Background
Among the chronic Philadelphia-negative myeloproliferative neoplasms (MPNs), myelofibrosis (MF) is the most aggressive disorder. Its hallmark is the extensive deposition of extracellular matrix fibers in the bone marrow (BM) that leads to the disruption of the microenvironment. Like other MPNs, MF is originated by the acquisition of somatic mutations in hematopoietic stem/progenitor cells (HSPCs). Mutations affecting JAK2, MPL and CALR have been discovered as driver events responsible for the onset of the myeloproliferative phenotype.
Hypothesis
Preliminary results, obtained in our laboratory, revealed that MF HSPCs express specific surface markers that distinguish them from healthy donor HSPCs. Recently NSG-SGM3 (NSGS) mice have been proven to allow the efficient engraftment of MF CD34+ HSPCs, giving rise to a myeloproliferative phenotype recapitulating the human disease in terms of clonal complexity and BM fibrosis development. Therefore, they represent a promising platform for the functional characterization of MF stem cells and for the preclinical evaluation of novel therapeutic strategies, such as targeting osteopontin (OPN), which we first identified as a secreted protein with a profibrotic role in MF.
Aims
We first aim at identifying and characterizing MF HSPCs whose expansion gives rise to the neoplastic clone. Following this, MF patient-derived xenografts (PDX) will be established to confirm that identified MF HSPC subpopulations propagate the disease in vivo and to evaluate the efficacy of therapeutic strategies targeting neoplastic stem cells or fibrosis development.
Experimental Design
We will characterize MF HSPC subsets isolated based on the expression of specific surface markers identified in preliminary experiments, such as CD133, CD244 and CD9. Marker expression will be correlated with the presence of disease-specific gene mutations through a multiomic approach. Moreover, we will define the molecular pathways that sustain neoplastic HSPC survival and expansion by evaluating their transcriptome and function in vitro. Once putative MF stem cells are identified, we will perform in vivo studies by transplanting HSPC subpopulations in NSGS mice to test whether they can recapitulate MF, including BM fibrosis. This PDX model will subsequently be used to: 1. test the efficacy of novel immunotherapy approaches targeting MF stem cells: 2. study the mechanisms responsible for fibrosis development; 3. assess the antifibrotic properties of molecules, which either inhibit OPN production or target other profibrotic pathways, in vitro.
Expected Results
Results obtained in this project will allow the identification of MF stem cells that can be distinguished from their normal counterparts based on surface marker expression. We anticipate that we will be able to design alternative therapeutic approaches targeting MF stem cells and fibrosis, whose efficacy will be evaluated using MF PDX mouse model.
Impact On Cancer
To date, approved targeted therapies, including JAK/STAT inhibitors, proved to only have a minor effect on reducing the disease burden and BM fibrosis in patients, therefore there is an urgent need for the development of more effective therapeutic approaches. A better characterization of MF stem cells is mandatory for the identification of novel actionable targets that may allow the eradication of the neoplastic
clone and treatment of BM fibrosis in patients.