Aplidin Improves Megakaryocytopoiesis and Halts Neo-Angiogenesis in the Gata1low Murine Model of Myelofibrosis
Authors: Verrucci Maria , Pancrazzi Alessandro , Aracil Miguel , Martelli Fabrizio , Guglielmelli Paola , Paz Maria Fe , Zingariello Maria , D’Amore Emanuela , Jimeno Jose , Migliaccio Anna Rita , Vannucchi Alessandro M. 
Published: April 2020

Abstract
Aplidin (plitidepsin) is a marine-derived depsipeptide currently in phase II clinical trials with evidence of activity in heavily pretreated multiple myeloma, T cell lymphoma, renal cell carcinoma and melanoma. Of importance, Aplidin lacks bone marrow toxicity. The molecular mechanisms of the anti-tumor activity of the drug have been mainly ascribed to induction of apoptosis of tumor cells through the extrinsic and intrinsic pathways, and inhibition of angiogenesis through an impact on the VEGF-VEGF-R loop. Primary myelofibrosis (MF) is a complex myeloproliferative disorder associated with abnormalities of megakaryocytic (MK) proliferation and maturation which result in increased release of several cytokines in the bone marrow microenvironment. VEGF and TGF-beta, in particular, are considered responsible for the profound abnormalities of the bone marrow stroma which include increased angiogenesis and fibrosis. Mice carrying the hypomorphic Gata1low mutation express MK abnormalities similar to those observed in MF patients and develop myelofibrosis with age, a syndrome that includes an increased angiogenesis process with striking similarities with that observed in human MF. While the molecular mechanism underlying MF in human and GATA1low mice are different, the cytokine-mediated events leading to the stromal changes are probably very similar. The aim of the present study was a pre-clinical assessment of the use of Aplidin as targeted therapeutic agent to halt development of myelofibrosis in Gata1low mice. Gata1low mice at an “early MF phase” (9-months age; n= 18) received Aplidin ip at 60 mg/kg/daily/9 days for two cycles 38 days a part; a second group of “late MF phase” mice (aged &gt;12 months; n= 18) received only one course of treatment. Equivalent numbers of age-matched Gata1low mice received saline only and were used as control. A significant increase of platelet count from 150±60 to 460±50×109/L (P&lt;.05) was observed in “early MF” Gata1low mice at day 16 after the first treatment. The increase was maintained after the second course. An increase in platelet count was also observed in “late MF” Gata1low mice. Moreover, an increase of hematocrit, although not statistically significant, was observed on day 16 (from 37% to 44%) and maintained a month later. There was a normalization of total femur cellularity, which is typically reduced in diseased mice, at the end of Aplidin treatment. It was particularly pronounced in the “early MF” mice: total cells/femur increased from a median of 6×106 in untreated Gata1low mice to 15×106 at 53 days after Aplidin, as compared to a median value of 16×106 (P&lt;0.01) in wild-type mice. Similar effects on platelet count and femur cellularity were observed in a third treatment where “early MF” mice (n=36) received Aplidin at 100 mg/kg/daily for 5 days 21 days apart for a total of four courses. In this case, there was a trend, though not statistically significant, towards less Mk number and reticulin fibers/mm2 of bone marrow area, while microvessel density, measured using immunostaining for CD34, was significantly reduced after the fourth cycle from 8±1.5 to 2.6±1.6 pixel arbitrary units (P&lt;.01). mRNA levels for both TGF-beta and VEGF, measured by quantitative PCR, were significantly reduced in the bone marrow of Aplid-intreated mice (P &lt;.01 for both). These data indicate that treatment with Aplidin ameliorated at least some of the traits of the myelofibrotic phenotype expressed by Gata1low mice. In particular, the observed inhibition of TGF-beta and VEGF expression, associated with reduced microvessel density, would suggest a possible activity of the drug in human MF where levels of these two cytokines are abnormally increased.