Ruxolitinib Does Not Affect Bone Marrow Stromal Cell (BMSC) Clonogenicity and Growth but Interferes with JAK Signaling Under Inflammatory Conditions - Implications for Fibrosis in Myeloproliferative Diseases?

The JAK2 V617F gain-of-function mutation is found in hematopoietic cells of nearly all patients with myeloproliferative diseases (MPDs). Ruxolitinib (Ruxo), a potent oral JAK1/JAK2 inhibitor, attenuates cytokine signaling by inhibition of JAK signaling, leading to anti-proliferative and pro-apoptotic effects. In patients with myelofibrosis (MF), effects of Ruxolitinib on splenomegaly, symptom improvement and possibly survival were successfully demonstrated in the two COMFORT studies. Furthermore, resolution of bone marrow fibrosis after long-term treatment with Ruxo was recently reported (Wilkins et al., Haematologica 2013). However, bone marrow stromal cells (BMSC), which contribute to fibrosis, do not harbor the JAK2 mutation or other chromosomal abnormalities that can be found in hematopoietic stem/progenitor cells. Therefore, the current study aimed to investigate possible effects of Ruxo on these non-hematopoietic cells. Bone marrow mononuclear cells were harvested from consenting donors and Ruxo effects on stroma progenitor cells were investigated using the standard CFU-F (colony-forming unit, fibroblast) assay. Our data showed that CFU-F numbers were not decreased by Ruxo at doses ranging from 0.2 to 10 μM (n = 9, p = 0.41, one-way ANOVA), but were even found to be increased at the 5 μΜ dose as indicated by a Ruxo/DMSO ratio of over one (p = 0.04, multiple comparisons test, control-vs-5 μΜ) (Figure 1). In contrast, statistically significant differences were observed in CFU-F colony size with a tendency to decreased sizes with higher doses of Ruxo (p < 0.0001, one-way ANOVA) (Figure 1). Next, we tested if Ruxo affected standard culture-derived bone marrow stromal cell (BMSC) growth, both, in short-term (6 h, 12 h, 24 h and 48 h, n = 3) and in long-term exposure experiments (up to a total of 21 days, n = 3). Neither short-term nor long-term exposure with Ruxo at 0.2 μΜ, 0.5 μΜ, 1 μΜ, 5 μΜ and 10 μΜ caused significant changes of BMSC numbers when compared to their corresponding DMSO controls (p > 0.05, two-way ANOVA), however, a trend to lower BMSC counts with higher Ruxo doses was observed. BMSC were reduced by maximum 25.3±5.7 % (mean ± SD) after 24-hour exposure with 10 μΜ Ruxo. After 21 days drug exposure, ratios of Ruxo-treated BMSC relative to their corresponding DMSO controls were 1.1±0.2, 1.1±0.3, 0.9±0.2, 0.7±0.4%, and 0.6±0.2 for 0.2 μΜ, 0.5 μΜ, 1 μΜ, 5 μΜ and 10 μΜ Ruxo, respectively. These data indicated that Ruxo might have a cytotoxic effect on BMSC, however, only at very high concentrations. We therefore went on to study Ruxo effects on JAK signaling in BMSC after stimulation with IL-6, in order to mimic the inflammatory environment in MPDs. Western blot assays revealed that IL-6 treatment (100 ng/ml) induced pJAK2 in BMSC, which was not detectable in unstimulated BMSC. Furthermore, exposure of IL-6 stimulated BMSC to Ruxo (1 μΜ) diminished pSTAT3 and reduced downstream STAT targets such as pAKT and pMAPK. Furthermore, preliminary results on the cytokine expression profile of supernatants from IL-6-activated BMSC treated with Ruxo showed significant differences compared to controls. Specifically, IL-23 and CCL4 were elevated in Ruxo-treated/IL-6 activated BMSC whereas MCP-1 was strongly reduced compared to the non-Ruxo treated controls. Taken together, these data show that clinically-relevant doses of Ruxo did not affect the clonogenic potential and proliferation of primary marrow stroma cells, indicating that Ruxo most likely has no or little direct effect on the fibrosis-causing cells in MPD. However, Ruxo considerably affects JAK-STAT signaling in activated BMSCs, leading to an altered cytokine expression profile which potentially contributes to the amelioration of fibrosis, and, accordingly, ongoing experiments address this question. Figure 1. Effects of increasing doses of Ruxo on a) clonogenic bone marrow stroma cells expressed as ratio of the number of Ruxo-treated CFU-F and corresponding DMSO controls, and b) colony size (*p < 0.05, **p < 0.01, ***p < 0.001) Figure 1. Effects of increasing doses of Ruxo on a) clonogenic bone marrow stroma cells expressed as ratio of the number of Ruxo-treated CFU-F and corresponding DMSO controls, and b) colony size (*p < 0.05, **p < 0.01, ***p < 0.001) Disclosures No relevant conflicts of interest to declare.

Aberrant Expression Of miRNA and mRNA Of Cell Cycle and Adhesion-Related Genes In Bone Marrow Stroma Cells Derived From Patients With Multiple Myeloma

Multiple myeloma (MM) is a B-cell malignancy characterized by accumulation of malignant plasma cells (PC) within the bone marrow. The bone marrow microenvironment such as bone marrow stroma cells (BMSC) supports MM disease progression, resistance to chemotherapy, protects the tumor cells against apoptosis and causes osteolytic bone disease and angiogenesis. The aim of this study was to identify constitutive genetic alterations in BMSC derived from patients with MM (MM-BMSC) in comparison to BMSC from healthy donors. For BMSC selection, mononuclear cells were isolated from fresh bone marrow aspirates and were further expanded in cell culture. We studied 25 MM patients and 5 healthy donors. Senescence status was determined in passage 1 of cell cultures. MM-BSMC displayed a considerably higher percentage of senescence cells (p<0,05). We investigated the expression of cell cycle and adhesion-associated genes (CCNE1, CCND1, CDKN1B, VCAM, ICAM, IKK-alpha) in BMSC (passage 4) using SYBR-Green Real-Time PCR and relative quantification by linear regression. A downregulation of CCNE1 (p=0,05), CDKN1B (p=0,29), and an upregulation of CCND1 (p=0,05), VCAM-1 (p=0,33), ICAM-1 (p=0,33), and IKK-alpha (p=0,05) was demonstrated. Furthermore, the expression profile of miRNAs, targeting the analyzed mRNA genes or correlating with senescence, was studied (miR-16, miR-221, miR-126, miR-223, miR-485-5p and miR-519d). For miRNA detection treatment with Poly(A)-Polymerase and cDNA-Synthesis with a Poly(T)VN-Adaptor primer were carried out following an amplification with an universal reverse primer corresponding to the adaptor sequence and a miRNA-specific primer. miR-16, miR-223, miR-485-5p and miR-519d were significantly upregulated, (p=0,02; p=0,004; p=0,02; and p=0,002, respectively), whereas miR-221 and miR-126 showed no considerable differences to BMSC obtained from healthy donors. Next we investigated incubation of immunmodulatory drug Lenalidomid in BMSC cultures. Cells were treated with 10 µM Lenalidomid over 72 hours and expression was normalized to a 0,01 % DMSO control. Significant difference in gene expression were visible for ICAM-1 (p=0,01). For CDKN1B (p=0,16) and VCAM-1 (p=0,12) we demonstrated changes in gene expression. Our results indicate aberrant expression of cell cycle and adhesion-related genes, such as CCNE1, CCND1 and CDKN1B VCAM-1, ICAM-1 and IKK-alpha in MM-BMSC as compared with healthy donors. Furthermore, we found significant upregulation of miR-16, miR-223, miR-485-5p and miR-519d. Further investigation are needed to determine molecular mechanisms in MM-BMSC and PC interaction that lead to constitutive changes in BMSC characteristics and gene expression patterns. Disclosures: No relevant conflicts of interest to declare.

Hemopoiesis Regulated Negatively In Aged Monkeys

Biological aging is associated with a progressive loss of regulation of cellular, tissue and organ interaction, ultimately resulting in senescence. In humans, accelerated marrow adipogenesis has been associated with aging and several chronic conditions, including diabetes mellitus and osteoporosis. Studies of the hemopoietic system using nonhuman primates have provided important information for understanding the mechanism of human hemopoiesis. Excellent reconstitution of donor hemopoietic cells in a collagen gel group has been observed in the long term in mice. We therefore investigated whether the method would be helpful for reconstituting hemopoiesis in the fatty tibias of monkeys. Nine- to 11-year-old monkeys were used for this study to examine if hemopoiesis could be restored in the fatty marrow of old monkeys. Bone marrow cells were collected from the humerus using the perfusion method, mixed with cultured bone marrow stroma cells in collagen gel, and then injected into the tibia of the same monkey. Clot sections were made from the tibias 1, 2 and 6 weeks after the bone marrow transplantation. However, no bone marrow cells were observed at any of the time points. These results suggested that adipocytes negatively regulate bone marrow reconstitution. Disclosures: No relevant conflicts of interest to declare.