MLL Rearrangement and Age At Diagnosis Are Strongly Associated with High Level Surface FLT3 Expression and Ex Vivo Sensitivity to FLT3 Inhibition: A Prospective Analysis of 54 Consecutive Infants with ALL Enrolled in Children's Oncology Group (COG) Trial AALL0631

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 568-568
Author(s):  
Allison J. Kaeding ◽  
Daniel Magoon ◽  
Yarden Fraiman ◽  
Tamekia Jones ◽  
Nyla A. Heerema ◽  
...  
Keyword(s):  
Protein Expression ◽  
Conflicts Of Interest ◽  
Lymphoblastic Leukemia ◽  
Predictive Biomarkers ◽  
Age At Diagnosis ◽  
Fusion Partner ◽  
Childhood All ◽  
Flt3 Inhibitor ◽  
Flt3 Expression

Abstract Abstract 568 Background: Infant acute lymphoblastic leukemia (ALL) is clinically and biologically distinct from ALL in older children. About 80% of infant ALL cases harbor MLL rearrangements (MLLr). MLLr infant ALL is an aggressive disease with poor prognosis, particularly in cases diagnosed at <90 days of age. Infant cases with wild type MLL (MLLwt) are clinically similar to childhood ALL. Retrospective studies of selected banked specimens have suggested that MLLr infant ALL cases express higher levels of FLT3 receptor tyrosine kinase mRNA and protein, and show enhanced in vitro FLT3 inhibitor-induced cytotoxicity compared to MLLwt childhood ALL, leading to an ongoing COG trial to determine if adding lestaurtinib to chemotherapy improves outcome for MLLr infant ALL (AALL0631). Methods: We prospectively characterized 54 consecutive diagnostic leukemia specimens from infants with ALL enrolled on AALL0631 with respect to: 1) Quantitative surface FLT3 protein expression (s-FLT3) using FACS CD135 mean fluorescence index (MFI); 2) FLT3 inhibitor (lestaurtinib: 0 to 100 nM) in vitro sensitivity using 48 hour WST-1 cytotoxicity assays; samples defined as “sensitive” if 100 nM produced ≥40% inhibition of cell viability compared to vehicle controls; 3) Identification and quantification of putative leukemia stem cell (LSC) subpopulation by flow cytometric immunophenotyping (% CD34+, CD38− viable cells). Studies were performed blinded to clinical information and MLL status; data were later correlated with age at diagnosis (<90 days vs. ≥90 days) and MLL-rearrangement status/fusion partner (cytogenetics/FISH). Results: Of 54 cases, 42 (78%) are MLLr and 12 (22%) are MLLwt. For MLLr cases, fusion partners are AF4 (n=13), ENL (n=17), AF9 (n=3), AF1p (n=2), and other (n=7). Of MLLr cases, 10 (24%) were <90d and 32 (76%) were ≥90 days. All MLLwt cases were ≥90 days.We analyzed s-FLT3 according to MLL genotype, MLL fusion partner and age, and found significantly higher s-FLT3 in MLLr than MLLwt cases (mean MFI 34.2 vs. 11.6, p=0.03). According to MLL fusion partner, we found the highest s-FLT3 in AF9, lowest in AF4, and intermediate in ENL and AF1p. The AF9 vs. AF4 comparison was significant (p=0.006). According to age in the MLLr cohort, we found strikingly higher s-FLT3 in infants diagnosed at <90days compared to those >90days (mean MFI 62.6 vs. 22.0, p<0.0001). We analyzed FLT3 inhibitor sensitivity according to MLL genotype and s-FLT3. All 42 of the MLLr samples were evaluable, with 35 (83%) sensitive and 7 (17%) resistant; 11 of 12 MLLwt samples were evaluable (1 had excessively poor viability), with 6 (55%) sensitive and 5 (45%) resistant. We thus found a significant association between MLLr and FLT3 inhibitor sensitivity (Chi square 4.125, p=0.042). In the s-FLT3 analysis, the sensitive samples trended towards higher s-FLT3 than the resistant samples (mean MFI 31.3 vs. 16.7, p=0.17). Finally, we compared s-FLT3 in the putative LSC-like subpopulation fraction (if present) with the “bulk” leukemia population. Of the 54 samples analyzed, 35 (65%) had a clearly identifiable LSC-like population (i.e., comprising >0.5% of the total viable leukemia population). Within these 35 samples, s-FLT3 was significantly higher in the LSC-like fraction than in the bulk leukemia population (mean MFI 40.4 vs. 31.2, p=0.05). Conclusions: We have for the first time prospectively compared FLT3 expression and FLT3 inhibitor sensitivity in unselected representative cohorts of MLLr and MLLwt infants, confirming that MLLr cases express significantly higher levels of surface FLT3 protein and are more sensitive to the cytotoxic effects of FLT3 inhibition. Novel findings include higher surface FLT3 protein expression in MLL-AF9 cases relative to MLL-AF4 cases, and the strikingly higher surface FLT3 protein expression in MLLr cases in the youngest infants. An intriguing finding is the presence of phenotypically-defined subpopulations of “LSC-like” cells in the majority of cases, and that this LSC-like population expresses significantly higher levels of FLT3 protein than the “bulk” population. These findings provide further evidence that FLT3 overexpression plays a role in MLLr leukemogenesis. If these findings correlate with clinical responses in COG AALL0631, they may prove to be useful predictive biomarkers in selecting patients for whom FLT3-targeted therapy is most appropriate. Disclosures: No relevant conflicts of interest to declare.

Genetic Mechanisms of Transcriptional Regulation in Childhood Acute Lymphoblastic Leukemia

2021 ◽  
Author(s):  
◽  
Xujie Zhao
Keyword(s):  
Transcriptional Regulation ◽  
Cell Line ◽  
Molecular Mechanism ◽  
Fusion Gene ◽  
Lymphoblastic Leukemia ◽  
Causal Variant ◽  
Childhood All ◽  
Transcriptional Regulatory ◽  
Flt3 Expression

Introduction. Advances in genomic profiling and sequencing studies have identified germline and somatic variations that are associated with childhood ALL, improving our understanding of the genetic basis of childhood acute lymphoblastic leukemia (ALL). Recent genome-wide association studies (GWAS) have identified germline genetic variations of ARID5B and, more recently, IGF2BP1 that are associated with susceptibility to ALL. Genome-wide sequencing studies also discovered a new ALL subtype characterized of ZNF384-mediated chromosomal translocations, providing new insights into genetic heterogeneity in childhood ALL. However, the underlying mechanism by which these genetic variants contribute to the transcriptional regulatory circuitries of ALL is still poorly understood. We tested these hypotheses: 1) A low ARID5B expression will increase the relapse risk of ALL, 2) Genetic variants of ARID5B will affect its expression and thus influence susceptibility to childhood ALL, 3) IGF2BP1 is transcriptionally suppressed by ETV6, 4) ZNF384-mediated fusion genes transcriptionally upregulate FLT3 expression as being a therapeutic target. Specific aims in this study include: 1) identifying the causal variant of ARID5B, 2) identifying molecular mechanism underlying drug resistance, 3) identifying molecular mechanism of transcriptional regulation of IGF2BP1 by ETV6, and 4) identifying molecular mechanism of transcriptional regulation of FLT3 by EP300-ZNF384 fusion protein. Methods. We analyzed association of ARID5B expression in primary human ALL blasts with different molecular subtypes and treatment outcomes. Subsequent mechanistic studies were performed in ALL cell lines by manipulating ARID5B expression isogenically, in which we evaluated drug sensitivity, metabolism, and molecular signaling events. We performed ARID5B targeted sequencing in 5,008 children with ALL and conducted high throughput CRISPR/dCas9 screening in an engineered ARID5B mCherry knock-in cell line. Effects of genetic polymorphism on binding affinity of transcription factor and chromatin accessibility were subsequently assessed. We applied CRISPR/dCas9 to investigate transcriptional regulation of IGF2BP1 by ETV6 in ALL cell lines. We stably knocked down EP300-ZNF384 fusion gene by CRISPR editing in ALL cell line, in which we analyzed FLT3 expression and drug sensitivity. Results. ARID5B expression varied substantially by ALL subtype, with the highest level being observed in hyperdiploid ALL. Lower ARID5B expression at diagnosis was associated with the risk of ALL relapse, and further reduction was noted at ALL relapse. In isogenic ALL cell models in vitro, ARID5B knockdown led to resistance specific to antimetabolite drugs. ARID5B downregulation significantly inhibited ALL cell proliferation and caused partial cell-cycle arrest partially through upregulating expression the cell-cycle checkpoint regulator p21 (encoded by CDKN1A). Using targeted sequencing in germline DNA of 5,008 children with ALL and high throughput CRISPR/dCas9 screening in an engineered ARID5B mCherry knock-in cell line, we nominated ALL risk variant (rs7090445, P = 1.82 × 10-10) as the causal variant. And its polymorphisms disrupted binding of transcription factor MEF2C and local chromosome accessibility as confirmed by ChIP-Sanger-seq and ATAC-seq. Although it was previously reported that IGF2BP1 expression was significantly higher in ETV6-RUNX1 ALL as well as other cancers, the underlying transcriptional regulatory mechanism remains elusive. In ALL cell models, we identified a cis-regulatory element (CRE) blocking of which by dCas9-KRAB strongly influenced transcription of IGF2BP1. Moreover, we presented a CRISPR-based approach to comprehensively investigate the transcriptional regulatory mechanism of IGF2BP1 by identifying its CREs and upstream transcriptional regulators. In tissue-specific overexpression mouse models, we demonstrated that role of Igf2bp1 in B-cell development was stage-specific. In a novel ALL subtype characterized of ZNF384-mediated rearrangements, for the first time we reported overexpression of FLT3 in this new ALL subtype, providing a novel therapeutic target for ALL patient with high expression of FLT3. Furthermore, we defined EP300-ZNF384 fusion protein as a transcriptional activator of FLT3 gene with direct binding at its 5’UTRand knocking down this fusion gene led to downregulation of FLT3 expression as well as decreased sensitivity to FLT3 inhibitor in vitro. Conclusions. Our studies have demonstrated that a causal variant of ARID5B affected its transcription in-cis and that a low expression of ARID5B increased ALL relapse risk. As a downstream effector of ETV6, IGF2BP1 expression influenced B-cell development in vivo in a stage-specific manner. Moreover, expression of FLT3 was transcriptionally upregulated by ZNF384-mediated fusion genes. This study sheds light on the underlying mechanism by which genetic variations altered transcriptional programs in childhood ALL and refined our understanding of the genetic basis of childhood ALL, providing new molecular targets which can be harnessed for development of new therapies for patients with ALL.

scholarly journals Whole Transcriptome Sequencing Identified a Distinct Subtype of Acute Lymphoblastic Leukemia with Abnormalities of CREBBP and EP300

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 3912-3912
Author(s):  
Hui Zhang ◽  
Maoxiang Qian ◽  
Shirley, Kow Yin Kham ◽  
Shuguang Liu ◽  
Chuang Jiang ◽  
...  
Keyword(s):  
Acute Lymphoblastic Leukemia ◽  
Conflicts Of Interest ◽  
Lymphoblastic Leukemia ◽  
Chromosomal Rearrangements ◽  
Ectopic Expression ◽  
Cytotoxic Agents ◽  
Hematopoietic Stem ◽  
Exon 2 ◽  
Childhood All

Abstract While acute lymphoblastic leukemia (ALL) is a prototype of cancer that can be cured by chemotherapy alone, current ALL treatment regimens rely primarily on conventional cytotoxic agents with significant acute and long-term side effects. Better understanding of genomic landscape of ALL is critical for developing molecularly targeted therapy and implementing genomics-based precision medicine in this cancer. In particularly, sentinel chromosomal translocations are common in ALL and often involve key transcription factors important for hematopoiesis. Epigenetic regulator genes are also frequently targeted by somatic genomic alterations such as sequence mutations (e.g., CREBBP) and gene fusions (e.g., MLL, EP300). To comprehensively define transcriptomic abnormalities in childhood ALL, we performed RNA-seq of an unselected cohort of 231 children enrolled on the MaSpore frontline ALL protocols in Singapore or Malaysia. In total, we identified 58 putatively functional and predominant fusion genes in 125 patients (54.1%), the majority of which have not been reported previously. In particular, we described a distinct ALL subtype with a characteristic gene expression signature driven by chromosomal rearrangements of the ZNF384 gene with different partners (i.e., histone acetyl-transferases EP300 and CREBBP, TAF15, and TCF3). In 9 of 11 ALL cases with ZNF384 rearrangements, the breakpoint in this gene was invariably between exon 2 and exon 3, resulting in deletion of the 5'-UTR and then in-frame fusion of the entire ZNF384 coding sequence with the partner genes. The top two most significantly up-regulated genes in the ZNF384-rearranged group were CLCF1 and BTLA, whose expression levels were 15.5- and 15.0-fold higher than in ALL cases with wildtype ZNF384, respectively. In fact, ZNF384 binding was identified within the CLCF1 and BTLA loci (particularly the promoter regions) by chromatin immunoprecipitation sequencing in B lymphoblasoid cells. Using luciferase transcription driven by CLCF1 promoter in HEK293T cells as a model system, we observed significantly greater transcription activity with EP300-ZNF384 fusion compared to cells expressing wildtype ZNF384, suggesting that this chimeric gene resulted in gain of ZNF384 function. Similar results were obtained with luciferase transcription assay driven by the BTLA promoter. In human ALL cells, CLCF1 and BTLA promoter activities were consistently and significantly higher in ZNF384-rearranged ALL than in ALL cell line with wildtype ZNF384. To examine the effects of ZNF384 fusion on hematopoietic stem and progenitor cell (HSPCs) function, we also evaluated colony forming potential of HSPC in vitro upon ectopic expression of ZNF384 fusions. While there was marked suppression of colonies from myeloid and erythoid lineages, expression of EP300-ZNF384 or CREBBP-ZNF384 significantly stimulated preB cell colony formation. However, neither EP300- nor CREBBP-ZNF384 fusion was able to transform mouse hematopoietic precursor cell Ba/f3 in vitro, but instead increased the transforming potential of other oncogenic mutations (NRASG12D). EP300-ZNF384 and CREBBP-ZNF384 fusion proteins lacked the histone acetyltransferase (HAT) domain, and showed only 25% and 10% of HAT activity of full-length EP300 and CREBBP, respectively, with dominant-negative effects. Also, expression of EP300-ZNF384 led to significant decrease in global H3 acetylation in Ba/f3 cells in vitro. Finally, in NRASG12D-transformed Ba/f3 cells, co-expression of EP300-ZNF384 or CREBBP-ZNF384 substantially potentiated cytotoxic effects of histone deacetylase inhibitor vorinostat. Similarly, in a panel of human ALL cell lines, ZNF384-rearrangement was also associated with increased sensitivity to vorinostat, suggesting that some ZNF384-rearranged ALL may benefit from therapeutic agents targeting histone acetylation regulation. In conclusion, our results indicate that gene fusion is the major class of genomic abnormalities in childhood ALL and chromosomal rearrangements involving EP300 and CREBBP may cause global epigenetic deregulation in ALL with potentials for therapeutic targeting. Disclosures No relevant conflicts of interest to declare.

The Kinase Inhibitor TKI258 Is Active Against the Novel CUX1-FGFR1 Fusion Detected In a Patient with T-Lymphoblastic Leukemia/Lymphoma and t(7;8)(q22;p11)

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 2715-2715
Author(s):  
Bartosz Wasag ◽  
Els Lierman ◽  
Peter Meeus ◽  
Jan Cools ◽  
Peter Vandenberghe
Keyword(s):  
Cell Line ◽  
Western Blotting ◽  
Kinase Inhibitors ◽  
Kinase Inhibitor ◽  
Conflicts Of Interest ◽  
Lymphoblastic Leukemia ◽  
Antiproliferative Effect ◽  
Fusion Partner ◽  
The Novel

Abstract Abstract 2715 The 8p11 myeloproliferative syndrome (EMS) is an aggressive, atypical stem cell disorder associated with chromosome translocations that constitutively activate FGFR1 by fusion to diverse partner genes. Here, we describe a case with a clinical and hematological diagnosis of T-lymphoblastic leukemia/lymphoma and with a t(7;8)(q22;p11) on cytogenetic analysis. We identified the fusion partner involved and characterized this translocation functionally in vitro using the interleukin 3 (IL3) dependent Ba/F3 cell line. The translocation was analyzed in more detail by FISH using FGFR1 flanking probes. We could confirm the 8p11 breakpoint and 7q as the partner chromosome. Using 5'-RACE CUX1 (7q22) was identified as the fusion partner of FGFR1 in this patient with T-lymphoblastic leukemia/lymphoma. CUX1 is a homeobox family DNA binding protein not previously described as a fusion partner in hematological malignancies. To evaluate the transforming potential of this novel fusion, the CUX1-FGFR1 fusion was cloned and used to transform Ba/F3 cells. CUX1-FGFR1 expressing Ba/F3 cells displayed IL3 independent proliferation thus demonstrating the oncogenic character of this fusion protein. Western blotting of the transformed Ba/F3 cells showed activation of FGFR1 as well as its downstream target STAT5. Treatment of the CUX1-FGFR1 expressing Ba/F3 cells with the kinase inhibitors PKC412 and TKI258 significantly inhibited cell growth with an IC50 of 483 and 489 nM respectively. With western blotting a direct effect of both inhibitors on FGFR1 kinase activity as well as on different downstream effectors was proven. Furthermore using an annexinV/propidium iodide-based apoptosis assay, we could show that PKC412 and TKI258 both induced apoptosis followed by cell death in inhibitor treated CUX1-FGFR1 transformed Ba/F3 cells. The antiproliferative effect of the inhibitors could be rescued by addition of IL3 for the TKI258 treated but not for PKC412 treated CUX1-FGFR1 expressing cells. This observation indicates a selective action of TKI258 on FGFR1 signaling at the concentrations used. In contrast, for PKC412 non-specific cytotoxicity is also contributing to the antiproliferative effect. In summary, we identified a novel CUX1-FGFR1 fusion in a case with EMS and a novel t(7;8)(q22;p11), and demonstrated the oncogenic potential of CUX1-FGFR1 in the Ba/F3 cell line. This new fusion partner CUX1 contains a potential coiled coil domain that can explain the observed constitutive FGFR1 activation, as has been elaborately demonstrated for other oncogenic kinase fusions. The in vitro data presented here using the inhibitor TKI258 support the use of this compound for the treatment of the novel CUX1-FGFR1 fusion as well as other constitutively active FGFR1 fusion proteins. Disclosures: No relevant conflicts of interest to declare.

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

Blood ◽  
2017 ◽  
Vol 130 (Suppl_1) ◽  
pp. 810-810 ◽  
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.

scholarly journals Phloridzin Docosahexaenoate (PZ-DHA) : A Novel Naturally Derived Drug Active in T-Cell Acute Lymphoblastic Leukemia (T-ALL)

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 5197-5197
Author(s):  
Niroshaathevi Arumuggam ◽  
Nicole Melong ◽  
Catherine K.L. Too ◽  
Jason N. Berman ◽  
H.P. Vasantha Rupasinghe
Keyword(s):  
Acute Lymphoblastic Leukemia ◽  
T Cell ◽  
Conflicts Of Interest ◽  
Lymphoblastic Leukemia ◽  
Xenograft Model ◽  
Jurkat Cells ◽  
Interferon Α ◽  
Omega 3 ◽  
Cell Acute Lymphoblastic Leukemia

Abstract T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive malignant disease that accounts for about 15% of pediatric and 25% of adult ALL. Although risk stratification has provided more tailored therapy and improved the overall survival of T-ALL patients, clinical challenges such as suboptimal drug responses, morbidity from drug toxicities, and drug resistance still exist. Plant polyphenols have therapeutic efficacy as pharmacological adjuvants to help overcome these challenges. They can be acylated with fatty acids to overcome issues concerning bioavailability, such as poor intestinal absorption and low metabolic stability. Phloridzin (PZ), a flavonoid found in apple peels, was acylated with an omega-3 fatty acid, docosahexaenoic acid (DHA), to generate a novel ester called phloridzin docosahexaenoate (PZ-DHA). The cytotoxic effect of PZ-DHA was studied in the human Jurkat T-ALL cell line. PZ-DHA significantly reduced the viability and cellular ATP levels of treated cells. PZ-DHA was found to selectively induce apoptosis in Jurkat cells, while sparing normal murine T-cells. Apoptosis was further confirmed by demonstrating the ability of PZ-DHA to induce morphological alterations, DNA fragmentation, caspase activation, and the release of intracellular lactate dehydrogenase. PZ-DHA also significantly inhibited cell division in Jurkat cells. Furthermore, interferon-α-induced phosphorylation of the transcription factor, STAT3, was downregulated following PZ-DHA treatment. The in vitro efficacy of PZ-DHA was recapitulated in vivo in an established zebrafish xenograft model, where the proliferation of transplanted Jurkat cells was inhibited when PZ-DHA was added to the embryo water. Overall, these findings provide evidence for PZ-DHA as a novel therapeutic agent with activity in T-ALL. Studies examining the effect of PZ-DHA on patient-derived ALL cells engrafted in zebrafish are currently underway. Disclosures No relevant conflicts of interest to declare.

scholarly journals TEL/AML-1 dimerizes and is associated with a favorable outcome in childhood acute lymphoblastic leukemia

Blood ◽  
1996 ◽  
Vol 88 (11) ◽  
pp. 4252-4258 ◽  
Author(s):  
TW McLean ◽  
S Ringold ◽  
D Neuberg ◽  
K Stegmaier ◽  
R Tantravahi ◽  
...  
Keyword(s):  
Acute Lymphoblastic Leukemia ◽  
Lymphoblastic Leukemia ◽  
Chromosomal Translocation ◽  
Fusion Transcript ◽  
Childhood Acute Lymphoblastic Leukemia ◽  
Childhood All ◽  
Polymerase Chain ◽  
B Lineage

Abstract Polymerase chain reaction-based screening of childhood acute lymphoblastic leukemia (ALL) samples showed that a TEL/AML1 fusion transcript was detected in 27% of all cases, representing the most common known gene rearrangement in childhood cancer. The TEL/AML1 fusion results from a t(12;21)(p13;q22) chromosomal translocation, but was undetectable at the routine cytogenetic level. TEL/AML1-positive patients had exclusively B-lineage ALL, and most patients were between the ages of 2 and 9 years at diagnosis. Only 3/89 (3.4%) adult ALL patients were TEL/AML1-positive. Most importantly, TEL/AML1-positive children had a significantly lower rate of relapse compared with TEL/AML1-negative patients (0/22 v 16/54, P = .004). Co- immunoprecipitation experiments demonstrated that TEL/AML-1 formed homodimers in vitro, and heterodimerized with the normal TEL protein when the two proteins were expressed together. The elucidation of the precise mechanism of transformation by TEL/AML1 and the role of TEL/AML1 testing in the treatment of childhood ALL will require additional studies.

scholarly journals In vitro cellular drug resistance in children with relapsed/refractory acute lymphoblastic leukemia

Blood ◽  
1995 ◽  
Vol 86 (10) ◽  
pp. 3861-3868 ◽  
Author(s):  
E Klumper ◽  
R Pieters ◽  
AJ Veerman ◽  
DR Huismans ◽  
AH Loonen ◽  
...  
Keyword(s):  
Drug Resistance ◽  
Acute Lymphoblastic Leukemia ◽  
Poor Prognosis ◽  
De Novo ◽  
Lymphoblastic Leukemia ◽  
Childhood All ◽  
Acquired Drug Resistance ◽  
The Poor ◽  
Response To Chemotherapy

Cellular drug resistance is thought to be an important cause of the poor prognosis for children with relapsed or refractory acute lymphoblastic leukemia (ALL), but it is unknown when, to which drugs, and to what extent resistance is present. We determined in vitro resistance to 13 drugs with the MTT assay. Compared with 141 children with initial ALL, cells from 137 children with relapsed ALL were significantly more resistant to glucocorticoids, L-asparaginase, anthracyclines, and thiopurines, but not to vinca-alkaloids, cytarabine, ifosfamide, and epipodophyllotoxins. Relapsed ALL cells expressed the highest level of resistance to glucocorticoids, with a median level 357- and >24-fold more resistant to prednisolone and dexamethasone, respectively, than initial ALL cells, whereas the resistance ratios for the other drugs differed from 0.8- to 1.9-fold, intraindividual comparisons between initial and relapsed samples from 16 children with ALL showed that both de novo and acquired drug resistance were involved. Specific in vitro drug-resistance profiles were associated with high-risk relapsed ALL groups. In vitro drug resistance was also related to the clinical response to chemotherapy in relapsed/refractory childhood ALL. We conclude that drug resistance may explain the poor prognosis for children with relapsed/refractory ALL. These day may be helpful to design alternative treatment regimens for relapsed childhood ALL.

Sensitivity of Childhood Acute Lymphoblastic Leukemia (ALL) to Idarubicin Is Significantly Higher Than to Daunorubicin Treatment Based on Ex Vivo Apoptosis Induction.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 4495-4495
Author(s):  
Aram Prokop ◽  
Banu Bagci ◽  
Guenaelle Lingfeld ◽  
Lucia Badiali ◽  
Karin Garbrecht ◽  
...  
Keyword(s):  
Acute Lymphoblastic Leukemia ◽  
Response Rate ◽  
De Novo ◽  
Ex Vivo ◽  
Lymphoblastic Leukemia ◽  
Cell Populations ◽  
Apoptosis Induction ◽  
Good Tolerability ◽  
Childhood All

Abstract Anthracyclines, especially daunorubicin, play a very important role in the treatment of acute lymphoblastic leukemia (ALL) and the relapsed ALL in childhood. In the present study, primary lymphoblasts isolated from 65 children with de novo ALL (median: 5.8 years; range: 1.9 – 16.9 years) and relapsed ALL (median: 12.7 years; range: 1.3 – 17.9 years) were treated with daunorubicin (10 mmol/l) or idarubicin (2 mmol/l) in vitro. We could show that both anthracylines induce apoptosis, as evidenced by measurement of genomic DNA fragmentation. Interestingly, daunorubicin only induced modest apoptosis, whereas idarubicin displayed a significantly stronger apoptosis inducing effect. Furthermore the treatment of daunorubicin-resistant lymphoblasts with idarubicin resulted in good response in most of the resistant cell populations. Out of the 65 patients analysed in this study 23 were female (13 de novo ALL, 10 relapsed ALL) and 42 were male (29 de novo ALL, 13 relapsed ALL). Primary lymphoblasts were obtained by bone marrow aspiration and separated by centrifugation over Ficoll. Within these cell populations following immunologic subgroups were found: 35 c-ALL, 10 pre-B-ALL, 7 pro-B-ALL, 10 T-ALL and 3 pre-T-ALL. Daunorubicin induced apoptosis in 33 out of 65 lymphoblast populations (response rate 50.8 %). Nevertheless, a far higher response rate was observed for idarubicin with 59/65 (90,8 %) (p < 0.008), if response is defined as apoptosis induction higher than 1 %. Daunorubicin-resistance was found in 32/65 (49,2 %), resistance to both was observed in 6/65 (9,2 %). Treatment of daunorubicin-resistant lymphoblasts with idarubicin resulted in significant apoptosis induction in 26 out of 32 cell populations (81,3 %). We clearly demonstrated here that the in vitro treatment of lymphoblasts from children with de novo or relapsed ALL with idarubicin induces significantly higher response rates than daunorubicin treatment. The ex vivo sensitivity of daunorubicin-resistant lymphoblasts of childhood ALL to idarubicin treatment reflects the better potency of idarubicin to induce apoptosis and to overcome daunorubicin resistance. These data prompted us to study the clinical relevance of idarubicin in ongoing clinical trials to improve existing therapeutic regiments. First clinical data point to a good tolerability of idarubicin in the treatment of relapsed ALL in childhood.

Glucocorticoid Induced Cell Death Is Mediated by Its Effect on Cellular Metabolism and Is Independent of Caspase Activity.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 3475-3475
Author(s):  
Sandeep Gurbuxani
Keyword(s):  
Cell Death ◽  
Cell Lines ◽  
Lymphoblastic Leukemia ◽  
Lactate Concentration ◽  
Cellular Metabolism ◽  
Mechanisms Of Resistance ◽  
Metabolic State ◽  
Childhood All ◽  
Mitochondrial Mass

Glucocorticoids (GCs) such as prednisone and dexamethasone are a crucial component of acute lymphoblastic leukemia (ALL) therapy protocols. Multiple studies in childhood ALL have demonstrated that resistance to GC mediated cell death in vitro and in vivo is the single most important predictor of treatment outcome in childhood ALL. However, the mechanisms of GC mediated cell death as well as the mechanisms of resistance are poorly understood. The present study was undertaken to better understand the mechanism of GC induced cell death and to delineate possible mechanisms of resistance. In the initial experiments performed, multiple ALL cell lines, when treated with dexamethasone, underwent a reduction in the amount of reactive oxygen species (ROS) followed by cell cycle arrest and finally cell death which was unaffected by the presence of a pan-caspase inhibitor z-VAD-fmk. Since the amount of ROS present in a cell is an indicator of the metabolic state of the cell, specifically the amount of oxidative phosphorylation going on in the mitochondria, additional experiments were performed to directly estimate the mitochondrial mass as well as the metabolic state of the cells treated with GCs. While the mitochondrial mass measured by Mitotracker green labeling of mitochondria in the viable cells remained unchanged in cell lines susceptible to low concentrations (nano or micromolar) of dexamethasone, there was a prominent reduction in mitochondrial mass 36 hours after dexamethasone exposure in MOLT-4 cell line that requires several fold higher (millimolar) concentration of dexamethasone to induce cell death. The reduction in ROS was not accompanied by an increase in glycolysis as determined by the measurement of lactate concentration in the culture supernatants either in the susceptible or the resistant cells. Since one possible mechanism of reduction in ROS is increased scavenging by molecules that are dependent on the presence of NADPH generated during glucose metabolism via the pentose phosphate pathway (PPP), additional experiments were performed to determine if chemical inhibition of this pathway could augment dexamethasone induced cell death in ALL cell lines. Indeed, addition of transandosterone, an inhibitor of G6PD, the rate limiting enzyme of the PPP, resulted in significantly increased dexamethasone toxicity. Based on these experiments it can be concluded that GC induced cell death is mediated by its effect on cellular metabolism. Furthermore, this cell death is caspase independent and likely proceeds via a pathway mechanistically distinct from classical apoptosis. Finally, cells resistant to GC induced cell death have evolved mechanisms to adapt to GC induced changes in cellular metabolism and may maintain energy production via alternative pathways such as the PPP shunt that are independent of mitochondria.

Transforming and Tumorogenic Activity of JAK2 by Fusion to BCR: Molecular Mechanisms of Action of a Novel BCR-JAK2 Tyrosin-Kinase.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 4683-4683
Author(s):  
Álvaro Cuesta-Domínguez ◽  
Mara Ortega ◽  
Cristina Ormazabal ◽  
Matilde Santos-Roncero ◽  
Marta Galán-Díez ◽  
...  
Keyword(s):  
Tyrosine Kinase ◽  
Nude Mice ◽  
Molecular Mechanisms ◽  
Conflicts Of Interest ◽  
Fusion Gene ◽  
Lymphoblastic Leukemia ◽  
Chimeric Protein ◽  
Tyrosine Kinase Domain ◽  
Molecular Response

Abstract Abstract 4683 Chromosomal translocations in human tumors frequently produce fusion genes whose chimeric protein products play an essential role in oncogenesis. Recent reports have found a BCR-JAK2 fusion gene in cases of chronic or acute myeloid leukemia, but the protein had not been characterized. We describe a BCR-JAK2 fusion gene by fluorescence in situ hybridization and RT-PCR amplification from bone marrow at diagnosis of a patient with acute lymphoblastic leukemia. After induction therapy, real time PCR showed persistent molecular response correlating with hematological remission maintained up to present. BCR-JAK2 is a 110 KDa chimeric protein containing the BCR oligomerization domain fused to the JAK2 tyrosine-kinase domain. In vitro analysis showed that BCR-JAK2 was constitutively phosphorylated and was located to the cytoplasm. BCR-JAK2 transformed the IL-3-dependent murine hematopoietic cell line Ba/F3 into IL-3 independent growth and induced STAT5b phosphorylation and translocation into the cell nuclei. The treatment with a JAK2 inhibitor abrogated BCR-JAK2 and STAT5b phosphorylation, leading to apoptosis of transformed Ba/F3 cells. To test whether BCR-JAK2 has tumorogenic ability in vivo, we performed experiments with nude mice, in which we injected subcutaneously cells transduced with the control vector and cells expressing BCR-JAK2. Notably, we only obtained tumors in the flank injected with BCR-JAK2 expressing cells, thus confirming the tumorogenic activity of the BCR-JAK2 fusion protein. We conclude that BCR-JAK2 is a new tyrosine-kinase that induces proliferation and cell survival, which can be abrogated by JAK2 inhibitors. In vitro studies demonstrate that BCR-JAK2 displays transforming activity. Moreover, the nude mice model reveals its ability to cause tumors. Disclosures: No relevant conflicts of interest to declare.

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