Role of Genetic Haploinsufficiency in the Biology and Targeted Therapy of Del(5q) Myelodysplastic Syndrome

textabstractThe studies presented in this thesis focused on the identification of genes that play a central role in the pathogenesis of myelodysplastic syndromes with a deletion of the short arm (q) of chromosome 5, the del(5q) MDS. The overall goal of the projects was to gain a better understanding of how gene haploinsufficiency in (del)5q MDS leads to a clonal advantage, ineffective hematopoiesis and how haploinsufficiency can be targeted. Heterozygous deletion of RPS14 occurs in del(5q) myelodysplastic syndrome (MDS) and has been linked to impaired erythropoiesis, characteristic of this disease subtype. So far, it was not well understood how ribosomal haploinsufficiency affects protein synthesis and if differentially translated proteins have a impact on the erythroid differentiation defect (chapter 1). We generated a murine model with conditional inactivation of Rps14 and demonstrated a p53-dependent erythroid differentiation defect with apoptosis at the transition from polychromatic to orthochromatic erythroblasts resulting in age- and erythroid stress dependent progressive anemia, megakaryocyte dysplasia, and loss of hematopoietic stem cell (HSC) quiescence (chapter 2). Protein synthesis was significantly reduced in Rps14 haploinsufficient hematopoietic stem cells and in particular in erythroid progenitor cells relative to wild-type cells. As assessed by quantitative proteomics, Rps14 haploinsufficient erythroblasts expressed higher levels of proteins involved in innate immune signaling, notably the heterodimeric S100 calcium-binding proteins S100a8 and S100a9 (alarmins). S100a8 is functionally involved in the erythroid defect caused by the Rps14 deletion, as addition of recombinant S100a8 was sufficient to induce an erythroid differentiation defect in wild-type erythroid cells (phenocopy), and genetic inactivation of S100a8 expression rescued the erythroid differentiation defect of Rps14-haploinsufficient HSCs. Our data link Rps14 haploinsufficiency in del(5q) MDS to activation of the innate immune system and induction of S100A8-S100A9 expression, leading to a p53-dependent erythroid differentiation defect. In future experiments I aim to focus on the role of chronic inflammation on myelodysplasia and on the role of monocytes and macrophages as disease-mediating, inflammatory bystander cells and ultimately, on how to therapeutically target this process. A key question in the field is, which genes within the commonly deleted region (CDR) are necessary and sufficient for del(5q) HSC to achieve clonal dominance over normal HSC. Csnk1a1 haploinsufficiency conferred increased intrinsic self-renewal of HSC and nuclear-Catenin accumulation (chapter 3). In striking contrast, Csnk1a1 homozygous inactivation is not tolerated in HSC due to activation of p53. The sensitivity of hematopoietic cells to Csnk1a1, -catenin and p53 gene dosage provides a therapeutic window for targeting haploinsufficiency in del(5q) MDS cells. The finding of targeting haploinsufficiency as a vulnerability of the hematopoietic stem cell in del(5q) MDS was a central concept in the study described in chapter 4. Here, we demonstrate that lenalidomide induces the ubiquitination of casein kinase 1A1 (CK1α) by the E3 ubiquitin ligase CRL4(CRBN), resulting in CK1α degradation. Haploinsufficient expression sensitizes cells to lenalidomide therapy, providing a mechanistic basis for the therapeutic window of lenalidomide in del(5q) MDS. In future experiments I would like to test candidate molecules to specifically target the gene dosage effect in del(5q) HSC with limited effects on normal hematopoiesis, overcoming the problems of resistance to lenalidomide therapy and maintenance of the del(5q) MDS clone under therapy. In the last part of the thesis (chapter 5) we aimed to create an artificial, transplantable BM niche that supports hematopoiesis while allowing for the genetic modification of both hematopoietic and mesenchymal cells as to dissect their interaction in steady state and under pathopyhsiological conditions (chapter 5). We established and characterized a multicomponent system that is optimal for studying hematopoietic-mesenchymal interactions and for the recapitulation of the native BM microenvironment in a transplantable extramedullary niche (mouse model). The combination of macro- and microporous β-TCP scaffolds and Matrigel optimally supports hematopoiesis, including hematopoietic recruitment, proliferation, and differentiation as well as ECM remodeling. In future experiments we intend to genetically modify stromal cells and study their impact on the HSC regulation in myeloproliferative neoplasms and their role in fibrotic transformation in myelofibrosis.