Myeloproliferative neoplasms (MPN) often respond well to symptomatic therapy, however, this therapy fails to eliminate the malignant clone, resulting in disease persistence as the major clinical challenge. We propose that persistence is mediated by immune escape both through the expression of immune-inhibitory molecules on the cell surface of MPN cells, which prevent immune-mediated elimination and the release of TGF-β.

Our preliminary data indicate that activating calreticulin (CALR) mutations can cause the expression of TGF-β. We used non-malignant 32D cells expressing human thrombopoietin receptor and induced human CalrWT (control), Calr ins5 or Calr del52 oncogenes and microarray based analysis revealed more than 6000 differentially regulated genes and an upregulation of TGF-β RNA together with pro-inflammatory cytokines.

To clarify the functional relevance of TGF-β in vivo, we further analyzed mice carrying Calr del52MPL bone marrow (BM) and observed an improved survival of the mice upon TGF-β neutralization with an increase in non-mutation carrying hematopoietic stem cells. These data support a functional role for CALR induced TGF-β causing immunosuppression in vivo. Furthermore, we could show that TGF-β reduced expression of cytolytic markers in T cells in a dose dependent manner.

To test if the findings made in the mouse model were reproducible in primary patient samples, we collected BM from patients diagnosed with myeloproliferative neoplasms. Indeed, MPN patient-derived cells exhibited increased TGF- β1 expression on CD110+ cells in the BM compared to healthy donor BM. In line with this, TGF-β mRNA was increased in whole BM and in CD34+ BM mononuclear cells compared to healthy donor BM. Furthermore, we could validate these findings through public data analysis using different openly accessible data sets.

Based on these findings, the aim of this project is to develop immuno-therapeutic approaches interfering with immune-suppressive ligands, metabolites and TGF-β, thereby promoting elimination of persisting MPN cells. For this, we want:

To assess which checkpoint molecules and soluble inhibitory factors are increasingly expressed by myeloid cells upon acquisition of an activating CALR mutation.

To investigate how the signaling pathways activated by the CALR mutation lead to activation of the promoters of checkpoint molecules, to TGF-β release and to metabolic alterations that cause LA release and persistence of MPN clones.

To define which immuno-therapeutic approaches that promote elimination of persisting MPN cells can be used therapeutically.