ALBERT

Description: Janus kinases (JAK) comprise a small family of cytoplasmic protein tyrosine kinases, which play an important role in the initiation of cytokine-triggered signaling events via signal transducer and activator of transcription (STAT) proteins. The recent reports of an activating somatic mutation in codon 617 of the JAK2 gene (JAK2V617F mutation) in patients with myeloproliferative disorders (MPDs), has opened new avenues for the development of targeted therapies for these malignancies and clinical trials with JAK2 inhibitors are underway. We report here the activity of Atiprimod (N,N-diethyl-8-dipropyl-2-azaspiro[4,5]decane-2-propanamine), a novel compound with anti-inflammatory properties, in retrovirus-transduced JAK2V617F mutant-expressing murine FDCP-EpoR cells, set-2 cells, and blood cells from patients with polycythemia vera (PV). We compared the growth inhibitory effect of Atiprimod against two mouse FDCP cell lines transfected with erythropoietin receptor (Epo-R), and either wild-type JAK2WT or mutant JAK2V617F, and human megakaryoblastic leukemia cells with mutated JAK2V617F (set-2 cells). The growth inhibitory effect was assessed using 3-days MTS assay. Atiprimod was more potent against FDCP cells carrying mutant JAK2V617F cells (IC50 0.42 μM) and set-2 cells (IC50 0.53 μM) than FDCP wildtype JAK2WT cells (IC50 0.69 μM). Atiprimod inhibited the phosphorylation of JAK2 and downstream STAT3, STAT5, and AKT proteins in a dose- and time-dependent manner. It induced apoptosis, as evidenced by increase in mitochondrial membrane potential, caspase3 activity, and cleavage of PARP protein. The anti-proliferative effect on expanded PV patient progenitor’s cells was paralleled by a decrease in JAK2V617F mutant allele frequency in BFU-E or CFU-GM clones in clonogenic assay. However, co-culturing of JAK2V617F mutant cells with three different bone marrow stromal cell lines (Hs5, ABM-MSC, NK-Tert) either directly (cell on cell) or indirectly (separated by 0.4 μm micropore membranes) for 48 hours resulted in a significant protection of mutant cells from the effect of Atiprimod. Co-culturing of bone marrow stromal cells prevented Atiprimod (0.4 and 0.8 μM) induced apoptosis, and reversed the inhibition of phosphorylation of STAT proteins. Our results suggest that cytokines secreted by stromal cells might play an important role in protecting the hematopoietic cells from a JAK2 inhibitor. Further dissection of the nature of interactions between JAK2V617F mutant cells and marrow stromal cells may lead to new therapeutic avenues for patients with MPD.

Description: Genes Involved in Maintaining the Bone Marrow Stroma Are Dysregulated in Patients with Myelofibrosis: Lenalidomide Treatment Upregulates SOCS3 Background: Primary myelofibrosis (PMF) is the most aggressive of the myeloproliferative neoplasms (MPNs), a group of genetically and clinically heterogenous yet-related diseases with a functionally disturbed hematopoietic niche. Egress of these cells from the BM in patients with PMF, suggests a change in the crosstalk between hematopoietic stem cells (HSCs) and the BM microenvironment (particularly BM stromal cells). Lenalidomide, an immunomodulatory agent, which modulates inflammatory cytokine secretion, angiogenesis and the expression of adhesion molecules, likely has effects on the BM microenvironment. Methods: We studied the expression of a set of genes involved in the organization of the hematopoietic niche in peripheral blood and bone marrow (BM) mononuclear cell (MNC) samples from 32 patients with PMF who participated in a phase 2 trial of lenalidomide plus prednisone (Quintas-Cardama et al., J Clin Oncol 2009;27:4760-766). Sequential samples taken at 3, 6, 9, 12 and 〉14 months after starting lenalidomide therapy were also analyzed. Baseline BM samples were available for 9 patients and baseline PB samples available for 12 patients and used for our analysis. Quantitative reverse transcriptase-polymerase chain reaction was performed to measure the expression levels of SPARC, COX-2, CXCR4, Pax5C-terminus, SOCS3 and HIF-1a transcripts. β-actin was used as an internal control. In addition, we compared the gene expression levels at baseline with healthy control samples. Results: We found that at baseline (before treatment) COX-2 was upregulated, while CXCR4, Pax5 C-terminus, SOCS3 and HIF-1a were downregulated when compared with expression in healthy BM MNCs. Expression of SPARC, K-RAS,FOS and SOCS2 were not significantly different. In addition, there were no significant differences in relative gene expression between BM MNC and PB samples. We found no correlation between gene expression and JAK2 mutational status or cytogenetic abnormalities. Treatment with LP significantly increased the expression of SOCS3, suggesting that some of the clinical effects of lenalidomide (i.e., reduction in splenomegaly) may be due to a SOCS3-mediated reduction in JAK signaling. Conclusions: Upregulation of COX-2 and downregulation of CXCR4, Pax5C, SOCS3, and HIF-1a may reflect disruptions in the interactions between cells in the BM microenvironment in MF. Increases in SOCS3 expression upon treatment with lenalidomide may lead to reduced JAK-STAT signaling, which may account for some of its clinical effects in myelofibrosis. Figure 1. Changes in SOCS3 gene expression with time on lenalidomide plus prednisone treatment. Expression of SOCS3 increases significantly with time on treatment (P = 0.0002 using one-way analysis of variance). Mean expression at 9, 12 and 〉 14 months is significantly higher than at baseline, as assessed by Dunnett's multiple comparisons test. Data shown represent inverse relative expression. P 〈 0.05 was considered statistically significant. Figure 1. Changes in SOCS3 gene expression with time on lenalidomide plus prednisone treatment. Expression of SOCS3 increases significantly with time on treatment (P = 0.0002 using one-way analysis of variance). Mean expression at 9, 12 and 〉 14 months is significantly higher than at baseline, as assessed by Dunnett's multiple comparisons test. Data shown represent inverse relative expression. P 〈 0.05 was considered statistically significant. Disclosures No relevant conflicts of interest to declare.