scholarly journals The Role of SMAD7 Downstream of TEL-AML1 Signalling in t(12;21) Acute Lymphoblastic Leukemia

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 4823-4823 ◽  
Author(s):  
Aishwarya Sundaresh ◽  
Maurizio Mangolini ◽  
Jasper de Boer ◽  
Mike Hubank ◽  
Nicholas Goulden ◽  
...  
Keyword(s):  
Gene Expression ◽  
Cell Lines ◽  
Conflicts Of Interest ◽  
Fusion Gene ◽  
Lymphoblastic Leukemia ◽  
Xenograft Model ◽  
Direct Role ◽  
Hematopoietic Stem ◽  
Childhood All

Abstract The single most frequent chromosomal translocation associated with childhood ALL is the t(12;21) rearrangement that creates a fusion gene between TEL (ETV6) and AML1 (RUNX1). Although TEL-AML1+ patients have very good prognoses, relapses occur in up to 20% of patients and many patients face long-term side effects of chemotherapy. Recent data has shown that TEL-AML1 has a direct role in inducing signal transducer and activator of transcription 3 (STAT3) activation in human t(12;21) leukemia. This activation has been shown to transcriptionally induce MYC and is critical for survival of TEL-AML+ leukemia cells. Here, we demonstrate that STAT3 also regulates SMAD7 gene expression. SMAD7 is an antagonist of TGF-β signaling, functioning through a negative feedback mechanism, but is also known to function in other biological pathways. Interestingly, SMAD7 has also been shown to play a role in promoting self-renewal of hematopoietic stem cells. We show that both pharmacological and mechanistic inhibition of STAT3 results in down regulation of SMAD7 gene expression in TEL-AML1+ cell lines. This result was specific to TEL-AML1+ cells and not found in cells of other ALL subtypes. To understand the role played by SMAD7 in TEL-AML1+ cells, we used lentiviral vectors expressing shRNA targeting SMAD7. Interestingly, SMAD7 silencing was found to inhibit proliferation of TEL-AML1+ cell lines, eventually leading to growth arrest and apoptosis. Furthermore, we have established that this effect is not mediated through TGF-β signalling. This poster highlights the results of RNA-seq performed on TEL-AML1+ cells with SMAD7 knockdown and in vivo xenograft model of SMAD7 shRNA in TEL-AML+ ALL. Disclosures No relevant conflicts of interest to declare.

Intermediate-Risk Acute Lymphoblastic Leukemia (ALL) Patients with and without Relapse Differentially Depend on Survival Signals From Microenvironment

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 752-752
Author(s):  
Peter Rhein ◽  
Rita Mitlohner ◽  
Renate Kirschner-Schwabe ◽  
Christian Hagemeier ◽  
Gunnar Cario ◽  
...  
Keyword(s):  
Gene Expression ◽  
Stromal Cells ◽  
Conflicts Of Interest ◽  
Lymphoblastic Leukemia ◽  
Leukemic Cells ◽  
Induction Treatment ◽  
Intermediate Risk ◽  
Childhood All ◽  
Survival Signals

Abstract Abstract 752 Risk stratification in the childhood ALL-BFM clinical trial has been based on molecular/cytogenetic markers and the in vivo response to treatment (blast reduction in peripheral blood after prednisone prephase, bone marrow clearance after induction treatment and minimal residual disease (MRD) after induction and induction consolidation). These criteria classify about 50% of ALL cases into standard-risk (SR) and high-risk (HR) groups. The intermediate-risk (IR) ALL group, showing moderate levels of MRD, provides the majority of relapses, but still lacks specific prognostic markers which could distinguish between those patients subsequently relapse and those remaining relapse-free (IR+r and IR-r). We established a bank of NOD/SCID ALL xenografts representing IR+r and IR-r subgroups (12 vs 13 patients) and characterized the samples in a broad series of functional assays. There was no difference in the sensitivity to drugs (dexamethasone, daunorubicin, L-asparaginase and vincristine) between the IR+r and IR-r groups, however, the rate of spontaneous apoptosis in vitro was significantly higher in the IR+r group than in the IR-r group (69 +/− 7% vs 39 +/− 6%, p=0.02). Given that in the presence of stromal cells the cell death level has been similar in both IR groups (9 +/− 5% vs 7 +/− 7%, p=0.8), these observations suggest that the IR+r ALL is more dependent on survival signals from microenvironment than IR-r ALL. In the NOD/SCID engraftment model, leukemic cells from IR+r patients engrafted more rapidly than cells from IR-r patients (93 +/− 12 days vs 170 +/− 24 days, p=0.006), pointing to a better in vivo cooperation between leukemia cells and microenvironment in the IR+r cases. In order to identify the underlying molecular pathways, gene expression changes induced in the presence and absence of stroma have been investigated at genome-wide level (Human Gene 1.0 ST Affymetrix array). Of the apoptosis-regulating genes, a caspase inhibitior from the IAP family, BIRC3, was found to be up-regulated significantly higher in the IR-r than in the IR+r group (3.7 +/− 1.1 vs 1.7 +/− 0.1-fold up-regulation, p=0.016). The adhesion molecule VCAM1 has been generally up-regulated by the co-incubation with stroma, with a tendency to a higher up-regulation in the IR-r (4.4 +/− 0.7-fold) than in the IR+r (2.7 +/− 1.0-fold) group. We further speculated that interaction with microenvironment is a mutual process which also implicates gene expression changes of the stroma. To this end, mouse stromal cells (MS5 cell line) were incubated with a series of ALL samples (n=3), purified by MACS sorting and investigated using Mouse Gene 1.0 ST Affymetrix arrays. The list of genes whose expression increased by at least 2-fold in all 3 cases (n=10), included the macrophage inflammatory peptide-1alpha (MIP1A/CCL3), a chemokine known to regulate chemotaxis of lymphocytes. Our data suggest that the cooperation of ALL with microenvironment may provide a promising approach to molecularly discriminate ALL cases with relapse and to identify potential relapse-associated targets within the IR-ALL group. Disclosures: No relevant conflicts of interest to declare.

Leukemogenic Fusion Gene (p190 BCR-ABL) Transduction Into Hematopoietic Stem/Progenitor Cells In the Common Marmoset

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 4323-4323
Author(s):  
Yamin Tian ◽  
Yan Dong ◽  
Seiichiro Kobayashi ◽  
Manabu Ozawa ◽  
Kiyoko Izawa ◽  
...  
Keyword(s):  
Gene Expression ◽  
Bone Marrow ◽  
Progenitor Cells ◽  
Fusion Gene ◽  
Lentiviral Vector ◽  
Lymphoblastic Leukemia ◽  
Common Marmoset ◽  
Hematopoietic Stem ◽  
The Common

Abstract Abstract 4323 [Introduction] Patients with Philadelphia chromosome (p190 BCR-ABL fusion gene)-positive acute lymphoblastic leukemia have a poor prognosis despite intensive therapeutic intervention. Although a rodent model of this leukemia was previously established, the genetic and physiological differences between humans and rodents make it difficult to extrapolate the results from these models and apply these findings to human cases. Primates are more genetically related to humans than rodents. In this study, we attempted to develop a leukemia non-human primate model that mimics various human systems. [Methods and results] (1) A third-generation VSV.G pseudotyped lentiviral vector expressing the p190 BCR-ABL fusion gene driven by CMV or PGK promoter was produced (HIV-CMV/PGK-BCR-ABL). Ba/F3 cells, a mIL-3-dependent murine hematopoietic cell line, were transfected with this vector and cultured without mIL-3. These cells rapidly expanded after 12 days, indicating that p190 BCR-ABL gene expression allowed the Ba/F3 cells to grow autonomously. Next, using a biotin-labeled anti-marmoset CD34 monoclonal antibody (clone MA24) which was produced in our laboratory, MACS-sorted bone marrow CD34+ cells were transduced with the lentiviral vector (HIV-CMV/PGK-BCR-ABL) and subjected to the colony formation assay. In the majority of examined colonies, p190 BCR-ABL gene was detected regardless of the promoter. Taken together, the above findings indicate that p190 BCR-ABL gene was efficiently transduced into marmoset hematopoietic stem/progenitor cells. (2) Peripheral blood mononuclear cells (PBMNCs) were collected from individual marmosets after mobilizing the hematopoietic stem/progenitor cells with G-CSF. These cells were stimulated with cytokines (hIL3, hSCF and hTPO), followed by the transduction with the lentiviral vector. These cells were transplanted into marmosets preconditioned with busulfan. In this ex vivo transduction method, p190 BCR-ABL gene expression which was detected in PBMNCs by nested RT-PCR disappeared after day 56 and 100 in two marmosets. (3) Concentrated lentiviral vector was directly injected into the bone marrow cavity of individual marmosets pretreated with 5-fluorouracil and prednisolone. In this in vivo direct injection method, p190 BCR-ABL gene expression was maintained for more than one year and a half. Transduction of p190 BCR-ABL gene into hematopoietic stem/progenitor cells was confirmed by colony forming assay. In this model, one marmoset unexpectedly developed myelofibrosis-like disease. However, none of the marmosets have developed leukemia to date. [Conclusion] We successfully achieved sustained p190 BCR-ABL gene expression in vivo. This novel in vivo approach will help to develop a marmoset leukemia model in the future. Because a multiple-hit model of oncogenesis has been proposed for various human cancers, a genetic mutation in addition to p190 BCR-ABL may be required for the malignant transformation of hematopoietic stem/progenitor cells in the common marmoset. Disclosures: No relevant conflicts of interest to declare.

Preclinical Investigations Demonstrate Effectiveness of Aplidin in Acute Leukemias and Lymphomas and Provide the Basis for Further Clinical Studies.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 4496-4496
Author(s):  
Debabrata Banerjee ◽  
Guray Saydam ◽  
Lata G. Menon ◽  
Giuseppe S.A. Longo ◽  
Daniel Medina ◽  
...  
Keyword(s):  
Gene Expression ◽  
Cell Lines ◽  
Phase Ii ◽  
Myeloid Leukemia ◽  
Xenograft Model ◽  
Vascular Endothelial ◽  
Endothelial Growth Factor ◽  
Leukemias And Lymphomas

Abstract Aplidin (dehydrodidemnin B, C57H89N7O15) (APLD) is a novel antitumor agent isolated from the Mediterranean tunicate (seasquirt) Aplidium albicans. APLD has shown impressive in vitro and in vivo activity against different human cancer cells and has recently entered Phase II clinical trials in a variety of solid tumors following promising toxicity and pharmacological properties seen in Phase I studies. Fatigue and muscular pain were the most prevalent toxicities at 5 mg/m2 iv 3 h every other week or 3.4 mg/m2/wk with little or no bone marrow toxicity. APLD inhibits protein synthesis via GTP-dependent elongation factors 1-alpha and ornithine decarboxylase (ODC) activity, induces rapid p53-independent apoptosis in vitro, cell cycle perturbation and alteration of gene expression at early times after treatment. APLD inhibits vascular endothelial growth factor (VEGF) secretion and vascular endothelial growth factor-receptor 1 (VEGF-R1/flt-1), preventing autocrine stimulation in the human lymphoid leukemic cell line MOLT-4 cells and in AML blasts. APLD is a potent inhibitor of human myeloid leukemia cell lines (K-562, HEL and HL60), as well as fresh blast cells obtained from patients with both ALL and AML and is more potent than Idarubicin. Cytototoxic doses effective against multiple myeloma cells and fresh pediatric and adult ALL/AML blasts are achievable in plasma and are well below the recommended dose, thus a positive therapeutic index is anticipated. Moreover, the lack of cross resistance with conventional agents against fresh pediatric and adult AML/ALL blasts except fludarabine and Gemcitabine makes APLD an attractive therapeutic choice. Characterization of gene expression profile is currently underway in an attempt to generate a molecular fingerprint of sensitivity/resistance to APLD that will be validated in phase II clinical studies. Based on in vitro antileukemic effect of APLD as well as early results of clinical trials, a systematic study of drug combinations with Aplidin (APLD), for use possible in hematologic malignancies was undertaken. Three cell lines viz. K562 (acute myeloid leukemia), CCRF-CEM (acute lymphocytic leukemia), and SKI-DLCL (diffuse large cell lymphoma) were used for combination studies. Cytarabine and mitoxantrone were found to be synergistic in combination with APLD in all 3 cell lines as assessed by the Chou-Talalay combination index analysis. Since cytarabine and APLD produced impressive synergistic cell kill in all three cell culture models, the combination was further tested in the CCRF-CEM ALL xenograft model in SCID mice. APLD (0.7 mg/Kg) potentiated the antitumoral effect of cytarabine (50mg/Kg) in vivo. Addition of APLD to cytarabine treatment in xenograft model resulted in greater than 50% reduction in tumor size as compared to the untreated group. T/C ratios indicated that the effect of the combination was maximal at day 5 but was still maintained on day 8 (T/C on day 3 = 0.614; day 5= 0.403 and day 8= 0.703). The preclinical results with APLD in leukemias and lymphomas, as a single agent and in combination with cytarabine provide the basis for implementation of a phase II program in resistant relapsed leukemias and lymphomas.

CD34+CD19−, CD34+CD19+ and CD34−CD19+ Cells May All Have Leukemia-Initiating Potential in NOD/Scid Mice.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 2542-2542
Author(s):  
Christoph Le Viseur ◽  
Marc Hotfilder ◽  
Annegret Rosemann ◽  
Ronald Stam ◽  
Andre Schrauder ◽  
...  
Keyword(s):  
Bone Marrow ◽  
High Risk ◽  
Lymphoblastic Leukemia ◽  
Xenograft Model ◽  
Current Data ◽  
Scid Mice ◽  
Functional Assay ◽  
Childhood All ◽  
Mouse Xenograft Model

Abstract Current data on the leukemic stem cell (LSC) compartment in childhood acute lymphoblastic leukemia (ALL) are conflicting. The traditional hypothesis supposed that childhood ALL originates in a lymphoid progenitor cell and this is assumed to be consistent with the overall good treatment responses in pediatric patients. In accordance with this hypothesis, our previous studies failed to detect involvement of immature CD34+CD19− progenitor cells in ALL/t(12;21) (Hotfilder et al., Blood 2002) while high-risk ALL/t(9;22) and t(4;11) appears to originate in a more primitive CD34+CD19− cell (Hotfilder et al., Cancer Res 2005). In order to characterize the leukemia-initiating cell in vivo, we established a mouse xenograft model by serial intrafemoral transplantation of NOD/scid mice with flow sorted subpopulations from childhood ALL. Samples were taken from the bone marrow of children with ALL/t(12;21) (n=1), t(4;11) (n=3) and t(11;19) (n=1) and B-cell precursor ALL without a marker translocation (n=2). Primary transplantations were performed with freshly thawed unsorted cells, followed by secondary, tertiary and quaternary transplantations with flow sorted populations. Human leukemic engraftment was defined by a proportion of >5% human CD45+ cells in the murine bone marrow that simultaneously express CD34 and/or CD19. From the bone marrow of leukemic mice, we isolated different leukemic populations and successfully re-transplanted 2×103 − 1×105 CD34+CD19− cells, 2×104 − 6×106 CD34+CD19+ lymphoid progenitors and 3×104 − 2×106 more differentiated CD34−CD19+ blasts onto secondary, tertiary and quaternary mice (average purity after flow sorting: >96%). So far, we detected leukemic engraftment in 60 of 161 (37%) transplanted mice (with many mice - having only recently been transplanted - still alive). These include 7 of 36 (19%) mice engrafted with CD34+CD19− cells, 33 of 72 (46%) mice engrafted with CD34+CD19+ cells and 20 of 53 (38%) mice engrafted with CD34−CD19+ cells. With as few as 2 × 103 CD34+CD19− cells being sufficient to re-initiate the leukemia, this intrafemoral ALL-NOD/scid mouse model represents a very sensitive functional assay for candidate LSC in childhood ALL. We have initiated limiting dilution experiments with the different subpopulations to quantify LSC frequency in the different compartments and to exclude that low levels of contaminating blasts with an immunophenotype different from the main transplanted cell population blurred the results. We are also currently investigating whether there is heterogeneity in the CD34+CD19− compartment in respect to standard and high-risk ALL. Altogether, our data indicate that all three subpopulations, CD34+CD19−, CD34+CD19+ and CD34−CD19+ cells, may have the capacity to transfer the leukemia onto NOD/scid mice and that lymphatic LSC may not loose their self-renewal potential with differentiation.

Prognostic Impact of Ikaros (IKZF1) Gene Alterations In Childhood ALL Treated with ALL IC-BFM 2002 Protocol: A Comparison of Gene Expression and Genomic-Based Methods.

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 1656-1656
Author(s):  
Jana Volejnikova ◽  
Ester Mejstrikova ◽  
Karel Svojgr ◽  
Jan Stary ◽  
Jan Trka ◽  
...  
Keyword(s):  
Gene Expression ◽  
Dna Binding ◽  
Gene Deletion ◽  
Conflicts Of Interest ◽  
Lymphoblastic Leukemia ◽  
Treatment Protocol ◽  
Induction Treatment ◽  
Prognostic Impact ◽  
Childhood All ◽  
Gene Alterations

Abstract Abstract 1656 Introduction: Recently, Ikaros (IKZF1) gene alterations were found to predict poor prognosis in childhood acute lymphoblastic leukemia (ALL). Thus, the implementation of IKZF1 status into the risk group stratification is discussed. So far, limited data are available concerning both IKZF1 importance in different treatment protocols for Ph-negative ALL and the choice of the best diagnostic method. In this study, we compared two methods based on either genomic DNA examination or gene expression analysis, and their prognostic impact within a treatment protocol for childhood ALL. Methods: Gene expression of functional (IK1, IK2) and non-DNA binding (IK4, IK6, IK8) IKZF1 isoforms was semi-quantitatively evaluated using Lab-on-a-chip (Agilent) electrophoresis and reported either as an absolute level or relatively to the total isoform signal. The thresholds for abnormal gene expression were set based on the analysis of peripheral blood (PB) of healthy donors, remission bone marrow (BM) samples of children with ALL, and sorted B- and T-cell precursor subpopulations. MLPA (multiplex ligation-dependent probe amplification) reaction including probes for Ikaros exons 1 to 8 was performed on BM DNA samples and the products were analyzed with the Coffalyser v9.4 software. Results: Results of both gene expression and MLPA analysis in the BM were available for 120 of 193 children diagnosed with ALL between 2002 and 2005. MLPA analysis revealed a deletion of at least one exon of IKZF1 gene in 17/120 (14%) patients. Of note, two patients with this deletion underwent a lineage switch (LS) from ALL to AML during the induction treatment. The entire IKZF1 gene was mononoallelically deleted in 3 patients. The ratio between non-DNA binding (IK4, IK4del, IK4A, IK6, Ik6del, IK8) and functional (IK1 and IK2) isoform expression was significantly elevated (non-DNA binding isoforms>70%) in 21 of 120 (18%) patients. The expression of a dominant-negative IK6 isoform was significantly elevated (>20% of total) in 7 of 120 (6%) patients. Surprisingly, gene deletion on one allele was not accompanied by decrease of total IKZF1 gene expression. On the contrary, patients with IKZF1 deletion had higher total IKZF1 transcript level than those without deletion (p=0.008, Mann Whitney), but it was not possible to set any reliable expression threshold for the prediction of gene deletion. The change on a DNA level was not always reflected in relative gene expression: Of 17 patients with gene deletion, only 7 had significantly altered short/long isoform ratio and 5 patients had an increased expression of IK6. Conversely, of 7 patients with IK6 overexpression, two patients had no DNA alteration, suggesting a different mechanism of altered gene expression. We next evaluated the prognostic impact of IKZF1 alterations in 113 patients treated with ALL IC-BFM 2002 protocol (5 patients were excluded due to Ph-positive ALL with imatinib-based treatment and 2 patients due to treatment change after LS). Patients with IKZF1 gene deletion had significantly worse relapse-free survival (RFS) than other patients (5-year RFS 50.0±14.4% vs. 90.8±2.9%, p=0.0002). The presence of IKZF1 deletion did not correlate with minimal residual disease (MRD) during induction treatment (days 8, 15, 33), neither in BM nor in PB. Patients with IK6 overexpression had 5-year RFS 50.0±20.4% compared to 88.4± 3.2% in those with low IK6 expression (p=0.004). The elevated short/long isoform ratio (>70%) had no prognostic impact. The best prediction of relapse was achieved via combining two factors: the presence of IKZF1 deletion detected by MLPA or the relative IK6 overexpression (>50% of total isoform signal). The 5-year RFS was 50.0±13.4% for this group (14 pts, 7 relapses) compared to 91.6±2.8% for other patients (99 pts, 8 relapses, p<0.0001). Conclusion: This study confirmed that the presence of Ikaros gene alterations was connected with a high risk of relapse also in a BFM-based protocol for Ph-negative childhood ALL treatment. The deletion within IKZF1 locus did not necessarily correlate with an altered Ikaros gene expression. Ideally, both genomic and gene expression-based approach should be applied together for the evaluation of prognosis. However, if this is not possible, the examination of DNA changes by MLPA identifies more patients who subsequently relapse than the gene expression-based approach. Support: VZ MSM 0021620813, P301/10/1877, IGA NS/10472-3 Disclosures: No relevant conflicts of interest to declare.

LYL1, Class II Basic Helix-Loop-Helix Transcription Factor, Induces the Differentiation of Common Marmoset Embryonic Stem Cells to Hematopoietic Stem Cells

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 2348-2348
Author(s):  
Hirotaka Kawano ◽  
Tomotoshi Marumoto ◽  
Michiyo Okada ◽  
Tomoko Inoue ◽  
Takenobu Nii ◽  
...  
Keyword(s):  
Gene Expression ◽  
Stem Cells ◽  
Lymphoblastic Leukemia ◽  
Embryonic Stem ◽  
Common Marmoset ◽  
Hematopoietic Stem ◽  
Helix Loop Helix ◽  
Cd34 Cells

Abstract Abstract 2348 Since the successful establishment of human embryonic stem cells (ESCs) in 1998, transplantation of functional cells differentiated from ESCs to the specific impaired organ has been expected to cure its defective function [Thomson JA et al., Science 282:1145–47, 1998]. For the establishment of the regenerative medicine using ESCs, the preclinical studies utilizing animal model systems including non-human primates are essential. We have demonstrated that non-human primate of common marmoset (CM) is a suitable experimental animal for the preclinical studies of hematopoietic stem cells (HSCs) therapy [Hibino H et al., Blood 93:2839–48, 1999]. Since then we have continuously investigated the in vitro and in vivo differentiation of CM ESCs to hematopoietic cells by the exogenous hematopoietic gene transfer. In earlier study, we showed that the induction of CD34+ cells having a blood colony forming capacity from CM ESCs is promoted by lentiviral transduction of TAL1 cDNA [Kurita R et al., Stem Cells 24:2014-22,2006]. However those CD34+ cells did not have a bone marrow reconstituting ability in irradiated NOG (NOD/Shi-scid/IL-2Rγnull) mice, suggesting that transduction of TAL1 gene is not enough to induce functional HSCs which have self-renewal capability and multipotency. Thus we tried to find other hematopoietic genes being able to promote hematopoietic differetiation more efficiently than TAL1. We selected 6 genes (LYL1, HOXB4, BMI1, GATA2, c-MYB and LMO2) as candidates for factors that induce the differentiation from ESCs to HSCs, based on the comparison of gene expression level between human ESCs and HSCs by Digital Differential Display from the Uni-Gene database at the NCBI web site (http://www.ncbi.nlm.nih.gov/UniGene/). Then, we transduced the respective candidate gene in CM ESCs (Cj11), and performed embryoid body (EB) formation assay to induce their differentiation to HSCs for 9 days. We found that lentiviral transduction of LYL1, a basic helix-loop-helix transcription factor, in EBs derived from Cj11, one of CM ESC lines, markedly increased the number of cells positive for CD34, a marker for hematopoietic stem/progenitors. The lymphoblastic leukemia 1 (LYL1) was originally identified as the factor of a chromosomal translocation, resulting in T cell acute lymphoblastic leukemia [Mellentin JD et al., Cell 58:77-83.1989]. These class II bHLH transcription factors regulate gene expression by binding to target gene sequences as heterodimers with E-proteins, in association with Gata1 and Gata2 [Goldfarb AN et al., Blood 85:465-71.1995][Hofmann T et al., Oncogene 13:617-24.1996][Hsu HL et al., Proc Natl Acad Sci USA 91:5947-51.1994]. The Lyl1-deficient mice display the reduction of B cells and impaired long-term hematopoietic reconstitution capacity [Capron C et al., Blood 107:4678-4686. 2006]. And, overexpression of Lyl1 in mouse bone marrow cells induced the increase of HSCs, HPCs and lymphocytes in vitro and in vivo [Lukov GL et al., Leuk Res 35:405-12. 2011]. These information indicate that LYL1 plays important roles in hematopoietic differentiation in primate animals including human and common marmoset. To examine whether overexpression of LYL1 in EBs can promote hematopoietic differentiation in vitro we performed colony-forming unit (CFU) assay, and found that LYL1-overexpressing EBs showed the formation of multi-lineage blood cells consisting of erythroid cells, granulocytes and macrophages. Next, we analyzed gene expression level by RT-PCR, and found that the transduction of LYL1 induced the expression of various hematopoietic genes. These results suggested that the overexpression of LYL1 can promote the differentiation of CM ESCs to HSCs in vitro. Furthermore we found that the combined overexpression of TAL1 and LYL1 could enhance the differentiation of CD34+ cells from CM ESCs than the respective overexrpession of TAL1 or LYL1. Collectively, our novel technology to differentiate hematopoietic cells from ESCs by the transduction of specific transcription factors is novel, and might be applicable to expand human hematopoietic stem/progenitor cells in vitro for future regenerative medicine to cure human hematopoietic cell dyscrasias. Disclosures: No relevant conflicts of interest to declare.

The Natural Product Borrelidin Induces UPR and Apoptosis in AML Cell Lines

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 4260-4260
Author(s):  
Leah Jackson ◽  
Shelby Bechler ◽  
Justin Miller ◽  
Amy Brownell ◽  
Danielle Garshott ◽  
...  
Keyword(s):  
Cell Death ◽  
Er Stress ◽  
Natural Product ◽  
Cell Lines ◽  
Time Course ◽  
Conflicts Of Interest ◽  
Xenograft Model ◽  
Myelogenous Leukemia ◽  
Ic50 Values

Abstract Abstract 4260 Acute Myelogenous Leukemia (AML) is the most common form of leukemia. Current therapies are intense and even those fortunate enough to achieve remission often relapse extending extremely poor prognoses to these patient. The most commonly used therapeutics, namely cytarabine aribinoside, the anthracyclines and etoposide, are decades old and target ubiquitous cellular processes. We have previously reported that small molecules and natural products that activate and exacerbate the unfolded protein response (UPR) can effectively and selectively induce cell death in a wide variety of solid tumor cells. We hypothesized that the UPR might be a viable new therapeutic target in AML and sought to determine whether or not the novel UPR-inducing natural product borrelidin might be used as such an agent. A luminescent proliferation assay performed with panel of four AML cell lines treated with the ER stress-inducing antibiotic tunicamycin (Tm) revealed that three of the cell lines displayed IC50 values between 0.47–2.5μ M, doses of Tm which are known to induce a low to moderate level of ER stress. We then repeated the experiment with the more general UPR-inducing natural product borrelidin, which has been shown to have potent anti-inflammatory properties in several murine assays in vivo. All four cell lines were sensitive to borrelidin, displaying IC50 values between 0.032–0.29 μ M. Time course assays performed with borrelidin revealed 4–20 fold increases in active caspase 3 and 7 indicating borrelidin-induced AML decreases in cell proliferation might be the result of apoptosis. Quantitative reverse-transcription real time PCR performed with mRNA isolated from two AML cell lines revealed an increase in the UPR-related transcripts CHOP, ATF4, and GADD34 and the cell death genes Noxa, Puma, DR5 and Bim confirming that borrelidin could induce the UPR and apoptosis in AML cells. Studies currently underway in our laboratory will determine the ability of borrelidin and other UPR-inducing agents to reduce leukemic burden in an in vivo xenograft model. Disclosures: No relevant conflicts of interest to declare.

Effects of G-CSF On Acute Lymphoblastic Leukemia.

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 2564-2564
Author(s):  
Jordan Basnett ◽  
Adam Cisterne ◽  
Kenneth F Bradstock ◽  
Linda J Bendall
Keyword(s):  
Bone Marrow ◽  
Disease Progression ◽  
Microarray Analysis ◽  
Environmental Changes ◽  
Conflicts Of Interest ◽  
Lymphoblastic Leukemia ◽  
Hematopoietic Stem ◽  
Childhood All ◽  
Extracellular Matrix Components ◽  
The Impact

Abstract Abstract 2564 G-CSF is commonly used to treat chemotherapy-induced neutropenia and for the mobilization of hematopoietic stem cells for transplantation in patients with leukemia. Administration of G-CSF has profound effects on the bone marrow microenvironment including the cleavage of molecules required for the maintenance of lymphopoiesis, including CXCL12 and VLA-4. We have recently reported that G-CSF results in the dramatic suppression of B-lymphopoiesis. This, together with previous reports by ourselves, and others, showing that disruption of CXCL12 or VLA-4 slow the progression of B-lineage ALL lead us to consider that G-CSF may similarly antagonize the progression of ALL. To explore this possibility, we examined the impact of G-CSF administration on six human ALL xenografts using a NOD/SCID mouse model. Mice were engrafted without radiation and G-CSF commenced when 1% of the bone marrow consisted of ALL cells. G-CSF was administered twice daily for 10 days, at which time all animals were culled and leukemia assessed in the blood, bone marrow and spleens. Surprisingly G-CSF was found to increase disease progression in two of xenografts investigated (1345 and 0398, referred to as G-CSF responsive xenografts hereafter), while the remainder demonstrated a small reduction in leukemia, with one showing a statistical significant decrease. No evidence for a direct mitogenic effect of G-CSF could be demonstrated in any of the xenografts using exogenous G-CSF in vitro cultures in the presence or absence of human or murine stromal support. Consistent with these findings, and previous reports, little to no G-CSF receptor was detected by flow cytometry or microarray analysis of xenografts. Microarray analysis of the xenografts revealed significant differences in gene expression between the G-CSF responsive xenografts and the remainder of the samples. A total of 83 genes were expressed at a higher level and 127 genes at a lower level in the G-CSF responsive xenografts. The more highly expressed genes included cell cycle regulators (eg cyclin A1), adhesion molecules (eg ALCAM), extracellular matrix components and surface receptors. Perhaps the most interesting was the exclusive expression of the acetylcholine receptor (cholinergic receptor, nicotinic, beta 4, nAChRb4) in the G-CSF responsive cases. Analysis of a large public dataset of childhood ALL samples revealed significantly higher expression of this gene in ALL samples with rearranged MLL (p<0.03). However, small numbers of cases in all ALL subgroups had greater than an 2 fold higher expression compared to normal B cell progenitors. The role of nAChR in the response of ALL cells to micro-environmental changes induced by G-CSF remains to be determined, however, nAChR has known roles in cell proliferation and inhibition of apoptosis. Furthermore G-CSF is known to induce acetylcholine production in other tissues. In summary, G-CSF inhibited leukemia progression in the majority of patient xenografts, however, in a subset of samples G-CSF accelerated disease progression. Clinically, G-CSF administration to ALL patients has not been associated with any major adverse outcomes. However our data suggest that a small subset of patients may experience accelerated disease. Identification of features associated with adverse responses to G-CSF will permit the identification of patients for whom G-CSF may present a risk for increased disease progression. Disclosures: No relevant conflicts of interest to declare.

Immunornase Therapy Of Lymphoma With Novel CD22-Targeting Quadruple Ranpirnase

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 3075-3075
Author(s):  
Donglin Liu ◽  
Thomas M Cardillo ◽  
David M Goldenberg ◽  
Chien-Hsing Chang
Keyword(s):  
Cell Lines ◽  
Targeted Delivery ◽  
Stock Options ◽  
Lymphoblastic Leukemia ◽  
Leukemia Cell ◽  
Xenograft Model ◽  
Stock Option ◽  
Amino Acid Sequences

Abstract Ranpirnase (Rap) is an amphibian ribonuclease showing anti-tumor activity in clinical studies. We have previously reported that targeted delivery of Rap by chemical conjugation or recombinant fusion with antibodies specific for CD22, CD74 and Trop-2 could enhance its in vitro cytotoxicity as high as 10,000-fold in selected malignant cell lines. The DOCK-AND-LOCKTM (DNLTM) platform technology is a powerful method to construct novel agents of defined composition and retained bioactivity by site-specific conjugation of two types of modules, one containing the dimerization and docking domain (DDD) of cAMP-dependent protein kinase A (PKA), referred to as the DDD module, and the other bearing the anchoring domain (AD) of an interactive A-kinase anchoring protein (AKAP), referred to as the AD module. Among the distinctive features of DNL are the spontaneous formation of a dimer of the DDD module and the self-assembly of the DDD module with the AD module into a non-covalent complex, which is subsequently rendered covalent with disulfide bonds to enhance stability in vivo. The amino acid sequences of a pair of DDD and AD linkers useful for the DNL conjugation are termed DDD2 and AD2, respectively. To further explore the potential of Rap-based immunotoxins, we expressed a DDD2-module of Rap in E. coli and linked the resulting dimer of Rap to an AD2-module of a humanized IgG (expressed in myeloma cells) at each of the carboxyl termini of either the light chain (the CK-format) or the heavy chain (the CH3-format), thus producing a class of novel immunoRNases with quadruple Rap. To date, we have evaluated a pair of such constructs, 22* -Rap and 22-Rap, comprising four copies of Rap linked to the CK or CH3 termini of epratuzumab (humanized anti-CD22), respectively, in a panel of CD22-expressing human lymphoma/leukemia cell lines, which include Burkitt lymphoma (Daudi, Raji, Ramos), acute lymphoblastic leukemia (REH, 697, and RS4;11), and mantle cell lymphoma (Granta-519 and Jeko-1). The results of the MTS assay indicated that 22* -Rap was highly active (EC50 ≤ 1 nM) against Daudi, Ramos, Raji, REH, 697, RS4;11, and Granta-519 cells. Comparable cytotoxicity with EC50 values in the subnanomolar range also was observed for 22-Rap in Ramos, Daudi and Jeko-1 cells. In contrast, neither the individual DNL components (IgG-AD2 or Rap-DDD2), tested alone or in combination, nor E1* -Rap, the counterpart of 22* -Rap, generated by substituting epratuzumab with hRS7 IgG (humanized anti-Trop-2), showed notable cytotoxicity. In a disseminated Daudi xenograft model in which treatment with 10 or 20 µg of 22* -Rap (q4dx4) started 7 days after intravenous inoculation of the tumor cells, all 10 mice (5 in each group) survived over 126 days and were tumor-free, whereas the control groups, treated with saline, epratuzumab (25 µg, q4dx4), or the same dose-schedule of nonspecific control, E1* -Rap, succumbed within 36 days. These promising results, together with the findings that 22* -Rap at 10 nM was marginally toxic to B cells and other hematological cells in PBMCs, encourage further development of 22* -Rap or 22-Rap for therapy of CD22-expressing lymphomas and leukemias. Disclosures: Liu: IBC Pharmaceuticals, Inc.: Employment, Stock option, Stock option Other; Immunomedics, Inc.: Employment, Stock option Other. Cardillo:Immunomedics, Inc.: Employment, Stock option Other. Goldenberg:Immunomedics: Employment, stock options, stock options Patents & Royalties. Chang:IBC Pharmaceuticals, Inc.: Employment, Stock option, Stock option Other; Immunomedics, Inc: Employment, Stock option Other.

Characterization of lincRNA BALIR-6 in MLL rearranged B-lymphoblastic leukemia

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 3730-3730
Author(s):  
Norma Iris Rodriguez-Malave ◽  
Weihong Yan ◽  
Giuseppe Basso ◽  
Martina Pigazzi ◽  
Dinesh S. Rao
Keyword(s):  
Gene Expression ◽  
B Cells ◽  
B Cell ◽  
Cell Lines ◽  
Microarray Data ◽  
Conflicts Of Interest ◽  
Lymphoblastic Leukemia ◽  
Regulate Gene Expression ◽  
In Cis ◽  
Regulate Gene

Abstract A new class of non-coding RNA, known as long intergenic non-coding RNAs (lincRNAs), has only recently been described. These lincRNAs have been found to play a role in various molecular processes within the cell including gene regulation, acting as sinks for microRNAs, and regulating splicing, implicating them in development and oncogenic processes. B lymphoblastic leukemia (B acute lymphoblastic leukemia; B-ALL), a malignancy of precursor B-cells, harbors mutations and translocations that result in a dysregulated gene expression. Interestingly, dysregulated expression of lincRNAs has been found in various cancers, but has not yet been described in B-ALL. Recently, we completed a gene expression profiling study in human B-ALL samples, which showed differential lincRNA expression in samples with particular cytogenetic abnormalities. This led us to hypothesize that lincRNAs may be related to disease pathogenesis. Here, we describe a promising lincRNA from our microarray data designated B-ALL associated long intergenic RNA 6 (BALIR-6). Expression of BALIR-6 is highest in patient samples carrying the MLL rearrangement (n=16; when compared to patients with TEL-AML1-translocated, n=39; E2A-PBX1-translocated, n=8; BCR-ABL-translocated, n=3; and cytogenetically normal cases, n=56; 1-way ANOVA p<0.0001) and showed significant variance in the expression level based on the immunophenotype (1-way ANOVA p=0.0004). BALIR-6 is located on chromosome 3p24.3 in humans, and exists in a syntenic gene block in with neighboring genes SATB1 and TBC1D5, and is conserved in mammals. Rapid Amplification of cDNA Ends (RACE) uncovered multiple transcript isoforms; from these, three were cloned out and sequenced, corresponding to the genomic locus as predicted. In B-ALL cell lines, BALIR-6 expression was highest in RS411 cells, which carry the MLL rearrangement, when compared to other B-ALL cell lines. This suggests that the cell lines may show a similar expression pattern to human B-ALL samples. To study the functional role of BALIR-6 we utilized siRNA in a mmu-miR-155 expression cassette to knockdown the transcript. In RS411 cells we observed a reduction in proliferation by MTS assay. Additionally, we observed an increase Sub-G0 cells and a decrease in G2-M phase cells by propidium iodide staining, suggesting an increase in apoptosis. Conversely, overexpression of BALIR-6 in a mouse pre-B cell line (70Z/3), leads to an increase in proliferation. Interestingly, during normal B cell development, BALIR-6 is dynamically expressed, with high expression in pre-B cells and subsequent downregulation, suggesting that a normal role during development is being hijacked in patients with B-ALL. Mechanistically, a few recent studies have described that lincRNAs can regulate gene expression in cis. To explore whether BALIR 6 regulates surrounding genes in cis, we analyzed microarray data of MLL rearranged B-ALL samples, finding that expression of BALIR-6 correlates with expression of surrounding genes SATB1 and TBC1D5. Interestingly for SATB1, this correlation is also seen in human B cell developmental stages. Altering BALIR-6 expression by siRNA mediated knockdown or overexpression causes an effect on the expression of surrounding genes SATB1 and TBC1D5. Previous findings have shown that dysregulated SATB1 has been seen in a variety of malignancies, suggesting a mechanism for how BALIR-6 may produce the changes in cell growth and apoptosis described above. Altogether, these results identify a novel and interesting RNA transcript with the potential to regulate gene expression and pathogenesis in B-ALL with MLL rearrangement, suggesting novel diagnostic, prognostic, and therapeutic implications. Disclosures: No relevant conflicts of interest to declare.

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