Flt3 Is Required for MLL-ENL-Induced Leukemogenesis In Vivo, but Not for LSC Function in Vitro

Abstract Abstract 2450 Flt3 is a type III tyrosine kinase receptor expressed on hematopoietic multipotential progenitors and more downstream progenitor cells in the mouse bone marrow (BM).Flt3 is one of the most frequently mutated genes in acute myeloblastic leukemia (AML) patients, leading to constitutive activation. Several Flt3 kinase inhibitors are in clinical development in AML with Flt3 mutations, although disappointing results suggest that Flt3-inhibitors as monotherapy are unlikely to become reality. Wild type (wt) Flt3, which differs in its signaling characteristics and downstream targets from mutated Flt3, is highly expressed in particular in AML and ALL (acute lymphoblastic leukemia) with MLL (Mixed Lineage Leukemia) rearrangements. These observations suggest a role for wt Flt3 in pathogenesis as well, although little experimental or clinical evidence has been published yet supporting this notion. To our knowledge, there is only one report suggesting that wt Flt3 is required for the development of leukemia in a murine model of c-cbl mutation-induced leukemogenesis (Rathinam C, et al. Cancer Cell 2010). For this reason, we focused on the role of wt Flt3 in leukemogenesis and using leukemia mouse models established by retroviral transduction of MLL-ENL, MLL-AF9 and AML1-ETO9a. Mice transplanted with MLL-ENL-transduced Flt3−/− BM cells showed a higher survival rate (wt 61.3%, n=34 vs Flt3−/− 96.8%, n=31, median follow-up period; 255 days, p=0.038). No difference in survival was observed in the MLL-AF9 and AML1-ETO9a models however. Surprisingly, MLL-ENL-transduced Flt3−/− BM cells showed no significant difference in serial replating capacity of BM cells in methylcellulose colony forming assays in vitro compared to wt BM cells, and putative lin-kit+ leukemic stem cells (LSCs) did not express Flt3. In vivo, however, putative MLL-ENL LSCs, but not MLL-AF9 LSCs expressed Flt3. Furthermore, in non- and pre-leukemic MLL-ENL-transduced mice BM cells the frequency of lin-kit+ putative LSCs among MLL-ENL positive populations was decreased in Flt3−/− group (wt 1.468% vs Flt3−/− 0.466%, p=0.031). Flt3+ lin-kit+ populations were increased in MLL-ENL transduced leukemic wt mice compared to non-leukemic mice (leukemic mice 26.7% vs non-leukemic mice 9.9%, p=0.028). These results suggest that Flt3 signaling is required for MLL-ENL-induced leukemogenesis and maintenance of LSCs in vivo, but not in vitro. Importantly, the data suggest that the in vitro replatable leukemia stem cell (LSC) paradigm might not reflect LSC function or phenotype in vivo. Disclosures: No relevant conflicts of interest to declare.

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Inhibition of Plasmin Generation in Plasma By Heparin, Low Molecular Weight Heparin and a Covalent Antithrombin-Heparin Complex (ATH)

Blood ◽

10.1182/blood.v124.21.4217.4217 ◽

2014 ◽

Vol 124 (21) ◽

pp. 4217-4217

Author(s):

Gabriela Chang ◽

Helen M. Atkinson ◽

Leslie R. Berry ◽

Anthony K.C. Chan

Keyword(s):

Molecular Weight ◽

Plasminogen Activator ◽

Low Molecular Weight Heparin ◽

Conflicts Of Interest ◽

Area Under The Curve ◽

Low Molecular Weight ◽

Plasminogen Activator Inhibitor 1 ◽

Significant Difference

Abstract Introduction: Unfractionated heparin (UFH) and low molecular weight heparin (LMWH) are widely used anticoagulants for thrombosis treatment. However, these anticoagulants have limitations such as increased bleeding, variable dose response, required frequent monitoring, and, in the case of LMWH, inability to inhibit thrombin. This has led to the development of a covalent complex of antithrombin and heparin (ATH), which has been shown to overcome many of these shortcomings. ATH has faster rates of inhibition of many coagulation factors, is able to inhibit clot-bound thrombin, and is a more effective inhibitor of both venous and arterial thrombosis in animal models. Moreover, in a rabbit thrombosis model, ATH has been shown to decrease clot mass and fibrin accretion, while the contrary was observed for UFH. From these observations, it was suggested that ATH may enhance fibrin breakdown and thus led to investigations into the effects of UFH and ATH on fibrinolysis. In vitro studies have shown that UFH enhances antithrombin inhibition of plasmin. In addition, ATH displays a slightly greater inhibition of plasmin generation and activity. Such studies were conducted in purified systems, in the absence of other plasmin inhibitors naturally present in plasma. Therefore, the aim of the present study was to compare the effects of UFH, LMWH, and ATH on plasmin generation in plasma. Methods: At 37°C tissue plasminogen activator (tPA) and soluble fibrin fragments (fib) were added to normal adult pooled platelet poor plasma supplemented with 0.35, 0.7, 1.4, or 2.1 U anti-Xa/ml UFH, LMWH, or ATH, to initiate plasmin generation (8.93nM tPA and 300µg/ml fib). At various time points, subsamples were mixed with excess plasminogen activator inhibitor 1 (PAI-1) (55.12nM) to stop further plasmin generation. The plasmin concentration at each time point was determined using a plasmin-specific chromogenic substrate and a standard curve produced from purified plasmin. Results: Comparisons of mean area under the curve (AUC) for plasmin generation displayed a significant decrease in plasmin generation in the presence of all three anticoagulants at all doses tested (p<0.05). Comparing the anticoagulants at similar doses, plasmin generation was significantly decreased in the presence of ATH (15384.66±1930.23nM/min) compared to LMWH (23892.28±3090.54nM/min) at 0.7 U/ml (p<0.05). At a dose of 1.4 U/ml, there was significantly less plasmin generated, over time, in the presence of UFH (20089.49±3022.1623nM/min) and ATH (19273.86±1805.7323nM/min) when compared to LMWH (24743.18±1265.1023nM/min) (p<0.05). There was no significant difference in plasmin inhibition between UFH and ATH at any of the doses tested. Conclusion: The present study supports previous findings that UFH and ATH can facilitate antithrombin inhibition of plasmin. It is also observed that LMWH catalyzes the inhibition of plasmin by antithrombin but possibly to a lesser extent. These findings suggest that ATH has a similar inhibitory effect on plasmin generation and activity in plasma compared to UFH, despite its overall superior anticoagulant properties. Therefore, previous in vivo observations displaying decrease in clot mass with administration of ATH was due to its enhanced anticoagulant abilities and not fibrinolysis enhancement. These findings add to our understanding of ATH mechanisms of action and aid in its development for clinical use. Disclosures No relevant conflicts of interest to declare.

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Novel CD19/CD22 Bicistronic Chimeric Antigen Receptors Outperform Single or Bivalent Cars in Eradicating CD19+CD22+, CD19-, and CD22- Pre-B Leukemia

Blood ◽

10.1182/blood.v130.suppl_1.810.810 ◽

2017 ◽

Vol 130 (Suppl_1) ◽

pp. 810-810 ◽

Cited By ~ 3

Author(s):

Haiying Qin ◽

Sang M Nguyen ◽

Sneha Ramakrishna ◽

Samiksha Tarun ◽

Lila Yang ◽

...

Keyword(s):

T Cell ◽

B Cell ◽

Conflicts Of Interest ◽

Lymphoblastic Leukemia ◽

Single Chain ◽

Dual Targeting ◽

Single Antigen ◽

Therapeutic Function

Abstract Treatment of pre-B cell acute lymphoblastic leukemia (ALL) using chimeric antigen receptor expressing T cells (CART) targeting CD19 have demonstrated impressive clinical results in children and young adults with up to 70-90% complete remission rate in multiple clinical trials. However, about 30% of patients relapse due to loss of the targeted epitope on CD19 or CART failure. Our CD22-targeted CAR trial has generated promising results in relapsed/refractory ALL, including CD19 antigen negative ALL, but relapse associated with decreased CD22 site density has occurred. Thus, developing strategies to prevent relapses due to changes in antigen expression have the potential to increase the likelihood of durable remissions. In addition, dual targeting of both CD19 and CD22 on pre-B ALL may be synergistic compared to targeting a single antigen, a potential approach to improve efficacy in patients with heterogeneous expression of CD19 and CD22 on leukemic blasts. We describe the systematic development and comparison of the structure and therapeutic function of three different types (over 15 different constructs) of novel CARs targeting both CD19 and CD22: (1) Bivalent Tandem CAR, (2) Bivalent Loop CAR, and (3) Bicistronic CAR. These dual CARs were assembled using CD19- and CD22-binding single chain fragment variable (scFv) regions derived from clinically validated single antigen targeted CARs. They are structurally different in design: both tandem and loop CARs have the CD19 and CD22 scFv covalently linked in the same CAR in different orders, whereas, bicistronic CARs have 2 complete CAR constructs connected with a cleavable linker. The surface expression on the transduced T cell of the CD19/CD22 dual CARs was detected with CD22 Fc and anti-idiotype of CD19 and compared to single CD19 or CD22 CARs. Activities of dual CARs to either CD19 or CD22 were evaluated in vitro with cytotoxicity assays or killing assays against K562 cells expressing either CD19 or CD22 or both antigens and also tested against a leukemia CD19+/CD22+ cell line, NALM6, and NALM6 with CRISPER/CAS9 knockout of CD19 or CD22 or both antigens. Therapeutic function of the top candidates of the dual CARs was then validated in vivo against these NALM6 leukemia lines. Some of these dual CARs were also further tested against patient-derived xenografts. Finally, we tested the dual targeting CARs in an artificial relapse model in which mice were co-injected with a mix of CD19 knockout and CD22 knockout NALM6 leukemia lines. From these studies, we established that the order of the scFv, size of the linker, type of leader sequence, and co-stimulatory domain in the CAR constructs all impact the efficacy of the dual targeting CARs. Tandem, Loop, and Bicistronic CARs all demonstrate some levels of in vitro and in vivo activities, but the bicistronic CAR was most effective at clearing leukemia and preventing relapse. In the CD19+/CD22+ NALM6 model, bicistronic CAR treated mice remain disease free while CD19 CAR or CD22 CAR treated mice already died or relapsed on day 27. In the relapse model, as expected, CD19 or CD22 single CAR T cell treatment resulted in progression of the corresponding antigen-negative NALM6. Treatment with dual targeted bicistronic CARs resulted in clearance of both CD19 and CD22 negative ALL with durable remission. In summary, we described novel CD19/CD22 dual targeting CARs with robust pre-clinical activity against pre-B cell ALL, and validated this approach in the prevention of resistance to single-antigen targeted CARs in preclinical models. Disclosures No relevant conflicts of interest to declare.

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Immunomodulation by 17β-estradiol in bivalve hemocytes

AJP Regulatory Integrative and Comparative Physiology ◽

10.1152/ajpregu.00139.2006 ◽

2006 ◽

Vol 291 (3) ◽

pp. R664-R673 ◽

Cited By ~ 38

Author(s):

Laura Canesi ◽

Caterina Ciacci ◽

Lucia Cecilia Lorusso ◽

Michele Betti ◽

Tiziana Guarnieri ◽

...

Keyword(s):

Kinase Inhibitors ◽

Hydrolytic Enzymes ◽

Physiological Role ◽

Estrogen Receptor Α ◽

Nitrite Accumulation ◽

No Production ◽

17Β Estradiol

In mammals, estrogens have dose- and cell-type-specific effects on immune cells and may act as pro- and anti-inflammatory stimuli, depending on the setting. In the bivalve mollusc Mytilus, the natural estrogen 17β-estradiol (E2) has been shown to affect neuroimmune functions. We have investigated the immunomodulatory role of E2 in Mytilus hemocytes, the cells responsible for the innate immune response. E2 at 5–25 nM rapidly stimulated phagocytosis and oxyradical production in vitro; higher concentrations of E2 inhibited phagocytosis. E2-induced oxidative burst was prevented by the nitric oxide (NO) synthase inhibitor NG-monomethyl-l-arginine and superoxide dismutase, indicating involvement of NO and O2−; NO production was confirmed by nitrite accumulation. The effects of E2 were prevented by the antiestrogen tamoxifen and by specific kinase inhibitors, indicating a receptor-mediated mechanism and involvement of p38 MAPK and PKC. E2 induced rapid and transient increases in the phosphorylation state of PKC, as well as of a aCREB-like (cAMP responsive element binding protein) transcription factor, as indicated by Western blot analysis with specific anti-phospho-antibodies. Localization of estrogen receptor-α- and -β-like proteins in hemocytes was investigated by immunofluorescence confocal microscopy. The effects of E2 on immune function were also investigated in vivo at 6 and 24 h in hemocytes of E2-injected mussels. E2 significantly affected hemocyte lysosomal membrane stability, phagocytosis, and extracellular release of hydrolytic enzymes: lower concentrations of E2 resulted in immunostimulation, and higher concentrations were inhibitory. Our data indicate that the physiological role of E2 in immunomodulation is conserved from invertebrates to mammals.

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TIE2 Induces Breast Cancer Cell Dormancy and Inhibits the Development of Osteolytic Bone Metastases

Cancers ◽

10.3390/cancers12040868 ◽

2020 ◽

Vol 12 (4) ◽

pp. 868

Author(s):

Florian Drescher ◽

Patricia Juárez ◽

Danna L. Arellano ◽

Nicolás Serafín-Higuera ◽

Felipe Olvera-Rodriguez ◽

...

Keyword(s):

Breast Cancer ◽

Bone Metastases ◽

Kinase Inhibitors ◽

Disseminated Tumor Cells ◽

Tyrosine Kinase Receptor ◽

In Vivo Models ◽

Hematopoietic Stem ◽

Cell Dormancy

Breast cancer (BCa) cells disseminating to the bone can remain dormant and resistant to treatments for many years until relapsing as bone metastases. The tyrosine kinase receptor TIE2 induces the dormancy of hematopoietic stem cells, and could also induce the dormancy of BCa cells. However, TIE2 is also a target for anti-angiogenic treatments in ongoing clinical trials, and its inhibition could then restart the proliferation of dormant BCa cells in bone. In this study, we used a combination of patient data, in vitro, and in vivo models to investigate the effect of TIE2 in the dormancy of bone metastases. In BCa patients, we found that a higher TIE2 expression is associated with an increased time to metastases and survival. In vitro, TIE2 decreased cell proliferation as it increased the expression of cyclin-dependent kinase inhibitors CDKN1A and CDKN1B and arrested cells in the G0/G1 phase. Expression of TIE2 also increased the resistance to the chemotherapeutic 5-Fluorouracil. In mice, TIE2 expression reduced tumor growth and the formation of osteolytic bone metastasis. Together, these results show that TIE2 is sufficient to induce dormancy in vitro and in vivo, and could be a useful prognostic marker for patients. Our data also suggest being cautious when using TIE2 inhibitors in the clinic, as they could awaken dormant disseminated tumor cells.

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Role of PARP1-mediated autophagy in EGFR-TKI resistance in non-small cell lung cancer

Scientific Reports ◽

10.1038/s41598-020-77908-z ◽

2020 ◽

Vol 10 (1) ◽

Author(s):

Zhimin Zhang ◽

Xiaojuan Lian ◽

Wei Xie ◽

Jin Quan ◽

Maojun Liao ◽

...

Keyword(s):

Lung Cancer ◽

Kinase Inhibitors ◽

Growth Factor Receptor ◽

Advanced Lung Cancer ◽

Tki Resistance ◽

Geo Dataset ◽

Epidermal Growth

AbstractResistance to epidermal growth factor receptor-tyrosine kinase inhibitors (EGFR-TKIs) has become the main clinical challenge of advanced lung cancer. This research aimed to explore the role of PARP1-mediated autophagy in the progression of TKI therapy. PARP1-mediated autophagy was evaluated in vitro by CCK-8 assay, clonogenic assay, immunofluorescence, and western blot in the HCC-827, H1975, and H1299 cells treated with icotinib (Ico), rapamycin, and AZD2281 (olaparib) alone or in combination. Our results and GEO dataset analysis confirmed that PARP1 is expressed at lower levels in TKI-sensitive cells than in TKI-resistant cells. Low PARP1 expression and high p62 expression were associated with good outcomes among patients with NSCLC after TKI therapy. AZD2281 and a lysosomal inhibitor reversed resistance to Ico by decreasing PARP1 and LC3 in cells, but an mTOR inhibitor did not decrease Ico resistance. The combination of AZD2281 and Ico exerted a markedly enhanced antitumor effect by reducing PARP1 expression and autophagy in vivo. Knockdown of PARP1 expression reversed the resistance to TKI by the mTOR/Akt/autophagy pathway in HCC-827IR, H1975, and H1299 cells. PARP1-mediated autophagy is a key pathway for TKI resistance in NSCLC cells that participates in the resistance to TKIs. Olaparib may serve as a novel method to overcome the resistance to TKIs.

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Functional Characterization of Brain Tumor-Initiating Cells: Implications for Preclinical Models and Drug Development

Neurosurgery ◽

10.1093/neuros/nyz310_807 ◽

2019 ◽

Vol 66 (Supplement_1) ◽

Author(s):

Cesar A Garcia ◽

Adip Guruprasad Bhargav ◽

Sujan K Mondal ◽

Karim ReFaey ◽

Natanael Zarco ◽

...

Keyword(s):

Brain Tumor ◽

Critical Role ◽

Functional Characterization ◽

Tumor Initiating Cells ◽

Retroviral Transduction ◽

Significant Difference ◽

Brain Tumor Initiating Cells ◽

Functional Features

Abstract INTRODUCTION Glioblastoma (GBM) is the deadliest and most common primary brain cancer in adults. Brain tumor-initiating cells (BTICs) are a heterogeneous subset of stem-like, invasive cells that play a critical role in treatment failure and recurrence. METHODS Here, we propose a system to functionally characterize patient-derived BTICs to identify features that will guide assessment of therapeutics in a BTIC subpopulation-specific manner. We established and evaluated 5 BTIC populations based on (1) proliferation, (2) stemness, (3) migration, (4) tumorigenesis, (5) clinical characteristics, and (6) therapeutic sensitivity. RESULTS Overall, in Vitro growth trends reflected in Vivo growth rates. However, a significant difference was found between tumor growth in male versus female mice in 3 BTIC lines (QNS108 P = .0011; QNS120 P < .0001; QNS 140 P < .0001). Differences in survival were observed, where BTICs derived from male and female patients produced faster morbidity in mice of the opposite sex (male derived QNS108 male vs female P = .0039; female derived QNS203 male vs female P = .029). QNS203, which was isolated from a tumor in contact with the anterior subventricular zone, decreased survival at a faster rate compared to other cell lines (n = 10 per line, 5 males/5 females, P < .0001). Stem-like properties of BTICs were assessed via differentiation marker expression, sphere-forming capacity, and detection of canonical marker CD133. Higher CD133 expression correlated with faster in Vitro doubling time and greater tumor burden. Histology reflected similar patient tumor features such as migration across the corpus callosum and cystic formation. BTICs revealed varying responses to therapies (TMZ, Radiation, TRAIL, BMP4) and varied competence to retroviral transduction. CONCLUSION By studying the functional features of BTICs within our model of GBM heterogeneity, it was shown that several factors influenced tumorigenesis and survival. These included original tumor location, stemness, variation in therapeutic sensitivity, and a critical finding for the role of sex, an unexplored area for creating next-generation, sex-specific, and BTIC-specific therapeutics.

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Serum interspecies differences in metabolic pathways of bradykinin and [des-Arg9]BK: influence of enalaprilat

AJP Heart and Circulatory Physiology ◽

10.1152/ajpheart.1996.271.4.h1340 ◽

1996 ◽

Vol 271 (4) ◽

pp. H1340-H1347 ◽

Cited By ~ 17

Author(s):

A. Decarie ◽

P. Raymond ◽

N. Gervais ◽

R. Couture ◽

A. Adam

Keyword(s):

Metabolic Pathways ◽

Neutral Endopeptidase ◽

Thrombin Inhibitor ◽

Rat Serum ◽

Highly Sensitive ◽

Significant Difference ◽

Hydrolysis Of

Among the different enzymes responsible for the metabolism of bradykinin (BK), three peptidases look relevant in vivo: kininase I (KI), which transforms BK into its active metabolite, [des-Arg9]BK; kininase II (KII); and neutral endopeptidase, which inactivate BK and [des-Arg9]BK. The in vitro incubation of BK and [des-Arg9]BK in the serum of four species with or without enalaprilat and the quantification of the immunoreactivity of both peptides at different time intervals allowed the measurement of the kinetic parameters characterizing their metabolic pathways. Highly sensitive chemiluminescent enzyme immunoassays were used to measure the residual concentrations of BK and [des-Arg9]BK. Half-life (t1/2) of BK showed significant difference among species: rats (10 +/- 1 s) = dogs (13 +/- 1 s) < rabbits (31 +/- 1 s) < humans (49 +/- 2 s). t1/2 values of [des-Arg9]BK were also species dependent: rats (96 +/- 6 s) < < rabbits (314 +/- 6 s) = dogs (323 +/- 11 s) = humans (325 +/- 12 s). Enalaprilat significantly prevented the rapid BK and [des-Arg9]BK degradation in all species except that of [des-Arg9]BK in rat serum. Relative amount of BK hydrolyzed by serum KII was given as follows: rabbits (93.7 +/- 14.8%) = rats (83.6 +/- 6.7%) = humans (76.0 +/- 7.5%) > dogs (50.0 +/- 3.9%). Its importance in the hydrolysis of [des-Arg9]BK was 5.2 +/- 0.5% in rats < < 33.9 +/- 1.5% in humans < 52.0 +/- 1.1% in rabbits < 65.1 +/- 3.4% in dogs. The participation of serum KI in the transformation of BK into [des-Arg9]BK was dogs (67.2 +/- 5.3%) > > humans (3.4 +/- 1.2%) = rabbits (1.8 +/- 0.2%) = rats (1.4 +/- 0.3%). Finally, no significant difference on t1/2 values for BK and [des-Arg9]BK could be demonstrated between serum and plasma treated with either sodium citrate or a thrombin inhibitor. These results revealed striking species differences in the serum metabolism of kinins that could address at least partially some of the controversial data related to the cardioprotective role of kinins.

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The Bone Marrow Niche of Patients with Acute Lymphoblastic Leukemia Produces No Increased Asparagine Levels In Vivo That May Lead to Clinical Asparaginase Resistance

Blood ◽

10.1182/blood.v118.21.1505.1505 ◽

2011 ◽

Vol 118 (21) ◽

pp. 1505-1505

Author(s):

Wing H. Tong ◽

Rob Pieters ◽

Wim C.J. Hop ◽

Claudia Lanvers-Kaminsky ◽

Joachim Boos ◽

...

Keyword(s):

Bone Marrow ◽

Aspartic Acid ◽

Peripheral Blood ◽

Lymphoblastic Leukemia ◽

Mesenchymal Cells ◽

Leukemic Cells ◽

Significant Difference ◽

Trough Levels

Abstract Abstract 1505 Asparaginase is an essential component of combination chemotherapy of acute lymphoblastic leukemia (ALL). Asparaginase breaks down asparagine into aspartic acid and ammonia. Because asparagine is necessary for protein synthesis, its depletion leads to cell death. Recently, it has been suggested that mesenchymal cells in the bone marrow may produce asparagine and form ‘protective niches’ for leukemic cells. In vitro, this led to high levels of asparagine and asparaginase resistance of the ALL cells (Iwamoto et al. (J Clin Invest. 2007)). However, it is unknown if this holds true for the clinical in vivo situation. The aim of our study is to analyse whether mesenchymal cells or other cells in the bone marrow indeed produce significant amounts of asparagine in vivo that may lead to clinical asparaginase resistance. Ten de novo ALL patients were enrolled in this study. All children received induction chemotherapy according to protocol 1-A and 1-B of the Dutch Childhood Oncology Group (DCOG) ALL-10 protocol. Asparaginase levels and amino acid levels (asparagine, aspartic acid, glutamine and glutamic acid) were measured in bone marrow (BM) and peripheral blood at diagnosis (day 1), days 15, 33 and 79. On days that asparaginase was administered (days 15 and 33) it was ensured that study material was obtained before the E-coli L-asparaginase infusions. Changes over time of asparaginase trough levels in BM and peripheral blood were evaluated using Mixed models ANOVA. The amino acids levels in 0.5 ml BM, 3 ml BM and peripheral blood at days 15 and 33 were also compared using Mixed models ANOVA. All these analyses were done after log transformation of measured values to get approximate normal distributions. A two-sided p-value < 0.05 was considered statistically significant. The asparaginase levels were all below detection limit (< 5 IU/L) in BM and peripheral blood at days 1 and 79. In both compartments, the median asparaginase trough levels were not significantly different at days 15 and 33. At diagnosis, no significant difference in asparagine level between 3 ml BM and peripheral blood was found (median: 44.5 μM (range 20.6–59.6 μM) and 43.9 μM (range 18.4 –58.5 μM), respectively). However, the median level of aspartic acid at diagnosis in 3 ml BM (19.2 μM; range 6.2–52.6 μM) was significantly higher as compared to median level of peripheral blood (5.7 μM; range 2.4–10.1 μM) (p=0.002). The aspartic acid levels were also higher in BM compared to peripheral blood at days 15 and 33 (both p=0.001) and at day 79 (p=0.002). Aspartic acid levels were significantly higher in 0.5 ml versus 3 ml BM (p=0.001) and this difference was also found when comparing 0.5 ml BM versus peripheral blood (p<0.001) suggesting dilution with peripheral blood when taking higher volumes of ‘bone marrow’. Asparagine levels were all below the lower limit of quantification (LLQ < 0.2 μM) in both BM and blood during asparaginase treatment at days 15 and 33. At day 79, no significant difference in asparagine levels between BM (37.7 μM; range 33.4–50.3 μM) and peripheral blood (38.9 μM; range 25.7 –51.3 μM) was seen. During the time course of asparaginase infusions, the glutamine and glutamic acid levels did not change significantly. In conclusion, we demonstrate higher aspartic acid levels in bone marrow compared to peripheral blood. The higher aspartic acid levels are detected at diagnosis, during asparaginase therapy at days 15 and 33, and also at day 79 at complete remission, showing that these do not originate from leukemic cells nor from asparagine breakdown by asparaginase but from cells in the microenvironment of the bone marrow. However, there is no increased asparagine synthesis in vivo in the bone marrow of ALL patients. Therefore, increased asparagine synthesis by mesenchymal cells may be of relevance for resistance to asparaginase of leukemic cells in vitro but not in vivo. Disclosures: No relevant conflicts of interest to declare.

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A Critical Role of Mir-9 in Myelopoesis and EVI1-Induced Leukemogenesis.

Blood ◽

10.1182/blood.v120.21.2332.2332 ◽

2012 ◽

Vol 120 (21) ◽

pp. 2332-2332

Author(s):

Vitalyi Senyuk ◽

Yunyuan Zhang ◽

Yang Liu ◽

Ming Ming ◽

Jianjun Chen ◽

...

Keyword(s):

Cell Transformation ◽

Conflicts Of Interest ◽

Critical Role ◽

Ectopic Expression ◽

Myeloid Differentiation ◽

Dna Hypermethylation ◽

Hematopoietic Stem

Abstract Abstract 2332 MicroRNA-9 (miR-9) is required for normal neurogenesis and organ development. The expression of miR-9 is altered in several types of solid tumors suggesting that it may have a function in cell transformation. However the role of this miR in normal hematopoiesis and leukemogenesis is unknown. Here we show that miR-9 is expressed at low levels in hematopoietic stem/progenitor cells (HSCs/HPCs), and that it is upregulated during hematopoietic differentiation. Ectopic expression of miR-9 strongly accelerates terminal myelopoiesis, while promoting apoptosis in vitro and in vivo. In addition, the inhibition of miR-9 in HPC with a miRNA sponge blocks myelopoiesis. EVI1, required for normal embryogenesis, and is considered an oncogene because inappropriate upregulation induces malignant transformation in solid and hematopoietic cancers. In vitro, EVI1 severely affects myeloid differentiation. Here we show that EVI1 binds to the promoter of miR-9–3 leading to DNA hypermethylation of the promoter as well as repression of miR-9. We also show that ectopic miR-9 reverses the myeloid differentiation block that is induced by EVI1. Our findings suggest that inappropriately expressed EVI1 delays or blocks myeloid differentiation, at least in part by DNA hypermethylation and downregulation of miR-9. It was previously reported that FoxOs genes inhibit myeloid differentiation and prevent differentiation of leukemia initiating cells. Here we identify FoxO3 and FoxO1 as new direct targets of miR-9 in hematopoietic cells, and we find that upregulation of FoxO3 in miR-9-positive cells reduces the acceleration of myelopoiesis. These results reveal a novel role of miR-9 in myelopoiesis and in the pathogenesis of EVI1-induced myeloid neoplasms. They also provide new insights on the potential chromatin-modifying role of oncogenes in epigenetic changes in cancer cells. Disclosures: No relevant conflicts of interest to declare.

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The Small Molecule Inhibitor of VLA4 TBC3486 Sensitizes Resistant ALL to Chemotherapy

Blood ◽

10.1182/blood.v120.21.1500.1500 ◽

2012 ◽

Vol 120 (21) ◽

pp. 1500-1500 ◽

Cited By ~ 2

Author(s):

Yao-Te Hsieh ◽

Eun Ji Gang ◽

Halvard Bonig ◽

Ronald J Biediger ◽

Peter Vanderslice ◽

...

Keyword(s):

Cell Viability ◽

Small Molecule ◽

Conflicts Of Interest ◽

Lymphoblastic Leukemia ◽

Chemotherapy Resistance ◽

Leukemia Cell ◽

The Novel ◽

Small Molecule Inhibition

Abstract Abstract 1500 Significant progress notwithstanding, drug resistant acute lymphoblastic leukemia (ALL) remains a therapeutic challenge, as well as acute and long-term off-target toxicity of anti-ALL therapies can be dose-limiting or debilitating. Therefore, the development of more targeted therapies is desirable. We recently provided evidence that chemotherapy resistance of ALL cells can be partly overcome by interfering with the function of VLA4, the alpha4beta1 integrin, in vivo. In those studies, we used the anti-functional antibody Natalizumab. We extended our studies to an alternative VLA4 inhibitor, the novel non-peptidic small molecule TBC3486. Previous in vitro assays and molecular modeling studies indicate that TBC3486 behaves as a ligand mimetic, competing with VCAM-1 for the MIDAS site of VLA-4. As such, the compound has been shown to be efficacious in VLA-4 dependent models of inflammatory and autoimmune disease. The potential usefulness of this novel inhibitor in leukemia treatment was tested in our established in vitro and in vivo assays. LAX7R cells, primary pre-B-ALL with a normal karyotype from a patient with an early relapse, were used throughout for the studies reported here. LAX7R cells were treated with 25μM TBC3486 or THI0012 control, the inactive enantiomer of TBC3486, and seeded onto plates coated with human VCAM-1. Adhesion, scored after 2 days, was significantly inhibited by TBC3486 compared to control treated cells (7.9%±4.0 vs 95.4%±8.0; p=0.003). Proliferation rate and cell viability were unaffected by the treatments. In a co-culture system of LAX7R cells with OP9 stroma cells, which we use as an in vitro model of stroma-mediated chemotherapy resistance, we assessed differential effects of VDL (Vincristine, Dexamethasone, L-Asparaginase) on leukemia cell survival in the presence or absence of TBC3486. Stromal adhesion significantly protected LAX7R cells against VDL chemotherapy; this effect was significantly attenuated by TBC3486 compared to the control as determined by Trypan blue exclusion of dead cells (Cell viability of 39.9%±5.1 vs. 57.2±1.8; p=0.02). After these encouraging observations, we next evaluated the benefit of TBC3486 on leukemia progression in a xenotransplant assay. LAX7R cells were lentivirally labelled with luciferase for in vivo tracking and injected into NOD/SCID hosts. Three days after leukemia cell transfer, mice received either TBC3486 or THI0012 (control) (10mg/kg/d) daily for 2 weeks (intraperitoneally), with or without VDL chemotherapy. This experiment is in progress, but already survival of leukemia-bearing mice was significantly prolonged, from a median survival time (MST) for control mice of 33 days post-leukemia injection to a MST of 47 days post-leukemia injection for TBC3486 treated mice (p=0.02). Similarly, bioluminescence imaging revealed a marked delay of leukemia cell dissemination (p<0.0001). Taken together, our data demonstrate that small molecule inhibition of VLA4 using the novel TBC3486 is a suitable approach for targeting of chemotherapy-resistant leukemia. Further studies are warranted to understand and evaluate preclinically adjuvant small molecule inhibition of integrins to overcome relapse of ALL. Disclosures: No relevant conflicts of interest to declare.

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