Liposomal melatonin targets mutant hematopoiesis and bone marrow remodeling in MPN

Abstract

Abstract IntroductionMyeloproliferative neoplasms (MPN) are chronic hematological disorders characterized by clonal expansion of the myeloid lineage, inflammation, and oxidative stress that may lead to bone marrow (BM) fibrosis. Allogeneic stem cell transplantation remains the only curative therapy for a limited subset of patients, pressing for the identification of novel therapeutic strategies. Melatonin (MT), a circadian rhythm regulator, has anti-inflammatory and antioxidant properties in various solid tumors. However, its therapeutic potential in hematological malignancies remains elusive. AimThis study aims to evaluate the therapeutic efficacy and elucidate the underlying molecular mechanisms of free and liposomal MT (nano-MT) as a novel therapeutic strategy for MPN. MethodsThe effect of MT was assessed in peripheral blood mononuclear cells (PBMNC) and induced-pluripotent stem cell (iPSC)-derived CD34+ hematopoietic stem and progenitor cells (HSPC) from MPN patients or healthy donors through clonogenic assays, cell viability, glucose uptake, and apoptosis assays. The underlying mechanisms were investigated via RT-qPCR, reactive oxygen species (ROS) measurement, 2D co-cultures of patient derived myeloid cells with healthy mesenchymal stromal cells (MSC), immunofluorescence, and 3'-mRNAseq of MT-treated patient-derived CD34+ cells. Disease progression was investigated longitudinally in SclCreER;JAK2V617F mice via micro-computed tomography (μCT). Therapeutic efficacy of MT (15 mg/kg per day, i.p.), nano-MT (37.5 mg/kg/2x and 3x per week respectively, i.v.) and nano-MT + ruxolitinib (37.5 mg/kg/3x, i.v. and 60 mg/kg/1x per day, oral) was assessed for six weeks by hematological analysis, flow cytometry, RT-qPCR, immunofluorescence and histological analyses of spleen and BM. ResultsMT selectively inhibited the clonogenic capacity of MPN PBMNC and MPN iPSC-derived CD34+ HSPC in comparison to healthy controls which was also confirmed in co-cultures of mutant and wild-type (WT) CD34+ HSPC (p<0.05). This effect was associated with increased apoptosis, reduced ROS levels and decreased glucose uptake in mutant HSPC (all p<0.05). Gene set enrichment analysis (GSEA) of treated MPN primary CD34+ HSPC revealed a significant decrease in MYC targets, G2M checkpoint, oxidative phosphorylation, ROS and glycolysis pathways (all p<0.05, q<0.05). Moreover, we observed a pronounced dose-dependent reduction of stromal α-SMA and pSMAD2/3 (all p<0.0001) by MT in a co-culture model of patient-derived myeloid cells and healthy MSC. In SclCreER;JAK2V617F mice with a strong polycythemia vera (PV) phenotype, in vivo µCT segmentation and histology revealed reduced long-bone and BM radiodensity over four weeks of disease progression, indicating bone remodeling, osteosclerosis and BM hypercellularity with increased lipid content. After 6 weeks of treatment, Nano-MT significantly reduced neoplastic blood parameters (RBC, HGB, and HCT; all p≤0.001), common myeloid progenitors (CMP; p=0.001) and granulocyte-monocyte progenitors (GMP; p=0.0004). Furthermore, late basophilic and orthochromatophilic erythroblasts in BM (p=0.01) and spleen (p=0.02) of JAK2V617F mice was significantly reduced. In primary samples, a pronounced reduction of BFU-E (p=0.029) was recapitulated. Nano-MT significantly reduced the elevated levels of Interleukin-1β (IL-1β) in the BM of diseased mice (p<0.05). At endpoint, CT signal intensity was significantly higher in the nano-MT group than the JAK2V617F control group (p=0.016), suggesting a normalizing effect. Importantly, nano-MT mitigated reticulin deposition (p=0.001) and megakaryocyte count (p<0.0001) in the BM. In a second in vivo experiment demonstrating an increased RBC and platelet (PLT) phenotype, nano-MT again restored RBC, HCT, PLT counts, and normalized the megakaryocyte-erythroid progenitor (MEP; p=0.003) population. In addition, when combined with ruxolitinib, the treatment specifically targeted the aberrant white blood cells (WBC; p<0.0001), neutrophils (p=0.0005), and monocytes (p=0.0019). ConclusionOur findings indicate that MT reduces the MPN phenotype by altering MEP differentiation and disrupting malignant clone support such as suppression of ROS and abnormal metabolism. Notably, nano-MT inhibited disease progression and BM fibrosis. To the best of our knowledge, this is the first study demonstrating a therapeutic benefit of (liposomal) MT in MPN, providing a compelling rationale for translational investigation.