Murine Model Of Myelofibrosis Research Articles

CXCL8/CXCR2 signaling mediates bone marrow fibrosis and is a therapeutic target in myelofibrosis

AbstractProinflammatory signaling is a hallmark feature of human cancer, including in myeloproliferative neoplasms (MPNs), most notably myelofibrosis (MF). Dysregulated inflammatory signaling contributes to fibrotic progression in MF; however, the individual cytokine mediators elicited by malignant MPN cells to promote collagen-producing fibrosis and disease evolution are yet to be fully elucidated. Previously, we identified a critical role for combined constitutive JAK/STAT and aberrant NF-κB proinflammatory signaling in MF development. Using single-cell transcriptional and cytokine-secretion studies of primary cells from patients with MF and the human MPLW515L (hMPLW515L) murine model of MF, we extend our previous work and delineate the role of CXCL8/CXCR2 signaling in MF pathogenesis and bone marrow fibrosis progression. Hematopoietic stem/progenitor cells from patients with MF are enriched for a CXCL8/CXCR2 gene signature and display enhanced proliferation and fitness in response to an exogenous CXCL8 ligand in vitro. Genetic deletion of Cxcr2 in the hMPLW515L-adoptive transfer model abrogates fibrosis and extends overall survival, and pharmacologic inhibition of the CXCR1/2 pathway improves hematologic parameters, attenuates bone marrow fibrosis, and synergizes with JAK inhibitor therapy. Our mechanistic insights provide a rationale for therapeutic targeting of the CXCL8/CXCR2 pathway among patients with MF.

Improved Repeatability of Mouse Tibia Volume Segmentation in Murine Myelofibrosis Model Using Deep Learning.

A murine model of myelofibrosis in tibia was used in a co-clinical trial to evaluate segmentation methods for application of image-based biomarkers to assess disease status. The dataset (32 mice with 157 3D MRI scans including 49 test-retest pairs scanned on consecutive days) was split into approximately 70% training, 10% validation, and 20% test subsets. Two expert annotators (EA1 and EA2) performed manual segmentations of the mouse tibia (EA1: all data; EA2: test and validation). Attention U-net (A-U-net) model performance was assessed for accuracy with respect to EA1 reference using the average Jaccard index (AJI), volume intersection ratio (AVI), volume error (AVE), and Hausdorff distance (AHD) for four training scenarios: full training, two half-splits, and a single-mouse subsets. The repeatability of computer versus expert segmentations for tibia volume of test-retest pairs was assessed by within-subject coefficient of variance (%wCV). A-U-net models trained on full and half-split training sets achieved similar average accuracy (with respect to EA1 annotations) for test set: AJI = 83-84%, AVI = 89-90%, AVE = 2-3%, and AHD = 0.5 mm-0.7 mm, exceeding EA2 accuracy: AJ = 81%, AVI = 83%, AVE = 14%, and AHD = 0.3 mm. The A-U-net model repeatability wCV [95% CI]: 3 [2, 5]% was notably better than that of expert annotators EA1: 5 [4, 9]% and EA2: 8 [6, 13]%. The developed deep learning model effectively automates murine bone marrow segmentation with accuracy comparable to human annotators and substantially improved repeatability.

TGF-β1 protein trap AVID200 beneficially affects hematopoiesis and bone marrow fibrosis in myelofibrosis

Myelofibrosis (MF) is a progressive chronic myeloproliferative neoplasm characterized by hyperactivation of JAK/STAT signaling and dysregulation of the transcription factor GATA1 in megakaryocytes (MKs). TGF-β plays a pivotal role in the pathobiology of MF by promoting BM fibrosis and collagen deposition and by enhancing the dormancy of normal hematopoietic stem cells (HSCs). In this study, we show that MF-MKs elaborated significantly greater levels of TGF-β1 than TGF-β2 and TGF-β3 to a varying degree, and we evaluated the ability of AVID200, a potent TGF-β1/TGF-β3 protein trap, to block the excessive TGF-β signaling. Treatment of human mesenchymal stromal cells with AVID200 significantly reduced their proliferation, decreased phosphorylation of SMAD2, and interfered with the ability of TGF-β1 to induce collagen expression. Moreover, treatment of MF mononuclear cells with AVID200 led to increased numbers of progenitor cells (PCs) with WT JAK2 rather than mutated JAK2V617F. This effect of AVID200 on MF PCs was attributed to its ability to block TGF-β1–induced p57Kip2 expression and SMAD2 activation, thereby allowing normal rather than MF PCs to preferentially proliferate and form hematopoietic colonies. To assess the in vivo effects of AVID200, Gata1lo mice, a murine model of MF, were treated with AVID200, resulting in the reduction in BM fibrosis and an increase in BM cellularity. AVID200 treatment also increased the frequency and numbers of murine progenitor cells as well as short-term and long-term HSCs. Collectively, these data provide the rationale for TGF-β1 blockade, with AVID200 as a therapeutic strategy for patients with MF.

Phase 1b Results of a Study to Assess the Efficacy and Safety of Vismodegib in Combination with Ruxolitinib in Patients with Intermediate- or High-Risk Myelofibrosis

Background: The JAK inhibitor (JAKi) ruxolitinib is approved for treatment of myelofibrosis (MF). Treatment for 5 years or more may be necessary to improve or stabilize bone marrow fibrosis, and a fraction of patients do not respond to ruxolitinib. The Hedgehog signaling pathway is involved in primitive and definitive bone marrow hematopoiesis, and maintenance of hematopoietic precursors, suggesting that addition of a Hedgehog pathway inhibitor (HPI) to ruxolitinib might improve responses. In a murine model of MF, a combination of a JAKi and HPI reduced fibrosis in the bone marrow and spleen and decreased splenomegaly. Vismodegib is a HPI approved for treatment of locally advanced and metastatic basal cell carcinoma. The MYLIE study aimed to assess the safety and efficacy of combined ruxolitinib and vismodegib for the treatment of MF. The pharmacokinetics (PK) of vismodegib were characterized in patients with intermediate or high-risk MF.Methods: In this phase 1b study, 10 patients with intermediate- or high-risk MF (per Dynamic International Prognostic Scoring System) received open-label vismodegib (150 mg/day orally) and ruxolitinib (15 or 20 mg orally twice daily, depending on baseline platelet count) for up to 48 weeks, or until withdrawal or discontinuation. PK samples were collected throughout the study. PK sampling was designed to characterize the PK of total and unbound vismodegib in this study for comparison with PK data collected in other patient populations. Efficacy outcomes assessed for phase 1b included spleen response (≥35% reduction in volume by computed tomography or magnetic resonance imaging) per central and investigator assessment, improvement in bone marrow fibrosis per central and investigator assessment (using European consensus grading system), symptom response (≥50% reduction in Myeloproliferative Neoplasm Symptom Assessment Form Total Symptom Score [MPN-SAF TSS]) and anemia response (per International Working Group for Myeloproliferative Neoplasms Research and Treatment [IWG-MRT] revised response criteria). We report results from the planned analysis of the phase 1b study for efficacy and safety at week 24.Results: Six patients with primary MF, 1 with post-polycythemia vera MF and 3 with post-essential thrombocythemia MF were treated with vismodegib and ruxolitinib for a median of 294.0 days. As of the interim analysis data cutoff (March 29, 2017), 4 patients had completed 48 weeks of study treatment with ruxolitinib and vismodegib, 5 patients were still receiving treatment, and 1 had discontinued treatment because of a vismodegib-related adverse event (AE) (dysgeusia). The most common AEs were muscle spasm (100% of patients), dysgeusia (50%), alopecia (50%), thrombocytopenia (50%), and nausea (40%). All were grade 1/2, except for 2 instances of grade 3 thrombocytopenia in 2 patients. Three patients experienced serious AEs: infection (3 patients), lung infiltration (1 patient), and vomiting (1 patient); none were considered by the investigators to be related to vismodegib. Because of AEs, 7 patients modified or interrupted their ruxolitinib dose, and 4 patients interrupted their vismodegib dose. At week 24, three patients achieved a ≥35% reduction in spleen volume (including the patient who discontinued early because the assessment occurred within the week 24 visit window) by central review (a spleen response occurred in 1 patient per the investigator assessment). According to the central reviews, no patients had improvement in bone marrow fibrosis (1 patient had improvement in bone marrow fibrosis per investigator assessment). Five patients experienced symptom response, and no patients experienced an anemia response. A further central review of bone marrow biopsy specimens from 5 patients at week 48 continued to show no improvement in fibrosis.Conclusions: There was no clear benefit of the addition of vismodegib to ruxolitinib in any of the phase 1b efficacy outcome measures assessed. Given the lack of evidence of a disease-modifying effect with addition of vismodegib to ruxolitinib, further evaluation of this combination will not be pursued.Source of funding: F. Hoffmann-La Roche DisclosuresKoschmieder:Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel support, Research Funding; Roche: Other: Clinical Trial participation; BMS: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel support, Research Funding; Incyte: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel support. Verstovsek:Incyte: Research Funding; NS Pharma: Research Funding; Blueprint Medicines Corp: Research Funding; Blueprint Medicines Corp: Research Funding; Galena BioPharma: Research Funding; Celgene: Research Funding; NS Pharma: Research Funding; Seattle Genetics: Research Funding; Genentech: Research Funding; Lilly Oncology: Research Funding; CTI BioPharma Corp: Research Funding; Astrazeneca: Research Funding; Promedior: Research Funding; Pfizer: Research Funding; Astrazeneca: Research Funding; Genentech: Research Funding; Gilead: Research Funding; Seattle Genetics: Research Funding; Bristol Myers Squibb: Research Funding; Bristol Myers Squibb: Research Funding; Gilead: Research Funding; Promedior: Research Funding; Celgene: Research Funding; CTI BioPharma Corp: Research Funding; Galena BioPharma: Research Funding; Roche: Research Funding; Pfizer: Research Funding; Lilly Oncology: Research Funding; Roche: Research Funding. Hooper:Roche Products Ltd: Employment. Tandon:F. Hoffmann-La Roche: Employment. Dimier:F. Hoffmann-La Roche: Employment, Other: Stock.

Abstract 1874: The PIM kinase inhibitor TP-3654 demonstrates efficacy in a murine model of myelofibrosis

Abstract Myelofibrosis (MF) is the deadliest form of myeloproliferative neoplasms (MPN). The JAK2V617F mutation has been found in ~50% patients with MF. Ruxolitinib, a JAK1/JAK2 inhibitor, has been approved for treatment of MF. However, Ruxolitinib treatment has failed to induce disease remission or cure fibrosis. Therefore, there is a need to develop novel targeted therapies for MF. We found that PIM1 expression is significantly increased in mouse and human MPN/MF hematopoietic progenitor cells. Moreover, PIM1 knockdown markedly inhibited proliferation in JAK2V617F-expressing cells but not in wild-type JAK2-expressing cells. In this study, we investigated the efficacy of the second-generation pan-PIM kinase inhibitor, TP-3654, in hematopoietic cells expressing JAK2V617F and in a murine model of MF. We found that TP-3654 (0.5-1.0 μM) treatment significantly reduced proliferation of murine Ba/F3-EpoR cells expressing JAK2V617F or human JAK2V617F-positive HEL and UKE-1 cells. TP-3654 Treatment also resulted in marked apoptosis in Ba/F3-EpoR-JAK2V617F, HEL and UKE-1 cells but not in wild-type JAK2 expressing Ba/F3-EpoR cells. Additionally, TP-3654 and Ruxolitinib synergistically induced apoptosis in JAK2V617F-expressing hematopoietic cells. Furthermore, TP-3654 significantly inhibited MPN/MF CD34+ hematopoietic progenitor colony growth. We previously reported the generation of JAK2V617F knock-in mice. Whereas heterozygous JAK2V617F knock-in mice exhibit a polycythemia vera phenotype, mice expressing homozygous JAK2V617F rapidly develop high-grade MF. We have utilized the homozygous JAK2V617F mice to test the in vivo efficacy of TP-3654 alone or in combination with Ruxolitinib against MF. Treatment of TP-3654 alone significantly reduced the increase in white blood cell (WBC) and neutrophil counts as well as spleen size in mice expressing homozygous JAK2V617F compared with vehicle treatment. Combined treatment of TP-3654 and Ruxolitinib almost completely normalized the WBC and neutrophil counts and the spleen size in homozygous JAK2V617F mice. Histopathologic analysis revealed marked reduction in fibrosis in the bone marrow and spleens of TP-3654-treated mice whereas Ruxolitinib treatment did not significantly reduce fibrosis. Combined TP-3654 and Ruxolitinib treatment almost completely eliminated fibrosis in the bone marrow and spleens of homozygous JAK2V617F mice. TP-3654 treatment was well tolerated and did not cause any significant toxicity in wild type mice. RNA-sequencing analysis on purified LSK (Lin-Sca-1+c-kit+) cells from these drug-treated mice revealed that the genes related to TNFα and WNT signaling pathways were significantly down-regulated by treatment of TP-3654 alone or TP-3654 and Ruxolitinib combination compared with vehicle. Overall, our results suggest that TP-3654 alone or in combination with Ruxolitinib may have therapeutic potential for treatment of MF. Citation Format: Avik Dutta, Dipmoy Nath, Yue Yang, Golam Mohi. The PIM kinase inhibitor TP-3654 demonstrates efficacy in a murine model of myelofibrosis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1874.

TGFb1 Antagonist Inhibits Fibrosis in a Murine Model of Myelofibrosis

Introduction: Myelofibrosis (MF) is a clonal stem cell disorder that originates from acquired mutations in the hematopoietic stem cells leading to abnormal kinase signaling, cell proliferation, cytokine expression, and splenomegaly and ultimately bone marrow (BM) fibrosis. Primary myelofibrosis (PMF), post-polycythemia vera (PV) MF and post-essential thrombocythemia MF are categorized under MF with overlapping disease phenotypes including progression to BM fibrosis. A genetic mutation in Janus kinase 2 (V617F) was identified as causative in ~95% PV, and ~50% of ET and PMF patients. Currently, treatment of MF patients with a JAK2 inhibitor offers symptomatic benefit, but does not alter the natural history of the disease or improve BM fibrosis. It is known that TGFβ1 is a critical regulator of fibrosis in many disease states. Elevated TGFβ1 levels were reported to be important for fibrosis in patients with MF. We hypothesize that inhibition of TGFβ1 signaling may prevent fibrosis and help reduce secondary morbidities associated with disease in MF patients. Therefore, we evaluated this hypothesis using a TGFβ1 antagonist in a murine model of MF. Methods: Transgenic JAK2 (V617F) mutant mice (MF model) and age-matched wild-type controls were used in the studies. Mice were dosed twice weekly with TGFβ1 antagonist (10 mg/kg). Complete blood counts (CBC), serum TGFβ1, bone metabolism and inflammatory cytokines levels were determined at different ages (2-12 months) during disease progression. Bone marrow and spleen cells were analyzed for different cell lineages by flow cytometry. Tissue sections were stained with H&E and reticulin to determine cellularity or degree of fibrosis respectively. Results: To understand the onset and progression of MF disease in JAK2 (V617F) mice, we initially analyzed the CBC and degree of fibrosis at various ages (2, 3, 4, 5, 8, 10 and 12 months) and compared the data with wild-type mice. These data were then correlated with the levels of TGFβ1 and other cytokines. As expected, red blood cells (RBC) and platelets were elevated in JAK2 mutant mice at all ages compared to wild-type mice, although a trend towards a progressive increase was observed between 2 to 5 months followed by a decrease from 8 to 14 months. Bone marrow fibrosis was detected starting at 5 months and worsened with age. JAK2 mutant mice displayed splenomegaly that increased as the disease progressed. Interestingly, serum levels of TGFβ1, TGFβ3 and bone metabolism cytokines (OPG, OPN, aFGF and Trance) displayed an increase at earlier ages (2-5 months) compared to the latter ages, a trend similar to RBC levels. These levels peaked during the initiation of fibrosis at 5 months. In contrast, inflammatory cytokines (such as IL6, IL-1β, and TNFα) were elevated at later ages consistent with disease progression. We initiated treatment with TGFβ1 antagonist in JAK2 (V617F) mice (N=8/treatment group) at 4 months of age, the age corresponding to elevated serum TGFβ1 levels and prior to the onset of fibrosis (at 5 months of age). Following 6 months of treatment, vehicle (VEH) treated JAK2 mutant mice displayed elevated RBC (+37.1%, P<0.001), platelets (+74.5%, P<0.001) and spleen weights (+9.5 fold, P<0.001) compared to wild-type mice. BM and spleen sections from VEH treated JAK2 mutant mice revealed severe fibrosis. TGFβ1 antagonist treatment of JAK2 mice displayed moderate effect on RBC (-8.4%, N.S) without any effect on platelet counts compared to VEH treatment. Flow-cytometry identified a reduced proportion of Ter119+ erythroid precursors in BM and spleen (-15%, P<0.05) and no change in CD41+ megakaryocytes. TGFβ1 antagonist treated mice displayed reduced spleen weights (-29%, P<0.01), and marked reduction in fibrosis in bone marrow (Figure) and spleen sections compared to VEH. Consistent with the reduction in fibrosis, TGFβ1 antagonist treated JAK2 mice displayed reduced IL-6 levels (-48.9%, P<0.05) compared to VEH treatment. Conclusion: Together, these data demonstrated that TGFβ1 levels were correlated with bone marrow fibrosis in a murine model of MF disease, and its inhibition using TGFβ antagonist reduces fibrosis, splenomegaly and inflammation in this murine model of myelofibrosis. [Display omitted] DisclosuresSuragani:Acceleron Pharma Inc: Employment, Equity Ownership, Patents & Royalties: No royalties. Martinez:Acceleron Pharma: Employment. Cawley:Acceleron Pharma Inc: Employment. Li:Acceleron Pharma: Employment, Equity Ownership. Pearsall:Acceleron Pharma Inc: Employment, Equity Ownership, Patents & Royalties. Kumar:Acceleron Pharma: Employment, Equity Ownership, Patents & Royalties.

Phase 1b/2 Study of the Efficacy and Safety of Sonidegib (LDE225) in Combination with Ruxolitinib (INC424) in Patients with Myelofibrosis

Phase 1b/2 Study of the Efficacy and Safety of Sonidegib (LDE225) in Combination with Ruxolitinib (INC424) in Patients with Myelofibrosis

Therapies Targeting the MAPK Pathway Improve Bone Marrow (BM) Fibrosis Induced By JAK2V617F

In primary myelofibrosis patients, somatic mutations such as JAK2V617F(JAKVF) and MPLW515 that activate JAK-STAT signaling are often seen. Small-molecule JAK2 inhibitors are effective for organomegaly and constitutional symptoms, but the drugs have little effect on BM fibrosis. To clarify the mechanism by which MPN cells with JAK2 mutations cause BM fibrosis, we compared the gene expression patterns of Lin−Sca1+ BM cells in JAK2VF transgenic mice (JAK2VF-TG), which develop myelofibrosis (MF), with that in WT mice. We found that TGFb1 and HOXB4, the target genes of transcription factor USF1 were highly expressed. TGFβ1, which is secreted by hematopoietic cells, is essential for fibrotic development in a murine model of MF (Chagraoui et al. Blood 2002), and increased expression of HOXB4 enhances human megakaryocytic development (Zhong et al. BBRC 2010).To investigate the mechanism of the high expression of these genes downstream of JAK2 signaling, USF1 and a cytokine receptor gene (MPL, EPOR or CSF3R) were co-transfected into 293T cells along with either a TGF-β1/HOXB4 promoter-driven or a STAT5 response element-driven luciferase reporter. Stimulation of MPL with TPO enhanced USF1 transcriptional activity about 3 fold, but stimulation of EPOR with EPO or of CSF3R with G-CSF did not change this activity. However, stimulation with any of the 3 types of cytokines enhanced STAT5 transcriptional activity. JAK2VF upregulated USF1 and STAT5 much more highly than JAK2WT without TPO stimulation. This USF1 upregulation specifically to TPO/MPL signaling was suppressed by a dominant negative mutant of USF1, JAK2 inhibitors (AG490, NS-018) or MEK inhibitors (U0126, PD325901). Inhibition of PI3K or p38MAPK did not affect the USF1 activation. Co-treatment with JAK2 and MEK inhibitors showed a synergistic effect in blocking both USF1 upregulation and STAT5 activation induced by JAK2VF.Next, we tested the MEK inhibitor, PD325901, in combination with the JAK2 inhibitor, NS-018, in the JAK2VF-TG mice. After disease was established 12 weeks after birth, JAK2VF-TG mice were divided into the following 4 groups: vehicle control; PD325901 monotherapy; NS-018 monotherapy; and combined therapy. PD325901 (5 mg/kg) and NS-018 (50 mg/kg) were orally administered once and twice daily, respectively. After 12 weeks of treatment, we evaluated the effect on BM fibrosis. The grading of MF in each group (n = 5-6) was as follows: vehicle control (MF-0: 0/6, MF-1 or 2: 6/6); PD325901 monotherapy (MF-0: 4/5, MF-1 or 2: 1/5); NS-018 monotherapy (MF-0: 0/6, MF-1 or 2: 6/6); and combined therapy (MF-0: 3/6, MF-1 or 2: 3/6). In the 2 groups treated with PD325901, 50~80% of mice showed MF-0. In contrast, in vehicle-treated or NS-018 monotherapy groups, all mice showed MF-1 or 2. Consistent with the MF grading, BM cellularity was significantly increased in the PD325901 monotherapy or combined therapy groups compared with the vehicle-treated group. A significant reduction was seen in the plasma TGFβ1 concentration in the PD325901 monotherapy and combined therapy groups compared with the vehicle-treated group (9.7 ng/ml, 8.1 ng/ml vs. 18.2 ng/ml, respectively). The TGFβ1 concentration in the extracellular fluid of BM (Wagner et al blood 2007) was also significantly reduced (5.6 ng/ml, 6.8 ng/ml vs. 9.1 ng/ml, respectively). BM cellularity and the TGFβ1 concentration in the NS-018 monotherapy group were comparable to those in the vehicle-treated group. Interestingly, megakaryocytes in the PD325901 monotherapy and combined therapy groups were decreased in number and were smaller than those in the vehicle-treated or NS-018 monotherapy groups. Regarding the effect on splenomegaly, spleen weight was significantly reduced in the NS-018 monotherapy and combined therapy groups compared with the vehicle-treated group (0.83 g, 0.69 g vs. 1.18 g, respectively). PD325901 monotherapy had little effect on splenomegaly.It is known that MEK-ERK1/2 pathway is critical in normal megakaryocyte development. In vitro data suggest that JAK2VF activates this pathway downstream of MPL and may contribute to TGFβ1 overproduction and dysmegakaryopoiesis, causing BM fibrosis via transcriptional enhancement of USF1. In vivo data suggest that MEK inhibition has the potential to improve dysmegakaryopoiesis and BM fibrosis. The combined therapy of JAK2 inhibitors with MEK inhibitors might be a promising therapy for improving both splenomegaly and BM fibrosis. DisclosuresNo relevant conflicts of interest to declare.

Effect of NS-018, a selective JAK2V617F inhibitor, in a murine model of myelofibrosis

A single somatic mutation, V617F, in Janus kinase 2 (JAK2) is one of the causes of myeloproliferative neoplasms (MPNs), including primary myelofibrosis, and the JAK2V617F mutant kinase is a therapeutic target in MPN. However, inhibition of wild-type (WT) JAK2 can decrease the erythrocyte or platelet (PLT) count. Our selective JAK2 inhibitor, NS-018, suppressed the growth of Ba/F3 cells harboring JAK2V617F more strongly than that of cells harboring WT JAK2. The 4.3-fold JAK2V617F selectivity of NS-018 is higher than the 1.0- to 2.9-fold selectivity of seven existing JAK2 inhibitors. NS-018 also inhibited erythroid colony formation in JAK2V617F transgenic mice at significantly lower concentrations than in WT mice. In keeping with the above results, in a JAK2V617F bone marrow transplantation mouse model with a myelofibrosis-like disease, NS-018 reduced leukocytosis and splenomegaly, improved bone marrow fibrosis and prolonged survival without decreasing the erythrocyte or PLT count in the peripheral blood. By exploring the X-ray co-crystal structure of NS-018 bound to JAK2, we identified unique hydrogen-bonding interactions between NS-018 and Gly993 as a plausible explanation for its JAK2V617F selectivity. These results suggest that NS-018 will have therapeutic benefit for MPN patients through both its efficacy and its reduced hematologic adverse effects.

Characterization of Targets of Plitidepsin In JAK2V617F-Mutated Cells From Myeloproliferative Neoplasms

AbstractAbstract 4093 Background:In a previous study (Verrucci M et al, J. Cell Physiol, 2010, Epub May10) we reported that the cyclic depsipeptide Aplidin®, a potent cytotoxic agent currently in phase II/III clinical trials for solid and hematologic neoplasia, displayed activity in a murine model of myelofibrosis, the GATA1-low mice. In fact, Aplidin improved the proliferation of Gata1-low hematopoietic cells, corrected abnormal microvessel density and reduced bone marrow fibrosis. These effects were largely attributed to improved maturation of megakaryocytes, as suggested by the increased platelet count in thrombocytopenic Gata1-low mice. It has been previously shown that a low expression of p27 in tumor cells might correlate with their response to Aplidin; of interest, GATA1-low megakaryocytes expressed reduced levels of p27. Overall, these results suggested that Aplidin could have the potential to alter the course of myelofibrosis-like disease in Gata1-low mice and could be useful for the treatment of myelofibrosis. Aims:to evaluate activity and targets of Aplidin in cellular models of myeloproliferative neoplasms (MPN). Methods:We measured the effect of Aplidin on the proliferation of JAK2V617F-mutated cell lines (UKE-1, HEL, SET2) and of primary cells from MPN patients in liquid cultures and semisolid medium, the rate of apoptosis using Annexin V flow cytometry analysis, and the cell cycle by propidium iodide staining. Expression of mRNA was quantified by real-time PCR, while protein level was measured by western blotting. Gene expression profiling was accomplished with Agilent Whole Human Genome Oligo Microarrays (44K). Results:Aplidin reduced the proliferation of all human V617F-mutated cell lines in the low nanomolar range, with IC50 from 0.5+/−0.03nM for UKE-1 to 1.5+/−0.05nM for HEL cells. Aplidin increased the proportion of cells in the G1/G0 phase of cell cycle, up to a mean of 80+/−5% from 60+/−3% in control cells (P<0.01). Also the proportion of apoptotic cells dose-dependently increased from 20+/−3% in control cultures to 50+/−6% at 10 nM in UKE-1 cells. After 24 h of incubation with Aplidin, the level of p27 mRNA was dose-dependently increased in all cell lines evaluated, while the BCR/ABL mutated K562 cells, whose proliferative rate was inhibited at higher Aplidin concentration, did not show significant changes of p27 mRNA. By western blotting analysis, level of p27 was overtly increased in UKE-1 cells treated with 5 and 10 nM Aplidin for 24h, and at the highest concentration we also observed reduction of phosphorylated STAT5. On the other hand, there was no change in JAK2, pJAK2, STAT5, STAT3, pSTAT3, Akt, pAkt. We then measured the level of p27 mRNA in peripheral blood cells obtained from patients with polycythemia vera (PV) or primary myelofibrosis (PMF). We found that PMF patients had significantly reduced p27 mRNA and protein level compared to controls and PV patients (P<.001 for both). After incubation with 1nM Aplidin there was a 2.5+/−2.0-fold increase in the p27 mRNA level and protein in cells of PMF patients while it was substantially unaffected in controls or PV patients. Gene expression analysis was performed in SET2 and UKE-1 cells that had been treated with 3nM of Aplidin for 24 hr. Aplidin treatment of SET2 cell line resulted in the up-regulation (UR) of 268 probsets and down-regulation (DR) of 247 (in total, about 1.5% of 40,961 genes called as “present”); corresponding figures in UKE-1 cells were 261 for UR (0.6%) and 364 (0.9%) for DR genes. MAPK signalling, TGF-beta signaling pathway, Cytokine-cytokine receptor interaction, cell adhesion molecules (CAMs), neuroactive ligand-receptor interaction, T cell receptor signaling, calcium signaling pathway, and regulation of actin cytoskeleton were the most involved pathways. Conclusions:Current results suggest that Aplidin has growth inhibitory activity and induces apoptosis in MPN cells. Response to Aplidin involves increasing p27 level, similar to findings in myelofibrotic GATA1-low mice; furthermore, we have identified low p27 expression as a characteristic of PMF cells compared to normal and PV cells. Finally, gene expression analysis allowed to identify a set of genes and involved pathways undergoing differential regulation in response to Aplidin. These information could be of value also for analyzing the response to Aplidin of PMF patients enrolled in an ongoing Phase II clinical trial. Disclosures:Aracil:PharmaMar: Employment. Vannucchi:PharmaMar: Research Funding.

Increased Myelofibrosis in Thrombospondin-1-Null Mice Is Associated with Enhanced TGF-β1-Mediated Response by Bone Marrow Fibroblasts

In Primary Myelofibrosis, several lines of evidence suggest that pleiotropic cytokine TGF-β1, released by clonal proliferation of pathological megakaryocytes and/or monocytes, plays a prominent role in reticulin fibers deposition. This cytokine is synthesized as a biologically inactive molecule that needs to be activated in order to trigger biological responses. However, the mechanisms involved in local TGF-β1 activation within the hematopoietic environment remain unclear. Since TGF-β1 and thrombospondin-1 (TSP-1) are synthesized and stored within the same organelles in megakaryocytes, one can speculate that the abnormal release of both molecules leads to pathological local TGF-β1 activation that becomes ultimately responsible of fibrosis development in the vicinity of these cells. To investigate the role of TSP-1 in local TGF-β1 activation, we used the TPOhigh murine model of bone marrow (BM) fibrosis. BM cells from wild-type (WT) or Tsp-1-null male littermates were infected with a retrovirus encoding the murine TPO protein and engrafted into lethally irradiated WT or Tsp-1-null female hosts, respectively, leading to the following engraftment combinations, WT/WT (WT TPOhigh mice, n=21) and Tsp-1-null/Tsp-1-null (Tsp-1-null TPOhigh mice, n=17). Lethally-irradiated hosts were engrafted with 4 to 8 × 106 cells in 3 independent experiments. Peripheral blood was analyzed every 4 weeks during 3 months and mice were killed for histological analysis at week 8 and 12 post-engraftment. The magnitude of plasma TPO level increase was comparable regardless of the TPOhigh mice groups. Chimerism levels, analyzed in recipients by FISH on the presence of the donor Y chromosome in whole nucleated BM cells, were more than 90% in either WT or Tsp-1-null TPOhigh mice. We report here that all TPOhigh mice developed a similar myeloproliferative syndrome associated with TGF-β1 overproduction. Surprisingly, we were able to detect the active form of TGF-β1 in BM and spleen extracellular fluids in all mice, including Tsp-1-null TPOhigh mice, suggesting that alternative mechanisms are mainly responsible for local TGF-β1 activation in this murine model of myelofibrosis. We then confirmed that Tsp-1-null platelets are able to activate TGF-β1 in vitro in response to thrombin. As predicted by the detection of the active form of TGF-β1, Tsp-1-null TPOhigh mice developed BM and spleen fibrosis which appears, intriguingly, to be of a greater grade than the one displayed by WT TPOhigh mice. Since TSP-1 is a potent inhibitor of angiogenesis, we investigate whether this increased fibrosis could be correlated with an augmentation of neoangiogenesis. The microvascular density (MVD) in control Tsp-1-null BM were higher than in control WT one (10±4.7 vs 0.6±0.2; p<0.001), as expected. However, MVD displayed by Tsp-1-null TPOhigh mice (8.3±4.4) did not rise above the one displayed by control Tsp-1-null mice and was similar to MVD observed in WT TPOhigh mice (5.7±2.9). Thus, the increase of myelofibrosis in Tsp-1-null TPOhigh mice cannot be explained by an augmentation of neoangiogenesis. Since TGF-β1 levels were similar in both TPOhigh groups, we hypothesized that this increase could be related to an enhanced TGF-β1-mediated response by Tsp-1-null BM fibroblasts. Indeed, we could show that Tsp-1 deficiency is associated with sustained phospho-Smad3 levels and a 10-fold increase in collagen III transcription level by BM fibroblasts in response to TGF-β1. Together, our resultsshow that TSP-1 is not the major activator of TGF-β1 in this in vivo model of myelofibrosis;suggest that other mechanisms are involved in this activation;shed light on a possible new mechanism of TGF-β1 regulation by one of its own activator.

Proteasome Inhibitor Bortezomib Can Inhibit Bone Marrow Fibrosis Development in a Murine Model of Myelofibrosis.

Several lines of evidence obtained from idiopathic myelofibrosis (IM) studies are in favour of a crucial role of the NF-κB pathway activation in myelofibrosis induction. It has been demonstrated that megakaryocytes, monocytes but also CD34+ cells from IM patients present a spontaneous NF-κB pathway activation associated with transforming growth factor-β1 (TGF-β1) secretion. This growth factor has been previously shown as the main fibrogenic cytokine involved in the myelofibrosis development. Mice exposed to high systemic levels of thrombopoietin (TPO) mediated by a retroviral vector (TPOhigh mice) develop a myeloproliferative disorder featuring numerous aspects of the human disease including bone marrow fibrosis, extramedullary hematopoiesis and dysmegakaryopoiesis. Moreover, TPOhigh mice display high plasma levels of IL-1α suggesting that the NF-κB pathway may play a role in this model of fibrosis. We then conducted a study to investigate whether NF-κB inhibition in this mice model could have an impact on myelofibrosis development using the proteasome inhibitor bortezomib. One month after engraftment with TPO-overexpressing hematopoietic cells, 2 groups of 20 immunocompetent C57BL/6J mice displaying similar myeloproliferation induced by TPO overexpression were constituted. Mice engrafted were treated twice a week, with either bortezomib (1 mg/kg) or a placebo for 4 weeks. At the end of this protocol, mice from both groups were examined for histological and haematological analysis including TGF-β1 and IL-1α levels determination. Here, we demonstrate that: i) the NF-κB pathway is activated in TPOhigh spleen cells and bortezomib treatment is able to inhibit this activation; ii) bortezomib treatment is able to decrease plasma concentration of TGF-β1 and IL-1α in mice as well as TGF-β1 content in extracellular fluids of marrow and spleen; iii) myelofibrosis development is inhibited after bortezomib treatment. These results emphasize the interest of developing bortezomib treatment in IM patients.

Adenoviral Mediated TGF-β1inhibition in a Murine Model of Myelofibrosis Can Prevent and Cure Bone Marrow Fibrosis Development.

Myelofibrosis is a clinical feature of several hematopoietic disorders and is most prominent in idiopathic myelofibrosis. It is characterized by excessive deposits of extracellular matrix proteins which occur as a marrow microenvironment reactive response to cytokines released from the clonal malignant myeloproliferation. The observation that mice exposed to high systemic levels of thrombopoietin (TPO) invariably develop a myelofibrosis within a period of 4 to 8 weeks has allowed the demonstration of the crucial role of TGF-b1 released by hematopoietic cells in the promotion of myelofibrosis (Chagraoui et al Blood, 2002 ; 100 :3495). The aim of this study was to investigate whether TGF-b1 inhibition could directly inhibit the fibrosis development in a curative approach of this murine model.An adenovirus encoding for TGF-b1 soluble receptor was constructed. SCID mice were infected with this adenovirus to measure the TGF-b1 inhibitory effect. We measure TGF-b1-Rs (TGF-b1 soluble receptor) concentration and TGF-b1 levels in 5 groups of mice injected with 0- 6.5 106- 6.5 107- 6.5 108-and 6.5 109 pfu/mice. We demonstrated that 6.5 108 pfu/mice was able to completely inhibit 10 fold increase of circulating TGF-b1 and that 1 microgram of soluble receptor was able to inhibit 1 ng of TGF-b1.All subsequent experiments were performed by injecting 1 109 pfu/mice either shortly after transplantation (preventive) or after 30 days (curative). Briefly, 46 mice (2 individuals experiments n= 16 and n= 30) were transplanted with 5-FU treated syngeneic bone marrow cells transduced with a retrovirus encoding for murine TPO. Retroviral transduction efficiency varied from 67% to 87%. All mice developed a myeloproliferative syndrome characterized by an increase in platelet count, a leukocytosis, an increase in progenitor cells in the blood circulation and a 1000-fold increase in TPO level. TGF-b Rs (13 to 32 micrograms/ml) was detected in the blood of all mice injected with the TGF-b1-Rs Adeno. A dramatic decrease in TGF-b1 level was noticed in all these treated mice in comparison to the control groups. Histological analysis demonstrated that the two approaches (curative or preventive) were identical to abolish fibrosis development in bone marrow as well as in the spleen. However, all mice which received the TGF-b1-RS adenovirus developed a hepatitis leading to death 6 weeks to 3 months after infections.In conclusion our approach support the major role of TGF-b1 in myelofibrosis development as previously demonstrated by our group using TGF-b1 KO mice. Moreover, the present results suggest that TGF-b1 inhibition could be a relevant approach to treat patients presenting a TGF-b1 mediated bone marrow fibrosis.

Polymorphonuclear Neutrophil and Megakaryocyte Mutual Involvement in Myelofibrosis Pathogenesis

The study presented here, performed on the bone marrow from patients with idiopathic myelofibrosis (MF) and on a murine model of MF, demonstrates a pathological interaction between PMN leukocytes and megakaryocyte (Mk), correlated with MF development. The data obtained revealed abnormal subcellular P-selectin distribution, which appeared to correlate with excessive and pathological emperipolesis of PMN leukocytes within Mk, leading to the destruction of Mk storage organelles and leakage of α -granular contents into the bone marrow microenvironment. The prominent role of growth factors, PDGF and TGF β, stored in the Mk α -granular compartment in the generation of MF has been previously largely documented. Both growth factors are essential for the Mk-dependent fibroblast proliferation. The destructive mutual cellular interaction of Mk and PMN leading to the pathological release of PDGF and TGF β within the bone marrow microenvironment may participate, through fibroblast activation, to the generation of MF. Therefore, this study provides insight into the possible pathophysiological mechanisms for the genesis of MF.