Colony Formation Of Hematopoietic Progenitors Research Articles

Micheliolide exerts effects in myeloproliferative neoplasms through inhibiting STAT3/5 phosphorylation via covalent binding to STAT3/5 proteins.

Ruxolitinib is a cornerstone of management for some subsets of myeloproliferative neoplasms (MPNs); however, a considerable number of patients respond suboptimally. Here, we evaluated the efficacy of micheliolide (MCL), a natural guaianolide sesquiterpene lactone, alone or in combination with ruxolitinib in samples from patients with MPNs, JAK2V617F-mutated MPN cell lines, and a Jak2V617F knock-in mouse model. MCL effectively suppressed colony formation of hematopoietic progenitors in samples from patients with MPNs and inhibited cell growth and survival of MPN cell lines in vitro. Co-treatment with MCL and ruxolitinib resulted in greater inhibitory effects compared with treatment with ruxolitinib alone. Moreover, dimethylaminomicheliolide (DMAMCL), an orally available derivative of MCL, significantly increased the efficacy of ruxolitinib in reducing splenomegaly and cytokine production in Jak2V617F knock-in mice without evident effects on normal hematopoiesis. Importantly, MCL could target the Jak2V617F clone and reduce mutant allele burden in vivo. Mechanistically, MCL can form a stable covalent bond with cysteine residues of STAT3/5 to suppress their phosphorylation, thus inhibiting JAK/STAT signaling. Overall, these findings suggest that MCL is a promising drug in combination with ruxolitinib in the setting of suboptimal response to ruxolitinib.

MLN0128, a Potential Target Therapy for Myeloid Proliferation Neoplasia (MPN) with a Benefit of Prolonging Survival

MLN0128 (TAK-228, INK128) is an investigational new drug (IND) that is currently being evaluated in clinical trials and has showed promising efficacies in patients with solid cancer. MLN0128 is one of the second generation of inhibitors for mammalian target of rapamycin (mTORi), which suppresses the dysregulated cell survival signaling activities in cancer cells by specifically inhibiting both mTORC1 and mTORC2 in PI3K/mTOR pathway. MLN0128 was reported inhibiting acute myeloid leukemia (AML) stem cells and progenitors [Zeng et al, Oncotarget, 2016]. However, it is yet to be clarified whether MLN0128 can improve survival time in leukemia patients. Here, we report data from the investigation of the impact of MLN0128 on leukemia survival in mice. We tested MLN0128 in a published mouse model which simultaneously produces experimental mice with genotypes that are associated with pediatric leukemia, including myeloproliferative neoplasm (MPN) and juvenile myelomonocytic leukemia (JMML), and adult chronic myelomonocytic leukemia (CMML) [Vara et al, Blood Adv.2020]. Specifically, mice with deletion of Phosphatase and tensin homolog gene (Pten) and loss-of-heterozygosity of Neurofibromin 1 (Nf1) (Pten Δ/ΔNf1 LOH) develop JMML, mice with Pten deletion and Nf1 heterozygosity (Pten Δ/ΔNf1 Δ/wt) develop pediatric MPN, both of which cause juvenile death; mice with a single copy of Pten deletion and Nf1 LOH (Pten Δ/wtNf1 LOH) develop CMML in adulthood. We previously reported that all of those diseased mice shared a feature with elevated activities in PI3K/MAPK/mTOR pathways. We first collected nucleated cells from bone marrow (BM) of the diseased mice and tested the inhibition of MLN0128 on colony formation of hematopoietic progenitors (CFU-GM) at a concentration range of 1-1000nM. Our data demonstrated that MLN028 significantly inhibited the CFU-GM colony formation of BM cells from mice with all three diseased genotypes, in a dose-dependent fashion. Importantly, the IC50 for CFU-GM was less than 100nM, which is in the concentration range of MLN0128 in the blood from patients who orally received MLN0128 at dosage range of 4-12mg/day [Ghobrial et al AJH 2016]. We then investigated the impact of MLN0128 on life span in diseased mice. We administrated MLN0128 in gavage at dosages of 0.5-1μg/g body weight, starting at an age of postnatal day 17-18 (PND17-18), 3-5 times a week, repeated for 5 weeks followed by a pause in 6 th week every course. Our data showed that MLN0128 significantly reduced the spleen sizes in treated mice with all diseased genotypes comparing to those treated with vehicle. The survival time was significantly prolonged in MLN0128-treated mice with genotypes of MNP (n=7 vs 3, median 150 vs 102 days) and CMML (n=7 vs 6, median 100 vs 56 days), comparing with the mice treated by vehicle. Unfortunately, we did not observe a survival improvement in treated JMML mice, but substantially swollen kidneys were found in the dead JMML mice, which was likely due to tumor-lysis syndrome because JMML mice carried a significantly worse leukemic burden. In conclusion, our data demonstrate that MLN0128 can significantly inhibit colony formation of BM cells from mice with genotypes of JMML, MPN, and CMML; and MLN0128 can significantly expand survival time of mice with genotypes of MPN and CMML, which has never been reported in previous studies that were related to mTORi in pediatric leukemia. Our data suggest that MLN0128 may be a potential target therapy for pediatric MPN and CMML with a benefit of prolonging survival. Further investigation is ongoing to evaluate the potential of MLN0128 in improving survival of JMML mice after minimizing the tumor-lysis syndrome by tipping down MLN0128 dosage. DisclosuresBertaina: Cellevolve Bio: Membership on an entity's Board of Directors or advisory committees; Neovii: Membership on an entity's Board of Directors or advisory committees; AdicetBio: Membership on an entity's Board of Directors or advisory committees.

Targeting Insulin-like Growth Factor/Insulin Receptor Signaling to Overcome JAK2 Inhibitor Persistence in Myeloproliferative Neoplasms

Classic Philadelphia chromosome-negative myeloproliferative neoplasms (MPNs) are driven by aberrant activity of the JAK2 tyrosine kinase. While JAK2 inhibitors rarely induce molecular remission, they provide quality of life improvements for patients and thus will be a mainstay for MPN treatment for the foreseeable future. This near ubiquitous upfront ineffectiveness of JAK2 inhibitors must be overcome if they are going to contribute to a significant disease-altering therapy for MPNs. The insulin-like growth factor-1 receptor (IGF1R) can contribute to resistance to kinase inhibitor cancer therapeutics and a recent study demonstrated a requirement of IGF1R in JAK2-V617F-induced MPN in mice. Furthermore, IGF is known to play a role in neoplastic erythroid colony formation of hematopoietic progenitors from MPN patients. Thus, there is evidence for a potential role for IGF1R signaling in the context of JAK2 inhibition, particularly in the setting of JAK2 inhibitor resistance/persistence, a concept that remains underexplored.We observed an increase in activated IGF1R and insulin receptor (IR), which is structurally similar to IGF1R, in JAK2-V617F-driven MPN model cells (UKE1, SET2, and HEL) that are persistently growing in high concentration of the JAK inhibitor ruxolitinib. This suggests cell signaling is re-wired to alter the activity of IGF1R/IR in this state. Interestingly, in ruxolitinib sensitive/drug naïve cells, we observed a rapid increase in IGF1R-beta and IR-beta protein levels in response to ruxolitinib treatment. These observations further suggest IGF1R/IR may contribute to the response of cells to ruxolitinib.To assess the role of IGF1R/IR activation in both ruxolitinib-persistent cells and drug naïve cells, we utilized the IGF1R/IR selective kinase inhibitor OSI-906. OSI-906 weakly inhibited growth of JAK2-V617F-driven cells (GI50s between 1 and 8 uM), and cells continued to proliferate at a reduced rate in the presence of a high concentration of drug. The IGF1R/IR inhibitor had little effect on cell viability, but in combination with ruxolitinib, it synergistically suppressed cell growth in part due to augmented induction of apoptosis. This combination also synergistically suppressed the neoplastic (erythropoietin-independent) growth of erythroid colonies of hematopoietic progenitors from MPN patients, which supports previous work indicating a role for serum IGF1 in this erythroid colony formation.Low concentration of IGF1R/IR inhibitor (0.1 uM) suppressed cell growth and viability of cells persistently growing in ruxolitinib yet had no effect on drug naïve cells. This along with our observation of enhanced activation of IGF1R/IR in these cells suggests that in the presence of ruxolitinib, cells may become more dependent on IGF1R/IR activity. We have always observed that high concentration of ruxolitinib is unable to reduce cell viability to zero in certain cell lines (UKE1 & HEL). Ruxolitinib (10 uM) induces a G1 cell cycle arrest in these cells and a sub-population of persister cells remains viable for several weeks. These persister cells remain competent for cell growth once ruxolitinib is removed. However, OSI-906 completely prevented the development of ruxolitinib persister cells. Culturing UKE1 cells in low serum sensitized cells to ruxolitinib and prevented the survival of persister cells. Supplementation of exogenous IGF-1 partially rescued this sensitivity and allowed for persister cells to survive, suggesting IGF-1 may contribute to the development of ruxolitinib persistence.Finally, in addition to an increase in IGF1R/IR protein, ruxolitinib (10 uM) treatment also led to long-term depletion of IRS1/2 levels, the main signaling docking protein that binds to IGF1R/IR. This correlated with re-activation of the ERK pathway, which has been reported to be due to SHC binding to IGF1R in the absence of IRS1/2. Because these persister cells remain arrested in G1, ERK activation is not sufficient to support cell growth, but perhaps supports cell survival. This may highlight potential therapeutic targets to prevent ruxolitinib persistence. Identifying mechanistic aspects of cell survival in the presence of ruxolitinib could play a significant role in the development of combination therapies for MPNs. Our data support the notion that ruxolitinib treatment may alter the dynamics of IGF1R/IR signaling leading to tolerance of cells to ruxolitinib. DisclosuresReuther:Incyte Corporation: Research Funding.

The CDK6 Inhibitor Palbociclib in Combination with Ruxolitinib Remarkably Improves Myelofibrosis in Murine Models

Myelofibrosis (MF) is the deadliest form of myeloproliferative neoplasms (MPN), characterized by deposition of fibrous tissues in the bone marrow, abnormal megakaryopoiesis, ineffective erythropoiesis and extramedullary hematopoiesis. The median survival for patients with MF is ~5 years. The JAK2V617F mutation, which results in constitutive activation of the JAK2 tyrosine kinase, has been found in 50-60% patients with MF. Additional mutations in the genes encoding thrombopoietin receptor (MPL) and calreticulin (CALR) have been found at a lower frequency than JAK2V617F in MF. The JAK1/JAK2 inhibitor ruxolitinib has been approved for treatment of MF. Although ruxolitinib can reduce splenomegaly and constitutional symptoms, it does not cure or prevent fibrosis in patients with MPN/MF. Additionally, ruxolitinib treatment does not improve survival in MF patients. So, there is a critical need to identify new therapeutic targets and develop novel therapies for MF. We have found that expression of CDK6 is significantly elevated in mouse and human MPN hematopoietic cells/progenitors. We also observed that knockdown of CDK6 significantly inhibited proliferation of BA/F3-EpoR-JAK2V617F and HEL cells expressing JAK2V617F but not wild-type JAK2 expressing BA/F3-EpoR cells, suggesting that CDK6 is a bona fide target in MPN. In this study, we investigated the efficacy of CDK4/6 inhibitor Palbociclib (PD-0332991) in hematopoietic cells expressing JAK2V617F and MPLW515L and murine models of MF. Since Palbociclib is an FDA-approved drug for treatment of breast cancer, information on the dose, pharmacokinetics and toxicity of Palbociclib in human is already available. Thus, it would be easier to move this drug into clinic and repurpose it for treatment of MPN/MF. We observed that Palbociclib treatment (0.25-0.5 uM) significantly reduced the proliferation of murine BA/F3 cells expressing JAK2V617F or MPLW515L. Palbociclib treatment also significantly inhibited human JAK2V617F-positive HEL, SET-2 and UKE-1 cells. However, Palbociclibonly modestly affected proliferation of wild type JAK2-expressing BA/F3 cells at higher concentrations. Palbociclib treatment also resulted in marked apoptosis in BA/F3-EpoR cells expressing JAK2V617F. Furthermore, Palbociclib and ruxolitinib synergistically induced apoptosis in JAK2V617F-expressing hematopoietic cells. Additionally, Palbociclib significantly inhibited hematopoietic progenitor colony formation in MPN/MF CD34+ cells. We previously generated conditional JAK2V617F knock-in mice. Whereas expression of heterozygous JAK2V617F induces a PV-like disease, homozygous JAK2V617F expression promotes rapid progression to myelofibrosis. We have utilized the homozygous JAK2V617F mice to test the in vivo efficacy of Palbociclib alone and in combination with ruxolitinib against MF. Treatment of Palbociclib alone significantly reduced the WBC and neutrophil counts and decreased the spleen size in homozygous JAK2V617F mice. Combined treatment of Palbociclib 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 of homozygous JAK2V617F mice treated with Palbociclib or Palbociclib and ruxolitinib combination. We also have tested the in vivo efficacy of Palbociclib and Palbociclib/ruxolitinib combination in MPL mutant (MPLW515L) mouse model of MF. We observed similar decrease in WBC and neutrophil counts and reduction in splenomegaly in MPL mutant mice upon treatment of Palbociclib alone or in combination with ruxolitinib. Combined treatment of Palbociclib and ruxolitinib remarkably reduced fibrosis in the bone marrow of MPL mutant mice. Overall, our results suggest that palbociclib in combination with ruxolitinib may have therapeutic potential against MF and support the clinical investigation of palbociclib and ruxolitinib combination in patients with MF. Disclosures No relevant conflicts of interest to declare.

The Activated TGFβ Pathway in Shwachman Diamond Syndrome Impairs Hematopoiesis and Is Down-Regulated By Deletion of 7q

Monosomy 7 or deletion of 7q (del(7q)) are common cytogenetic abnormalities in pediatric MDS. Monosomy 7/del(7q) frequently arises in the context of inherited bone marrow failure (BMF) syndromes such as Shwachman Diamond Syndrome (SDS), an autosomal recessive disorder caused by biallelic mutations in the SBDS gene. Monosomy 7/del(7q) is associated with high grade MDS and a high risk of malignant transformation to acute myelogenous leukemia, a major cause of morbidity and mortality for patients with inherited BMF. The basis for this propensity to develop monosomy 7/del(7q) remains unclear. Whether monosomy 7/del(7q) functions as a driver of MDS, or is merely an associated marker of clonal progression in BMF, remains a critical question. The aim of this study is to investigate the molecular consequences of del(7q) in the context of BMF with the goal of developing more effective treatments.The lack of synteny between murine and human chromosome 7 has posed a major barrier to modeling monosomy 7/del(7q). Therefore, we utilized SDS patient-derived induced pluripotent stem cells (SDS-iPSC), which recapitulate the hematopoietic defects of this BMF syndrome. Independent SDS-iPSC lines were derived from two patients with SDS. Proliferationof SDS-iPSCs was reduced relative to wild type controls without a concomitant increase in cell death. Compared to normal iPSC controls, SDS iPSC-derived CD34+ cells exhibited reduced hematopoietic differentiation to CD45+ cells, decreased erythroid and myeloid hematopoietic progenitor colony formation in methylcellulose, and impaired terminal myeloid differentiation.To study the molecular and biologic effects of del(7q), a deletion of the MDS-associated region of the long arm of chromosome 7 was genomically engineered using a previously published modified Cre-Lox approach. The deletion of 7q in SDS iPSCs spanned band q11.23 to q36.3. The SDS-del(7q) iPSCs expressed stem cell markers and formed teratomas as efficiently as their isogenic SDS iPSC controls.Deletion of 7q failed to confer a relative growth advantage even within the context of BMF. Indeed, proliferation of the SDS-del(7q) iPSCs was reduced below that of both the isogenic SDS iPSCs and normal controls without an increase in cell death. To investigate the effect of del(7q) on hematopoiesis, iPSC-derived CD34+ cells were assayed for their ability to undergo hematopoietic differentiation. SDS-del(7q) CD34+ cells demonstrated reduced differentiation to CD45+ cells, reduced methylcellulose hematopoietic progenitor colony formation and impaired terminal myeloid differentiation compared with isogenic SDS CD34+ cells. These data demonstrate that deletion of 7q fails to confer a relative growth advantage relative to isogenic SDS iPSCs and results in a further impairment of hematopoiesis.To gain insight into the mechanisms of del7q-associated clonal evolution in SDS, we performed RNA sequencing (RNAseq) of SDS and SDS-del(7q) iPSC. Expression of TGFβ pathways and their downstream targets were reduced in SDS del(7q) iPSCs compared to isogenic SDS iPSC controls. This observation was intriguing because single cell RNAseq analysis of primary SDS bone marrow cells revealed that TGFβ pathway is hyperactivated in SDS. Additionally, a quantitative DNA aptamer-based proteomic analysis demonstrated elevated levels of TGFβ ligands in plasma from six SDS patients compared to 6 age-matched controls. Western blot analysis showed increased P-SMAD2 levels in SDS iPSCs compared to SDS-del(7q) and normal controls, while total levels of SMAD2 were unchanged. Pharmacological targeting of TFGβ with small molecule inhibitors resulted in selective improvement of SDS hematopoietic colony formation without stimulating outgrowth of the isogenic SDS-del(7q) cells. Although del(7q) does not seem to confer an advantage in growth or hematopoiesis in vitro, we are testing whether the deletion of 7q might confer relative resistance to elevated TGFβ levels in vivo, resulting in an overall survival advantage of the del7q clone.These results demonstrate that the TGFβ pathway is activated in SDS, and that deletion of 7q reverses the TGFβ pathway hyperactivation in SDS. Inhibition of TGFβ selectively rescues hematopoiesis in SDS but not in isogenic del7q cells, suggesting a potential strategy to treat bone marrow failure without stimulating del7q clonal outgrowth. DisclosuresStegmaier:Novartis: Consultancy, Research Funding. Shimamura:TransCellular Therapeutics: Other: Spouse is majority shareholder.

Changes in Several Disease Parameters Including Abzymes and Hematopoietic Progenitor Colony Formation in Brain Inflammation and Demyelination

Changes in Several Disease Parameters Including Abzymes and Hematopoietic Progenitor Colony Formation in Brain Inflammation and Demyelination

Abstract 5376: Requirement of TET2 in MLL-AF9 mediated leukemogenesis

Abstract TET methylcytosine dioxygenase 2 (TET2) catalyzes the conversion of 5-methylcytosine to 5-hydroxymethylcytosine resulting in DNA demethylation. Loss-of-function mutations or deletions of TET2 have been found in several myeloid malignancies including myelodysplastic syndrome (MDS), myeloproliferative neoplasms (MPN), chronic myelomonocytic leukemia (CMML) and acute myeloid leukemia (AML). Moreover, deletion of TET2 leads to myeloid malignancies in mice. Therefore, TET2 has been suggested to play a tumor suppressor role in myeloid malignancies. In this study, unexpectedly, we have uncovered a tumor promoter function for TET2 in MLL rearranged leukemia. Chromosomal rearrangements of the Mixed Lineage Leukemia (MLL) gene have been found in ~10% of acute leukemia and are associated with poor prognosis. We observed that depletion of TET2 significantly inhibited the proliferation of MLL-AF9 positive MOLM14 leukemia cells. Similarly, knockdown of TET2 markedly reduced proliferation in murine Ba/F3 cells stably expressing MLL-AF9. Conversely, overexpression of TET2 significantly increased proliferation in MLL-AF9 expressing MOLM14 and Ba/F3 cells. In addition, deletion of TET2 significantly inhibited MLL-AF9 mediated myeloid colony formation in the bone marrow (BM) cells ex vivo. Next, we assessed the in vivo effects of TET2 deletion in MLL-AF9-mediated leukemogenesis using retroviral bone marrow transduction and transplantation assays. Whereas recipients of MLL-AF9-transduced wild type BM exhibited marked increase in white blood cells (WBC) and neutrophils in their peripheral blood and significant increase in the spleen size, recipients of MLL-AF9-transduced TET2-deficient BM showed significantly reduced WBC and neutrophil counts and reduced spleen size. Flow cytometric analysis showed significantly increased myeloid precursors (Gr-1+/Mac1+) and granulocyte macrophage progenitors (GMP) in the BM of transplanted animals receiving MLL-AF9 expressing wild type BM compared with recipients of MLL-AF9 expressing TET2-deficient BM. Furthermore, transplanted animals receiving MLL-AF9-transduced wild type BM showed marked increase in hematopoietic stem/progenitor cells (LSK; Lin-Sca-1+c-kit+), which was significantly reduced upon TET2 deletion. Deficiency of TET2 also significantly reduced MLL-AF9-mediated increase in hematopoietic progenitor colony formation in the BM. In addition, deletion of TET2 resulted in increased overall survival in mice expressing MLL-AF9. Together, these data indicate that TET2 is required for leukemic transformation mediated by MLL-AF9. Our results suggest a novel tumor promoter function for TET2 in MLL rearranged leukemia. Citation Format: Dipmoy Nath, Golam Mohi. Requirement of TET2 in MLL-AF9 mediated leukemogenesis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5376. doi:10.1158/1538-7445.AM2017-5376

Abstract 223: Brain-derived Neurotrophic Factor Promotes Regenerative Functions of Human Endothelial Progenitor Cells - Roles of miRNAs

Our previous study has demonstrated that brain-derived neurotrophic factor (BDNF) enhances anti-oxidant capacity of endothelial progenitor cells (EPCs). Since BDNF is produced in cerebrovascular endothelial cells, and functions as a vasoprotective molecule, we hypothesized that BDNF might enhance regenerative capacity of EPCs. Early EPCs (circulating angiogenic cells) were derived from healthy male subjects (48±1.5 years old, n=10). EPCs were treated with BDNF 100 ng/ml for 7 hours, and miRNA transcriptome was examined using Affymetrix miRNA array (version 3.0). BDNF significantly changed 31 miRNAs (n=4, P<0.05, fold-change cut off=1.5). Notably, miR-181c level was increased by 1.9 fold (P<0.05) after BDNF treatment. Since miR-181c is known to down-regulate TNF-α expression, BDNF may protect EPCs by promoting anti-inflammatory phenotype. We also found that BDNF decreased miR-433 level by 1.6 fold (P<0.05). We further evaluated miR-433 function in human early EPCs. Knock-down of miR-433 in EPCs using siRNA significantly increased expression of guanylate binding protein 2 (GBP2) (n=6, P<0.05), an established direct target for miR-433. In contrast, treatment with miR-433 mimic significantly reduced GBP2 protein expression in EPCs (n=4, P<0.05). Since GBP2 has been shown to play an important role in the growth and colony formation of hematopoietic progenitors, BDNF/miR-433/GBP2 signaling may enhance angiogenic capacity of EPCs. In addition, neither miR-433-siRNA nor miR433 mimic significantly changed protein expressions of other targets such as growth factor receptor-binding protein2, cAMP response element-binding protein-1, cyclin-dependent kinase 6, and γ glutamyl cysteine synthetase (n=4-6, P>0.05). In summary, changes in miRNA transcriptome induced by treatment with BDNF may promote anti-inflammatory and angiogenic phenotype of EPCs. These effects of BDNF may have important implications for therapeutic enhancement of regenerative capacity of EPCs.

Changes in different parameters, lymphocyte proliferation and hematopoietic progenitor colony formation in EAE mice treated with myelin oligodendrocyte glycoprotein.

Myelin oligodendrocyte glycoprotein (MOG) is an antigen of the myelin sheath, which may trigger immune cell responses and the production of auto-antibodies in multiple sclerosis (MS). In this study, we used MOG(35-55) -induced experimental autoimmune encephalomyelitis (EAE), a model of human MS, to assess the production of catalytically active immunoglobulin G (IgG) antibodies or abzymes which have been shown to be present in sera of patients with several autoimmune diseases. Here, we show that IgGs from the sera of control C57BL/6 mice are catalytically inactive. During development of EAE, a specific reorganization of the immune system of mice occurred leading to a condition which was associated with the generation of catalytically active IgGs hydrolysing DNA, myelin basic protein (MBP) and MOG which was associated with increased proteinuria, changes in differentiation of mice bone marrow hematopoietic stem cells (HSCs) and an increase in proliferation of lymphocytes in bone marrow, spleen and thymus as well as a significant suppression of cell apoptosis in these organs. The strongest alterations were found in the early disease phase (18-24 days after immunization) and were less pronounced in later EAE stages (40 days after EAE induction). We conclude that a significant increase in DNase and proteolytic activities of antibodies may be considered the earliest statistically significant marker of MOG-induced EAE in mice. The possible differences in immune system reorganizations during preclinical phases of the disease, acute and late EAE, leading to production of different auto-antibodies and abzymes as well other changes are discussed.

Jak2 V617F Induced TNF Resistance as a Mechanism of Clonal Expansion In Myeloproliferative Neoplasm

AbstractAbstract 795Elevated levels of pro-inflammatory cytokines such as TNF-alpha (TNF) have been implicated in the constitutional symptoms commonly seen in JAK2 V617F MPN. However, it is unclear to what extent they contribute to the disease process itself. We hypothesized that JAK2 V617F may confer a selective advantage on hematopoietic progenitors residing in the high cytokine milieu uniquely present MPN patients, focusing on the pro-inflammatory cytokine TNF.We used ELISA and qPCR to measure TNF in plasma and neutrophils of MPN patients and normal controls. All MPN subtypes had increased plasma concentrations of TNF, with primary myelofibrosis, polycythemia vera and essential thrombocythemia displaying 6, 4 and 2 fold higher mean concentrations of plasma TNF as compared to normal controls, respectively. Median neutrophil TNF mRNA was 2 fold higher in PV patients as compared to normal controls (p<0.001). We then tested the effect of TNF on CFU-GM and BFU-E formation from MPN vs normal mononuclear cells. At low concentrations (1ng/ml) of TNF we observed a decrease in BFU-E from normal controls to 82% of that without TNF but an increase in MPN BFU-E to 120% (p<0.05) of that without TNF. We also observed a blunting of TNF induced CFU-GM suppression in MPN versus normal. We genotyped colonies and found that in the presence of TNF colony formation was almost exclusively JAK2 V617F, demonstrating that TNF selects for JAK2 V617F colony growth. To distinguish between direct and indirect TNF effects (i.e. effects mediated through bystander cells) as well as to potentially augment the differential effects of TNF on normal and MPN by the use of a more homogenous cell population we performed equivalent colony formation experiments on CD34+ cells. The most notable difference observed was the effect 10ng/ml TNF on CFU-GM. TNF reduced CFU-GM colony formation by normal CD34+ cells to 50%, but increased CFU-GM colony formation by MPN cells to 120% (p<0.001). The suppressive effect of 10ng/ml on BFU-E was also blunted in MPN versus normal CD34+ cells (49% vs 30% of that without TNF) (p=0.054). Genotyping of MPN colonies allowed us to compare the effect of TNF on colony formation on three distinct populations: (1) JAK2WT MPN CD34+ cells, (2) JAK2V617F MPN CD34+ cells and (3) normal CD34+ cells. As compared to normal controls (59% decrease), JAK2 non-mutated MPN CD34+ cells are hypersensitive to myeloid colony suppression (78% decrease) whereas TNF paradoxically enhances colony formation by JAK2 V617F CD34+ cells (180% increase). This suggests that “pre-JAK2” CD34+ cells are functionally disadvantaged when compared to those from normal controls and implicates TNF in the selection and clonal expansion of the JAK2 V617F clone. To confirm the latter we tested whether ectopic expression of JAK2 V617F in murine hematopoietic progenitors blunts TNF-induced inhibition of myeloid colony formation. In the presence of TNF, colony formation of hematopoietic progenitors expressing empty vector or BCR-ABL (included as an additional control) was decreased to 60%, while TNF had no effect on progenitors with ectopic expression of JAK2 V617F. This suggests that JAK2 V617F directly imparts TNF resistance. To interrogate the role of TNF for the development of MPN in vivo, we infected TNF+/+ and TNF−/− bone marrow with JAK2 V617F-GFP retrovirus and transplanted the cells into genotypically matched recipients. Two strains of mice were used in independent experiments (C57BL6 and B6129S6). Equal numbers of TNF+/+ and TNF−/− GFP+ cells per mouse were injected. Although peripheral blood counts were comparable, JAK2 V617F burden (assessed by GFP%) was significantly lower in recipients of TNF−/− knockout bone marrow compared to recipients of TNF+/+ marrow. This difference in JAK2 V617F burden between the two groups was apparent in peripheral blood (47% vs 3.7%, p <0.05) and bone marrow (52% vs 2%, p<0.05) and the spleens of the TNF−/− were smaller (0.46g vs 0.11g, p=0.078).Our data suggest that JAK2 V617F-induced TNF resistance may drive the expansion of the mutant clone by conferring a growth advantage to JAK2 V617F cells over their JAK2 wild-type counterparts in the high cytokine microenvironment of MPN patients. Further, JAK2WT MPN cells exhibit intrinsic TNF hypersensitivity compared to normal cells, which greatly enhances this selective advantage. This JAK2V617F-negative state may play a role in the predisposition to acquire MPN. Disclosures:No relevant conflicts of interest to declare.

Mitotic Spindle Dysfunction Promotes Genomic Instability In Marrow Failure

AbstractAbstract 880Shwachman-Diamond syndrome (SDS) is an autosomal recessively inherited disorder associated with bone marrow failure and leukemia predisposition. The majority of patients harbor biallelic mutations in the SBDS gene. The SBDS protein has been implicated in several cellular functions including ribosome biogenesis and microtubule stabilization during mitosis. We have previously found that SBDS deficiency results in multipolar spindles, centrosome amplification and aneuploidy, implicating a role for SBDS in cell division. The mechanism by which SBDS functions to ensure proper spindle assembly and DNA segregation during mitosis remains unknown. Here we present evidence that SBDS functions to promote mitotic spindle stability both by directly modifying microtubule dynamics and through a microtubule crosslinking activity. Importantly, the microtubule stabilizing effects of SBDS appear to be essential for the growth and differentiation of hematopoietic progenitor cells. Specifically, we found that SBDS deficiency resulted in shortened mitotic spindle length and decreased spindle acetylation, a marker of microtubule stability. The loss of microtubule stability in the absence of SBDS function may be due to changes in microtubule dynamics or reduction in microtubule crosslinking activity, as we found that addition of recombinant purified wild-type SBDS to polymerized microtubules in vitro increases their polymerization rate and strongly promotes microtubule bundling. Interestingly, recombinant patient-derived missense mutant SBDS proteins showed a marked decrease in their microtubule bundling ability. To assess whether spindle destabilization contributes to marrow failure, we modeled hematopoiesis in the absence of SBDS in vitro. When SBDS expression was knocked down in human CD34+ cells, proliferation, differentiation, and hematopoietic progenitor colony formation were impaired, consistent with published data on primary marrow from SDS patients. The addition of taxol at concentrations that significantly impaired hematopoiesis in control CD34+ cells resulted in stable to improved hematopoiesis in the SBDS-deficient CD34+ cells. Based on these data, we hypothesize that spindle destabilization by SBDS loss promotes genomic instability, which in turn, contributes to marrow failure and leukemia predisposition. Disclosures:No relevant conflicts of interest to declare.

Role of Schlafen 2 (SLFN2) in the Generation of Interferon α-induced Growth Inhibitory Responses

The precise STAT-regulated gene targets that inhibit cell growth and generate the antitumor effects of Type I interferons (IFNs) remain unknown. We provide evidence that Type I IFNs regulate expression of Schlafens (SLFNs), a group of genes involved in the control of cell cycle progression and growth inhibitory responses. Using cells with targeted disruption of different STAT proteins and/or the p38 MAP kinase, we demonstrate that the IFN-dependent expression of distinct Schlafen genes is differentially regulated by STAT complexes and the p38 MAP kinase pathway. We also provide evidence for a key functional role of a member of the SLFN family, SLFN2, in the induction of the growth-suppressive effects of IFNs. This is shown in studies demonstrating that knockdown of SLFN2 enhances hematopoietic progenitor colony formation and reverses the growth-suppressive effects of IFNalpha on normal hematopoietic progenitors. Importantly, NIH3T3 or L929 cells with stable knockdown of SLFN2 form more colonies in soft agar, implicating this protein in the regulation of anchorage-independent growth. Altogether, our data implicate SLFN2 as a negative regulator of the metastatic and growth potential of malignant cells and strongly suggest a role for the SLFN family of proteins in the generation of the antiproliferative effects of Type I IFNs.

Reconstitution of erythroid, megakaryocyte and myeloid hematopoietic support function with neutralizing antibodies against IL-4 and TGFβ1 in long-term bone marrow cultures infected with LP-BM5 murine leukemia virus

Murine acquired immunodeficiency syndrome (MAIDS) induced by a defective LP-BM5 murine leukemia virus (MuLV) produces hematopoietic cytopenias similar to HIV in patients with AIDS. The pathogenesis of MAIDS induced cytopenias remains obscure; however, direct retroviral infection of bone marrow stroma has been implicated to play a role. To evaluate the consequential effect of viral infection, primary stromal cell cultures were transiently incubated in vitro with LP-BM5 MuLV viral supernatant. Reverse transcription polymerase chain reaction (RT-PCR) and Southern blot hybridization revealed that defective LP-BM5 MuLV infection resulted in elevated levels of IL-4 and TGFβ1 transcript expression in infected stromal cells. The increased expression of both IL-4 and TGFβ1 transcripts was associated with enhanced production of corresponding proteins as determined by quantitative western blot analyses. Hematopoietic reconstitution assays revealed that the hematopoietic support function of stromal cells was significantly reduced following transient exposure to LP-BM5 MuLV. The production of nonadherent mononuclear cells and the growth of myeloid, megakaryocyte and erythroid lineages were all suppressed in infected cultures. Culture supernatant conditioned by infected stromal cells demonstrated growth-inhibitory activity for hematopoietic progenitor colony formation. This growth-inhibitory activity could be significantly abolished by addition of anti-IL-4 and/or anti-TGFβ1 neutralizing antibodies to the culture supernatant or directly to the stromal cell cultures. This study demonstrates LP-BM5 MuLV increases two known cytokines to suppress hematopoiesis implicating viral infection can directly suppress hematopoiesis mediated by inhibitors released from marrow stroma.

Thrombopoietin Enhances Proliferation and Differentiation of Murine Yolk Sac Erythroid Progenitors

AbstractThrombopoietin (TPO), the ligand for the receptor proto-oncogene c-Mpl, has been cloned and shown to be the critical regulator of proliferation and differentiation of megakaryocytic lineage. Initially, TPO was not considered to have the activity on hematopoietic lineages other than megakaryocytes. Recently, however, TPO was reported to enhance the in vitro erythroid colony formation from human bone marrow (BM) CD34+ progenitors or from mouse BM cells in combination with other cytokines. We examined the effects of TPO on the colony formation of hematopoietic progenitors in mouse yolk sac. TPO remarkably enhanced proliferation and differentiation of erythroid-lineage cells in the presence of erythropoietin (Epo). This effect was observed even in the absence of Epo. Compared with adult BM, yolk sac turned out to have relatively abundant erythroid and erythro-megakaryocytic progenitors, which responded to TPO and Epo stimulation. TPO similarly stimulated erythroid colony formation from in vitro differentiation-induced mouse embryonic stem (ES) cells whose hematopoietic differentiation status was similar to that of yolk sac. These findings help to understand the biology of hematopoietic progenitors of the early phase of hematopoiesis. Yolk sac cells or in vitro differentiation-induced ES cells would be good sources to analyze the TPO function on erythropoiesis.

Raf-1 protein is required for growth factor-induced proliferation of primitive hematopoietic progenitors stimulated with synergistic combinations of cytokines.

Raf-1 is a serine/threonine kinase that has been identified as a component of growth factor-activated signal transduction pathways, and is required for growth factor-induced proliferation of leukemic cell lines and colony formation of hematopoietic progenitors stimulated with single colony-stimulating factors, which promote the growth of committed hematopoietic progenitor cells. However, it is known that the most primitive progenitors in the bone marrow require stimulation with multiple cytokines to promote cell growth. We have determined that c-raf antisense oligonucleotides inhibit the growth of murine lineage-negative progenitors stimulated with two-, three- and four-factor combinations of growth factors, including GM-CSF + interleukin (IL)- 1, IL-3 + steel factor (SLF), IL-3 + IL-11 + SLF and IL-3 + IL-11 + SLF + G-CSF. In addition, c-raf antisense oligonucleotides inhibit the synergistic response of the MO7e human progenitor cell line induced to proliferate with IL-3 + SLF (99%) or GM-CSF + SLF (99%). In contrast, c-raf antisense oligonucleotides only partially inhibited day 14 colony formation of CD34+ human progenitors stimulated with IL-3 + SLF (50%) or GM-CSF + SLF (55%) but completely inhibited day 7 colony formation. However, pulsing CD34+ cells with additional oligonucleotides on day 7 of the colony assay further inhibited day 14 colony formation (70%-80%). Furthermore, a comparison of the effect of c-raf antisense oligonucleotides on the synergistic response of normal human fetal liver cells in [3H]thymidine incorporation assays and colony assays showed strong inhibition in short-term proliferation assays and partial inhibition in 14-day colony assays. Taken together, these results demonstrate that partial inhibition of colony formation of primitive human progenitors stimulated with multiple growth factors is a result of the length (14 days) of the human colony assay and does not represent a differential requirement of primitive progenitors for Raf-1. Thus Raf-1 is required for the proliferation and differentiation of primitive hematopoietic progenitor cells stimulated with synergistic combinations of cytokines.

In vitro potency of inhibition by antiviral drugs of hematopoietic progenitor colony formation correlates with exposure at hemotoxic levels in HIV positive humans

In vitro potency of inhibition by antiviral drugs of hematopoietic progenitor colony formation correlates with exposure at hemotoxic levels in HIV positive humans

Human interleukin-4 enhances stromal cell-dependent hematopoiesis: costimulation with stem cell factor

Interleukin-4 (IL-4) has distinct hematopoietic activities, primarily as a costimulant with other cytokines to enhance colony formation of hematopoietic progenitors. We investigated the influence of IL-4 on stromal cell-supported long-term cultures (LTCs) of normal human bone marrow. Addition of IL-4 to LTCs of unseparated bone marrow or highly enriched CD34+ cells resulted in a significant increase of myeloid progenitors in the nonadherent, as well as in the stromal cell-adherent cell populations. In contrast, the total cell number was not influenced by IL-4, suggesting a selective effect on primitive progenitor cells. Cord blood cells or CD34+ bone marrow cells were incubated with stem cell factor (SCF) and/or IL-4 in stromal cell-free cultures. In these experiments, a twofold to fivefold increase of myeloid progenitor cells was observed in the presence of SCF and IL-4 as compared with SCF alone. Preincubation of the stromal cell cultures with IL-4 resulted in an enhanced adherence of CD34+ cells to the stromal layer. Secretion of hematopoietic growth factors produced by the stromal cells, such as granulocyte-macrophage colony-stimulating factor (G-CSF), and IL-1, was inhibited by IL-4. Thus, the increase of hematopoietic progenitors in LTCs, as observed in the presence of IL-4, can be at least partially explained by a costimulation of SCF and IL-4 on primitive progenitor cells and by an enhancement of hematopoietic cells to stroma. The downregulation of CSFs by IL-4 might prevent the expansion of the mature hematopoietic cell compartment.

Human Interleukin-4 Enhances Stromal Cell-Dependent Hematopoiesis: Costimulation With Stem Cell Factor

Abstract Interleukin-4 (IL-4) has distinct hematopoietic activities, primarily as a costimulant with other cytokines to enhance colony formation of hematopoietic progenitors. We investigated the influence of IL-4 on stromal cell-supported long-term cultures (LTCs) of normal human bone marrow. Addition of IL-4 to LTCs of unseparated bone marrow or highly enriched CD34+ cells resulted in a significant increase of myeloid progenitors in the nonadherent, as well as in the stromal cell-adherent cell populations. In contrast, the total cell number was not influenced by IL-4, suggesting a selective effect on primitive progenitor cells. Cord blood cells or CD34+ bone marrow cells were incubated with stem cell factor (SCF) and/or IL-4 in stromal cell-free cultures. In these experiments, a twofold to fivefold increase of myeloid progenitor cells was observed in the presence of SCF and IL-4 as compared with SCF alone. Preincubation of the stromal cell cultures with IL-4 resulted in an enhanced adherence of CD34+ cells to the stromal layer. Secretion of hematopoietic growth factors produced by the stromal cells, such as granulocyte-macrophage colony-stimulating factor (G-CSF), and IL-1, was inhibited by IL-4. Thus, the increase of hematopoietic progenitors in LTCs, as observed in the presence of IL-4, can be at least partially explained by a costimulation of SCF and IL-4 on primitive progenitor cells and by an enhancement of hematopoietic cells to stroma. The downregulation of CSFs by IL-4 might prevent the expansion of the mature hematopoietic cell compartment.