LAUSR

Several evidences indicate that abnormal megakaryocyte (MK) maturation drives the development of Myelofibrosis (MF), the end stage of Philadelphia-negative Myeloproliferative neoplasms: a) presence in bone marrow (BM) of numerous clusters of MK with ultrastructural abnormalities indicating retarded maturation was the first cell hallmark reported for MF (Zucker-Franklin, Dahlem Workshop on Myelofibrosis-Osteosclerosis Syndrome Germany-West: Pergammon Press; 1975); b) studies in mouse models with MK-restricted JAK2V617F expression demonstrated that abnormal MK are necessary and sufficient to induce MF (Zhan, Leukemia 2016;30:23324) and c) both in patients and mouse models, the driver mutations induce a RSP14 ribosomopathy that reduces expression of GATA1 in MK, leading to retarded maturation (Vannucchi, AmJPathol 2005;167:849; Gilles, JCI 2017;127:1316). Due to fibrosis and hematopoietic failure in BM, MF patients develop hematopoiesis in spleen which also is rich of MK. Whether in MF MK in BM and spleen express similar ultrastructural abnormalities has not been investigated as yet. By transmission electron microscopy, we compared the ultrastructural features of MK from BM of 5 MF patients and spleen of 11 MF patients (9 JAK2V617Fhomo, 1 JAK2V617Fhomo/ASXl1R1068X, 2 JAK2V617Fhetero, 1 JAK2V617F hetero/ASXl1R693X, 1 CARLtyp1/TET2R1214W and 2 triple negative; 12 primary and 4 post-PV, IPSS score: low-intermedium 2, DIPSS score: intermedium 1-2). MF underwent splenectomy to reduce discomfort of large spleen size (>20 cm from the left costal margin). BM from 2 healthy controls (HC) and post-trauma spleen from 1 HC were analyzed as control. MK were numerous both in BM (43±5 vs 30±2/area in MF vs HC, p=0.027, Anova) and spleen (47±18 in MF vs below detection in HC). The majority (98%) of MK from MF BM had immature ultrastructural morphology while 59% of MK from HC BM were mature (p=0.00003). To a surprise, the majority (69%) of MK from MF spleen had also mature morphology with developed platelet territories. However, by contrast with the heavy electron dense granules found in the cytoplasm of HC MK, the cytoplasm of MK from both BM (56%) and spleen (32%) contained granules with light electron density, an indication of reduced protein content. MK from MF BM and spleen consistently expressed distinctive ultrastructural abnormalities: 100% of BM MK presented dilated nuclear membranes with abnormally large nuclear pores which in 33% of the cases were associated with thickening of the plasma membrane with extrusion of pseudopods and presence of glycogen droplets. By contrast, these abnormalities were not presented by spleen MK, 49% of which presented instead mitochondria with reduced crests. These results suggest that MK from BM and spleen express distinctive metabolic abnormalities: those from BM express insufficient lipid metabolism impairing membrane biosynthesis while those from spleen have impaired aerobic metabolism. As predicted by the impaired RSP14 signature reported in MF (Gilles, JCI 2017;127:1316), the cytoplasm of MK from MF BM contained reduced number of ribosomes (455±125 vs 1059±41 in MF vs HC, p=0.004) but the percent of ribosomes engaging with the rough endoplasmic reticulum (RER) was the same in both cases (41±2 vs 46±10%, p=0.80). The number of ribosomes was lower also in MK from spleen (157±85, p=0.0003 ) but in addition, MK from the spleen were poorly engaged with the RER (33±22%, p=0.008). These results indicate that in addition to low number of ribosomes resulting in defective mRNA translation, MK from spleen express ribosomes that engage poorly with RER leading to defective folding check and post-translational modification of nascent proteins. These results predict that partially different mechanisms drive the alterations of platelet territories observed in MK from BM and spleen in MF: defective membrane biosynthesis drives the paucity of the granules and the retarded maturation of the platelet territories observed in MK from BM. Insufficient proteins synthesis drives the poor protein content of granules found in MK both from BM and spleen but in addition the function of the proteins in the granules of MK from spleen may be impaired by improper protein folding check/post-translational modification. These results suggests lipid metabolism and RER functions as two potential novel therapeutic targets to restore MK function in MF. Vannucchi: Incyte: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Celgene: Membership on an entity's Board of Directors or advisory committees; Italfarmaco: Membership on an entity's Board of Directors or advisory committees; CTI BioPharma: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau.