Over Expression of MN1 Accelerates Leukemia Onset and Confers Resistance to Chemotherapy by Suppression of p53 and Bim

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 2501-2501
Author(s):  
Timothy Pardee
Keyword(s):  
Bone Marrow ◽  
Conflicts Of Interest ◽  
The United States ◽  
Retroviral Vectors ◽  
Genetic Alterations ◽  
P Value ◽  
Over Expression ◽  
Resistance To Chemotherapy ◽  
Worse Prognosis

Abstract Abstract 2501 Acute myeloid leukemia (AML) is an aggressive malignancy of immature myeloid precursors that leads to progressive marrow failure and death. This disease will affect approximately 12,950 people this year in the United States, causing 9,050 deaths. The most common treatment is combination chemotherapy containing cytarabine (Ara-C) and an anthracycline. Resistance to these therapies is a major problem and most patients diagnosed with AML will ultimately die from resistant disease. AML is a genetically diverse malignancy and karyotype can be used to delineate prognosis. There is a clear link between chromosomal abnormalities and resistance to chemotherapy as complete remission rates are significantly different between groups. Additionally, there are now multiple submicroscopic genetic alterations that have been found to effect prognosis. These alterations can be mutations, over or under expression of a particular gene. MN1 is a transcription co-factor and several studies have demonstrated its over-expression confers a worse prognosis. High MN1 expressers were less likely to achieve a remission and had lower 3 year survival rates. Additionally, over expression of MN1 in murine bone marrow leads to AML in transplanted recipients and predicts for resistance to ATRA in elderly AML patients. The effect of MN1 on response to standard chemotherapy is currently unknown. To determine the effect of MN1 expression on therapy response we infected murine MLL-ENL driven AML cells with retroviral vectors that expressed MN1. When partially infected populations were exposed to a titration of either Ara-C or doxorubicin MN1 expressing cells were significantly enriched compared to untreated controls. When cells were exposed to a titration of Ara-C the MN1 expressing cells were enriched up to1.69 fold and when exposed to doxorubicin were enriched up to 3.80 fold. Both results were highly statistically significant with p values of 0.004 and < 0.0001. Consistent results were obtained with repeated infections and with separately derived MLL-ENL lines. Additionally, MN1 was able to confer therapy resistance to anthracycline resistant Flt3-ITD expressing cells suggesting non-overlapping mechanisms. Purified populations of cells expressing MN1 were resistant to Ara-C when compared to the parental leukemia (IC50 175.6nM vs 67.28nM) and highly resistant to doxorubicin. Consistent with these results human OCI-AML3 cells expressing MN1were enriched by 1.6 fold when exposed to doxorubicin, a highly significant result with a p value of 0.0002. In contrast a control vector without MN1 was not significantly enriched. In vivo when mixed leukemia cells were injected into syngeniec recipients MN1 expressers were significantly enriched in the femoral bone marrow of treated animals compared to controls. Treated animals had 90.58% (+/−0.66) MN1 expressing blasts compared to 55.38% (+/−5.25) in controls. This result was highly statistically significant with a p value of < 0.0001. This observation was reproducible in a separately derived MLL-ENL driven cell line. Additionally, the engraftment of MLL-ENL and Flt3-ITD expressing cells was significantly increased by MN1 expression leading to shorter survival in recipient animals despite the already highly aggressive nature of the parental leukemia. When MN1 expressing cells were exposed to doxorubicin or Ara-C they displayed significantly lower Annexin V positivity consistent with an attenuated apoptotic response (3.65 vs 34.79, p=<0.0001). When we examined BH3 only family member induction following exposure to Ara-C and doxorubicin we found significantly decreased levels of Bim induction by QPCR in cells expressing MN1. Similarly, stabilization of p53 following treatment was blunted in MN1 expressers as was induction of its downstream targets p21 and MDM2. Importantly the amount of DNA damage induced by doxorubicin as assessed by γH2AX foci was not different between MN1 expressing cells and the parental leukemia. These data suggest that over expression of MN1 confers resistance to both Ara-C and doxorubicin in vitro and in vivo by suppression of Bim induction and p53 response. These observations suggest a biological explanation for the clinical observation that it confers a worse prognosis. Disclosures: No relevant conflicts of interest to declare.

Over Expression of MN1 Confers Resistance to Chemotherapy In Vitro and In Vivo

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 969-969
Author(s):  
Timothy Pardee ◽  
Teresa Mascenik ◽  
Britt H. Bolemon ◽  
Guerry J Cook
Keyword(s):  
Bone Marrow ◽  
The United States ◽  
Genetic Alterations ◽  
T Test ◽  
Over Expression ◽  
Student’S T ◽  
Worse Prognosis ◽  
Student’S T Test

Abstract Abstract 969 Acute myeloid leukemia (AML) is an accumulation of immature myeloid precursors that leads to progressive marrow failure and death. This disease affects approximately 12,000 people per year in the United States, causing 9,000 deaths. Despite decades of active research the overall 5 year survival remains a dismal 30–40%. The backbone of initial therapy for the last 30 years is combination chemotherapy containing cytarabine (Ara-C) and an anthracycline. Resistance to these therapies is a major problem and most patients diagnosed with AML will ultimately die from resistant disease. AML is characterized by heterogeneous genetic alterations that can be used to delineate prognosis. Using standard karyotyping techniques patients can be divided into good, intermediate and poor prognostic categories. There is a clear link between these chromosomal aberrations and response to chemotherapy as complete remission rates are significantly different between groups. Patients with no detectable cytogenetic abnormality fall into an intermediate prognostic group with a very heterogeneous outcome. Recent work has begun to uncover submicroscopic genetic alterations that effect prognosis for these patients. These alterations can be mutations, over or under expression of a particular gene. The MN1 gene encodes a transcription co-factor first identified by its involvement in a balanced translocation in a patient with a meningioma. Since its initial description it has been found over-expressed in multiple AML patient samples. There are several reports that over-expression of MN1 confers a worse prognosis in AML. High MN1 expressers were less likely to achieve a remission and had a lower 3 year survival rate. Additionally, over expression of MN1 in murine bone marrow leads to AML in transplanted recipients and predicts for resistance to ATRA in elderly AML patients. However, the effect of MN1 over expression on response to standard chemotherapy is currently unknown. To answer this question we used a murine model of AML driven by MLL-ENL. AML blasts were infected with retroviral vectors that contained MN1 and a GFP reporter. Partially infected blast populations were then exposed to various concentrations of either Ara-C or doxorubicin and the ratio of GFP positive and negative cells was compared to untreated controls. When blasts were exposed to 150 nM Ara-C the GFP+ percentage went from 21.10 (+/− 0.5302) in the control samples to 35.68 (+/−1.230) in the treated samples. This result was even more profound when cells were treated with 15 ng/ml doxorubicin where the percentage went from 21.10 (+/− 0.5302) to 80.27 (+/−1.615). Both results were highly statistically significant by two tailed student's t test with p values of 0.004 and <0.0001 respectively. Consistent results were obtained in multiple different infections and with separately derived MLL-ENL lines. These data demonstrate that blasts expressing MN1 had an advantage when exposed to either Ara-C or doxorubicin although the effect was far more pronounced with doxorubicin exposure. MN1 expressing blasts were also resistant to the combination of Ara-C and doxorubicin. In order to determine if MN1 conferred resistance to Ara-C and doxorubicin in vivo we injected sublethally irradiated, Ly5.1+ C57Bl6 recipients with a partially infected population of blasts. Ly5.1+ animals do not express the Ly5.2 allele; thus, staining cells for Ly5.2 allows differentiation of leukemic cells from endogenous marrow. Eight days after injection of blasts animals were treated with 100 mg/kg Ara-C plus 3 mg/kg doxorubicin daily for 5 days or observed. On day 6 animals were sacrificed and bone marrow from bilateral femurs was harvested, stained for Ly5.2 and analyzed by flow cytometery. Animals treated with Ara-C plus doxorubicin had 90.58% (+/−0.6638) Ly5.2+, GFP+ blasts compared to 55.38% (+/−5.245) in control animals. This result was highly statistically significant with a p value of <0.0001 by two tailed student's t test. This observation was reproducible in a separately derived MLL-ENL driven cell line. These data suggest that over expression of MN1 in this murine AML model confers resistance to both Ara-C and doxorubicin in vitro and in vivo and provides a biological explanation for the clinical observation that it confers a worse prognosis. The mechanisms involved in this resistance are currently under study. Disclosures: No relevant conflicts of interest to declare.

How Does the Selection of Laboratory Mice Affect the Results of Physiological Distribution of Radiopharmaceuticals?

2021 ◽  
Vol 14 ◽  
Author(s):  
Urszula Karczmarczyk ◽  
Piotr Ochniewicz ◽  
Ewa Laszuk ◽  
Kamil Tomczyk ◽  
Piotr Garnuszek
Keyword(s):  
Quality Control ◽  
Statistical Significance ◽  
The United States ◽  
Radioactive Isotopes ◽  
In Vivo Studies ◽  
P Value ◽  
Preclinical Studies ◽  
In Vivo Experiments ◽  
Physicochemical Methods

Background: The choice of mice strain can significantly influence the physiological distribution and may lead to an inadequate assessment of the radiopharmaceutical properties. Objective: This work aims to present how the legal requirements that apply to radiopharmaceuticals contained in the various guidelines determine the choice of the mouse strain for quality control and preclinical studies and affect the results of physiological distribution. Methods: Swiss and BALB/c mice were chosen as commonly used strains in experiments for research and quality control purposes. Radiopharmaceuticals, i.e., preparations containing one or more radioactive isotopes in their composition, are subject to the same legal regulations at every stage of the research, development and routine quality control as all other medicines. Therefore, in vivo experiments are to be carried out to confirm the pharmacological properties and safety. Moreover, if a radiopharmaceutical's chemical structure is unknown or complex and impossible to be determined by physicochemical methods, an analysis of physiological distribution in a rodent animal model needs to be performed. Results: In our studies, thirty-six mice (Swiss n=18, BALB/c n=18) were randomly divided into six groups and injected with the following radiopharmaceuticals: [99mTc]Tc-Colloid, [99mTc]Tc-DTPA and [99mTc]Tc-EHIDA. Measurement of physiological distribution was conducted following the requirements of European Pharmacopoeia (Ph. Eur.) monograph 0689, internal instructions and the United States Pharmacopeia (USP) monograph, respectively. Additionally, at preclinical studies, ten mice (Swiss n=5, BALB/c n=5) were injected with the new tracer [99mTc]Tc-PSMA-T4, and its physiological distribution has been compared. The p-value <0.05 proved the statistical significance of the radiopharmaceutical physiological distribution. Conclusion: We claim that mice strain choice can significantly influence the physiological distribution and may lead to inaccurate quality control results and incomprehensible interpretation of the results from preclinical in vivo studies of a new radiopharmaceutical.

Exhaustion of Donor Hematopoietic Stem Cells in Lethally-Irradiated Hosts Can Be Sbstantially Mitigated by Targeting p18INK4C.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 1200-1200
Author(s):  
Hui Yu ◽  
Youzhong Yuan ◽  
Xianmin Song ◽  
Feng Xu ◽  
Hongmei Shen ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Hematopoietic Stem Cells ◽  
Kinase Inhibitor ◽  
Hematopoietic Cells ◽  
Hematopoietic Stem ◽  
Total Period ◽  
Over Expression

Abstract Hematopoietic stem cells (HSCs) are significantly restricted in their ability to regenerate themselves in the irradiated hosts and this exhausting effect appears to be accelerated in the absence of the cyclin-dependent kinase inhibitor (CKI), p21. Our recent study demonstrated that unlike p21 absence, deletion of the distinct CKI, p18 results in a strikingly positive effect on long-term engraftment owing to increased self-renewing divisions in vivo (Yuan et al, 2004). To test the extent to which enhanced self-renewal in the absence of p18 can persist over a prolonged period of time, we first performed the classical serial bone marrow transfer (sBMT). The activities of hematopoietic cells from p18−/− cell transplanted mice were significantly higher than those from p18+/+ cell transplanted mice during the serial transplantation. To our expectation, there was no detectable donor p18+/+ HSC progeny in the majority (4/6) of recipients after three rounds of sBMT. However, we observed significant engraftment levels (66.7% on average) of p18-null progeny in all recipients (7/7) within a total period of 22 months. In addition, in follow-up with our previous study involving the use of competitive bone marrow transplantation (cBMT), we found that p18−/− HSCs during the 3rd cycle of cBMT in an extended long-term period of 30 months were still comparable to the freshly isolated p18+/+ cells from 8 week-old young mice. Based on these two independent assays and the widely-held assumption of 1-10/105 HSC frequency in normal unmanipulated marrow, we estimated that p18−/− HSCs had more than 50–500 times more regenerative potential than p18+/+ HSCs, at the cellular age that is equal to a mouse life span. Interestingly, p18 absence was able to significantly loosen the accelerated exhaustion of hematopoietic repopulation caused by p21 deficiency as examined in the p18/p21 double mutant cells with the cBMT model. This data directly indicates the opposite effect of these two molecules on HSC durability. To define whether p18 absence may override the regulatory mechanisms that maintain the HSC pool size within the normal range, we performed the transplantation with 80 highly purified HSCs (CD34-KLS) and then determined how many competitive reconstitution units (CRUs) were regenerated in the primary recipients by conducting secondary transplantation with limiting dilution analysis. While 14 times more CRUs were regenerated in the primary recipients transplanted with p18−/−HSCs than those transplanted with p18+/+ HSCs, the level was not beyond that found in normal non-transplanted mice. Therefore, the expansion of HSCs in the absence of p18 is still subject to some inhibitory regulation, perhaps exerted by the HSC niches in vivo. Such a result was similar to the effect of over-expression of the transcription factor, HoxB4 in hematopoietic cells. However, to our surprise, the p18 mRNA level was not significantly altered by over-expression of HoxB4 in Lin-Sca-1+ cells as assessed by real time PCR (n=4), thereby suggesting a HoxB4-independent transcriptional regulation on p18 in HSCs. Taken together, our current results shed light on strategies aimed at sustaining the durability of therapeutically transplanted HSCs for a lifetime treatment. It also offers a rationale for the feasibility study intended to temporarily target p18 during the early engraftment for therapeutic purposes.

Abnormal Distribution of Erythroid Progenitors Populations in Mice Lacking p45 NF-E2.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 1988-1988
Author(s):  
Jadwiga Gasiorek ◽  
Gregory Chevillard ◽  
Zaynab Nouhi ◽  
Volker Blank
Keyword(s):  
Bone Marrow ◽  
Red Blood Cells ◽  
Blood Cells ◽  
Conflicts Of Interest ◽  
Globin Genes ◽  
Erythroid Progenitors ◽  
Erythroid Progenitor ◽  
Adult Mice ◽  
Deficient Mice

Abstract Abstract 1988 Poster Board I-1010 The NF-E2 transcription factor is a heterodimer composed of a large hematopoietic-specific subunit called p45 and widely expressed 18 to 20-kDa small Maf subunits. In MEL (mouse erythroleukemia) cells, a model of erythroid differentiatin, the absence of p45 is inhibiting chemically induced differentiation, including induction of globin genes. In vivo, p45 knockout mice were reported to show splenomegaly, severe thrompocytopenia and mild erythroid abnormalities. Most of the mice die shortly after birth due to haemorrhages. The animals that survive display increased bone, especially in bony sites of hematopoiesis. We confirmed that femurs of p45 deficient mice are filled with bone, thus limiting the space for cells. Hence, we observed a decrease in the number of hematopoietic cells in the bone marrow of 3 months old mice. In order to analyze erythroid progenitor populations we performed flow cytometry using the markers Ter119 and CD71. We found that p45 deficient mice have an increased proportion of early erythroid progenitors (proerythroblasts) and a decreased proportion of late stage differentiated red blood cells (orthochromatic erythroblasts and reticulocytes) in the spleen, when compared to wild-type mice. We showed that the liver of p45 knockout adult mice is also becoming a site of red blood cell production. The use of secondary sites, such as the spleen and liver, suggests stress erythropoiesis, likely compensating for the decreased production of red blood cells in bone marrow. In accordance with those observations, we observed about 2 fold increased levels of erythropoietin in the serum of p45 knockout mice.Overall, our data suggest that p45 NF-E2 is required for proper functioning of the erythroid compartment in vivo. Disclosures: No relevant conflicts of interest to declare.

Histone Deacetylase Inhibitors Demonstrate Significant Preclinical Activity as Single Agents, and in Combination with Bortezomib in Waldenstrom's Macroglobulinemia.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 4785-4785
Author(s):  
Jenny Sun ◽  
Lian Xu ◽  
Hsiuyi Tseng ◽  
Bryan Ciccarelli ◽  
Mariateresa Fulciniti ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Histone Deacetylase Inhibitors ◽  
Conflicts Of Interest ◽  
Trichostatin A ◽  
Hdac Inhibitors ◽  
Waldenström’S Macroglobulinemia ◽  
Over Expression ◽  
Waldenstrom's Macroglobulinemia ◽  
Waldenstrom’S Macroglobulinemia ◽  
Waldenström's Macroglobulinemia

Abstract Abstract 4785 Waldenstrom's Macroglobulinemia (WM) is a B-cell lymphoproliferative disorder characterized by bone marrow infiltration of CD19+ cells and production of a monoclonal IgM protein. Despite advances in treatment, WM remains incurable. As part of these efforts we sought to define the role of HDAC-inhibitors in WM. Gene expression profiling of bone marrow CD19+ cells from 30 WM patients and 10 healthy donors showed over-expression of HDAC4, HDAC9, and Sirt5 in WM patients. Evaluation of the HDAC inhibitors suberoylanilide hydroxamic acid (SAHA or Vorinostat), Trichostatin A (TSA), LBH-589 (Panobinostat), and sirtinol demonstrated dose dependent killing of BCWM.1 cells with IC50 of 3.5 uM, 70 nM, 0.8 uM, and 30 uM, respectively, whilst the combination of these agents with bortezomib resulted in at least additive tumor cell killing. TSA is more potent than bortezomib in inducing apoptosis in primary WM tumor cells in patients with prior treatment. TSA and bortezomib showed synergistic effect in 25% of the patients samples tested. We also observed that TSA and bortezomib-induced apoptosis of BCWM.1 cells depended on the activation of a similar set of caspases. Conversely, changes in cell cycle regulators were distinctly different between TSA and bortezomib treated BCWM.1 cells. The results of these studies demonstrate over-expression of distinct members of HDAC in WM cells, and provide a framework for the examination of HDAC-inhibitors as monotherapy, as well as combination therapy with bortezomib in the treatment of WM. Disclosures: No relevant conflicts of interest to declare.

Thrombopoietin Stimulation Leads to Enhanced Polyploidization in p53-/- Bone Marrow Megakaryocytes: In Vivo and in Vitro Studies.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 2531-2531
Author(s):  
Pani A. Apostolidis ◽  
Stephan Lindsey ◽  
William M. Miller ◽  
Eleftherios T. Papoutsakis
Keyword(s):  
Bone Marrow ◽  
Conflicts Of Interest ◽  
Platelet Surface ◽  
Platelet Counts ◽  
Reticulated Platelets ◽  
High Ploidy ◽  
And Control ◽  
Platelet Formation

Abstract Abstract 2531 Poster Board II-508 BACKGROUND AND HYPOTHESIS. We have previously shown that tumor suppressor p53 is activated in differentiating megakaryocytic (Mk) cells and its knock-down (KD) leads to increased polyploidization and delayed apoptosis in CHRF, a human Mk cell line. Furthermore, bone marrow (BM)-derived Mks from p53−/− mice reach higher ploidy classes in culture. Accordingly, we hypothesized that the role of p53 during megakaryopoiesis is to delimit polyploidization and control the transition from endomitosis by inhibiting DNA synthesis and promoting apoptosis. Here, we test this hypothesis by examining the differential effect of mouse thrombopoietin (rmTpo) on the ploidy of p53−/− and p53+/+ mouse Mk cells. METHODS. 8–10 week-old, male p53−/− mice and p53+/+ littermates were injected once with 1.2 μg rmTpo or saline. On days 2 and 5 after Tpo/saline treatment, tail-bleeding assays were performed to measure bleeding times/volumes, mice were bled for platelet counts and sacrificed to harvest BM. We employed flow cytometry to examine baseline ploidy in BM-resident Mks in p53−/− and p53+/+ mice as well as Mk cells generated from BM progenitors after 4 and 6 days of culture with rmTpo. RESULTS. At steady state, ploidy in BM-resident CD41+ Mk cells was similar in p53−/− and p53+/+ mice: 11.8±2.3% and 10.7±1.3% of p53−/− and p53+/+ Mks, respectively, reaching a ploidy of ≥32N (n=3-4). Platelet counts were 1.3×106±1×105/μl (12.5±1.0% reticulated) and 1.1×106±5×104/μl (12.4±1.3% reticulated) in p53−/− and p53+/+ mice, respectively (n=8). Two days following Tpo treatment of the mice, we did not observe significantly increased platelet levels, while ploidy was marginally affected. However, 5 days following Tpo treatment, we found greater ploidy in the BM in the absence of p53: 22±1.6% 16N and 10.1±0.8% ≥32N Mks in the p53−/− versus 18.6±3.3% 16N and 7.1±1.4% ≥32N Mks in the p53+/+ (n=2). This was accompanied by increased platelet formation: 23.6±8.3% reticulated platelets in the p53−/− versus 17.8±2.6% in the p53+/+ (n=2). Culture of BM cells from non-Tpo treated mice with 50ng/ml rmTpo resulted in a 50% increase in total Mks and increased polyploidy by day 6 of culture: 38.6±4.6% of p53−/− versus 19.2±2.3% of p53+/+ Mks reached ploidy classes of ≥32N (n=3-4, p < 0.01). Lack of p53 led to hyperploid Mk cells; by day 6 of culture 10.3±2.2% of p53−/− Mks were in ploidy classes of 128N and higher, while only 0.6±0.1% p53+/+ Mks achieved such high ploidy (n=3-4). In addition, a 6 day culture with Tpo of BM cells derived from p53−/− and p53+/+ mice pre-treated with Tpo 5 days prior to sacrifice led to more profound polyploidization compared to Mks generated from the non-Tpo treated mice but only in the p53−/− Mks: 48.8±1.1% of p53−/− versus only 17.6±0.2% of p53+/+ Mks reached ploidy ≥32N (n=2). Microarray analysis comparing p53KD to control CHRF cells undergoing Mk differentiation revealed down-regulation of genes coding for platelet surface complex CD41/CD61 and CD62P in the p53KD cells. To examine the possibility of altered functionality of platelets in p53−/− mice, we performed tail-bleeding assays on the mice that did not receive Tpo. Bleeding times and volumes were generally prolonged in the absence of p53 (all p53−/− mice exceeded the 10 min duration of the assay; mean p53−/− and p53+/+ blood loss was 17μl and 10μl, respectively, n=3-4). CONCLUSIONS. Our data indicate that in vivo polyploidization and platelet formation from Mks is increased in the p53−/− relative to p53+/+ mice after Tpo administration. These data are in line with our hypothesis that p53 activation decreases the ability of Mks to respond to Tpo and undergo polyploidization. Additionally, our preliminary data on platelet functionality suggest that p53 may have a role in hemostasis. Disclosures: No relevant conflicts of interest to declare.

The Role of gas6 in Venous Thrombosis In Vivo.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 3057-3057
Author(s):  
Richard Robins ◽  
Peter Carmeliet ◽  
Mark Blostein
Keyword(s):  
Bone Marrow ◽  
Venous Thrombosis ◽  
Bone Marrow Cells ◽  
Conflicts Of Interest ◽  
Thrombus Formation ◽  
Recovery Period ◽  
Vena Cava ◽  
Specific Gene ◽  
Combined Genotypes

Abstract Abstract 3057 Poster Board II-1033 Gas6 is the vitamin-K dependent protein product of growth arrest specific gene 6. A genetic deficiency of this protein protects mice against experimentally induced thrombosis without causing a bleeding diathesis. Protection from thrombosis results from a deficiency in platelet aggregation and secretion. In addition to being expressed by platelets, Gas6 and its receptors are also expressed by vascular cells including the endothelium, an organ known to play a role in the hemostatic balance. While endothelial Gas6 has been shown to promote inflammation and cell survival, it remains unknown if it contributes to the pathophysiology of venous thrombosis. To answer this question, we employed a bone marrow transplantation (BMT) strategy using wild type and Gas6 null mice to create chimeric mice with combined genotypes in the vascular and platelet compartments. Mice were exposed to a dose of radiation optimized to maximize both survival and ablation of recipient marrow. Irradiated mice were then infused with bone marrow cells isolated from the femurs and tibias of donor mice and were allowed a one month recovery period for hematologic reconstitution. Success of marrow uptake was confirmed by PCR. They were then subjected to the Ferric Chloride model of venous thrombosis in the Inferior Vena Cava (IVC). Four groups of transplanted mice were studied. Results from these BMT experiment show a contributing effect by both endothelial as well as platelet Gas6 to thrombus formation (n=8, p<0.01). Mice with combined genotypes (Gas6-/- into WT and WT into Gas6 -/-) show an intermediate thrombus weight suggesting that both vascular and platelet derived Gas6 are both responsible for thrombosis pathology. Therefore, Gas6 at both sites could be potential targets in treating venous thrombosis. Disclosures No relevant conflicts of interest to declare.

Mesenchymal Stem Cells From Patients with Acute Myeloid Leukemia (AML) Can Differentiate Into B-Cells in NOD/SCID/IL-2Rγ-/- Mice.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 4573-4573
Author(s):  
Rui-Yu Wang ◽  
Yue-Xi Shi ◽  
Zhihong Zeng ◽  
Wendy D. Schober ◽  
Teresa J. McQueen ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Mesenchymal Stem Cells ◽  
B Lymphocyte ◽  
Conflicts Of Interest ◽  
Lymphoid Cells ◽  
Proliferative Potential ◽  
Unexpected Finding ◽  
Nog Mice

Abstract Abstract 4573 Human mesenchymal stem cells (MSCs) derived from bone marrows are characterized by high proliferative potential and pluripotentiality to differentiate into multiple lineages such as osteo-, chondro-, and adipogenic cells. MSC express CD105, CD73 and CD90, but not CD45, CD34, CD14 or CD11b, CD79alpha or CD19 and HLA-DR surface molecules. In this study, we observed that MSC derived from the bone marrow of four AML patients differentiated into B-cell lymphoblasts with NOD/SCID/IL-2Rg-/- engraftment potential. MSC cell lines were established by culturing adherent cells from newly diagnosed AML (n=4) age 20 to 74 years in alpha-DMEM medium supplement with 20% fetal bovine serum. Surface antigen phenotype analysis and G-banding karyotype analysis were performed in passage 2 to 4. FACS-sorted CD90 positive cells were then intravenously (I.V.) injected into NOD/SCID/IL-2Rg-/- (NOG) mice via tail vein (n=9) or into the bone marrow (n=3). Circulating cells were analyzed for CD19, CD33, CD34, and CD90 expression on day 36, 45, 60, 75 after injection of MSC. Results 1) G-banding showed normal karyotype in all MSC; 2) Injected MSC engrafted and differentiated in NOG mice. Surprisingly, CD19 positive cells were found in all samples starting on day 36 (table) and increased on day 60 and 75 (from d36: 6.9±3.5%, d45:0.7±0.1%, d60:2.6 ± 1.6% and d75: 9.3 ± 1.0%); 3) CD90 positive cells were found on day 45 (range from 0.07-3.96% and decreased to 0.1-0.5% on day 75). Low percentage of CD33 (day 45: 0.19-0.78% and day 60: 0.12-2.53%) and CD34 positive cells (day 45: 0.32-1.9% and day 60: 0.21-2.39%) were observed before day 60 and were undetectable by day 75. Table shows the percentages of CD19+ cells found in circulation in NOD/SCID/IL-2Rg-/- (NOG) mice after MSC I.V. or intra-bone marrow injection. (* Mice died after phlebotomy.) Conclusion Human MSC derived from AML bone marrows have the capacity to differentiate into CD19 positive B lymphocyte in NOG mice in vivo. It has previously been reported that AML can be propagated by a leukemic stem cell with lymphoid characteristics (Cancer Cell 2006, 10, 363-74). Data reported here suggest the possibility that AML-derived MSC give rise to lymphoid cells that engraft in NOG mice. This unexpected finding could shed light on the role of stroma cells in the pathogenesis and propagation of leukemias. Disclosures: No relevant conflicts of interest to declare.

Bone Marrow Failure with 13q Deletion: A Distinct Clinicopathological Entity with Immune Pathophysiology,

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 3420-3420
Author(s):  
Kohei Hosokawa ◽  
Takamasa Katagiri ◽  
Naomi Sugimori ◽  
Ken Ishiyama ◽  
Yumi Sasaki ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Conflicts Of Interest ◽  
Bone Marrow Failure ◽  
Survival Rates ◽  
Snp Array ◽  
Normal Karyotype ◽  
Genetic Alterations ◽  
Fish Analysis ◽  
Hematopoietic Stem ◽  
Who 2008

Abstract Abstract 3420 Background: Numerical karyotypic abnormalities such as −7/del(7q) and del(13q) are occasionally seen in patients with bone marrow (BM) failure who do not have typical signs of myelodysplasia. The WHO 2008 defined this subset of BM failure as MDS-U because of its likely association with a risk of evolving into leukemia, while the presence of isolated abnormalities including +8, del(20q), and -Y was not considered to be presumptive evidence of MDS. Previous studies showed that BM failure patients with del(13q) responded to immunosuppressive therapy (IST) and had a favorable prognosis (Ishiyama K et al, Br J Haematol; 117: 747. 2002; Sloand, JCO 2010). However, the clinical features of del(13q) BM failure remain unclear due to its low incidence as well as the frequently associated karyotypic abnormalities. Objectives/Methods: To characterize the clinicopathological features of patients with BM failure with del(13q), this study reviewed the clinical data of 1705 BM failure patients (733 with AA, 286 with MDS-RCUD, 149 with RCMD, 60 with MDS-U) whose blood was examined for the presence of glycosylphosphatidyl-inositol anchored protein (GPI-AP)-deficient granulocytes and erythrocytes from May 1999 through July 2010. Genomic DNA was isolated from the peripheral blood cells of 7 patients with 13q- and was subjected to SNP array-based genome-wide analysis for genetic alterations using GeneChip® 250K arrays to identify the gene locus that is commonly deleted as a result of 13q-. Results: The 13q- clone was found in 25 (1.5%) of the 1705 patients. All the 13q- patients were classified as MDS-U, due to the absence of significant dysplasia to fulfill the criteria for MDS defined by the WHO 2008 classification. BM was hypocellular in 17 patients and normocellular in 6. Seventeen patients had a clone with 13q- alone, while the remaining 8 patients had a clone with 13q- and other numerical abnormalities including –Y, +mar in 2, and −20, del(7q), +8, der(1;7) in 1. A significant increase in the percentage of GPI-AP- granulocytes was detected in 366 (50%) of 733 patients with AA and 115 (23%) of 495 patients with MDS. GPI-AP- cells were detected in all (100%) of the 17 patients with 13q- alone. On the other hand, the prevalence of increased GPI-AP− cells in patients with 13q- plus other abnormalities and in those with the normal karyotype was 38% (3/8) and 43% (405/937), respectively. Fifteen patients with 13q- alone were treated with IST (ATG + cyclosporine in 6 and cyclosporine ± anabolic steroid in 9) and all of them achieved either a PR or a CR, while in the patients with 13q- plus other abnormalities, the response rate to IST was 40%. A total of 106 patients with the normal karyotype were treated with ATG+CsA (48) or CsA±AS (58) and the response rates were 73% and 85%, respectively. None of the 17 13q- patients progressed to advanced MDS or AML during the follow-up period of 3 to 108 months (median: 52 months) while 2 of 8 patients with 13q- plus other abnormalities developed AML. The 5-year overall survival rates of the patients with 13q-, those with 13q- plus other abnormalities, and patients with a normal karyotype were 84%, 45%, and 91%, respectively. The percentage of 13q- clones increased in 5 patients, and decreased in 3 patients after successful IST. When GPI-AP- and GPI-AP+ granulocytes were subjected to a FISH analysis using a 13q probe (13q14.3), the 13q- clones were detected only in of GPI-AP+ granulocytes, suggesting that 13q- cells are derived from non-PIG-A mutant HSCs. SNP arrays identified 13q13.3 to 13q14.3 regions in all cases. Conclusions: MDS-U with 13q- is a benign BM failure syndrome characterized by a good response to IST and a markedly high prevalence of GPI-AP cells. Patients with this type of BM failure may be inappropriately treated with hypomethylating agents or hematopoietic stem cell transplantation from unrelated donors, which is associated with high therapy-related mortality. Therefore, del(13q) should be eliminated from the intermediate prognosis group defined by the IPSS, and BM failure with del(13q) should be managed as idiopathic AA. Disclosures: No relevant conflicts of interest to declare.

Profilin 1 Is Essential for Retention and Metabolism of Hematopoietic Stem Cells in Bone Marrow

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 1224-1224
Author(s):  
Junke Zheng ◽  
Chengcheng Zhang
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Conflicts Of Interest ◽  
Bone Marrow Failure ◽  
Actin Binding ◽  
Wild Type ◽  
Cell Fates ◽  
Hematopoietic Stem ◽  
Multiple Cell

Abstract Abstract 1224 How stem cells interact with the microenvironment to regulate their cell fates and metabolism is largely unknown. Here we show that, in a hematopoietic stem cell (HSC) -specific inducible knockout model, the cytoskeleton-modulating protein profilin 1 (pfn1) is essential for the maintenance of multiple cell fates and metabolism of HSCs. The deletion of pfn1 in HSCs led to bone marrow failure, loss of quiescence, increased apoptosis, and mobilization of HSCs in vivo. In reconstitution analyses, pfn1-deficient cells were selectively lost from mixed bone marrow chimeras. By contrast, pfn1 deletion did not significantly affect differentiation or homing of HSCs. When compared to wild-type cells, levels of expression of Hif-1a, EGR1, and MLL were lower and an earlier switch from glycolysis to mitochondrial respiration with increased ROS level was observed in pfn1-deficient HSCs. This switch preceded the detectable alteration of other cell fates. Importantly, treatment of pfn1-deficient mice with the antioxidant N-acetyl-l-cysteine reversed the ROS level and loss of quiescence of HSCs, suggesting that pfn1 maintained metabolism is required for the quiescence of HSCs. Furthermore, we demonstrated that expression of wild-type pfn1 but not the actin-binding deficient or poly-proline binding-deficient mutants of pfn1 rescued the defective phenotype of pfn1-deficient HSCs. This result indicates that actin-binding and proline-binding activities of pfn1 are required for its function in HSCs. Thus, pfn1 plays an essential role in regulating the retention and metabolism of HSCs in the bone marrow microenvironment. Disclosures: No relevant conflicts of interest to declare.

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