliki Stamatopoulou
  8. Konstantina Nika

  • Curated by eLife

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    **eLife assessment
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    This study presents valuable findings on how the ectopic expression of the Lck protein tyrosine kinase in Chronic Lymphocytic Leukemias (CLL) contributes to malignant transformation. The evidence supporting the claims of the authors is solid, although the use of gene editing to directly explore the functions unique to LCK in a CLL model would increase the appeal of the work.

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Abstract

The Src family kinases (SFKs) Lck and Lyn are crucial for lymphocyte development and function. Albeit tissue-restricted expression patterns the two kinases share common functions; the most pronounced one being the phosphorylation of ITAM motifs in the cytoplasmic tails of antigenic receptors. Lck is predominantly expressed in T lymphocytes; however, it can be ectopically found in B-1 cell subsets and numerous pathologies including acute and chronic B-cell leukemias. The exact impact of Lck on the B-cell signaling apparatus remains enigmatic and is followed by the long-lasting question of mechanisms granting selectivity among SFK members. In this work we sought to investigate the mechanistic basis of ectopic Lck function in B-cells and compare it to events elicited by the predominant B-cell SFK, Lyn. Our results reveal substrate promiscuity displayed by the two SFKs, which however, is buffered by their differential susceptibility toward regulatory mechanisms, revealing a so far unappreciated aspect of SFK member-specific fine-tuning. Furthermore, we show that Lck- and Lyn-generated signals suffice to induce transcriptome alterations, reminiscent of B-cell activation, in the absence of receptor/co-receptor engagement. Finally, our analyses revealed a yet unrecognized role of SFKs in tipping the balance of cellular stress responses, by promoting the onset of ER-phagy, an as yet completely uncharacterized process in B lymphocytes.

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  1. Version published to 10.3389/fimmu.2023.1224520
  2. Version published to 10.1101/2022.09.30.510317v2 on bioRxiv
  3. eLife

    Authorr Response

    Reviewer #1 (Public Review):

    1. The study finds Lyn to be degraded more efficiently via the proteasome and to be more tightly controlled by phosphatases when compared to Lck. However, rather than interpreting the findings as distinct kinase-intrinsic properties, one could attribute the slower degradation and stricter PTP control of Lyn to the fact that Lyn is the principal and predominant SFK in B cells and thus a "standard target" of the B-lymphoid molecular machinery, to which it is better adapted to.

    We respectfully disagree with the reviewer’s comment that our interpretation is limited to “kinase-intrinsic properties”. In many points within the manuscript we refer to the “B-lymphoid molecular machinery”. More specifically:

    • Lines 62-64 in the original submission (lines 60-61 in the revised manuscript): “….enzymatic promiscuity of SFKs can be buffered by their differential susceptibility to regulatory control mechanisms designed for keeping global SFK activity levels under strict control….”

    • Lines 113-114 in the original submission (lines 137-138 in the revised manuscript): “Lck and Lyn differ in the efficiency for signal ignition and in their susceptibility to regulatory mechanisms in B-cells”

    • Lines 135-136 in the original submission (lines 159-160 in the revised manuscript): “Thus, the proteasomal degradation machinery constrains the abundance of Lyn, but not Lck, within B-cells.”

    • Lines 162-163 in the original submission (lines 185-186 in the revised manuscript): “Collectively these data show that the BCR signaling machinery is more responsive to the action of Lyn, at the same time imposing stricter regulation on its expression and activity levels.”

    • Lines 475-477 in the original submission (lines 527-528 in the revised manuscript): “…identified specialized control mechanisms designed to keep Lyn, but not Lck, activity levels under strict control.”

    However, we cannot rule out, as a mutually inclusive scenario, that intrinsic SFK features contribute to their differential regulation by cellular mechanisms, a possibility that we also refer to in the manuscript. More specifically:

    • Lines 335-337 in the original submission (modified text in the revised version, lines 372-374): “On one hand there is the total amount of SFK activity within the cell, and on the other the individuality of SFK family members, dictated by intrinsic molecular features.”

    • Lines 477-478 in the original submission (lines 528-529 in the revised manuscript): “These data may signify that SFKs have been evolutionarily diversified to best suit the needs of the cellular environment they are expressed in…”

    Based on the reviewer’s comment, and to clarify further, we have modified the revised version of the manuscript (lines 372-374) as follows:

    “On one hand there is the total amount of SFK activity within the cell, and on the other the individuality of SFK family members, dictated by intrinsic molecular features and/or adaptation to cell-specific regulatory mechanisms.”

    We hope that our clarifications, satisfy the reviewer.

    1. Venn diagram depicting differentially regulated transcripts between Lck- and Lyn-expressing cells, it does not seem like Lck is able to regulate pathways which are not "canonically" regulated by Lyn.

    and

    As a distinct functional difference between Lck and Lyn is not established in this work, said SFKs' largely exclusive expression in T and B cells remains enigmatic.

    We thank the reviewer for the comment. We address this issue on the discussion section of the revised manuscript (lines 514-519).

    1. There is also the persisting problem of Lck being expressed to a much higher extent and the effect of the endogenously expressed Lyn since the model systems are not based on a Lyn-deficient cell line.

    For the purpose of the analysis, we tried to circumvent the discrepancies between Lck and Lyn expression levels by our equal GFP gating strategy (explained in Figure 1-figure supplement 3E/Fig.S3E in the original submission). Nevertheless, as shown in Figure 1C there is a physiological reason for the two SFKs not being equally expressed, and we refer to the biological implications of these individualities in the Discussion.

    The effect of endogenously expressed Lyn is represented by the phenotype of -Dox cells which we use as background in all our studies, especially since we show that there are no alterations on Lyn or any other SFK activation status resulting from Lck overexpression (Figure 1-figure supplement 2B/ Fig.S2B in the original submission), so we do not believe this is a problem. Additionally, a Lyn-deficient environment would also not be perfect, since very plausibly it could have undergone further signaling and survival adaptations that we could not account for.

    1. Lastly, the authors follow up their finding of deregulated transcripts belonging to the ER/UPR ontology cluster. Flow cytometric analysis indeed shows an influence of Lck and Lyn expression on ER homeostasis, which can be reverted with SFK inhibitors. Alas, additional follow-up experiments to functionally investigate the deregulated pathways suggested by the RNAseq analysis are not included in this study.

    We thank the reviewer for the comment, and we agree. However, its beyond of our capabilities and manpower and the scope of the present work to perform numerous functional or semi-functional studies for every GO analysis pathway that emerged from the transcriptomics studies. Although follow up work from our group will focus on comprehensive and meticulous analyses of gene expression profiles, currently such an effort would require long-lasting studies which would also significantly extend the size of the manuscript but also distort the focus from the effects we wish to pinpoint with the present work i.e. the unique adaptation of SFKs within the lymphocyte environment and gene expression profile tendencies exclusively controlled by SFK-generated signals.

    In an effort to satisfy the reviewer, we performed focused follow up studies specifically on the ER effect of SFK-transduced signals, since it appears to be a so-far unknown aspect of their function. The new data are presented in the revised version of Figure 4 (panels C and D) and Supplementary Figure 4-figure supplement 1. Corresponding text can be found in lines 323-345 of the revised manuscript (results section) and lines 499-512 and line 531 of the discussion. In brief, we show an SFK kinase-activity dependent activation of the ER-phagy receptor FAM134B, which is not accompanied by recruitment of LC3B, as dictated by the currently known canonical ER-phagy pathway. This is the first report of SFKs’ involvement in ER-phagy process and first time FAM134B activation is described in B-cells. Since this field is relatively new, and the role and regulation of ER-phagy is almost unexplored in B-cells, we hope that the reviewers will appreciate the novelty of the finding and its sufficiency for the current manuscript. We do realize that these initial data prompts for more detailed mechanistic investigation, which we are pursuing in the form of a more complete and comprehensive future study.

    Reviewer #2 (Public Review):

    1. Studies reveal no qualitative functional differences in Lck and Lyn that are likely to explain its unique ectopic expression of Lck in CLL

    and

    If Lck promotes pathophysiology by transduction of a qualitatively unique signal, one would expect that transcriptome analysis should reveal this difference.

    We thank the reviewer for the comment. We address this issue on the discussion section of the revised manuscript (lines 514-519).

    1. It is unclear from the material and methods whether the overexpressed Lyn is LynA or Lyn B. It appears in the text (lines 130-133) that they overexpress LynB specifically. A recent paper from Tania Freedman (Sci Adv 2022 PMID:35452291) suggests that LynA is more activating whereas LynB is more balanced with an inhibitory bias. The point is that it is important to discuss this because they may not be making a relevant comparison.

    We thank the reviewer for the comment, to clarify this, we added in the Materials and Methods section of the revised manuscript (under “Cloning and Plasmids”) the use of Lyn isoform B.

    We initially attempted to produce BJAB lines overexpressing LynA, however expression levels of this isoform was particularly low and we could not proceed with further analyses, so we cannot comment on how LynA might behave in an overexpression model in B-cells, especially given the absence of relevant information in the existing literature.

    The recent Sci Adv 2022 PMID:35452291 study deals with germline LynA and LynB isoform-specific knockouts and their propensity towards autoimmunity in mice. The authors compared the single isoform (LynA or LynB) and total Lyn knockouts by performing systemic phenotypic analyses of autoimmunity features (splenomegaly, myeloid cell profiles, proinflammatory markers on myeloid cells, B cell development, expansion of activated and autoimmunity-associated B cell subsets, autoimmunity scores). Differences they pinpoint between LynA and LynB are summarized as follows:

    1. “It was found that LynB has the dominant regulatory role in mice of both sexes, but that LynA expression is uniquely required to prevent autoimmunity in female mice”. The etiology of which is unclear.

    2. “LynB generally appears to be the dominant immunosuppressive isoform, with LynB deletion causing severe autoimmune disease in male and female mice. For some indicators (splenomegaly, glomerular IgG and C3 deposition, and kidney fibrosis), LynBKO and total LynKO mice developed equally severe phenotypes. In other cases (serum IgM and BAFF, glomerular immune infiltration, myeloid cell polarization, and monocyte/granulocyte expansion), LynBKO mice had less severe phenotypes than total LynKO mice, suggesting an additive effect with LynA”.

    3. “LynA and LynB seemed equally capable of promoting B cell development, regulating myeloid cell polarization and restraining myeloid-driven inflammation. Given the increased number of activated/inflammatory B cell types in LynAKO and LynBKO mice, future studies will be aimed at determining whether the single-isoform knockouts have a more B cell–initiated than myeloid cell–initiated form of autoimmune disease”.

    After careful reading of the manuscript, we could not find any functional analyses on the activation status of the distinct isoforms, or signaling events they elicit. Furthermore, the authors do not report any conclusions that LynA is more activating at the molecular level. Based on the above, we cannot connect the data published in PMID:35452291 paper and our results for discussing “LynA being more activating” and implications this might have on our studies.

    To comply with the reviewer’s suggestion, in our revised manuscript we cite this study (ref number 29) in the following sentence appearing in lines 380-383:

    “Lyn exists as two alternatively spliced variants LynA and LynB. Distinct biological functions between the two isoforms still remain poorly understood. A recent study (29) documented that LynB provides an advantage in protecting against autoimmunity compared to LynA; however, the underlying mechanisms for this phenotype are unclear.”

  4. eLife

    **eLife assessment
    **
    This study presents valuable findings on how the ectopic expression of the Lck protein tyrosine kinase in Chronic Lymphocytic Leukemias (CLL) contributes to malignant transformation. The evidence supporting the claims of the authors is solid, although the use of gene editing to directly explore the functions unique to LCK in a CLL model would increase the appeal of the work.

  5. eLife

    Reviewer #1 (Public Review):

    Nikolaos Koutras et al shed light on potential distinct functions of the Src family kinases (SFKs) Lck and Lyn in lymphoid signal transduction. The authors therefore overexpress Lyn and ectopic Lck in the B lymphoid cell line BJAB in an elegant Dox-inducible manner and compare the SFK's ability to trigger and shape B-lymphoid signal transduction. The findings indicate that ectopic expression of Lck is sufficient to phosphorylate the B cell receptor (BCR) ITAMs in BJAB cells. In these cells, constitutive ITAM and ITIM phosphorylation by both overexpressed Lck and Lyn induces BCR signaling, as demonstrated by phosphorylation of Syk and Akt, as well as CD22 inhibitory signaling, as shown by SHP-1 phosphorylation. In direct comparison, the influence of Lyn on said phosphorylation is stronger when it is (over-)expressed in the same amounts as Lck. This outcome was somewhat expected, since ITIM/ITAM phosphorylation is considered to be the principal function of Lyn in B cells.

    The study finds Lyn to be degraded more efficiently via the proteasome and to be more tightly controlled by phosphatases when compared to Lck. However, rather than interpreting the findings as distinct kinase-intrinsic properties, one could attribute the slower degradation and stricter PTP control of Lyn to the fact that Lyn is the principal and predominant SFK in B cells and thus a "standard target" of the B-lymphoid molecular machinery, to which it is better adapted to.

    Next, the authors present a RNAseq transcriptome analysis of Lck- and Lyn-expressing B cells and validate selected findings via qPCR. The data show Lyn and Lck to regulate pathways and biological functions of critical importance to B lymphocytes. Generally, most of the Lck/Lyn-regulated biological functions and pathways shown here (antigen presentation, cytokine production, migration, apoptosis, autophagy, etc.) are well known to be controlled by BCR signaling, which the overexpression of SFKs are constitutively activating, as shown earlier. While the authors draw a Venn diagram depicting differentially regulated transcripts between Lck- and Lyn-expressing cells, it does not seem like Lck is able to regulate pathways which are not "canonically" regulated by Lyn. There is also the persisting problem of Lck being expressed to a much higher extent and the effect of the endogenously expressed Lyn, since the model systems are not based on a Lyn-deficient cell line.

    Lastly, the authors follow up their finding of deregulated transcripts belonging to the ER/UPR ontology cluster. Flow cytometric analysis indeed shows an influence of Lck and Lyn expression on ER homeostasis, which can be reverted with SFK inhibitors. Alas, additional follow-up experiments to functionally investigate the deregulated pathways suggested by the RNAseq analysis are not included in this study.

    While there definitely are implications for the role of ectopic expression of Lck in CLL cells, this work however presents no direct comparison of expression strength or signaling outcomes between the study's BJAB (Burkitt lymphoma) cell line-based model and a model of CLL - be it a mouse model, human patient samples or a CLL cell line. Since the B-lymphoid cell line used, the Burkitt lymphoma line BJAB, is not CLL-derived, the conclusions that can be drawn for the pathophysiology of CLL is limited.

    In principal, the authors show that the Src kinase Lck - when ectopically expressed - largely fills out the role of the predominant B-lymphoid Src kinase Lyn, namely phosphorylation of the CD79-ITAMs and induction of constitutive antigen receptor signaling. Given that the established role of Lck is the phosphorylation of ITAMs and activation of the T cell receptor in T cells, where it is predominantly expressed, these findings provide limited advancement of our current understanding of antigen receptor signal transduction. As a distinct functional difference between Lck and Lyn is not established in this work, said SFKs' largely exclusive expression in T and B cells remains enigmatic.

  6. eLife

    Reviewer #2 (Public Review):

    The normally T cell restricted Src family tyrosine kinase Lck is ectopically expressed in most B cell Chronic Lymphocytic Leukemias. This, along with the fact that ectopic expression of other SFKs, such are Fyn and Fgr, are not seen, suggests that Lck may have some unique function, distinct from the endogenous Lyn SFK, that promotes malignant transformation. Using inducible expression in a human B cell lymphoma, the study explores this possibility. Studies reveal no qualitative functional differences in Lck and Lyn that are likely to explain its unique ectopic expression of Lck in CLL.

    The strengths of this study include the use of Lentiviral transfer of genes encoding SFKs in conjunction with Doxycycline inducible expression. This allows comparative analysis of acute Lyn and Lck overexpression effects, free of cell resetting artifacts consequent to long term expression of the SFK. Strength is also seen in the authors fluorescent tagging of the SFK so analysis could be gated on ectopic expression level. Strength exists in the authors dissection of SFK effects on early events in the BCR signaling pathway, which reveal the ability of both overexpressed SFKs to drive receptor ITAM tyrosine phosphorylation and initiating BCR signaling. These studies reveal little difference in the function of the SFKs, though it appears that Lck may be less sensitive to phosphatase regulation.

    It is unclear from the material and methods whether the overexpressed Lyn is LynA or Lyn B. It appears in the text (lines 130-133) that they overexpress LynB specifically. A recent paper from Tania Freedman (Sci Adv 2022 PMID:35452291) suggests that LynA is more activating whereas LynB is more balanced with an inhibitory bias. The point is that it is important to discuss this because they may not be making a relevant comparison.

    If Lck promotes pathophysiology by transduction of a qualitatively unique signal, one would expect that transcriptome analysis should reveal this difference. The authors look for this signal using transcriptome analysis of bulk populations expressing similar levels of SFK. Although differences were seen in the transcriptome, finding were not consistent with a qualitatively unique function. However, bulk transcriptomic analysis may miss important differences. Single cell RNAseq, e.g., by 10x, may have been more incisive because gene expression could have been normalized to SFK expression in individual cells.

    Finally, while some interesting differences are seen in the biology of Lyn and Lck, weakness exists in the failure to explore the causality of these differences in driving CLL phenotype. A final thought relevant to this comment. It is a truism that "absence of proof is not proof of absence".

  7. Version published to 10.1101/2022.09.30.510317v1 on bioRxiv