Combined Inhibition of JAK2 and mTOR Signaling Results in Enhanced Efficacy in in-Vitro and Preclinical Mouse Models of JAK2V617F-Driven Myeloproliferative Disease

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 708-708
Author(s):  
Alessandro M. Vannucchi ◽  
Niccolò Bartalucci ◽  
Costanza Bogani ◽  
Serena Martinelli ◽  
Lorenzo Tozzi ◽  
...  
Keyword(s):  
Clinical Trials ◽  
Mouse Models ◽  
Drug Combination ◽  
Colony Formation ◽  
Myeloproliferative Neoplasms ◽  
Solid Cancer ◽  
Synergistic Activity ◽  
Inhibition Of Proliferation ◽  
Cd34 Cells

Abstract Abstract 708 Background and Aims. The JAK1/JAK2 inhibitor Ruxolitinib (ruxo) produced rapid and sustained responses in splenomegaly and symptomatic improvement in patients (pts) with myelofibrosis (MF), supporting the central role of dysregulated JAK2 signaling in myeloproliferative neoplasms (MPN). Splenomegaly and constitutional symptoms improved also after treatment with Everolimus (RAD001), a rapamycin-derivative inhibitor of the serine/threonine kinase mTOR, in a phase I/II study, pointing to mTOR pathway as a novel target for MPN therapy. BEZ235 is a dual phosphatidylinositol-3-kinase (PI3K) and mTOR inhibitor currently in clinical trials in solid cancer. Aim of the study was to investigate the efficacy of BEZ235, alone and in combination with ruxo, against MPN cells in vitro and in preclinical mouse models. Methods. We used mouse (Ba/F3 and Ba/F3-EPOR expressing wild-type (WT) or V617F(VF) mutated JAK2) and human (VF HEL and SET2 or WT K562) cell lines and primary MPN CD34+ cells from pts with MF or polycythemia vera (PV); cell proliferation, colony formation, apoptosis, cell cycle and protein phosphorylation status were evaluated. Effects of drug combination were analyzed according to Chou and Talalay to calculate the combination index (CI); a CI <1.0 indicates synergistic activity. For in vivo studies, two mouse models were used. (1) SCID mice receiving iv Ba/F3-EPOR VF-luciferase (luc) cells (gift of T. Radimerski) were randomized on d6 to treatment groups (either drugs alone and in combination or vehicle (VE)) based on baseline luminescence. Bioluminescence measurement was done at week intervals until death. (2) A C57Bl6/J JAK2 V617F Knock-in mouse model was generated by the flex switch strategy with insertion of the reversed JAK2V617F exon 13 sequence; mating with Vav-Cre transgenic mice activates the VF allele producing a MPN phenotype in progenies from VF heterozygous expression. Mice were treated for 5d, then blood, spleen and bone marrow cells were analyzed. Results. We found that BEZ235 inhibited proliferation of Ba/F3 VF and Ba/F3-EPOR VF cells at concentrations significantly lower than the wt counterparts (IC50 was 64±10nM vs 10,000±500nM and 87±50nM vs 676±200nM; P<0.01); similar preferential inhibition was observed in HEL and SET2 cells compared to K562 cells (IC50, 387±90nM and 334±40nM vs 5,000±1,000nM; P<0.01). BEZ235 dose-dependently increased the percentage of cells in G0/G1 and induced apoptosis. Western blot analysis showed marked reduction of the mTOR target p4EBP1 as well as appreciable downregulation of pSTAT5 and pSTAT3 at 6 to 24h of treatment. BEZ235 impaired the proliferation of CD34+ cells from MF pts with an IC50= 43±20nM vs 780±150nM in healthy donors (P<0.01), and reduced colony formation of MF and PV hematopoietic progenitors at doses statistically lower (2 to 15-fold) than normal cells. The growth of EPO-independent colonies (EEC) in PV pts was potently inhibited (IC50=20±10nM). Co-treatment of BEZ235+ruxo resulted in synergistic inhibition of proliferation in SET2 (median CI=0.37) and BaF3-EPOR VF (CI=0.77) cells and increased apoptosis rate in SET2 (CI=0.25); drug combination was highly effective also in the EEC assay (CI=0.17). In the Ba/F3VF luc model, the median bioluminescence index (No. of pixel) on day 7 of treatment was 4.7×106 in VE animals, 7.2×106 in ruxo (P=ns), 1.1×106 in BEZ235 (P=0.04) and 4.6×105 in animals receiving the BEZ+ruxo combination (P<0.01). The median survival in mice treated with the combination of BEZ+ruxo was 30d, significantly longer than either ruxo (18.5d; P<0.01) or BEZ (24d; P<0.04) alone (15.0d in VE animals). In JAK2V617F KI mice treated for 5d, we found that drug combination was significantly more effective in reducing enlarged spleen (median spleen index (spleen weight/body weightx100): 38, 35, 27 and 7 for VE, BEZ, Ruxo and BEZ+Ruxo) and reticulocyte count (median No. per HPF: 48, 50, 44, and 3 for VE, BEZ, Ruxo and BEZ+Ruxo) than either drugs alone. The phosho levels of STAT5 and 4EBP1 in the spleen were significantly reduced in mice receiving BEZ+Ruxo as compared to single treatment. Conclusions. Combined inhibition of PI3K/mTOR and JAK2 signaling resulted in enhanced activity in preclinical models of MPN compared with either treatment alone, providing a rationale for the development of combination clinical trials. Disclosures: Vannucchi: Novartis: Membership on an entity's Board of Directors or advisory committees.

Specific MicroRNA Deregulated in Myleoproliferative Neoplasm Are Regulated by JAK2V617F and May Contribute to Aberrant Hematopoiesis.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 965-965
Author(s):  
Xiaoqing Lin ◽  
Monica Buzzai ◽  
Martin Carroll ◽  
Elizabeth Hexner ◽  
Fabricio F Costa ◽  
...  
Keyword(s):  
Progenitor Cells ◽  
Mirna Expression ◽  
Colony Formation ◽  
Lentiviral Vector ◽  
Myeloproliferative Neoplasms ◽  
Rna Extraction ◽  
Differentially Expressed ◽  
Loss Of Function ◽  
Cd34 Cells

Abstract Abstract 965 The myeloproliferative neoplasms (MPN), PV, ET and IMF, harbor the same gain-of-function mutation JAK2V617F at a high frequency (∼100%, 70% and 50% respectively). Accumulating evidence suggest that JAK2V617F may not be the initiating event in MPN, and other genetic anomalies play an important role in MPN pathogenesis. We hypothesized that miRNA deregulation contributes to the development of MPN. To test this idea, miRNA expression in CD34+ cells isolated from 8 patient samples (4 PV with JAK2V617F, 3 ET with wild-type JAK2 and 1 IMF with unknown JAK2 status) and 4 healthy controls was determined using a Taqman Low Density Array (TLDA) representing 667 known miRNAs. PV (JAK2V617F) and ET cases (JAK2WT) showed 14 and 78 differentially expressed miRNAs, respectively, when compared to controls. 6 miRNAs were commonly deregulated in PV and ET, while the majority were unique to each disease type. When all MPN patients were grouped and compared to controls, 28 miRNAs were significantly deregulated (p<0.05). These miRNAs differ from those previously reported to be differentially expressed in the peripheral blood of PV patients. Among these 28, mir-214 was down-regulated and mir-410, mir-22* and mir-505* were up-regulated most consistently. Several miRNAs, including mir-135b, mir-542-5p, mir-149, mir-133b and mir-134 were undetectable in normal CD34+ cells and activated in MPN patients. We further hypothesized that some miRNAs are regulated through the action of the mutant JAK2V617F kinase. To test this, miRNA levels were assessed by TaqMan array in HEL and UKE-1 cells (harboring JAK2V617F) treated with 2 μM JAK inhibitor I (Calbiochem) for 20h before RNA extraction. In parallel, miRNA expression as determined by TLDA in TF-1 cells rendered cytokine independent by stable expression of JAK2V617F was compared to that of control TF-1 cells, both cultured overnight in the absence of cytokines. A total of 24 miRNAs were significantly deregulated (>2 fold) in at least two cell line systems. To test which deregulated miRNAs in MPN patients were JAK2 responsive, JAK2 activity was manipulated in HEL and TF-1 cells as described above, and the expression of miRNAs was determined by individual Taqman miRNA assays. mir-1, mir-200a, mir-9, mir-133b, mir-22* and mir-155 were responsive to manipulation of JAK2 activity. miR-155 expression was repressed 50% with the inhibition of JAK2 in HEL cells and stimulated almost 2 fold with the overexpression of JAK2V617F in TF-1 cells. By contrast, mir-214 (downregulated in MPN) and mir-134 (upregulated in MPN) were not responsive to manipulation of JAK2V617F activity in either the gain or loss-of-function systems. To further confirm the ability of JAK2V617F to regulate specific miRNAs, lineage negative (lin-) murine marrow progenitor cells were transduced with JAK2V617F or empty vector, allowed to form colonies for 7 days and miRNA levels in the colonies were determined. Again miR-200a, miR-9 and miR-22* and miR-155 were responsive to JAKV617F overexpression, while mir-134 was not. Transduction of lineage negative murine marrow progenitor cells with a lentiviral vector harboring mir-155 yielded a 30% increase in a myeloid colony formation in vitro. The effect is consistent with the reported ability of mir-155 to induce myeloproliferation in mice. Transduction of marrow progenitors with miR-133b, which is activated in MPN patients, responsive to JAK2V617F manipulation and not previously reported to have a role in hematopoiesis, led to an increase in both erythroid and myeloid colony formation. Taken together we conclude that at least 4 miRNAs are deregulated in CD34+ cells of MPN patients as a result of aberrant JAK2 activity. Two of these tested so far have a role in hematopoiesis. Part of the action of JAK2V617F in myeloproliferation may be mediated by specific miRNA, thus representing alternative therapeutic targets in MPN. Disclosures: Carroll: Sanofi Aventis Corp: Research Funding; Cephalon Oncoloy: Consultancy.

The PI3K Specific Inhibitor BKM120 Results Effective and Synergizes With Ruxolitinib In Preclinical Models Of Myeloproliferative Neoplasms

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 1599-1599 ◽  
Author(s):  
Niccolò Bartalucci ◽  
Costanza Bogani ◽  
Serena Martinelli ◽  
Carmela Mannarelli ◽  
Jean-Luc Villeval ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Colony Formation ◽  
Myeloproliferative Neoplasms ◽  
Janus Kinase ◽  
Preclinical Models ◽  
Mtor Inhibition ◽  
Jak2 Inhibitor ◽  
Cd34 Cells

Abstract Background and Aims A gain-of-function mutation in Janus kinase 2 (JAK2V617F) is at the basis of the majority of chronic myeloproliferative neoplasms (MPN). The dual JAK1/JAK2 inhibitor ruxolitinib (ruxo) determined rapid and sustained responses in splenomegaly and symptomatic improvement in patients with myelofibrosis (MF), supporting the central role of dysregulated JAK2 signaling. Enhanced activation of other downstream pathways including the PI3K/mTOR pathway has been documented as well. We previously reported (Bogani et al, PlosOne 2013;8:54828) that targeting mTOR by the allosteric inhibitor RAD001 resulted in inhibition of JAK2VF mutated cells and produced clinical benefits in a phase I/II trial (Guglielmelli et al, Blood 2011;118:2069). In this study we evaluated the effects of BKM120, a specific PI3K inhibitor, alone and in combination with ruxolitinib, in in-vitro and in-vivo MPN models. Methods To evaluate cell proliferation, colony formation, apoptosis, cell cycle and protein phosphorylation status we used mouse BaF3 and BaF3-EPOR cells expressing wild type (WT) or VF mutated JAK2, the human VF-mutated HEL and SET2 cell lines, and primary MPN CD34+ cells from patients with MF or polycythemia vera (PV). Effect of drug combination was analyzed according to Chou and Talalay calculating the combination index (CI); a CI <1 indicates synergistic activity. For in vivo studies we used two mouse models: (1) SCID mice receiving iv BaF3-EPOR VF-luciferase (luc) cells (gift of T. Radimerski) were randomized on day 6 to different treatment groups based on baseline luminescence. (2) C57Bl6/J JAK2 VF Knock-in mice were generated by insertion of the reversed JAK2V617F exon 13 sequence; mating with Vav-Cre transgenic mice activates the VF allele producing a MPN phenotype in progenies with VF heterozygous expression (Hasan et al, Blood 2013;Epub). Mice were treated for 15 days, then blood, spleen and bone marrow cells were analyzed. Results We found that BKM120 preferential inhibited BAF3 VF and BaF3-EpoR VF cells (IC50: 364±200nM and 1100±207nM, respectively) compared to their respective WT counterpart (5300±800nM and 3122±1000nM: p<.05). HEL and SET2 cells resulted also sensitive to BKM120 (2000±500nM and 1000±300nM). Interestingly we found that BKM120 significantly increased G2/M phase and decreased S phase of cell cycle (p<.01) and induced apoptosis (IC50, SET2=10µM, BaF3-EPOR VF=1.8 µM). Western blot analysis showed marked reduction of phospho-mTOR and its target phospho-4EBP1 as well as downregulation of phospho-STAT5 at 6 and 24h of treatment. BKM120 impaired colony formation from MF and PV CD34+ cells at doses 2 to 8-fold lower than healthy controls (p<.01). BKM120 strongly inhibited EEC colony growth from PV pts (IC50, 9±4nM). Co-treatment of BKM120+ruxo resulted in synergistic inhibition of proliferation of SET2 (median CI=0.45) and BaF3-EPOR VF (median CI=0.8) cells. Triple combinations including BKM120/ruxo plus either RAD001 (Torc1 inhibitor) or PP242 (Torc1/2 inhibitor) resulted highly synergistic (median CI=0.27 and 0.52) to indicate the importance of complete mTOR inhibition. BKM120 at 45mpk and 60mpk increased mean lifespan of BaF3 VF luc mouse model from 21d in control mice to 27.2d and 28d in BMK120 treated mice. In KI mice, co-treatment with 60mpk BKM120 + 60mpk ruxo resulted in improvement of splenomegaly (median spleen weight: 1.4, 0.82, 0.8 and 0.6 g respectively for controls, 60mpk BKM120, 60mpk ruxo and 60mpk BKM120+60mpk ruxo) and reduction of leukocytosis and reticulocyte count. The level of phosho-STAT5 and -4EBP1 in the spleen was significantly reduced in mice receiving BKM120+ruxo as compared to single drug treatment. We finally analyzed the effects of BKM120+/-ruxo on the in-vitro clonogenic growth of BM cells from VF and WT KI mice mixed in a 1:1 ratio. The proportion of VF-positive colonies resulted reduced in a dose dependent manner by 19%, 33% and 44% (p<.03) compared to controls with 50nM, 100nM and 300nM BKM120 respectively. A 25% and 39% of VF-positive colonies reduction was achieved with 50nM and 100nM ruxolitinib. The combined treatment with 100nM BKM120 + 50nM ruxo resulted in a 50% decrease of the number of mutated colonies (p<.02). Conclusions Inhibition of PI3K by BKM120 alone and combined with JAK2 inhibitor ruxolitinib resulted in enhanced activity in preclinical models of MPN, providing a rationale for the ongoing combination clinical trial. Disclosures: Vannucchi: Novartis: Membership on an entity’s Board of Directors or advisory committees.

Pre-Stimulation of CML CD34+ Cells with High Concentrations of Growth Factors Counters Imatinib-Mediated Proliferation Suppression and Enhances Apoptosis of Non-Dividing Cells.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 2479-2479
Author(s):  
Melissa S. Holtz ◽  
Stephen J. Forman ◽  
Ravi Bhatia
Keyword(s):  
Growth Factors ◽  
Imatinib Treatment ◽  
Hematopoietic Growth Factors ◽  
Inhibition Of Proliferation ◽  
Gm Csf ◽  
Cd34 Cells ◽  
Stem And Progenitor Cells ◽  
Dividing Cells ◽  
High Concentrations

Abstract While imatinib mesylate is a highly effective treatment for CML, there is accumulating evidence that it may not adequately target quiescent malignant HSCs. In vitro exposure to imatinib inhibits CML progenitor growth primarily through suppression of abnormally enhanced proliferation. Apoptosis appears to be restricted to dividing cells while quiescent progenitors are resistant to apoptosis. One approach to more effectively enhance the sensitivity of HSCs to imatinib may be to induce them to cycle using hematopoietic growth factors (GF). We have shown that exposure of CML CD34+ progenitors to imatinib (1μM) in high GF conditions (100ng/ml SCF and FL3, 20ng/ml IL6, G-CSF and IL3) reduced the total number of viable, undivided cells compared to control cells cultured in 100-fold lower GF conditions (low GF). High GF treated cells were more proliferative but less sensitive to imatinib-mediated apoptosis (Blood2004, 104:2967). We hypothesized that pre-stimulation with high GF prior to imatinib exposure would further reduce viable, non-dividing CML progenitors. CML CD34+ cells were cultured in high GF for 48 hours and then exposed to imatinib (1μM) for 48 hours in either high or low GF conditions. Compared to cells exposed to imatinib without any pre-stimulation, high GF pre-stimulation significantly reduced imatinib-mediated inhibition of proliferation in both low GF (22±5%, p=0.0009) and high GF (18±3%; p=0.0003). Pre-stimulation decreased imatinib-mediated apoptosis when compared to the same conditions with no pre-stimulation [19±2% for imatinib treatment in low GF (p&lt;0.0001) and 7±3% in high GF (p=0.064)]. However, although overall apoptosis decreased, pre-stimulation resulted in increased apoptosis of undivided cells exposed to imatinib in either low GF (14±5%; p=0.022) or high GF (13±6%, p=0.065). These results are notable since increased apoptosis of undivided cells was not previously observed in any other condition. Importantly, the percent of input cells remaining viable and undivided decreased significantly for pre-stimulated cells exposed to imatinib in low GF (19±3%; p&lt;0.0001) or high GF (7±2%; p=0.016). These results highlight the potential use of GF stimulation to enhance targeting of CML HSC. Additional studies examined whether GF readily available for clinical use (G-CSF and/or GM-CSF) could also enhance imatinib targeting of quiescent CML progenitors. CML CD34+ cells were exposed to 1mM imatinib for 96 hours in a basal low GF cocktail (250pg/ml G-CSF, 10pg/ml GM-CSF, 200pg/ml SCF, 1ng/ml IL6, 200pg/ml MIP1α, 50pg/ml LIF) alone or with the addition of G-CSF (50ng/ml) and/or GM-CSF (10ng/ml). While overall apoptosis decreased, apoptosis of undivided cells significantly increased for cells exposed to imatinib in high concentrations of G-CSF + GM-CSF compared to those in basal GF alone (8.7±1.3%; p=0.007). The percent of input cells remaining viable and undivided in the presence of imatinib significantly decreased with high G-CSF + GM-CSF compared to basal GF alone (7.2%±1.1; p=0.007). In conclusion, pre-stimulation with high concentrations of GF can lead to increased proliferation and enhance reduction of non-dividing CML CD34+ cells by imatinib. These results are of significance because non-dividing primitive cells have previously proven highly resistant to elimination by imatinib and support translational clinical studies to investigate whether intermittent GF administration can enhance elimination of residual CML stem and progenitor cells in patients in remission on imatinib treatment.

Further Preclinical Studies with APR-246, a Novel Anticancer Compound Currently in Clinical Trials for Hematological Malignancies and Prostate Cancer.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 3773-3773
Author(s):  
Nina Mohell ◽  
Charlotta Liljebris ◽  
Jessica Alfredsson ◽  
Ylva Lindman ◽  
Maria Uustalu ◽  
...  
Keyword(s):  
Clinical Trials ◽  
Cancer Cells ◽  
Cell Lines ◽  
Cancer Cell ◽  
Ex Vivo ◽  
Cancer Cell Lines ◽  
Solid Cancer ◽  
Dependent Manner

Abstract Abstract 3773 Poster Board III-709 Introduction The tumor suppressor protein p53 induces cell cycle arrest and/or apoptosis in response to various forms of cellular stress, through transcriptional regulation of a large number of down stream target genes. p53 is frequently mutated in cancer, and cancer cells carrying defects in the p53 protein are often more resistant to conventional chemotherapy. Thus, restoration of the wild type function to mutant p53 appears to be a new attractive strategy for cancer therapy. APR-246 is a novel small molecule quinuclidinone compound that has been shown to reactivate non-functional p53 and induce apoptosis. Although the exact molecular mechanism remains to be determined, recent results suggest that an active metabolite of APR-246 alkylates thiol groups in the core domain of p53, which promotes correct folding of p53 and induces apoptosis (Lambert et al., Cancer Cell 15, 2009). Currently, APR-246 is in Phase I/IIa clinical trials for hematological malignancies and prostate cancer. In the present abstract results from in vitro, ex vivo and in vivo preclinical studies with APR-246 are presented. Results The lead compound of APR-246, PRIMA-1 (p53 reactivation and induction of massive apoptosis), was originally identified by a cellular screening of the NCI library for low molecular weight compounds (Bykov et al., Nat. Med., 8, 2002). Further development and optimization of PRIMA-1 led to the discovery of the structural analog APR-246 (PRIMA-1MET), with improved drug like and preclinical characteristics. In in vitro experiments APR-246 reduced cell viability (WST-1 assay) in a large number of human cancer cell lines with various p53 status, including several leukemia (CCRF-CEM, CEM/VM-1, KBM3), lymphoma (U-937 GTP, U-937-vcr), and myeloma (RPMI 8226/S, 8226/dox40, 8226/LR5) cell lines, as well as many solid cancer cell lines, including osteosarcoma (SaOS-2, SaOS-2-His273,U-2OS), prostate (PC3, PC3-His175, 22Rv1), breast (BT474, MCF-7, MDA-MB-231), lung (H1299, H1299-His175) and colon cancer (HT-29). In human osteosarcoma cell lines APR-246 reduced cell viability and induced apoptosis (FLICA caspase assay) in a concentration dependent manner being more potent in the p53 mutant (SaOS-2-His273) than in the parental p53 null (SaOS-2) cells. The IC50 values (WST-1 assay) were 14 ± 3 and 27 ± 5 μM, respectively (n=35). In in vivo subcutaneous xenograft studies in SCID (severe combined immunodeficiency) mice APR-246 reduced growth of p53 mutant SaOS-2-His273 cells in a dose-dependent manner, when injected i.v. twice daily with 20 -100 mg/kg (64 – 76% inhibition). An in vivo anticancer effect of APR-246 was also observed in hollow-fiber test with NMRI mice using the acute myeloid leukemia (AML) cell line MV-4-11. An ex vivo cytotoxic effect of APR-246 and/or its lead compound PRIMA-1 has also been shown in primary cells from AML and CLL (chronic lymphocytic leukemia) patients, harbouring both hemizygously deleted p53 as well as normal karyotype (Nahi et al., Br. J. Haematol., 127, 2004; Nahi et al., Br. J. Haematol., 132, 2005; Jonsson-Videsater et al., abstract at this meeting). APR-246 was also tested in a FMCA (fluorometric microculture assay) test using normal healthy lymphocytes (PBMC) and cancer lymphocytes (CLL). It was 4-8 fold more potent in killing cancer cells than normal cells, indicating a favorable therapeutic index. This is in contrast to conventional cytostatics that often show negative ratio in this test. Furthermore, when tested in a well-defined panel of 10 human cancer cell lines consisting of both hematological and solid cancer cell lines, the cytotoxicity profile/activity pattern of APR-246 differed from common chemotherapeutic drugs (correlation coefficient less than 0.4), suggesting a different mechanism of action. Conclusion In relevant in vitro, in vivo and ex vivo cancer models, APR-246 showed unique pharmacological properties in comparison with conventional cytostatics, by being effective also in cancer cells with p53 mutations and by demonstrating tumor specificity. Moreover, in experimental safety/toxicology models required to start clinical trials, APR-246 was non toxic at the predicted therapeutic plasma concentrations. Thus, APR-246 appears to be a promising novel anticancer compound that may specifically target cancer cells in patients with genetic abnormality associated with poor prognosis. Disclosures: Mohell: Aprea AB: Employment. Liljebris:Aprea AB: Employment. Alfredsson:Aprea AB: Employment. Lindman:Aprea AB: Employment. Uustalu:Aprea AB: Employment. Wiman:Aprea AB: Co-founder, shareholder, and member of the board. Uhlin:Aprea AB: Employment.

Crucial Factors of the Inflammatory Microenvironment (IL-1 beta/TNF-alpha/TIMP-1) Promote Maintenance of the Malignant Hemopoietic Clone of Myelofibrosis By Stimulating the in Vitro Survival/Proliferation/Migration of Circulating CD34+ stem/Progenitor Cells

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 4094-4094
Author(s):  
Dorian Forte ◽  
Daria Sollazzo ◽  
Nicola Polverelli ◽  
Romano Marco ◽  
Lara Rossi ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Inflammatory Cytokines ◽  
Stromal Cells ◽  
Colony Formation ◽  
Inflammatory Factors ◽  
Inflammatory Microenvironment ◽  
Cd34 Cells ◽  
Tnf Α ◽  
Pro Inflammatory Cytokines

Abstract Introduction. Myelofibrosis (MF), an acquired clonal disorder of the hematopoietic stem/progenitor cell (HSPC) with a dysregulation in JAK/STAT signalling (mutations in JAK2, MPL and Calreticulin (CALR) genes), is characterized by a state of chronic inflammation. It is argued that the up-regulated production of proinflammatory cytokines by both HSPCs and the surrounding stromal cells generates a microenvironment that selects for the malignant clone. Only recently, it has been hypothesized that the sustained inflammatory microenvironment of MF can alter crucial biological processes, leading to genomic instability and cancer progression. Here we tested the in vitro functional effects of pivotal players of the inflammatory microenvironment (the extracellular ATP nucleotide and selected cytokines, such as Interleukin (IL)-1β, Tumor Necrosis Factor (TNF)-α or the Tissue Inhibitor of Metalloproteinases-1 (TIMP-1)) on the HSPCs from MF patients. Methods: Circulating CD34+/CD34+ CD38- cells from MF patients (JAK2V617F (17 cases) and CALR (9 cases) mutations) or cord blood (CB; 8 samples) were phenotypically and functionally characterized after in vitro incubation with or without ATP (1000 μM), IL-1β (10 ng/mL), TNF-α (10 ng/mL) or TIMP-1 (100 ng/mL) (alone or in combination). Cells were then analyzed for survival/apoptosis (Annexin-V/Propidium Iodide staining), phenotype (evaluation of CD63 (TIMP-1 receptor), CXCR4 and CD38 expression), cell cycle and clonogenic capacity. Migration was assessed first towards a CXCL12 gradient in the presence or absence of the pro-inflammatory factors. In parallel experiments, CD34+ cells from MF patients were co-cultured with normal mesenchymal stromal cells (MSCs) in the presence or absence of the pro-inflammatory cytokines and then evaluated for their ability to migrate towards a CXCL12 gradient. Plasma TIMP-1, TNF-α, IL-1β and CXCL12 were measured by ELISA assay. Results: The plasma levels of TIMP-1, TNF-α, IL-1β, CXCL12 and the number of circulating CD34+, CD34+ CD38-, CD34+ CD63+, CD34+ CD184+ cells were increased in MF patients. According to mutational status, the CD34+ CD63+ cells were higher in the CALR+ patients. The survival of MF CD34+ cells was strongly stimulated by in vitro incubation with TNF-α or IL-1β as compared with the CB-derived CD34+ cells or untreated cells. By multiple cytokine combinations, IL-1β/TIMP-1, IL-1β /ATP or IL-1β /TNF-α treatments significantly promote the survival of MF CD34+ cells as compared with the normal counterparts or the untreated cells. Various combinations with IL-1β were also effective in stimulating survival of CD34+CD38- cells. IL-1β/TIMP-1 and IL-1β/TNF-α/TIMP-1, but not factors alone, significantly increased the CFU-C growth of MF patients as compared with the CB-derived counterparts and the untreated cells. Moreover, comparing CALR+ vs JAK2V617F+ patients, the colony formation of JAK2V617F+ patients was mainly promoted by the IL-1β/TNF-α treatment. Along with clonogenic capacity stimulation, exposure of CD34+ cells from MF patients to IL-1β/TNF-α/TIMP-1 significantly increases the S-phase cells, suggesting that these pro-inflammatory factors stimulated cell-cycle progression in dormant CD34+ MF cells. Migration of CD34+ cells from MF was significantly increased in CXCL12 treated cells. In addition, exposure of MF CD34+ cells to IL-1β/TNF-α, IL-1β/TIMP-1 or IL-1β/TNF-α/TIMP-1 significantly promotes cell migration in comparison with the CB-derived counterparts or SDF-1 alone. MF migrated cells in the presence of IL-1β/TNF-α significantly upregulate CD63 expression. Intriguingly, colony formation of MF migrated CD34+ cells in the presence of IL-1β/TNF-α or IL-1β/TNF-α/TIMP-1 was potently increased. Finally, co-culture systems with normal MSCs in the presence of pro-inflammatory factors revealed that MF CD34+ cells display increased migration ability toward CXCL12 gradient. Conclusions: Altogether our findings suggest that in MF the inflammatory niche plays a key role in the maintenance of the malignant clone. Thus, the interplay between the pro-inflammatory cytokines promote and select the HSPCs with higher proliferative activity, clonogenic potential and migration capability. Targeting these microenvironmental interactions may be a clinically relevant approach. D.F. and D.S. equally contributed Disclosures Martinelli: Pfizer: Consultancy; Ariad: Consultancy; Novartis: Consultancy, Speakers Bureau; MSD: Consultancy; AMGEN: Consultancy; BMS: Consultancy, Speakers Bureau; ROCHE: Consultancy.

Loss of ASXL1 Triggers an Apoptotic Response in Human Hematopoietic Stem and Progenitor Cells

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 4107-4107
Author(s):  
Susan Hilgendorf ◽  
Hendrik Folkerts ◽  
Jan Jacob Schuringa ◽  
Edo Vellenga
Keyword(s):  
Progenitor Cells ◽  
Colony Formation ◽  
Erythroid Differentiation ◽  
Lentiviral Vectors ◽  
Apoptotic Response ◽  
Double Positive ◽  
Hematopoietic Stem ◽  
Cd34 Cells ◽  
Stem And Progenitor Cells

Abstract In recent clinical studies, it has been shown that ASXL1 is frequently mutated in myelodysplastic syndrome (MDS), in particular in high-risk MDS patients who have a significant chance to progress to acute myeloid leukemia (AML). The majority of ASXL1 mutations leads to truncation of the protein and thereby to loss of its chromatin interacting and modifying domain, possibly facilitating malignant transformation. However, the functions of ASXL1 in human hematopoietic stem and progenitor cells are not well understood. In this study, we addressed whether manipulation of ASXL1-expression in the hematopoietic system in vitro mimics the changes observed in MDS-patients. We downregulated ASXL1 in CD34+ cord blood (CB) cells using lentiviral vectors containing several independent shRNAs and obtained a 40-50% reduction of ASXL1 expression. Colony Forming Cell (CFC) assays revealed that erythroid colony formation was significantly impaired (p<0.01) and, to some extent, granulocytic and macrophage colony formation as well (p<0.09, p<0.05 respectively). In myeloid suspension culture assays, we observed a modest reduction in expansion (two-fold at week 1) upon ASXL1 knockdown under myeloid conditions. In erythroid conditions, shASXL1 CB CD34+ cells showed a strong four-fold growth disadvantage, with a more than two-fold delay in erythroid differentiation. The reduced expansion was partly due to a significant increase in apoptosis (5.9% in controls vs. 14.0% shASXL1, p<0.02). The increase in cell death was restricted to differentiating cells, defined as CD71 bright- and CD71/GPA-double positive. In addition, we tested whether HSCs were affected by ASXL1 loss. Long-term culture-initiating cell (LTC-IC) assays revealed a two-fold decrease in stem cell frequency. To test dependency of shASXL1 CB 34+ cells on the microenvironment, transduced cells were cultured on MS5 bone marrow stromal cells with or without additional cytokines. shASXL1 CB CD34+ cells cultured on MS5 showed a modest two-fold reduction in cell growth at week 4. In the presence of EPO and SCF, we detected a growth disadvantage (three-fold at week 2) and a delay in erythroid differentiation, similar to what was observed in liquid culture. ASXL1 has been proposed to be an epigenetic modifier by recruiting/stabilizing the polycomb repressive complex 2 (PRC2). Active PRC2 can lead to trimethylation of H3K27 and silencing of certain loci. It has been proposed that perturbed ASXL1 activity may disturb PRC2 function, leading to reduced H3K27me3 and increased gene expression. Using an erythroid leukemic cell line, we downregulated ASXL1 and as a positive control EZH2, one of the core subunits of PRC2. We then performed ChIP and did PCR for several loci. Upon knockdown of ASXL1, we did not observe changes in H3K27me3 on any of he investigated loci. However, upon knockdown of EZH2 we observed more than 50% loss of the H3k27m3 mark for many of the loci. This implies that our observed phenotypes may not be conveyed via the PRC2 complex but maybe via an alternative pathway. Preliminary data revealed an increase in H2AK119ub, suggesting that the BAP1-ASXL1 complex may be involved. In patients, mutations in ASXL1 are frequently accompanied by a mutation of TP53. Possibly, this additional mutation is necessary to allow ASXL1-mutant induced transformation thereby bypassing the apoptotic response. Therefore, we modeled simultaneous loss of ASXL1 and TP53 using shRNA lentiviral vectors. Our data showed that while in primary CFC cultures shASXL1/shTP53 did not give rise to more colonies, an increase in colony-forming activity was observed upon replating of the cells. Furthermore, shASXL1/shTP53 transduced cells grown in erythroid liquid conditions revealed a decrease in apoptosis compared to the ASXL1 single mutation and an outgrowth of these double positive cells. Nevertheless, no transformation occurred in vitro. We therefore injected shASXL/TP53 transduced CB CD34+ in a humanized scaffold model in mice to determine whether transformation can occur in vivo. In conclusion, our data indicate that mutations in ASXL1 trigger an apoptotic response in CB CD34+ cells with a delay in differentiation, which leads to reduced stem and progenitor output in vitro without affecting H3K27me3. Disclosures No relevant conflicts of interest to declare.

scholarly journals Clomipramine and Benznidazole Act Synergistically and Ameliorate the Outcome of Experimental Chagas Disease

2016 ◽  
Vol 60 (6) ◽  
pp. 3700-3708 ◽  
Author(s):  
Mónica Cristina García ◽  
Nicolás Eric Ponce ◽  
Liliana Maria Sanmarco ◽  
Rubén Hilario Manzo ◽  
Alvaro Federico Jimenez-Kairuz ◽  
...  
Keyword(s):  
Synergistic Effect ◽  
Chagas Disease ◽  
Drug Combination ◽  
Specific Inhibitor ◽  
Chronic Phase ◽  
Control Group ◽  
Histopathological Analysis ◽  
Synergistic Activity ◽  
Content Type

Chagas disease is an important public health problem in Latin America, and its treatment by chemotherapy with benznidazole (BZ) or nifurtimox remains unsatisfactory. In order to design new alternative strategies to improve the current etiological treatments, in the present work, we comprehensively evaluated thein vitroandin vivoanti-Trypanosoma cruzieffects of clomipramine (CMP) (a parasite-trypanothione reductase-specific inhibitor) combined with BZ.In vitrostudies, carried out using a checkerboard technique on trypomastigotes (T. cruzistrain Tulahuen), revealed a combination index (CI) of 0.375, indicative of a synergistic effect of the drug combination. This result was correlated with the data obtained in infected BALB/c mice. We observed that during the acute phase (15 days postinfection [dpi]), BZ at 25 mg/kg of body weight/day alone decreased the levels of parasitemia compared with those of the control group, but when BZ was administered with CMP, the drug combination completely suppressed the parasitemia due to the observed synergistic effect. Furthermore, in the chronic phase (90 dpi), mice treated with both drugs showed less heart damage as assessed by the histopathological analysis, index of myocardial inflammation, and levels of heart injury biochemical markers than mice treated with BZ alone at the reference dose (100 mg/kg/day). Collectively, these data support the notion that CMP combined with low doses of BZ diminishes cardiac damage and inflammation during the chronic phase of cardiomyopathy. The synergistic activity of BZ-CMP clearly suggests a potential drug combination for Chagas disease treatment, which would allow a reduction of the effective dose of BZ and an increase in therapeutic safety.

scholarly journals Systematic measurement of combination drug landscapes to predict in vivo treatment outcomes for tuberculosis

2021 ◽  
Author(s):  
Jonah Larkins-Ford ◽  
Talia Greenstein ◽  
Nhi Van ◽  
Yonatan N. Degefu ◽  
Michaela C. Olson ◽  
...  
Keyword(s):  
Combination Therapy ◽  
Treatment Outcomes ◽  
Mouse Models ◽  
Drug Combination ◽  
Combination Therapies ◽  
Therapy Outcomes ◽  
Combination Drug ◽  
Shorten Treatment Duration

AbstractA lengthy multidrug chemotherapy is required to achieve a durable cure in tuberculosis. Variation in Mycobacterium tuberculosis drug response is created by the differing microenvironments in lesions, which generate different bacterial drug susceptibilities. To better realize the potential of combination therapy to shorten treatment duration, multidrug therapy design should deliberately explore the vast combination space. We face a significant scaling challenge in making systematic drug combination measurements because it is not practical to use animal models for comprehensive drug combination studies, nor are there well-validated high-throughput in vitro models that predict animal outcomes. We hypothesized that we could both prioritize combination therapies and quantify the predictive power of various in vitro models for drug development using a dataset of drug combination dose responses measured in multiple in vitro models. We systematically measured M. tuberculosis response to all 2- and 3-drug combinations among ten antibiotics in eight conditions that reproduce lesion microenvironments. Applying machine learning to this comprehensive dataset, we developed classifiers predictive of multidrug treatment outcome in a mouse model of disease relapse. We trained classifiers on multiple mouse models and identified ensembles of in vitro models that best describe in vivo treatment outcomes. Furthermore, we found that combination synergies are less important for predicting outcome than metrics of potency. Here, we map a path forward to rationally prioritize combinations for animal and clinical studies using systematic drug combination measurements with validated in vitro models. Our pipeline is generalizable to other difficult-to-treat diseases requiring combination therapies.One Sentence SummarySignatures of in vitro potency and drug interaction measurements predict combination therapy outcomes in mouse models of tuberculosis.

scholarly journals Azacitidine and Ascorbate Inhibit the Competitive Outgrowth of Human TET2 Mutant HSPCs in a Xenograft Model of Pre-Leukemia

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 887-887
Author(s):  
Yusuke Nakauchi ◽  
Daniel Thomas ◽  
Rajiv Sharma ◽  
M. Ryan Corces ◽  
Andreas Reinisch ◽  
...  
Keyword(s):  
Colony Formation ◽  
Erythroid Differentiation ◽  
High Dose ◽  
Myeloid Lineage ◽  
Hematopoietic Stem ◽  
Nsg Mice ◽  
Base Pair Deletion ◽  
Cd34 Cells

Abstract The TET2 gene is frequently mutated in pre-leukemic hematopoietic stem cells in human acute myeloid leukemia (AML) and encodes for an enzyme that catalyzes the conversion of DNA 5-methylcytosine to 5-hydroxymethylcytosine. Recent studies suggest that (i) the product of this reaction can be enhanced using high dose ascorbate, and (ii) formation of the substrate 5-methylcytosine can be blocked with azacitidine. To understand the mechanisms of TET2 mutation-driven leukemogenesis, we developed two CRISPR/Cas9 approaches to disrupt the TET2 gene in primary human CD34+ HSPCs to mimic TET2-mutated pre-leukemia. First, in "Hit & Run," we use Cas9 with two single-guide RNAs (sgRNAs) to disrupt the TET2 gene within exon 3 (average indel frequencies=94.3%). Second, we using homology directed repair (HDR) of Cas9-mediated dsDNA breaks to disrupt the TET2 gene within exon 7 by inserting a GFP expression cassette to generate in vivo traceable cells. Thus, we have developed a tractable and cell-traceable model that recapitulates TET2-mutated pre-leukemia and clonal hematopoiesis. First, we examined the effects of TET2 disruption on human erythroid differentiation in vitro by culturing bulk CD34+ cells for 10 days under conditions that promote erythroid differentiation. Both Hit & Run and HDR (GFP+) TET2 disruption decreased CD71+CD235+ erythroid differentiation compared to control cells. Exposure to high dose ascorbate partially rescued the erythroid defect in TET2-disrupted cells (Hit & Run, n=3 independent experiments, p<0.02). This underscores the importance of TET2 in promoting erythroid differentiation and suggests TET2 mutations can exert a myeloid lineage skewing sensitive to ascorbate. Next, we investigated the effects of TET2 disruption on hematopoietic colony formation in methylcellulose. Both methods resulted in increased numbers of TET2-disrupted colonies compared to control (Hit & Run, n=4 independent experiments, p<0.0001; HDR, n=3 independent experiments, p<0.0001) and absence of erythroid BFU-E. Interestingly, analysis of indels in Hit & Run colonies showed that serial replating enriched for a 65 base pair deletion that results in a null allele, suggesting that TET2-disrupted cells outcompete normal HSPCs in vitro. Next, we transplanted control or TET2-disrupted Hit & Run CD34+ cells into NSG mice. Primary transplantation at 4 months showed no statistical differences in either engraftment rate (human CD45+) or differentiation (T/ B/ Myeloid cells), although the frequency of TET2 indels increased gradually in CD33+ cells. Intriguingly, 36 weeks after secondary transplantation, we detected a marked expansion of human myeloid lineage cells (lymphoid=22.1%, myeloid=73.0%, Mann-Whitney U, p=0.0485) and a particular increase in a CMML-like CD33highCD14+CD16- population. Furthermore, preliminary data from tertiary transplantation (8 weeks after transplantation) indicates persistent myeloid skewing in the bone marrow in some mice and expansion of TET2-mutant cells, suggesting a CMML-like disease. Finally, we used in vivo competition studies to determine if TET2-disrupted HSPCs are selectively targeted by azacitidine or ascorbate treatment compared to controls. NSG mice were intrafemorally transplanted with a one-to-one ratio of control and TET2-disrupted HSPCs, and 4 months later, these mice were treated with azacitidine (2.5mg/kg/dose, i.p. daily on days 1-5 of a 14-day cycle for 2 cycles) or ascorbate (4g/kg/dose, i.p. twice daily for a month). In PBS control treated mice, the percentage of TET2-disrupted cells increased from 29.3 to 71.6 over 4 weeks. Intriguingly, azacitidine slowed the expansion of TET2-disrupted cells in evaluable mice (delta increase of 42% in PBS vs 5% in azacitidine, p=0.036), but did not eradicate established TET2 pre-leukemia in all evaluable mice. Similarly, high dose ascorbate treatment slowed the rate of expansion to a lesser degree (delta increase of 42% in PBS vs 18.3% in ascorbate, p=0.14). Our data show that TET2 disruption in primary human HSPCs blocked erythroid differentiation, increased colony formation and replating, and caused myeloid skewing and a CMML-like disease in vivo after an extended period of time. In this model, azacitidine or ascorbate treatment slowed expansion of TET2-mutant human pre-leukemic clones raising the intriguing possibility of preventing CHIP progression to de novo AML. Disclosures No relevant conflicts of interest to declare.

A Novel 4-anilino-3-quinolinecarbonitrile Dual Src and Abl Kinase Inhibitor (SKI-606) Has In Vitro Activity on CML Ph+Blast Cells Resistant to Imatinib.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 1991-1991 ◽  
Author(s):  
Tiziana Grafone ◽  
Manuela Mancini ◽  
Emanuela Ottaviani ◽  
Matteo Renzulli ◽  
Frank Boschelli ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Kinase Inhibitor ◽  
Blast Crisis ◽  
G1 Phase ◽  
Inhibition Of Proliferation ◽  
Cd34 Cells ◽  
Dependent Inhibition ◽  
Dose Dependent Inhibition ◽  
Dose Dependent

Abstract The tyrosine kinase Bcr-Abl is the fusion product of a reciprocal translocation between chromosomes 9 and 22, known as Philadelphia chromosome and it is present in the leukemic cells of more than 95% of patients with chronic myeloid leukemia (CML). Overexpression of Bcr-Abl in myeloid cells activates various signaling pathways. Previous studies have demonstrated that certain Src family kinases, such as Hck and Lyn, are also targets of Bcr-Abl activity. Hck and Lyn are expressed and activated in CML blast-crisis patients and their increased expression correlates with disease progression or STI571 resistance in some CML patients. Resistance to STI571 seems to be mediated by amplification of or mutations in the Bcr-Abl gene, reducing sensitivity to this inhibitor; newer Abl inhibitors may be susceptible to the same mechanism of resistance. Alternative strategies for control of CML, including the biological relevance of the Bcr-Abl - Src family kinase pathway, are necessary. One such strategy is the use of a specific small molecule Src kinase inhibitor. Recently, a new class of compounds, 4-anilino-3-quinolinecarbonitrile Src kinase inhibitors, has been synthesized. One member of this class, SKI606, is a dual-specificity inhibitor of both Src family and Abl kinases. To investigate the effect in vitro of SKI-606, we analyzed human cell lines from CML patients in blast crisis (K562, MK2, LAMA) and CD34+ from 9 patients in CML blast crisis using a wide range of concentrations (0.01μM-10μM) of this novel agent. Cell cycle analysis, in particular for the cell lines, showed that a major effect of SKI606 is to alter cell cycle progression, producing G1/S arrest. SKI606 induced dose-dependent inhibition of proliferation with IC50 of 1μM at 24hr. Flow cytometric analysis with Annexin-V showed that SKI-606 induced apoptosis of 50% of cells at 48hr. Western blotting and immuno-blotting analyses showed reduced phosphorylation of Bcr-Abl and also of Lyn and Hck. We also demonstrated activation of Caspase-9, an effector cysteine-protease, after exposure to SKI606. These drug effects also reduced the oncogenic effects of the Bcr-Abl gene product in CD34+ cells from patients with CML blast crisis. SKI606 induced a dose-dependent inhibition of proliferation with an IC50 of 1μM at 48hr and induction of apoptosis at 72hr. Cytofluorimetric analysis after 72hr of exposure revealed marked accumulation of cells in the G1 phase of cell cycle, accompanied by a significant increase in the number of apoptotic cells. In some of these patient samples, we observed hypophosphorylation of Bcr-Abl, Hck and Lyn at low concentration of SKI606 (1uM at 24h, 10uM at 48h). Interestingly, CD34+ cells taken from two of our imatinib-resistant patients with Bcr-Abl point mutations (E255K and Y253H) in the P-loop region of the protein exhibited a significant increase of apoptosis (50%) and a block in G1 phase of the cell cycle after treatment with 1 μM SKI606 for 48h. Our study thus showed a potential therapeutic usefulness of the drug in treatment of CML, particularly in blast crisis phase. Ongoing gene expression profiles will contribute to further understanding of the drug mechanism.

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