Inflammatory response in hematopoietic stem and progenitor cells triggered by activating SHP2 mutations evokes blood defects
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Curated by eLife
Evaluation Summary:
The authors of this paper model the D61G mutation in the gene PTPN11 that encodes the protein-tyrosine phosphatase SHP2 in zebrafish, creating a model consistent with the human Noonan syndrome (NS), which is predisposed to a juvenile myelomonocytic leukemia (JMML) and myeloproliferative neoplasm (MPN)-like syndrome. The study nicely provides a new model that can be used as the basis of future studies in the field. Because the mutant variably displays phenotypes along a spectrum from NS to MPN, different researchers can choose to focus on this as they see fit.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. The reviewers remained anonymous to the authors.)
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Abstract
Gain-of-function mutations in the protein-tyrosine phosphatase SHP2 are the most frequently occurring mutations in sporadic juvenile myelomonocytic leukemia (JMML) and JMML-like myeloproliferative neoplasm (MPN) associated with Noonan syndrome (NS). Hematopoietic stem and progenitor cells (HSPCs) are the disease propagating cells of JMML. Here, we explored transcriptomes of HSPCs with SHP2 mutations derived from JMML patients and a novel NS zebrafish model. In addition to major NS traits, CRISPR/Cas9 knock-in Shp2 D61G mutant zebrafish recapitulated a JMML-like MPN phenotype, including myeloid lineage hyperproliferation, ex vivo growth of myeloid colonies, and in vivo transplantability of HSPCs. Single-cell mRNA sequencing of HSPCs from Shp2 D61G zebrafish embryos and bulk sequencing of HSPCs from JMML patients revealed an overlapping inflammatory gene expression pattern. Strikingly, an anti-inflammatory agent rescued JMML-like MPN in Shp2 D61G zebrafish embryos. Our results indicate that a common inflammatory response was triggered in the HSPCs from sporadic JMML patients and syndromic NS zebrafish, which potentiated MPN and may represent a future target for JMML therapies.
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Author Response:
JMML is a rare pediatric leukemia, emanating from mutations in the RAS pathway. One of the most frequent genetic causes are loss of function mutations in PTPN11. Using genetically-engineered zebrafish, the investigators show that a chronic inflammatory state is present.
Comments:
- A mouse model for Noonan Syndrome with overlap with JMML, PTPN11 D61G , displays myeloproliferative, cardiac, and craniofacial disease. Here, the zebrafish ptpn11 D61G displayed a wide penetrance depending on allelic burden.
- The hematopoietic effects in the affected fish involve the myeloid compartment. There appears to be no zebrafish ortholog for GM-CSF, and instead the author look at effects of Gcsf. They note enhanced GM colony formation -- but Gcsf should not promote macrophage development. In addition, the effect in human is that …
Author Response:
JMML is a rare pediatric leukemia, emanating from mutations in the RAS pathway. One of the most frequent genetic causes are loss of function mutations in PTPN11. Using genetically-engineered zebrafish, the investigators show that a chronic inflammatory state is present.
Comments:
- A mouse model for Noonan Syndrome with overlap with JMML, PTPN11 D61G , displays myeloproliferative, cardiac, and craniofacial disease. Here, the zebrafish ptpn11 D61G displayed a wide penetrance depending on allelic burden.
- The hematopoietic effects in the affected fish involve the myeloid compartment. There appears to be no zebrafish ortholog for GM-CSF, and instead the author look at effects of Gcsf. They note enhanced GM colony formation -- but Gcsf should not promote macrophage development. In addition, the effect in human is that of spontaneous growth of CFU-GM. The authors would need to address the differences with human JMML malignant hematopoiesis.
- The use of MEK or PI3K inhibitors do not themselves implicate proinflammatory response (line 273). These agents are not anti-inflammatory but have a wide range of effects. They are as much anti-proliferative. To demonstrate inhibition of proinflammatory response would require true anti-inflammatory agents. Dexamethasone targets lymphocytes, and may also reduced cytokine release by macrophages in fish. Controls should include genes that are biomarkers for inflammation (i.e., not l-plastin or c-myb).
We have rephrased the text to make sure we did not unintentionally suggest that MEK or PI3K inhibitors are anti-inflammatory agents. We used MEK and PI3K inhibitors, because these are known to inhibit signaling downstream of SHP2. As anti-inflammatory agent, we used dexamethasone, which rescued many of the observed blood defects in Shp2-D61G mutant zebrafish embryos. We observed a rescue of the number of c-myb and l-plastin expressing cells (read-out for HSPCs). We have done additional experiments and assessed that the increase in the number of neutrophils and macrophages was largely rescued by dexamethasone, showing that dexamethasone targets the myeloid lineage. Moreover, dexamethasone rescued the inflammatory response in Shp2- D61G expressing embryos as assessed by expression of the inflammatory response genes, tnfa, gcsfb and il1b (new Fig. 6D-G). Based on these data, we conclude that the inflammatory response in Shp2- D61G mutant zebrafish embryos may have a causal role in the pathogenesis of the NS/JMML-like MPN blood phenotype.
Reviewer #3:
The authors of this paper model the D61G mutation in zebrafish, creating a model consistent with the human Noonan syndrome, which is predisposed to a JMML and MPN like syndrome. They use RNA-seq to identify the potential cellular abnormalities in either the HSPC or monocyte/macrophage clusters, which nominates an inflammatory signature as being pathogenic. They complement this with analysis of human JMML patients, showing a similar inflammatory signature.
The study nicely provides a new model that can be used as the basis of future studies in the field. Because the mutant variably displays phenotypes along a spectrum from NS to MPN, different researchers can choose to focus on this as they see fit. Where the manuscript falls short is in more clearly delineating the defect in HSPC vs. monocyte/macrophages (especially in comparing fish to human) and at least a hint of the involved mechanisms.
In fish and human, we are analyzing RNA expression in HSPCs, consisting of HSC- like cells and early progenitors, but not fully differentiated monocytes and/or macrophages. We have used the single cell sequencing dataset of zebrafish HSPCs to determine when pro-inflammatory gene expression was evoked. We used Monocle trajectory inference analysis and found that differentiation started in HSC-like cells and progressed in two directions in pseudotime, towards monocyte/macrophage progenitors in one direction and towards thrombocyte and erythrocyte progenitors in the other direction. The analysis showed that proinflammatory genes are evoked during early differentiation of monocyte/macrophage progenitor cells specifically in the Shp2D61G mutants. We have included this analysis in the new panels C-E in Fig. 4 and Fig.4 – figure supplement 1 of the revised version of the manuscript and accompanying textual changes in the Results section.
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Evaluation Summary:
The authors of this paper model the D61G mutation in the gene PTPN11 that encodes the protein-tyrosine phosphatase SHP2 in zebrafish, creating a model consistent with the human Noonan syndrome (NS), which is predisposed to a juvenile myelomonocytic leukemia (JMML) and myeloproliferative neoplasm (MPN)-like syndrome. The study nicely provides a new model that can be used as the basis of future studies in the field. Because the mutant variably displays phenotypes along a spectrum from NS to MPN, different researchers can choose to focus on this as they see fit.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. The reviewers remained anonymous to the authors.)
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Reviewer #1 (Public Review):
JMML is a rare pediatric leukemia, emanating from mutations in the RAS pathway. One of the most frequent genetic causes are loss of function mutations in PTPN11. Using genetically-engineered zebrafish, the investigators show that a chronic inflammatory state is present.
Comments:
1. A mouse model for Noonan Syndrome with overlap with JMML, PTPN11 D61G , displays myeloproliferative, cardiac, and craniofacial disease. Here, the zebrafish ptpn11 D61G displayed a wide penetrance depending on allelic burden.
2. The hematopoietic effects in the affected fish involve the myeloid compartment. There appears to be no zebrafish ortholog for GM-CSF, and instead the author look at effects of Gcsf. They note enhanced GM colony formation -- but Gcsf should not promote macrophage development. In addition, the effect in …
Reviewer #1 (Public Review):
JMML is a rare pediatric leukemia, emanating from mutations in the RAS pathway. One of the most frequent genetic causes are loss of function mutations in PTPN11. Using genetically-engineered zebrafish, the investigators show that a chronic inflammatory state is present.
Comments:
1. A mouse model for Noonan Syndrome with overlap with JMML, PTPN11 D61G , displays myeloproliferative, cardiac, and craniofacial disease. Here, the zebrafish ptpn11 D61G displayed a wide penetrance depending on allelic burden.
2. The hematopoietic effects in the affected fish involve the myeloid compartment. There appears to be no zebrafish ortholog for GM-CSF, and instead the author look at effects of Gcsf. They note enhanced GM colony formation -- but Gcsf should not promote macrophage development. In addition, the effect in human is that of spontaneous growth of CFU-GM. The authors would need to address the differences with human JMML malignant hematopoiesis.
3. The use of MEK or PI3K inhibitors do not themselves implicate proinflammatory response (line 273). These agents are not anti-inflammatory but have a wide range of effects. They are as much anti-proliferative. To demonstrate inhibition of proinflammatory response would require true anti-inflammatory agents. Dexamethasone targets lymphocytes, and may also reduced cytokine release by macrophages in fish. Controls should include genes that are biomarkers for inflammation (i.e., not l-plastin or c-myb).
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Reviewer #2 (Public Review):
In Solman's et al study they explore the impact of PTPN11 mutations (gene that encodes for the protein-tyrosine phosphatase SHP2) associated with Noonan syndrome (NS), juvenile myelomonocytic leukemia (JMML) and JMML-like myeloproliferative neoplasm. They use a zebrafish model and JMML patient cells. They create a zebrafish model using CRISPR/cas9 introducing D61G mutation, a common mutation present in these patients. These mutants showed a phenotype similar to NS in homozygosis and heterozygosis with different penetrance. Mutants are smaller, presented craniofacial defects, heart edema and absence of the swim bladder. They also presented hematopoietic abnormalities including myeloid hyperproliferation. Single cell mRNA sequencing of HSPCS from zebrafish mutants and JMML patients showed an excessive …
Reviewer #2 (Public Review):
In Solman's et al study they explore the impact of PTPN11 mutations (gene that encodes for the protein-tyrosine phosphatase SHP2) associated with Noonan syndrome (NS), juvenile myelomonocytic leukemia (JMML) and JMML-like myeloproliferative neoplasm. They use a zebrafish model and JMML patient cells. They create a zebrafish model using CRISPR/cas9 introducing D61G mutation, a common mutation present in these patients. These mutants showed a phenotype similar to NS in homozygosis and heterozygosis with different penetrance. Mutants are smaller, presented craniofacial defects, heart edema and absence of the swim bladder. They also presented hematopoietic abnormalities including myeloid hyperproliferation. Single cell mRNA sequencing of HSPCS from zebrafish mutants and JMML patients showed an excessive proinflammatory response. Interestingly they found the same response in both species. With these results, they conclude the study using anti-inflammatory agents to prove that the inflammatory response can be ameliorated in JMML-like MPN patients. Using the zebrafish shp2 mutants they showed improvement of c-myb and l-plastin expression in Shp2 mutants. Finally, they suggest that pharmacological inhibition of the inflammatory response can be a therapeutic option for human patients. The conclusions of this study are well supported for the data provided.
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Reviewer #3 (Public Review):
The authors of this paper model the D61G mutation in zebrafish, creating a model consistent with the human Noonan syndrome, which is predisposed to a JMML and MPN like syndrome. They use RNA-seq to identify the potential cellular abnormalities in either the HSPC or monocyte/macrophage clusters, which nominates an inflammatory signature as being pathogenic. They complement this with analysis of human JMML patients, showing a similar inflammatory signature.
The study nicely provides a new model that can be used as the basis of future studies in the field. Because the mutant variably displays phenotypes along a spectrum from NS to MPN, different researchers can choose to focus on this as they see fit. Where the manuscript falls short is in more clearly delineating the defect in HSPC vs. monocyte/macrophages …
Reviewer #3 (Public Review):
The authors of this paper model the D61G mutation in zebrafish, creating a model consistent with the human Noonan syndrome, which is predisposed to a JMML and MPN like syndrome. They use RNA-seq to identify the potential cellular abnormalities in either the HSPC or monocyte/macrophage clusters, which nominates an inflammatory signature as being pathogenic. They complement this with analysis of human JMML patients, showing a similar inflammatory signature.
The study nicely provides a new model that can be used as the basis of future studies in the field. Because the mutant variably displays phenotypes along a spectrum from NS to MPN, different researchers can choose to focus on this as they see fit. Where the manuscript falls short is in more clearly delineating the defect in HSPC vs. monocyte/macrophages (especially in comparing fish to human) and at least a hint of the involved mechanisms.
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