Correlations of ΔNp73 and TAp73 Expression Pattern with Specific Genetic Rearrengents in AML.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 4501-4501
Author(s):  
Antonio Roberto L. Araújo ◽  
Ana Silvia G. Lima ◽  
Rodrigo A. Panepucci ◽  
Eduardo M. Rego
Keyword(s):  
Bone Marrow ◽  
De Novo ◽  
Expression Patterns ◽  
Reference Sample ◽  
Distinct Pattern ◽  
Dominant Negative ◽  
Leukemic Cells ◽  
Transactivation Domain ◽  
Expression Levels ◽  
Significant Difference

Abstract The p73 protein is a p53 homolog and acts on cell cycle and apoptosis regulation. Resistance to apoptosis is a common feature of Acute Myeloid Leukemia (AML), but mutations on the genes p53 and p73 are rare. It is translated in two distinct isoforms: TAp73 and ΔNp73. The later does not possess the N-terminal transactivation domain and exerts a dominant negative action over TAp73 and p53 functions. Theoretically an aberrant high expression of ΔNp73 may lead to a block of p53 and TAp73, thus conferring a proliferative advantage to the leukemic cells. In order to evaluate this issue, we proposed to: Compare the gene expression levels of TAp73 and ΔNp73 isoforms in the bone marrow from de novo AML patients and normal individuals; Correlate these expression patterns with the presence of the rearrangements PML-RARα, AML1-ETO and CBFβ-MHY11, (previously determined by RT-PCR according the BIOMED-1 protocol). From 137 AML patients whose samples were evaluated by Real Time PCR, 78 harbored the genetic rearrangements (referred to as RP group): PML-RARα (n = 30), AML1-ETO (n = 16) or CBFβ-MHY11 (n = 32), whereas in the 59 remaining samples these rearrangements were not detected (RN group). Additionally, CD34+ cell samples of 22 normal bone marrow donors were also evaluated. Sample input was normalized by GAPDH expression and the relative expression was calculated using the cell line k562 as reference sample. The mean expression of TAp73 and ΔNp73 was significantly lower on normal CD34+ cell compared to leukemic samples [(TAp73: mean (m) = 0.0162 ± standard deviation = 0.004 vs m = 0.623 ± 0.0845, p = 0,0047); (ΔNp73: m = 0.277 ± 0.09 vs m = 8.09 ± 1.34, p = 0,0215)]. A higher expression of TAp73 and ΔNp73 was observed on RN compared to RP samples [(TAp73: m = 0.992 ± 0.171 vs m = 0.344 ± 0.055, p < 0,0001); (ΔNp73: m = 12.44 ± 2.434 vs m = 4.80 ± 1.382, p = 0,0046)]. There was no difference in the expression of TAp73 between PML-RARα positive samples (m = 0.391 ± 0.095) and the remaining leukemic samples (m = 0.688 ± 0.104, p = 0,1476). However, the expression levels of ΔNp73 were significantly lower in the PML-RARα positive samples (m = 2.656 ± 0.370 vs m = 9.62 ± 1.69, p = 0,0317). No significant difference was observed in ΔNp73 and TAp73 expression between PML-RARα positive samples and the remaining samples with gene rearrengements (TAp73: m = 0.391 ± 0.095 vs m = 0.3144 ± 0.0671, p = 0,4990; ΔNp73: m = 2.656 ± 0.37 vs m = 6.153 ± 2.221, p = 0,2205). When compared to AML1-ETO and CBFβ-MHY11, the RN samples had a higher expression level of TAp73 (m = 0.3144 ± 0.0672 vs m = 0.992 ± 0.1717, p = 0.001), while there was no significant difference on the expression levels of ΔNp73 (m = 6.15 ± 2.22 vs m = 12.44 ± 2.43, p = 0.0642). These findings suggest that both p73 isoforms pathways are involved in the leukemogenic process. Moreover, the lower expression of ΔNp73 in the group with gene rearrangements may contribute to its better prognosis. The distinct pattern of ΔNp73 isoforms expression in AML with PML-RARα rearrangements suggests that it may be associated to a distinct response to apoptotic stimuli and to treatment outcome.

scholarly journals Adhesion Signaling States in AML

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 2386-2386 ◽  
Author(s):  
Chenyue W Hu ◽  
Amina A Qutub ◽  
Yihua Qiu ◽  
Suk Young Yoo ◽  
Nianxiang Zhang ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Protein Expression ◽  
Signaling Pathway ◽  
Conflicts Of Interest ◽  
Expression Patterns ◽  
Principal Component ◽  
Leukemic Cells ◽  
Holistic View ◽  
Npm1 Mutation ◽  
Expression Levels

Abstract Background Stromal contact in the bone marrow microenvironment is known to affect the resistance of leukemic cells to therapy, in particular the homing and engraftment of leukemic stem cells. This stromal interaction is mediated by the adhesion signaling pathway including extracellular matrix proteins (e.g. FN1, SPP1), cell surface and transmembrane proteins (e.g. CD44, integrin, CAV1), as well as intracellular binding proteins and enzymes (e.g. IGFBP2, PTK2, TGM2). Previous studies mostly examined these proteins in isolation, and hence they were unable to capture the coordination among subpathways and within patient subpopulations. Therefore, it is of key interest to study these proteins in their ensemble and obtain a holistic view of how adhesion signaling pathway gives rise to and affects different AML subpopulations. Methods To profile protein expressions in AML, we made a reverse phase protein array (RPPA) with proteins from leukemia enriched cells from 511 new AML patients. Both bone marrow (n=387) and peripheral blood (n=283) samples were used, with 140 cases having both. The RPPA was probed with 231 strictly validated antibodies, including antibodies against ITGA2, ITGB3, FN1, ITGAL, PTK2, IGFBP2, CD44, SPP1, CAV1 and TGM2. The normal bone marrow derived CD34+ cells were used for comparison. The protein expression data generated from this RPPA was then analyzed by the Standard Proteomic Analysis (SPA), a combination of computational methods including clustering, principal component analysis, network reconstruction (glasso), survival analysis, correlation tests and data mining from public databases. Results Based on the expression levels of ten proteins in the adhesion pathway, we first built a heatmap (Figure A) using “Prototype Clustering” that grouped all patients into six distinct clusters featured by C1) pan low, C2) high SPP1-CD44; C3) high CAV1; C4) high PTK2-ITGA2-ITGB3-FN1-IGFBP2-TGM2; C5) high TGM2; C6) high ITGAL and pan intermediate high expression levels. The adhesion pathway in AML showed literature-consistent patterns, e.g. the coupling between CD44 and SPP1 and the co-expression among integrin subunits, FN1 and PTK2, but also demonstrated new patterns, e.g. independent regulation of CAV1, as well as decoupled expression of TGM2 from the integrins. Each patient cluster represents an adhesion signaling state that can be seen in AML. As shown in this transition map (Figure B), there is an OFF state (C1), two isolated activation states of either CAV1 (C3) or CD44-SPP1 (C2), two intermediate activation states of either TGM2 (C5) or ITGAL (C6), and a combined activation state (C4) from the two intermediate states. By combining both connections inferred from the data and interactions collected from public databases (e.g. String, KEGG), we were able to expand the protein network beyond adhesion pathway and examine their expression levels in each adhesion signaling state. We observed positive co-regulation of SRC and PRKCA with the integrin subunits, connections between IGFBP2 and metabolism/synthesis proteins (e.g. GADPH, EIF4E, GSK), as well as the association of CD44 with histone modification (H3K4Me2, H3K4Me3), most of which have not been reported before. The adhesion activation states are not associated with most clinical correlates, including FLT3 and NPM1 mutation, gender and response status, with the exception of the CAV1 activation state (C3). A significant amount of patients with high CAV1 expression levels are in the favorable cytogenetics group (35% vs. 8% in general, p=0.00001), thus have fewer relapses (relapse rate of 26% vs. 64% in general, p=0.001) and superior overall survival (Figure C) and remission duration (Figure D). Conclusions We have discovered previously unrecognized protein expression patterns and activation states that control stromal contact and adhesion in AML. This includes independent activation of SPP1-CD44 and CAV1, intermediate activation of TGM2 and ITGAL and combined activation of integrin-FN1-PTK2, indicating diverse stromal interaction states in the bone marrow. In particular, the activation of CAV1 is prognostically favorable, suggesting a potential target for future therapeutics in AML. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

scholarly journals Evaluation of circulating miRNAs and mRNAs expression patterns in autism spectrum disorder

2021 ◽  
Vol 22 (1) ◽  
Author(s):  
Amany H. Abdelrahman ◽  
Ola M. Eid ◽  
Mona H. Ibrahim ◽  
Safa N. Abd El-Fattah ◽  
Maha M. Eid ◽  
...  
Keyword(s):  
Autism Spectrum Disorder ◽  
Gene Family ◽  
Complex Disease ◽  
Expression Patterns ◽  
Normal Volunteer ◽  
Disease Diagnosis ◽  
Autism Spectrum ◽  
Spectrum Disorder ◽  
Expression Levels ◽  
Significant Difference

Abstract Background Autism spectrum disorder is a condition related to brain development that affects a person’s perception and socialization, resulting in problems in social interaction and communication. It has no single known cause, yet several different genes appear to be involved in autism. As a genetically complex disease, dysregulation of miRNA expression and miRNA–mRNA interactions might be a feature of autism spectrum disorder. The aim of the current study was to investigate the expression profile of circulating miRNA-128, miRNA-7 and SHANK gene family in ASD patients and to assess the possible influence of miRNA-128 and miRNA-7 on SHANK genes, which might provide an insight into the pathogenic mechanisms of ASD and introduce noninvasive molecular biomarkers for the disease diagnosis and prognosis. Quantitative real-time PCR technique was employed to determine expression levels of miRNA-128, miRNA-7 and SHANK gene family in blood samples of 40 autistic cases along with 30 age- and sex-matched normal volunteer subjects. Results Our study revealed a statistical significant upregulation of miRNA-128 expression levels in ASD cases compared to controls (p value < 0.001). A statistical significant difference in SHANK-3 expression was encountered on comparing cases to controls (p value < 0.001). However, miRNA-7 expression showed no significant difference between the studied groups. Conclusions MiRNA-128 and SHANK-3 gene are emerging players in the field of ASD. They are promising candidates as noninvasive biomarkers in autism. Future studies are needed to emphasize their pivotal role.

Characteristics of De Novo Acute Leukemia Patients with Glycosylphosphatidylinositol (GPI) Protein-Negative Population of Leukemic Cells.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 4462-4462
Author(s):  
Hideyoshi Noji ◽  
Tsutomu Shichishima ◽  
Masatoshi Okamoto ◽  
Kazuhiko Ikeda ◽  
Akiko Nakamura ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Acute Leukemia ◽  
De Novo ◽  
Bone Marrow Failure ◽  
Flow Cytometric Analysis ◽  
Leukemic Cells ◽  
Remission Induction ◽  
Color Flow ◽  
Flow Cytometric ◽  
Number Of Patients

Abstract Paroxysmal nocturnal hemoglobinuria (PNH) is considered to be an acquired stem cell disorder affecting all hematopoietic lineages, which lack GPI-anchored membrane proteins, such as CD59, because of abnormalities in the phosphatidylinositol glycan-class A (PIG-A) gene. Also, PNH is one disorder of bone marrow failure syndromes, including aplastic anemia and myelodysplastic syndrome, which are considered as pre-leukemic states. In this study, to know some characteristics of patients with de novo acute leukemia, we investigated expression of CD59 in leukemic cells from 25 patients (female: male=8: 17; mean age ± standard deviation, 57.8 ± 19.5 years) with de novo acute leukemia by single-color flow cytometric analysis. In addition, the PIG-A gene from CD59− leukemic cells sorted by FACS Vantage in 3 patients with acute leukemia was examined by sequence analysis. All the patients had no past history of PNH. Based on the French-American-British criteria, the diagnosis and subtypes of acute leukemia were determined. The number of patients with subtypes M1, M2, M3, M4, M5, and M7 was 1, 14, 2, 4, 2, and 2, respectively. Two of the patients were classified into acute myeloid leukemia with trilineage myelodysplasia from morphological findings in bone marrow. Chromosomal analyses presented abnormal karyotypes in 14 of 25 patients. Flow cytometric analyses showed that leukemic cells from 16 of 25 patients (64%) had negative populations of CD59 expression and the proportion of the populations was 63.3 ± 25.7%, suggesting the possibility that CD59− leukemic cells from patients with de novo acute leukemia might be derived from PNH clones. In fact, the PIG-A gene analyses showed that monoclonal or oligoclonal PIG-A mutations in coding region were found in leukemic cells from 3 patients with CD59− leukemic cells and all of the clones with the PIG-A mutations were minor. Then, various clinical parameters, including rate of complete remission for remission-induction chemotherapy, peripheral blood, bone marrow blood, and laboratory findings, and results of chromosomal analyses were statistically compared between 2 groups of patients with (n=16) and without (n=9) CD59− leukemic cells. The reticulocyte counts (10.5 ± 13.0 x 104/μl) and proportions of bone marrow erythroblasts (17.5 ± 13.9%) in patients with only CD59+ leukemic cells were significantly higher than those (2.5 ± 1.7 x 104/μl, p&lt;0.05; and 5.6 ± 6.2%, p&lt;0.01, respectively) in patients with CD59− leukemic cells. The proportions of bone marrow blasts (69.3 ± 21.1%) in patients with CD59− leukemic cells were significantly higher than those (45.5 ± 19.3%, p&lt;0.02) in patients with only CD59+ leukemic cells. In conclusion, our findings indicate that leukemic cells derived from PNH clones may be common in de novo acute leukemia patients, suggesting that bone marrow failure may have already occurred in localized bone marrow even in de novo acute leukemia.

Expression of P73 Splice Variants Correlates to Resistance to DNA Damaging Drugs in Childhood T-Lineage Acute Lymphoblastic Leukemia.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 995-995
Author(s):  
Marrit Meier ◽  
Monique L. Den Boer ◽  
Jules P.P. Meijerink ◽  
Monique Passier ◽  
Elisabeth R. Van Wering ◽  
...  
Keyword(s):  
Acute Lymphoblastic Leukemia ◽  
Mononuclear Cells ◽  
Splice Variants ◽  
Lymphoblastic Leukemia ◽  
Mrna Level ◽  
Dominant Negative ◽  
Leukemic Cells ◽  
Mrna Levels ◽  
Transactivation Domain ◽  
Loss Of Function

Abstract Children with T-lineage Acute Lymphoblastic Leukemia (T-ALL) have a higher relapse-risk and are in-vitro more resistant to therapeutic drugs compared to ALL patients with a precursor-B phenotype. Cellular resistance to anti-cancer agents has previously shown to be associated with failure of P53 family member signaling by abrogation of P53 function due to loss-of-function mutations or dominant-negative inhibition by isoforms of P73 lacking (part of) the N-terminal transactivation domain (P73ΔEX2, P73ΔEX2/3, ΔN-P73 and ΔN’-P73). Since p53 mutations are not commonly found in T-ALL, we investigated the expression levels of p73 splice variants in relation to drug resistance in children with T-ALL. Splice variants were quantitatively measured at the mRNA level in leukemic cells of 55 T-ALL patients and mononuclear cells of 12 non-leukemic controls. TA-p73 (transactivation competent), p73Δex2, p73Δex2/3, ΔN-p73 and ΔN’-p73 were all found to be present at a relatively higher mRNA level in T-ALL patients than controls (P < 0.05 for all), suggesting that expression of the TP73 gene is deregulated in T-ALL. Resistance of T-ALL cells to the DNA damaging drug daunorubicin correlated with mRNA levels of the dominant-negative variants of p73, i.e. ΔN-p73 and ΔN’-p73 (Rs = 0.38, P = 0.03). In contrast, expression of none of the variants, including ΔN-p73 and ΔN’-p73, was related to resistance of T-ALL cells to non-DNA damaging drugs (prednisolone, vincristine and L-asparaginase). In conclusion, high expression of ΔN-p73 and ΔN’-p73 variants possibly contributes to resistance to DNA damaging drugs in childhood T-ALL.

Clinical Activity, Pharmacokinetics, and Pharmacodynamics of Sorafenib In Pediatric Acute Myeloid Leukemia.

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 1073-1073
Author(s):  
Hiroto Inaba ◽  
Jeffrey E Rubnitz ◽  
Elaine Coustan-Smith ◽  
Lie Li ◽  
Brian D Furmanski ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Acute Myeloid Leukemia ◽  
Myeloid Leukemia ◽  
De Novo ◽  
Leukemic Cells ◽  
Clinical Activity ◽  
Newly Diagnosed ◽  
Pediatric Acute Myeloid Leukemia ◽  
Rtk Signaling ◽  
Acute Myeloid

Abstract Abstract 1073 Background: Aberrant receptor tyrosine kinase (RTK) signaling arising from genetic abnormalities, such as FLT3-internal tandem duplications (FLT3-ITD), is an important mechanism in the development and growth of acute myeloid leukemia (AML) and is often associated with a poor outcome. Hence, inhibition of RTK signaling is an attractive novel treatment option, particularly for disease that is resistant to conventional chemotherapy. We evaluated the clinical activity of the multikinase inhibitor sorafenib in children with de novo FLT3-ITD–positive AML or relapsed/refractory AML. Methods: Fourteen patients were treated. Six patients with newly diagnosed FLT3- ITD–positive AML (aged 9–16 years; median, 12 years) received 2 cycles of remission induction therapy and then started sorafenib (200 mg/m2 twice daily for 20 days) the day after completing induction II (low-dose cytarabine, daunorubicin, and etoposide). Nine patients (aged 6–17 years; median, 9 years) with relapsed AML (including one treated on the above regimen) received sorafenib alone (2 dose levels; 200 and 150 mg/m2) twice daily for the first week of therapy, concurrently with clofarabine and cytarabine on days 8–12, and then alone from days 13 to 28. Sorafenib pharmacokinetics were analyzed at steady-state on day 8 of sorafenib in patients with newly diagnosed AML and on day 7 in patients with relapsed AML. In patients with relapsed AML, the effect of sorafenib on signaling pathways in AML cells was assessed by flow cytometry. Results: All 6 newly diagnosed patients, including 2 whose AML was refractory to induction I, achieved a complete remission (CR) after induction II; 5 had negative minimal residual disease (MRD; <0.1% AML cells in bone marrow) after induction II. Both patients in this group who relapsed achieved second remissions, one with sorafenib alone and one on the relapse regimen described above. Of the 9 patients with relapsed AML, 6 (4 with FLT3-ITD) were treated with sorafenib 200 mg/m2. All 6 had a >50% decrease in blast percentage and/or bone marrow cellularity after 1 week of sorafenib. After concurrent sorafenib and chemotherapy, 5 of the 9 patients with relapsed AML achieved CR (2 had negative MRD) and 2 achieved a partial remission (PR; 5%-25% AML cells in bone marrow); all 4 patients with FLT3-ITD had a CR or PR. After sorafenib treatment, 6 patients underwent HSCT while 2 with FLT3-ITD who could not receive HSCT were treated with single-agent sorafenib and have maintained CR for up to 8 months. Hand-foot skin reaction (HFSR) or rash occurred in all patients and improved with cessation of sorafenib. Dose-limiting toxicity (DLT, grade 3 HFSR and/or rash) was observed in 3 of the 6 patients with relapsed AML treated with 200 mg/m2 of sorafenib; no DLT was observed at 150 mg/m2. The effect of sorafenib on downstream RTK signaling was tested in the leukemic cells of 4 patients: in most samples, phosphorylation of S6 ribosomal protein and 4E-BP1 was inhibited. The mean (± SD) steady-state concentration (Css) of sorafenib was 3.3 ± 1.2 mg/L in the newly diagnosed group and 6.5 ± 3.6 mg/L (200 mg/m2) and 7.3 ± 3.6 mg/L (150 mg/m2) in those with relapsed AML. In both groups, the mean conversion of sorafenib to sorafenib N-oxide was 27%-35% (approximately 3 times greater than previously reported), and mean sorafenib N-oxide Css was 1.0–3.2 mg/L (2.1-6.7 μM). In a 442-kinase screen, the inhibitory profiles of sorafenib N-oxide and sorafenib were similar, and FLT3-ITD phosphorylation was potently inhibited by both forms (sorafenib N-oxide Kd = 0.070 μM; sorafenib Kd = 0.094 μM). Sorafenib N-oxide inhibited the growth of an AML cell line with FLT3-ITD (IC50 = 0.026 μM) and 4 AML cell lines with wild-type FLT3 (IC50 = 3.9–13.3 μM) at approximately half the potency of sorafenib. Conclusion: In children with de novo FLT3-ITD and relapsed/refractory AML, sorafenib given alone or with chemotherapy induced dramatic responses and inhibited aberrant RTK signaling in leukemic cells. Sorafenib and its active metabolite (sorafenib N-oxide) likely contribute to both efficacy and toxicity. These results warrant the incorporation of sorafenib into future pediatric AML trials. Disclosures: Inaba: Bayer/Onyx: Research Funding. Off Label Use: Sorafenib and clofarabine: both used for treatment of pediatric acute myeloid leukemia.

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 ◽  
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.

Thymic Stromal Lymphopoeitin Signaling Contributes To Increased Risk Of Relapse Of Pediatric Acute Lympoblastic Leukemia Through Enhanced Survival Of Leukemic Cells That Overexpress Thymic Stromal Lymphopoeitin Receptor

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 2543-2543
Author(s):  
Christopher D Chien ◽  
Matthew Kreitman ◽  
Haiying Qin ◽  
Terry J Fry
Keyword(s):  
Bone Marrow ◽  
Annexin V ◽  
Thymic Stromal Lymphopoietin ◽  
Leukemic Cells ◽  
Leukemia Cells ◽  
Early Stages ◽  
Increased Risk ◽  
Significant Difference ◽  
Risk Of Relapse

Abstract Pediatric acute lymphoblastic leukemia (ALL) is the most common childhood malignancy. Although the cure rate for this disease is greater than 85%, ALL remains the number one cause of cancer-related deaths in children due to relapse of ALL. Therefore, there is a great need to identify new therapies for patients who have recurrent disease. Recently, a subset of pediatric ALL patients whose leukemic cells express high levels of thymic stromal lymphopoietin receptor (TSLPR/CRLF2) have been shown to have an increased risk of relapse and shorter disease free and poorer overall survival. Overexpression of TLSPR occurs in 8% of unscreened pediatric precursor B ALL and occurs by genomic rearrangement of the TSLPR gene, which fuses the unmutated TSLPR gene to altered transcriptional control or by other yet to be described means. The mechanism by which Thymic Stromal Lymphopoietin (TLSP) signaling contributes to increased risk of relapse is unknown. Studies have shown aberrant signaling in high TSLPR expressing ALL patient derived cell lines relying heavily on in vitro experiments. As no pre-clinical model of high TSLPR ALL has been published, we created a model of high TSLPR expressing leukemia to study TSLPR overexpressing leukemia progression. We have created a high TSLPR expressing leukemia cell line through retroviral transduction of a transplantable syngeneic mouse leukemia model in which the leukemic progression can be studied with physiologic levels of TSLP. This high TSLPR leukemia has levels of expression of TSLPR comparable to what is found on human leukemia that overexpress TSLPR. The TSLPR is functional in these cells and we see increased phosphorylation of STAT5 protein in response to IL-7 or TSLP stimulation. When we introduce the leukemia into mice and look at disease progression, we observed an 8 fold difference in the numbers of cells in the bone marrow 5 days after intravenous injection corresponding to an early stage of leukemia progression (Figure 1. high TSLPR 1.61%+/-0.95 vs. low TSLPR 0.20%+/-0.16). Interestingly we find no significant difference in long term survival of mice injected with either low or high TSLPR leukemia lines. The increased numbers of leukemic cells in the bone marrow at early stages of leukemic progression could be due to an increased rate of proliferation or better survival/engraftment. Low and high TSLPR expressing cells show no significant difference in growth rate in vitro or in vivo in dye dilution assays (Figure 2) suggesting that the increase in leukemic cells in the bone marrow is through enhanced survival. To test this, we treated low and high TSLPR leukemia lines with the steroid dexamethasone in the absence or presence of TSLP. We found that the addition of TSLP significantly reduced the Annexin V positive relative to cells not treated with TSLP in the high TSLP expressing leukemia cells, while in low TSLPR expressing cells we observed no decrease in Annexin V positive cells (Figure 3). This suggests that high TSLPR expression sensitizes leukemia cells to TSLP in the leukemia microenvironment. To confirm that this is the case we have found by gene expression analysis that we can detect TSLP in mouse bone marrow. We hypothesize that therapies targeting the TSLP signaling axis in ALL would decrease the risk of relapse. To test this hypothesis we have generated TSLPR-Fc conjugates to block TSLP signaling. We plan on using these reagents to block TSLP signaling to see if we can reverse the increased amounts of leukemia we find in mice at early stages of leukemic progression as well as the eliminate the survival advantage provided by TSLP to high TSLPR expressing leukemic cells in response to chemotherapeutic agents. Disclosures: No relevant conflicts of interest to declare.

Selectins and Their Ligands Are Required for Homing and Engraftment of BCR-ABL+ Leukemia-Initiating Cells.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 697-697 ◽  
Author(s):  
Daniela S. Krause ◽  
Ulrich H. von Andrian ◽  
Richard A. Van Etten
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Autologous Transplantation ◽  
Leukemic Cells ◽  
Leukemic Stem Cells ◽  
Hematopoietic Stem ◽  
Retroviral Transduction ◽  
Expression Levels ◽  
Selectin Ligand ◽  
Beta 1 Integrin

Abstract Autologous hematopoietic stem cell (HSC) transplantation is a feasible form of treatment for many types of leukemias and lymphomas, including chronic myeloid leukemia (CML). Malignant cells contaminating the graft, however, can engraft and lead to relapse of the original disease. Previous studies have demonstrated that BCR-ABL+ leukemic progenitors have defects in the adhesive function of beta-1 integrins and in their response to the chemokine SDF-1alpha, pathways that are critical for homing and engraftment of normal HSC. We hypothesized that BCR-ABL-expressing leukemic stem cells differ from normal HSC in their homing and engraftment properties. Using a retroviral transduction/transplantation model of CML and donor/recipient mice with mutations in adhesion molecules, we investigated the role of specific adhesion pathways in the engraftment of CML-like leukemia. We found no difference in the expression levels of integrins, LFA-1, and CXCR4 between normal and BCR-ABL+ c-Kit+ Lin- cells, but lower expression levels of P-selectin glycoprotein ligand-1 (PSGL-1) and of L-selectin. In transplantation experiments, VCAM-1, the principal bone marrow ligand for beta-1 integrin, was not required in the bone marrow endothelium of the recipient for efficient engraftment of CML-like disease, confirming that progenitors capable of initiating CML-like leukemia upon transplantation are independent of the beta-1 integrin pathway for engraftment. Likewise, recipient P-selectin was also not required for the engraftment of CML-like leukemia. By contrast, deficiency of PSGL-1 in the leukemic cells or of E-selectin in the recipient significantly reduced engraftment by BCR-ABL-expressing stem cells, as assessed by Southern blot quantitation of proviral clone frequency. The requirement for recipient E-selectin could be bypassed by direct intrafemoral injection of BCR-ABL-expressing cells, leading to polyclonal leukemia. BCR-ABL-expressing cells that were deficient for the selectin ligand-synthesizing enzymes Core-2 or Fucosyltransferases IV and VII also exhibited decreased engraftment and increased disease latency. Treatment of BCR-ABL-transduced cells with neuraminidase, which destroys selectin binding sites, completely blocked leukemic engraftment. Whereas L-selectin has no role in homing and engraftment of normal HSC, BCR-ABL-expressing L-selectin-deficient progenitors were profoundly defective for engraftment, with decreased disease clonality, increased disease latency, and frequent death of recipients from graft failure. Importantly, efficient engraftment and leukemogenesis of BCR-ABL-expressing L-selectin-deficient cells was restored by co-expression of a chimeric E/L-selectin molecule that is resistant to cell surface shedding. These results establish that BCR-ABL-expressing leukemic stem cells rely to a greater extent on selectins and their ligands for homing and engraftment than normal HSC. Specific blocking of selectin-ligand interactions is a novel clinical strategy to exploit the differences in normal and Ph+ stem cells that may be beneficial in an autologous transplantation setting.

Essential Role of Gab2 in CSF-1 Dependent Macrophage Development in the Bone Marrow.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 2197-2197
Author(s):  
Angel W. Lee ◽  
David J. States ◽  
Heather Grifka
Keyword(s):  
Bone Marrow ◽  
Colony Formation ◽  
Fold Increase ◽  
Dominant Negative ◽  
Mononuclear Phagocytes ◽  
Dominant Negative Effect ◽  
Fold Reduction ◽  
Significant Difference

Abstract Mononuclear phagocytes (MNPs) are critical in health to maintain tissue homeostasis and in disease as major effectors of innate immunity. In the adult, MNPs develop from bone marrow (BM) progenitors that differentiate to monocytes, tissue macrophages (Mϕs), and specialized cells (dendritic cells, microglia and osteoclasts). Colony Stimulating Factor-1 (CSF-1) acts through the CSF-1R to regulate proliferation, survival and differentiation of MNPs. GAB2, a member of the GAB family of scaffolding proteins (GAB1-3), modulates and amplifies signals from numerous receptors, through recruitment of phosphatidylinositol 3-kinase (PI3K) and Shp2 phosphatase. Knockdown studies in the 32D myeloid cell line from our lab showed that GAB2 is required for CSF-1 induced mitogenesis and activation of Akt, a PI3K effector. To test the hypothesis that the GAB2-PI3K axis is important for MNP development in vivo, we examined Mϕ development in GAB2 +/+ and −/− mice (gift of Josef Penninger). GAB2 is upregulated 14-fold during CSF-1-induced differentiation of primary BM cells from GAB2+/+ mice. A significant difference is detected in the steady state percentage of F4/80+ BM cells (F4/80 is a mature Mϕ marker): 17.5 ± 1.6 (GAB2+/+, n=8) vs. 11.4 ± 1.6 (GAB2–/−, n=6) (p=0.025, 2-sided t-test). Using the CFU-C progenitor assay with CSF-1 as the only growth factor, primary BM cells from GAB2 −/− mice show a striking 7-fold reduction in colony numbers compared to those from GAB2 +/+ mice (p=0.004) and the colonies were much smaller. Thus GAB2 is essential for optimal CSF-1-dependent Mϕ colony formation. We then used CD31 and Ly6C and flow cytometry to follow the kinetics of Mϕ formation during BM differentiation. These markers monitor sequential stages of Mϕ development: CD31highLy6C– -&gt; CD31+Ly6C+ -&gt; CD31-Ly6Chigh (Eur. J. Immunol.24:2279). As early as 2 days after differentiation induction, GAB2−/− BM cells show a 2-fold reduction in the CD31+Ly6C+ subset (p=6×10−6) and a 6-fold increase in the CD31-Ly6Chigh subset (p=1×10−4), indicating that in the absence of GAB2, CSF-1 promotes a smaller increase in myeloid progenitors and an earlier appearance of more mature cells. To assess proliferation in the progenitor population, day 2 BM cells were labeled with CFSE. Consistent with decreased cell division during early stages of Mϕ development in the absence of GAB2, we observed a 66% reduction in CFSE intensity in GAB2+/+ compared to −/− cells after 3 days in culture. A 2-fold reduction in proliferation by the MTS assay is similarly observed during late Mϕ development (days 5-7) (p=10−4). No difference in viability or expression of CSF-1R or CD11b is found in day 7 Mϕs from GAB2+/+ and −/− mice, excluding increased cell death or arrested differentiation as causes. To investigate the role of GAB2-PI3K, we transduced BM cells with viruses expressing WT-GAB2, 3YF-GAB2 (defective in PI3K binding), both in MSCV-IRES-GFP, or empty MSCV. WT- and 3YF-GAB2 expression in GAB2−/− cells increase the numbers of CFU-Cs by 5- and 2-fold respectively and by 8- and 2.4-fold in GFP+ colonies ≥ 500 μ. Conversely, 3YF-GAB2 exerts a dominant-negative effect on GAB2+/+ cells (a decrease of 30% and 76% in unsorted cells and GFP+ colonies ≥ 500 μ respectively). Therefore PI3K recruitment by GAB2 is required for CSF-1-induced Mϕ colony formation but other GAB2 effector pathways are also important. Our findings support the conclusion that GAB2 is crucial for CSF-1 mediated Mϕ development in the BM, by regulating monocyte/Mϕ progenitor expansion and Mϕ proliferation, in part through PI3K.

A Phase II Study of the Combination of Oral Rigosertib and Azacitidine in Patients with Myelodysplastic Syndromes (MDS)

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 910-910 ◽  
Author(s):  
Shyamala C. Navada ◽  
Lewis R. Silverman ◽  
Katherine P. Hearn ◽  
Rosalie Odchimar-Reissig ◽  
Erin P. Demakos ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Phase I ◽  
Phase Ii ◽  
Research Funding ◽  
De Novo ◽  
Standard Dose ◽  
Single Agent ◽  
Leukemic Cells ◽  
Bone Marrow Blast ◽  
Recommended Phase Ii Dose

Background: Rigosertib (RIG) is a Ras-mimetic that inhibits the PI3K and PLK cellular signaling pathways by binding directly to the Ras-binding Domain found in Ras effector proteins. It has been tested as a single agent in patients (pts) after failure of hypomethylating agents (HMAs). In vitro, the combination of RIG with azacitidine (AZA) inhibits growth and induces apoptosis of leukemic cells in a sequence-dependent fashion (RIG administered prior to AZA) (Skidan et al 2006). Phase I results of this study in pts with MDS or AML showed combination of oral RIG and standard-dose AZA to be well-tolerated with evidence of efficacy (Navada et al, Blood 2014). Phase II was initiated to further study the combination in pts with MDS. Methods: Results from pts in Phase II with MDS previously untreated with an HMA, or who had failed to respond to or progressed on a prior HMA, are presented, while response data from Phase I MDS pts are updated. Pts with CMML are analyzed separately. Oral RIG was administered twice daily on Day 1-21 of a 28-day cycle at the recommended Phase II dose (RPTD: 560 mg qAM and 280 mg qPM). AZA 75 mg/m2/d SC or IV was administered for 7 days starting on Day 8. A CBC was performed weekly and a bone marrow aspirate and/or biopsy was performed at baseline, day 29, and then every 8 weeks thereafter. Results: The combination of oral RIG and AZA has been administered to a total of 45 pts within Phase I (N=18) and Phase II (N=27). Pts were classified into the following MDS risk categories per the IPSS (Greenberg et al, Blood 1997): intermediate-1 (4), intermediate-2 (10), high-risk (14), and IPSS classification pending (4). Five pts had CMML and 8 had AML. Median age was 66 years; 69% of pts were male; and ECOG performance status was 0, 1, and 2 in 27%, 67%, and 6%, respectively. Twelve pts [MDS (9), CMML (3)] received prior HMA therapy: AZA (11 pts), decitabine (1 pts). Patients have received 1-21+ cycles of treatment to date (median, 3 cycles), with median duration of treatment of 14 weeks. Among 15 evaluable MDS pts treated with the RPTD (1 pt in Phase I, 14 pts in Phase II), marrow responses were observed in 10: marrow CR (mCR) (8), marrow PR (mPR) (2). Responses according to IWG criteria were observed in 10 pts: complete remission (CR) (1), mCR (7), hematologic improvement (HI) (2). Table 1. Responses for MDS Patients Treated at the Recommended Phase II Dose Pt Prior HMA Best BMBL at Nadir1 IWG Response2 Hematologic Improvement 102-008 None mCR mCR Platelet 101-010 None mCR CR Erythroid & Neutrophil 101-011 None mCR mCR None 101-013 None mCR mCR Erythroid 102-010 None SD SD None 101-014 AZA PD PD None 102-011 AZA mPR HI Erythroid & Platelet 101-016 AZA SD SD None 101-017 AZA mCR mCR None 102-013 None NE NE NE 101-019 None SD SD None 101-021 None PD PD None 101-024 None mCR mCR None 101-022 AZA mCR mCR None 101-025 None mCR mCR None 101-026 AZA NE NE NE 101-027 None NE NE NE 102-016 None mPR HI Platelet 1 Silverman et al, Hematol Oncol 2014 2 IWG = International Working Group (Cheson et al, Blood 2006) NE = not evaluable BMBL = bone marrow blast Overall, in pts with MDS treated on Phase I and Phase II, marrow responses were observed in 15 out of 20 evaluable pts: mCR (13), mPR (2). Responses according to IWG 2006 criteria were observed in 14 out of 19 evaluable MDS pts: CR (2), mCR (10), HI (2). Among the 7 evaluable pts with MDS in both the Phase I and Phase II who had failed to respond or progressed on prior treatment with an HMA, 5 had a response after RIG was added: CR (1), mCR (3), HI (1). Analyzed as a separate subgroup, 2 out of 5 (40%) pts with CMML had a mCR. The most frequent adverse events (AEs) in Cycle 1 included nausea (21%) and fatigue (15%), which were also the most frequent AEs in all cycles (fatigue, 28%; nausea, 26%). Six deaths have been observed so far. Three pts were treated for more than 1 year and continue on study. Conclusions: The combination oforalrigosertib and standard-dose AZA was well tolerated in repetitive cycles in pts with MDS. Marrow CR was observed in 65% of pts, both with de novo MDS and after failure of prior HMA therapy. In pts who received the RPTD, 67% of pts with MDS had a bone marrow blast and IWG response. These results suggest potential synergistic interaction of the combination and support continued study of this unique combination in patients with MDS. Disclosures Silverman: Onconova Therapeutics Inc: Honoraria, Patents & Royalties: co-patent holder on combination of rigosertib and azacitdine, Research Funding. Daver:ImmunoGen: Other: clinical trial, Research Funding. DiNardo:Novartis: Research Funding. Konopleva:Novartis: Research Funding; AbbVie: Research Funding; Stemline: Research Funding; Calithera: Research Funding; Threshold: Research Funding. Pemmaraju:Stemline: Research Funding; Incyte: Consultancy, Honoraria; Novartis: Consultancy, Honoraria, Research Funding; LFB: Consultancy, Honoraria. Fenaux:CELGENE: Honoraria, Research Funding; JANSSEN: Honoraria, Research Funding; AMGEN: Honoraria, Research Funding; NOVARTIS: Honoraria, Research Funding. Fruchtman:Onconova Therapeutics Inc: Employment. Azarnia:Onconova Therapeutics Inc: Employment.

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