Irinotecan Induces Disease Remission in Xenograft Mouse Models of Pediatric MLL-Rearranged Acute Lymphoblastic Leukemia

Acute lymphoblastic leukemia (ALL) in infants (<1 year of age) remains one of the most aggressive types of childhood hematologic malignancy. The majority (~80%) of infant ALL cases are characterized by chromosomal translocations involving the MLL (or KMT2A) gene, which confer highly dismal prognoses on current combination chemotherapeutic regimens. Hence, more adequate therapeutic strategies are urgently needed. To expedite clinical transition of potentially effective therapeutics, we here applied a drug repurposing approach by performing in vitro drug screens of (mostly) clinically approved drugs on a variety of human ALL cell line models. Out of 3685 compounds tested, the alkaloid drug Camptothecin (CPT) and its derivatives 10-Hydroxycamtothecin (10-HCPT) and 7-Ethyl-10-hydroxycamtothecin (SN-38: the active metabolite of the drug Irinotecan) appeared most effective at very low nanomolar concentrations in all ALL cell lines, including models of MLL-rearranged ALL (n = 3). Although the observed in vitro anti-leukemic effects of Camptothecin and its derivatives certainly were not specific to MLL-rearranged ALL, we decided to further focus on this highly aggressive type of leukemia. Given that Irinotecan (the pro-drug of SN-38) has been increasingly used for the treatment of various pediatric solid tumors, we specifically chose this agent for further pre-clinical evaluation in pediatric MLL-rearranged ALL. Interestingly, shortly after engraftment, Irinotecan completely blocked leukemia expansion in mouse xenografts of a pediatric MLL-rearranged ALL cell line, as well as in two patient-derived xenograft (PDX) models of MLL-rearranged infant ALL. Also, from a more clinically relevant perspective, Irinotecan monotherapy was able to induce sustainable disease remissions in MLL-rearranged ALL xenotransplanted mice burdened with advanced leukemia. Taken together, our data demonstrate that Irinotecan exerts highly potent anti-leukemia effects against pediatric MLL-rearranged ALL, and likely against other, more favorable subtypes of childhood ALL as well.

Download Full-text

Clinical relevance of BCL-2 overexpression in childhood acute lymphoblastic leukemia

Blood ◽

10.1182/blood.v87.3.1140.bloodjournal8731140 ◽

1996 ◽

Vol 87 (3) ◽

pp. 1140-1146 ◽

Cited By ~ 145

Author(s):

E Coustan-Smith ◽

A Kitanaka ◽

CH Pui ◽

L McNinch ◽

WE Evans ◽

...

Keyword(s):

Acute Lymphoblastic Leukemia ◽

Peripheral Blood ◽

Lymphoblastic Leukemia ◽

Philadelphia Chromosome ◽

Leukemic Cell ◽

Trophic Factors ◽

Chromosomal Translocations ◽

Childhood All ◽

Protein Levels

Enforced BCL-2 gene expression in leukemic cell lines suppresses apoptosis and confers resistance to anticancer drugs, but the clinical significance of increased BCL-2 protein levels in acute lymphoblastic leukemia (ALL) is unknown. Among 52 children with newly diagnosed ALL, BCL-2 expression in leukemic lymphoblasts ranged widely, from 4,464 to 59,753 molecules of equivalent soluble fluorochrome per cell (MESF), as determined by flow cytometry. The mean (+/- SD) level of MESF in 43 cases of B-lineage ALL (19,410 +/- 11,834) was higher than that detected in CD10+ B-lymphoid progenitors from normal bone marrow (450 +/- 314; P < .001), and CD19+ peripheral blood B lymphocytes (7,617 +/- 1,731; P = .02). Levels of BCL-2 in T-ALL cases (17,909 +/- 18,691) were also generally higher than those found in normal CD1a+ thymocytes (1,762 +/- 670), or in peripheral blood T lymphocytes (9,687 +/- 3,019). Although higher levels of BCL-2 corresponded to higher leukemic cell recoveries after culture in serum-free medium, they did not correlate with higher cell recoveries after culture on stromal layers, or with in vitro resistance to vincristine, dexamethasone, 6- thioguanine, cytarabine, teniposide, daunorubicin or methotrexate. BCL- 2 protein levels did not correlate with presenting clinical features. Unexpectedly, however, lower-than-median MESF values were significantly associated with the presence of chromosomal translocations (P = .010). Notably, all six cases with the Philadelphia chromosome, a known high- risk feature, had low levels of BCL-2 expression (P = .022). Higher levels of BCL-2 were not associated with poorer responses to therapy among 33 uniformly treated patients, and were not observed in three patients studied at relapse. In conclusion, increased BCL-2 expression in childhood ALL appears to enhance the ability of lymphoblasts to survive without essential trophic factors, and is inversely related to the presence of chromosomal translocations. However, it does not reflect increased disease aggressiveness or resistance to chemotherapy.

Download Full-text

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

Blood ◽

10.1182/blood.v88.11.4252.4252 ◽

1996 ◽

Vol 88 (11) ◽

pp. 4252-4258 ◽

Cited By ~ 33

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.

Download Full-text

Poor prognosis of children with pre-B acute lymphoblastic leukemia is associated with the t(1;19)(q23;p13): a Pediatric Oncology Group study

Blood ◽

10.1182/blood.v76.1.117.117 ◽

1990 ◽

Vol 76 (1) ◽

pp. 117-122 ◽

Cited By ~ 157

Author(s):

WM Crist ◽

AJ Carroll ◽

JJ Shuster ◽

FG Behm ◽

M Whitehead ◽

...

Keyword(s):

Treatment Outcome ◽

Acute Lymphoblastic Leukemia ◽

Pediatric Oncology ◽

Poor Prognosis ◽

Lymphoblastic Leukemia ◽

Chromosomal Translocations ◽

Oncology Group ◽

Childhood All ◽

Pediatric Oncology Group ◽

Worse Prognosis

Abstract The prognostic significance of chromosomal translocations, particularly t(1;19) (q23;p13), was evaluated in children with pre-B and early pre-B acute lymphoblastic leukemia (ALL). Patients were treated on a risk- based protocol of the Pediatric Oncology Group (POG) between February 1986 and May 1989. An abnormal clone was detected in 46% (130 of 285) of pre-B cases and 56% (380 of 679) of early pre-B cases. Translocation of any type was associated with a worse treatment outcome than other karyotypic abnormalities: 15 of 66 versus 3 of 64 failed therapy in the pre-B group (P = .001), and 37 of 141 versus 23 of 239 failed in the early pre-B group (P less than .001). The t(1;19) (q23;p13) occurred significantly more often in cases of pre-B ALL with a clonal abnormality than in early pre-B ALL cases (29 of 130 v 5 of 380, P less than .001). Among the 285 pre-B cases in which bone marrow was studied cytogenetically, those with t(1;19) had a significantly worse treatment outcome than all others (11 of 29 v 27 of 256 have failed therapy, P less than .001). This difference is significant (P less than .001) after adjustment for leukocyte count, age, and other relevant features. Cases with the t(1;19) also had a worse prognosis than pre-B patients with other translocations (4 of 37 have failed, P less than .01) or with any other karyotypic abnormality (7 of 101 have failed, P less than .001). We conclude that chromosomal translocations confer a worse prognosis for non-T, non-B-cell childhood ALL, and that the t(1;19) is largely responsible for the poor prognosis of the pre-B subgroup.

Download Full-text

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

Blood ◽

10.1182/blood.v86.10.3861.bloodjournal86103861 ◽

1995 ◽

Vol 86 (10) ◽

pp. 3861-3868 ◽

Cited By ~ 148

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.

Download Full-text

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

Blood ◽

10.1182/blood.v104.11.4495.4495 ◽

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.

Download Full-text

Transient Responses to Notch and Tlx1/Hox11 Inhibition In T-Cell Acute Lymphoblastic Leukemia/Lymphoma.

Blood ◽

10.1182/blood.v116.21.1026.1026 ◽

2010 ◽

Vol 116 (21) ◽

pp. 1026-1026

Author(s):

Erica A. Lehotzky ◽

Mark Y. Chiang

Keyword(s):

Acute Lymphoblastic Leukemia ◽

T Cell ◽

Cell Line ◽

Conflicts Of Interest ◽

Lymphoblastic Leukemia ◽

Ectopic Expression ◽

Leukemia Cells ◽

Inhibitory Effects ◽

Notch Inhibition

Abstract Abstract 1026 Despite numerous advances in the past few decades, treatment of acute lymphoblastic leukemia/lymphoma (ALL) remains a common and considerable challenge. Further efforts to define the molecular lesions that drive ALL are needed to improve clinical management. The Hox subfamily of T-cell ALL (T-ALL) represents 30–40% of pediatric and adult cases. TLX1/HOX11 is the prototypical member of the Hox group. To generate a resource for developing targeted therapies for Hox T-ALLs, we developed a doxycycline-regulated mouse model of Tlx1-initiated T-ALL. Dysregulated thymic expression of Tlx1 induces T-ALL after ∼5-7 months with penetrance of 15–60%. The lymphoblasts are arrested at the early CD4+/CD8+/CD24hi stage of T-cell development, similar to human T-ALLs of the TLX1 subtype. Spontaneous activation of the Notch1 oncogene occurred in the tumors. In about two-thirds of samples, Notch was activated through acquired mutations in the heterodimerization and PEST domains that resemble the Notch1 mutations found in human patients. Inhibition of Notch signaling with g-secretase inhibitors completely abrogated cell line growth and induced apoptosis. Notch inhibition also transiently delayed leukemia progression by ∼17 days in vivo. In contrast, suppression of Tlx1 expression had more moderate inhibitory effects on cell line growth in vitro. However, suppression of Tlx1 expression in transgenic mice transiently delayed leukemia progression by ∼11 days. Tlx1 suppression had the strongest inhibitory effects on expression of CCR7 and lymph node size. These effects were fully reversed with ectopic expression of Tlx1. These data show that Tlx1 can convert normal thymocytes into leukemia cells, but the leukemia cells are not fully dependent on continued Tlx1 expression. The leukemia cells recruit secondary factors and pathways such as Notch and c-Myc to sustain growth and survival. Our study highlights a strong resiliency of T-ALL cells to both Tlx1 and Notch inhibition. Our study has important implications for targeting these pathways for the treatment of T-ALL. Disclosures: No relevant conflicts of interest to declare.

Download Full-text

Vincristine and Prednisolone Combination Reduces MDR1 and Microenvironment-Mediated Treatment Resistance In Acute Lymphoblastic Leukemia

Blood ◽

10.1182/blood.v122.21.2529.2529 ◽

2013 ◽

Vol 122 (21) ◽

pp. 2529-2529

Author(s):

Nan Jiang ◽

Zhenhua Li ◽

Grace Shimin Koh ◽

Yi Lu ◽

Shirley K.Y. Kham ◽

...

Keyword(s):

Treatment Outcome ◽

Acute Lymphoblastic Leukemia ◽

Treatment Response ◽

Cell Line ◽

Cell Lines ◽

Lymphoblastic Leukemia ◽

Treatment Resistance ◽

Intrathecal Methotrexate ◽

The Cost

Abstract Acute lymphoblastic leukemia (ALL) is the most common form of childhood cancer with excellent treatment outcome where >80% are cured. However, relapse and therapy-related toxicities limit further improvements and greatly increase the cost of therapy. Vincristine (VCR) is cheap, well tolerated, and highly effective. Using VCR optimally will help improve the cost-benefit ratio favorably by allowing us to reduce toxicities like infections from myelosuppression and yet improving cure. The highly successful BFM-ALL treatment backbone starts with a single intrathecal methotrexate on Day 1 followed by 7 days of oral prednisolone (PRED). The persistence of absolute blasts count >1,000/µL at Day 8 (D8), known as PRED poor response, confers a significantly poorer treatment outcome. To avoid seeding the CNS with leukemia from traumatic taps, the new Ma-Spore ALL 2010 treatment protocol, omitted intrathecal methotrexate at Day 1 and replaced with VCR at Day 0. By June 2013, a total of 133 patients have been enrolled. We found that the number of poor PRED responders was halved from the historical 9.5% in the previous Ma-Spore ALL 2003 study (Yeoh et al. J Clin Oncol 2013) to only 4.7% of patients in the ALL 2010 study. In addition, the percentage of MRD standard risk patients (Day 33 blast count ≤1x10-4) increased from 38.9% in the Ma-Spore ALL 2003 to 51.8% in the Ma-Spore ALL 2010 study (P<0.001). The 2-year event-free survival (EFS) for good and poor D8 response patients under the Ma-Spore ALL 2010 trial remained similar to the ALL 2003 study despite only half the number of PRED poor responders (Fig. 1). These data taken together suggests that VCR and PRED combination is highly synergistic and can improve early treatment response. We investigated VCR and PRED combination in PRED and VCR-resistant (VCR-R) cell lines. Specifically, REH cell line is intrinsically resistant to PRED in vitro because of a mutation in its glucocorticoid receptor. We exposed the REH cell line to increasing concentrations of VCR over 6 months and generated a VCR resistant REH cell line (Fig. 2). This VCR-R REH cell line is resistant to both PRED or VCR when exposed individually in vitro. However when exposed to both PRED and VCR in combination, only 30% of the resistant cells survived (P<0.01). We found that the drug efflux transporter multi-drug resistance protein 1 (MDR1) was preferentially highly expressed in our VCR-R cell line models. To determine if the highly expressed MDR1 is responsible for treatment resistance, we exposed the VCR-R cell lines to VCR, verapamil (an MDR1 inhibitor) and combination of both VCR and verapamil. The combination of VCR and verapamil increased the G2 cell cycle arrest by 3- folds compared to when VCR was used alone (Fig. 3), supporting the role of MDR1 in treatment resistance. Interestingly we also found that the combination of VCR and PRED led to a decrease in levels of MDR1 expression by western blot, suggesting that depletion of MDR1 may be a mechanism through which VCR and PRED combination therapy enhances leukemic cell killing. We also investigated microenvironment-mediated resistance to VCR and PRED using mesenchymal stromal cells (MSC) co-culture systems. It was found that after co-culture with MSC or in conditional medium containing soluble factors secreted by MSC, leukemic cells were resistant to VCR and PRED mono-treatment, but the resistance was abrogated after combinatorial therapy. In conclusion, VCR in combination with PRED improves D8 peripheral blood treatment response during early induction in our Ma-Spore 2010 trial. This synergistic combination results from its ability to reverse resistance from intrinsic overexpression of MDR1 in resistant leukemia cells and decrease microenvironment-contributed resistance by mesenchymal cells. Disclosures: No relevant conflicts of interest to declare.

Download Full-text

Genetic Mechanisms of Transcriptional Regulation in Childhood Acute Lymphoblastic Leukemia

10.21007/etd.cghs.2021.0531 ◽

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.

Download Full-text

Anti-Leukemic Property of Sulphoraphane In Precursor B Cell Acute Lymphoblastic Leukemia

Blood ◽

10.1182/blood.v116.21.3974.3974 ◽

2010 ◽

Vol 116 (21) ◽

pp. 3974-3974

Author(s):

Koramit Suppipat ◽

Xiao Zhu ◽

Chun Shik Park ◽

H. Daniel Lacorazza

Keyword(s):

Acute Lymphoblastic Leukemia ◽

B Cells ◽

B Cell ◽

Cell Line ◽

Cell Lines ◽

Lymphoblastic Leukemia ◽

Annexin V ◽

Dependent Manner ◽

Carcinogenic Activity ◽

Childhood All

Abstract Abstract 3974 Acute lymphoblastic leukemia (ALL) is the most common form of hematologic malignancy in children. In spite of significant advances achieved in the treatment of childhood ALL, one fifth of these patients still relapse after the standard treatment. Moreover, relapse ALL is the second most common cause of cancer-related deaths in children. The development of novel therapies is prevented by a limited understanding of the exact pathobiology. There are emerging evidences that the transcription factor KLF4 has a tumor suppressor property in ALL. Recently, a gene expression classifier study in pediatric precursor B-cell ALL (pre-B ALL) showed that KLF4 expression was significantly reduced in high risk ALL patients with positive MRD after induction. This finding suggests a possible role of this cell cycle inhibitor on the development, expansion and drug-resistant of leukemic cells. Several studies demonstrate that overexpression of KLF4 in normal B cells and BCR transformed B cells show increased apoptosis and reduced proliferation. Furthermore, we recently described that KLF4 inhibits proliferation of naïve lymphocytes by activating p21 (Yamada, et al, 2009). Sulphoraphane (SF; 4-methylsulfonylbutyl isothiocyanate) is a dietary compound derived from Cruciferae vegetables with anti-carcinogenic activity in colon cancer by upregulating KLF4 and p21 among other genes. Thus, we hypothesized that SF could also exhibit anti-leukemic activity in human-derived acute lymphoblastic leukemia cells via the activation of KLF4. The pre-B ALL cell lines (Nalm6, REH, RS-4, SUP-B15) and an EBV transformed B cell line were treated with different concentrations of SF (0-40 μM) for 24–48 hours. Then, cell number was estimated using an ATP-based viability method. Flow cytometric analysis of ANNEXIN-V/7-AAD binding as well as CFSE dilution was used to measure apoptosis and proliferation respectively. We found that SF induced cytotoxicity in Nalm-6, REH and RS-4 cell lines in a dose and time dependent manner. This cytotoxic effect was less pronounced in EBV-transformed B cell line. SF treatment led to increased ANNEXIN-V and 7-AAD positive cells (82% apoptotic cells in SF-treated versus 9% in DMSO control). Further, SF-treated cells displayed significantly less proliferation in comparison to DMSO controls thus suggesting that SF inhibits cellular proliferation. Preliminary data also suggest that SF-mediated apoptosis is caused by upregulation of KLF4. In conclusion, Sulphoraphane exhibits an anti-leukemic property by inducing apoptosis and abrogating proliferation in pre-B ALL cell lines. Thus, sulphoraphane could potentially be used as an adjunctive therapy in a subgroup of pre-B ALL patients who have decreased expression of KLF4. Disclosures: No relevant conflicts of interest to declare.

Download Full-text

Quantitative Proteomic Analysis Reveals Maturation As a Mechanism Underlying Glucocorticoid Resistance in Childhood Acute Lymphoblastic Leukemia and PAX5 As a Re-Sensitising Therapeutic Target

Blood ◽

10.1182/blood.v118.21.1411.1411 ◽

2011 ◽

Vol 118 (21) ◽

pp. 1411-1411

Author(s):

Lindsay Nicholson ◽

Caroline Evans ◽

Elizabeth C Matheson ◽

Lynne Minto ◽

Christopher Keilty ◽

...

Keyword(s):

Acute Lymphoblastic Leukemia ◽

Cell Viability ◽

B Cell ◽

Cell Line ◽

Cell Lines ◽

Therapeutic Target ◽

Proteomic Analysis ◽

Lymphoblastic Leukemia ◽

Childhood All ◽

Resistant Phenotype

Abstract Abstract 1411 Glucocorticoids (GC), such as prednisolone and dexamethasone, are an integral component of the multi-agent treatment of childhood acute lymphoblastic leukemia (ALL). GC-resistance is a significant prognostic indicator of a poor treatment outcome and remains a clinical problem, with the underlying mechanisms still unclear. Mutation or loss of the primary mediator of GC-action, the glucocorticoid receptor (GR), underlies the GC-resistant phenotype in several commonly used leukemic cell lines. However, these events are rare in primary leukemic cells, with relatively few examples in vivo. This suggests that it may be possible to reverse the GC-resistant phenotype pharmacologically. We have used an iTRAQ proteomics approach for hypothesis generation of potential mechanisms for GC-resistance in childhood ALL. To achieve this, we compared a well-characterized GC-sensitive cell line, PreB 697, and a GC-resistant sub-clone (R3F9), both bearing wildtype GR, in a comparative proteomic experiment using 4-channel isobaric tagging for relative and absolute quantification (iTRAQ). A comparison of protein profiles before and after dexamethasone exposure of the two cell lines identified two transcription factors involved in B-cell differentiation, PAX5 and IRF4, to be differentially upregulated in the PreB 697 compared to the R3F9 cell line in response to GC. Experimentally, there was approximately 50% reduction in PAX5 basal protein expression in R3F9 compared to its GC-sensitive parent, a finding which was also evident in four other resistant sub-lines. This was accompanied by a decreased expression of CD19 and CD10, indicative of an increased B-cell maturation state. The reduced PAX5 level in the GC-resistant cell lines was not due to mono-allelic loss or mutation and mRNA levels were not significantly altered, suggestive of a post-transcriptional mechanism for PAX5 protein reduction. Paradoxically, knockdown of PAX5 reversed the GC-resistant phenotype of the R3F9 cell line such that the apoptotic response to dexamethasone was similar to that of the GC-sensitive parent line as measured by Annexin V staining (R3F9: mean 52.22%, SD 12.54%, n=3; PreB 697: mean 67.23%, SD 9.96%, n=3) and cell viability assays. This chemosensitization after PAX5 knockdown was specific to GC, with no difference in cell viability observed in either cell line after exposure to daunorubicin, vincristine or L-asparaginase when compared to negative siRNA or mock controls. This increase in GC-sensitivity was coupled with a significant upregulation of GR and its transcriptional target, GILZ. We also showed an enhanced GC response after PAX5 knockdown in two out of eight primary, diagnostic pre-B lineage ALL patient samples. Thus, in this ALL cell line model, quantitative proteomic analysis revealed increased maturation as a recurrent mechanism underlying GC-resistance and identifies PAX5 as a possible therapeutic target to fully re-sensitise GC-response in childhood ALL. Disclosures: No relevant conflicts of interest to declare.

Download Full-text