ORCID

http://orcid.org/0000-0002-2041-9867

Date of Award

Winter 1-15-2021

Author's School

Graduate School of Arts and Sciences

Author's Department

Biology & Biomedical Sciences (Molecular Cell Biology)

Degree Name

Doctor of Philosophy (PhD)

Degree Type

Dissertation

Abstract

TGF-b expression is increased in most cases of myeloproliferative neoplasms (MPNs); however, its contribution to disease pathogenesis is not well understood. Here, we explore two specific hypotheses. First, we hypothesize that increased TGF-b signaling in mesenchymal stromal cells contributes to the development of myelofibrosis. Second, we hypothesize that Jak2 mutated hematopoietic stem cells (HSCs) are resistant to the growth suppressive effect of TGF-b, conferring a fitness advantage that contributes to their expansion in MPNs and clonal hematopoiesis. To test the first hypothesis, we abrogated TGF-b signaling in mesenchymal stem/progenitor cells by deleting Tgfbr2 using a doxycycline-repressible Osterix-Cre transgene (Osx-Cre), which targets all mesenchymal lineage cells in the bone marrow. We transplanted hematopoietic stem/progenitor cells (HSPCs) transduced with MPLW515L retrovirus or bone marrow cells from Jak2V617F mice into irradiated wild type or Osx-Cre; Tgfbr2f/- recipients. MPLW515L induced a rapidly fatal MPN with reticulin fibrosis in the bone marrow. A similar hematopoietic phenotype and survival was observed in Osx-Cre; Tgfbr2f/- recipients. However, myelofibrosis, as measured by reticulin staining, collagen I and III immunofluorescent staining, and Col3a1 and Acta2 mRNA expression, was significantly reduced in Osx-Cre; Tgfbr2f/- mice. Likewise, in the Jak2V617F MPN model, expression of collagen I and III in the bone marrow was significantly reduced in Osx-Cre; Tgfbr2f/- recipient mice. Surprisingly, induction of myelofibrosis by MPLW515L was intact in Osx-Cre; Smad4f/f recipients, suggesting that SMAD4-independent (non-canonical) TGF-b signaling mediates the myelofibrosis phenotype. Consistent with these findings, TGF-b1 induced expression of fibrosis associated genes, Col1a1, Acta2 and Loxl1, was intact in primary cultures of Smad4-deleted bone marrow derived mesenchymal stromal cells (MSCs). However, treatment with a JNK inhibitor, but not inhibitors of MEK, p38, or NFkB, abrogated the TGF-b1-induced expression of fibrosis markers in vitro, and preliminary data suggests that treatment with a JNK inhibitor attenuates myelofibrosis induced by MPLW515L in vivo. Together, these data show that TGF-b signaling contributes to the development of myelofibrosis in MPN through activation of the JNK pathway in MSCs. To test the second hypothesis, we first assessed TGF-b signaling in wild type and Jak2V617F HS Cs. Whereas TGF-b1 induced robust Smad2/3 phosphorylation in wild type HSCs, it was barely detectable in Jak2V617F HSCs. Interestingly, this phenotype was lost in lineage-restricted progenitors, where TGF-b-induced Smad2/3 phosphorylation was comparable in wild type and Jak2V617F cells. These data suggest that TGF-b signaling, at least Smad2/3 phosphorylation, is selectively loss in Jak2V617F HSCs. Competitive repopulation assays (CRAs) were performed using wild type and Jak2V617F HSPCs, in which Tgfbr2 was deleted to abrogate TGF-b signaling in HSCs. CRAs comparing wild type and Tgfbr2-/- HSPCs showed that TGF-b signaling negatively regulates repopulating activity. When competing Jak2V617F with Jak2V617F; Tgfbr2-/-, the Tgfbr2 deleted group no longer had repopulating advantage, confirming Jak2V617F is resistant to TGF-b. As expected, Jak2V617F HSCs had a competitive repopulating advantage to wild type. However, this fitness advantage was loss when the competitor HSPCs lack Tgfbr2. Moreover, no competitive advantage of Jak2V617F was observed when both wild type and Jak2V617F HSCs are Tgfbr2-deficient. This data suggest that TGF-b contributes to clonal expansion of Jak2V617F by inhibiting wild type HSCs. Using cell cycle analysis and transcriptome analysis, we further showed that, opposite to published in vitro data, TGF-b may promote cell cycle progression in a non-cell intrinsic manner in vivo. This may contribute to the less quiescent phenotype in Jak2V617F HSCs. Collectively, our data show that TGF-b plays an essential role in the pathogenesis of MPNs. We identify that TGF-b signaling in mesenchymal stromal cells is required for the development of myelofibrosis through the non-canonical JNK pathway. TGF-b signaling in JAK2V617F HSPCs also plays a key role in the clonal expansion in MPNs. Specifically, our data support a model in which increased local production of TGF-b by JAK2V617F hematopoietic cells selectively inhibits wild type HSPCs, thereby promoting the expansion of JAK2V617F HSPCs. Targeting TGF-b or JNK signaling in patients with myelofibrosis may be beneficial in the treatment of bone marrow fibrosis. Targeting TGF-b signaling early in the course of MPN or in persons with JAK2V617F clonal hematopoiesis may prevent or delay clonal progression.

Language

English (en)

Chair and Committee

Daniel C. Link

Committee Members

Timothy J. Ley, Grant A. Challen, David G. DeNardo, Matthew J. Walter,

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