Contributions
Abstract: S421
Type: Oral Presentation
Presentation during EHA22: On Saturday, June 24, 2017 from 12:30 - 12:45
Location: Hall C
Background
The hematopoietic stem cell (HSC) compartment in mice encompasses a broad range of heterogeneous cell types including highly lineage-biased HSCs, such as platelet-biased HSCs (PMID:23934107). Myeloproliferative neoplasms (MPNs) are a heterogeneous spectrum of clonal hematopoietic disorders, that includes essential thrombocythemia (ET), a MPN-subtype usually presenting with isolated thrombocytosis. Most ET patients carry a gain-of-function point mutation in JAK2 (JAK2V617F), with several other collaborating hits reported to co-occur with JAK2V617F at lower frequencies, including loss-of-function mutations of the epigenetic regulator EZH2, which are more frequent in advanced MPN.
Aims
Although it is broadly accepted that MPNs are propagated by counterparts of HSCs, the impact of collaborating MPN-associated mutations arising in different HSC subsets remains unclear. We aimed to explore the possibility that platelet-biased HSCs might selectively promote development of an ET phenotype.
Methods
We generated a novel mouse model of MPN that carries a conditional knock-in of heterozygous human JAK2V617F (hJAK2V617F) and the conditional knock-out (KO) of EZH2 together with an inducible Mx1-Cre transgene. To analyse platelet-biased HSC subsets upon onset of the mutation(s), we also crossed in the vwf-eGFP transgene, which is selectively expressed in platelet-biased HSCs.
Results
Compared to wild-type and single mutant mice, EZH2-KO hJAK2V617F mice showed increased platelet counts, including a subset of mice which became acutely unwell with an extreme thrombocytosis. Strikingly, in serial bone marrow (BM) transplantation assays, EZH2-KO fully rescued the previously described hJAK2V617F-associated transplantation defect (PMID:20489053). EZH2-KO hJAK2V617F BM recipients showed long-term serial engraftment that was fully restricted to the platelet and myeloid lineages with a persistent thrombocytosis and absence of lymphoid reconstitution. RNA-sequencing revealed upregulation of several signaling pathways, including Hedgehog, and increased inflammation associated gene expression in EZH2-KO hJAK2V617F HSCs. Unexpectedly in this mouse model of thrombocytosis, phenotypic analysis of the HSC compartment in the BM showed that vwf-eGFP+ve HSCs were selectively lost (fold change[FC]=0.12 p=0.009), while vwf-eGFP–ve HSC numbers remained unaffected (FC=1.06 p=0.88) in Ezh2-KO hJAK2V617F mice. To assess a differential contribution of vwf-eGFP+ve HSCs vs vwf-eGFP-ve HSCs in the ability to propagate MPN, we sorted HSCs according to vwf-eGFP expression and transplanted them into recipient mice. Unlike their normal counterparts, which showed lymphoid-biased reconstitution, vwf-eGFP–ve HSCs from Ezh2-KO hJAK2V617F mice primarily gave rise to platelets and myeloid cells. In contrast, vwf-eGFP+ve HSCs from Ezh2-KO hJAK2V617F mice engrafted poorly without recapitulating the disease in recipients.
Conclusion
In this novel Ezh2-KO hJAK2V617F mouse model, Ezh2 loss collaborates to worsen thrombocytosis and rescue the HSC function defect in hJAK2V617F mice. We also observed a striking disruption of phenotypic and functional HSC heterogeneity in Ezh2-KO hJAK2V617F mice with an unexpected and selective loss of vwf-eGFP+ve HSCs together with subversion of vwf-eGFP–ve HSCs towards platelet-myeloid lineage committment. This previously undescribed disruption of HSC heterogeneity in myeloid malignancy together with the clonal advantage conferred to HSCs by EZH2-KO helps to explain how this collaborating mutation might promote the development of more advanced MPN.
Session topic: 15. Myeloproliferative neoplasms - Biology
Keyword(s): Stem cell, Myeloproliferative disorder
Abstract: S421
Type: Oral Presentation
Presentation during EHA22: On Saturday, June 24, 2017 from 12:30 - 12:45
Location: Hall C
Background
The hematopoietic stem cell (HSC) compartment in mice encompasses a broad range of heterogeneous cell types including highly lineage-biased HSCs, such as platelet-biased HSCs (PMID:23934107). Myeloproliferative neoplasms (MPNs) are a heterogeneous spectrum of clonal hematopoietic disorders, that includes essential thrombocythemia (ET), a MPN-subtype usually presenting with isolated thrombocytosis. Most ET patients carry a gain-of-function point mutation in JAK2 (JAK2V617F), with several other collaborating hits reported to co-occur with JAK2V617F at lower frequencies, including loss-of-function mutations of the epigenetic regulator EZH2, which are more frequent in advanced MPN.
Aims
Although it is broadly accepted that MPNs are propagated by counterparts of HSCs, the impact of collaborating MPN-associated mutations arising in different HSC subsets remains unclear. We aimed to explore the possibility that platelet-biased HSCs might selectively promote development of an ET phenotype.
Methods
We generated a novel mouse model of MPN that carries a conditional knock-in of heterozygous human JAK2V617F (hJAK2V617F) and the conditional knock-out (KO) of EZH2 together with an inducible Mx1-Cre transgene. To analyse platelet-biased HSC subsets upon onset of the mutation(s), we also crossed in the vwf-eGFP transgene, which is selectively expressed in platelet-biased HSCs.
Results
Compared to wild-type and single mutant mice, EZH2-KO hJAK2V617F mice showed increased platelet counts, including a subset of mice which became acutely unwell with an extreme thrombocytosis. Strikingly, in serial bone marrow (BM) transplantation assays, EZH2-KO fully rescued the previously described hJAK2V617F-associated transplantation defect (PMID:20489053). EZH2-KO hJAK2V617F BM recipients showed long-term serial engraftment that was fully restricted to the platelet and myeloid lineages with a persistent thrombocytosis and absence of lymphoid reconstitution. RNA-sequencing revealed upregulation of several signaling pathways, including Hedgehog, and increased inflammation associated gene expression in EZH2-KO hJAK2V617F HSCs. Unexpectedly in this mouse model of thrombocytosis, phenotypic analysis of the HSC compartment in the BM showed that vwf-eGFP+ve HSCs were selectively lost (fold change[FC]=0.12 p=0.009), while vwf-eGFP–ve HSC numbers remained unaffected (FC=1.06 p=0.88) in Ezh2-KO hJAK2V617F mice. To assess a differential contribution of vwf-eGFP+ve HSCs vs vwf-eGFP-ve HSCs in the ability to propagate MPN, we sorted HSCs according to vwf-eGFP expression and transplanted them into recipient mice. Unlike their normal counterparts, which showed lymphoid-biased reconstitution, vwf-eGFP–ve HSCs from Ezh2-KO hJAK2V617F mice primarily gave rise to platelets and myeloid cells. In contrast, vwf-eGFP+ve HSCs from Ezh2-KO hJAK2V617F mice engrafted poorly without recapitulating the disease in recipients.
Conclusion
In this novel Ezh2-KO hJAK2V617F mouse model, Ezh2 loss collaborates to worsen thrombocytosis and rescue the HSC function defect in hJAK2V617F mice. We also observed a striking disruption of phenotypic and functional HSC heterogeneity in Ezh2-KO hJAK2V617F mice with an unexpected and selective loss of vwf-eGFP+ve HSCs together with subversion of vwf-eGFP–ve HSCs towards platelet-myeloid lineage committment. This previously undescribed disruption of HSC heterogeneity in myeloid malignancy together with the clonal advantage conferred to HSCs by EZH2-KO helps to explain how this collaborating mutation might promote the development of more advanced MPN.
Session topic: 15. Myeloproliferative neoplasms - Biology
Keyword(s): Stem cell, Myeloproliferative disorder