Common B-cell Acute Lymphoblastic Leukemia Research Articles

Adaptation to Metabolic Stress By Mondoα in Common B-Cell Acute Lymphoblastic Leukemia

Malignant cells evolve adaptive mechanisms to survive metabolic stress to drive progression. We previously described the stress sensor MondoA as promoting malignancy of common acute lymphoblastic leukemia (cALL). MondoA knockdown (MKD) in cALL cell lines xenografted into mice reduced the number of leukemic blasts compared to control cells (Sipol 2014). Next we investigated potential mechanisms of MondoA mediated malignancy. Microarray analyses of MKD cALL lines revealed an induction of glycolytic and hypoxia response associated gene sets. To validate and expand these results we generated cALL lines with MondoA knockout (MKO) via CRISPR/cas9 gene editing. Colony forming assays and direct cell counting demonstrated reduced proliferation of MKO cells under normoxic conditions compared to controls. However, under hypoxia, there was a diminished MondoA dependent growth advantage. This suggests that MondoA proficient cells may have a selective growth advantage in the presence of oxygen, and that MondoA might confer the ability to utilize oxidative phosphorylation and thus increase metabolic activity. We therefore measured mitochondrial respiration by determining oxygen consumption rates (OCR) in control and MKO cells using an extracellular flux analyzer. Surprisingly, MKO cells displayed a higher basal OCR as well as significantly increased extracellular acidification rate (ECAR) and glycolysis. This suggested that MondoA might actually limit oxidative phosphorylation and glycolysis in response to glucose. We therefore hypothesized that MondoA might remodel cALL metabolism towards an alternative energy source. Indeed, Kegg pathway analysis of microarray data showed upregulation of fatty acid synthesis (FAS) and fatty acid oxidation (FAO) genes by MondoA. This suggested that MondoA might facilitate sustained fatty acid metabolism to maintain high proliferative rates. Key regulators of citrate to oleate conversion, fatty acid elongation and cholesterol synthesis (ACLY, ELOVL5, ELOVL6, ELOVL1, FASN, HMGCR, HMGCS1) were stimulated by MondoA. Colorimetric assays demonstrated decreased NADPH and Acetyl-CoA levels corresponding to disregulated fatty acid metabolism by MKO. In addition, ROS measurements by electron paramagnetic resonance (EPR) using the spin trap CMH in the presence or absence of NADPH oxidase NOX2 inhibitory peptide gp91ds-Tat, a readout of NOX activity, demonstrated that MKO cells increase cytoplasmic ROS production, mediated by NOX2. Moreover, MondoA depleted leukemia cells generate more ROS in glutamine-restricted media. In summary, (1) MondoA increases leukemic cell proliferation under both normoxia and hypoxia; (2) MondoA increases metabolic activity, in particular fatty acid metabolism; and (3) MondoA decreases cytoplasmic ROS production in glutamine-deprived conditions. We conclude that MondoA has multiple functions in supporting malignancy of cALL, most likely by increasing fatty acid metabolism while simultaneously providing adaptation to oxidative stress. DisclosuresNo relevant conflicts of interest to declare.

Abstract 4515: Metabolic stress sensor MondoA mediates in vivo aggressiveness of common ALL by induction of HIF1α

Abstract Tumor cells evolve adaptive mechanisms to survive hypoxia, nutrient deprivation, oxidative or genotoxic stress to ultimately drive tumor progression (Sorensen et al. 2015). We previously described MondoA (also known as MLXIP, MAX like protein X interacting protein) as a metabolic stress sensor, required for leukemogenesis. Lymphocytes, in particular B Lymphocytes are adapted to hypoxic environments from their very beginning. They are destined to travel from the hypoxic bone marrow via normoxic peripheral blood to hypoxic lymph nodes. They thus are specialized in glycolysis to compensate the lack of oxidative phosphorylation under hypoxic conditions. Leukemic counterparts of B-lymphocytes exploit these features for their survival. Glucose-derived metabolites control the nuclear activity of the transcription factor MondoA. Here we report on the expression of MondoA in common B-cell acute lymphoblastic leukemia (cALL) compared to other malignancies, its role in malignancy of cALL in vivo, downstream pathways and correlation with relapse risk. Methods. Our human/murine xenotransplantation model with immunodeficient RAG2-/-gc-/- mice was used (Richter et al. 2009). NALM6 and 697 cALL lines were lentivirally transduced with MondoA short hairpin RNA. Upon successful MondoA knock down (KD), KD and control lines were injected into the mice; CD10+ blasts in blood, spleen and marrow were assessed. Results. We found MondoA to be most strongly expressed in pediatric cALL and AML. Moreover MondoA expression was high in gastrointestinal stromal tumors and alveolar rhabdomyosarcoma. MondoA KD in cALL cell lines and their subsequent analysis in xenograft mice resulted in a reduced number of leukemic blasts in blood, marrow and spleen. Spleen size and weight normalized in mice after MondoA KD. Further microarray analysis revealed an enrichment of glycolytic and hypoxia response gene sets by MondoA. Moreover, HIF1a induction under hypoxia required MondoA. We demonstrate that hypoxia facilitates vincristine resistance of cALL. MondoA is induced under hypoxia and confers cALL cells chemotherapeutic resistance. Tied to these results, MondoA overexpression correlated with relapse risk; its expression was 63% higher in the very high-risk group as compared to the non-high-risk group of cALL. In conclusion, our findings demonstrate that MondoA maintains leukemic burden and aggressiveness of cALL in vivo possibly by modulating metabolic and hypoxia stress response, in particular by induction of HIF1a. Citation Format: Alexandra A. Sipol, Thomas G. Grunewald, Juliane Schmaeh, Monique L. den Boer, Rebeca Alba Rubío, Michaela Baldauf, Caroline Wernicke, Martin Horstmann, Gunnar Cario, Günther Richter, Stefan Burdach. Metabolic stress sensor MondoA mediates in vivo aggressiveness of common ALL by induction of HIF1α [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4515. doi:10.1158/1538-7445.AM2017-4515

In vitro study of BRD4 inhibitor GSK525762A against primary adult common B-cell acute lymphoblastic leukemia cells in vitro

目的探讨4型含Bromo结构域蛋白（BRD4）拮抗剂对普通型急性B淋巴细胞白血病（B-ALL）患者原代白血病细胞的作用及可能机制。方法采用流式细胞术分选14例普通型B-ALL患者（Ph+ B-ALL 4例，Ph−B-ALL 10例）白血病细胞，在模拟骨髓微环境条件下进行短期培养；给予BRD4拮抗剂GSK525762A处理后，采用CCK-8法检测细胞增殖抑制率，用AnnexinⅤ/7-AAD法检测细胞凋亡率，荧光定量PCR法检测c-MYC、CDK6、Bcl-2 mRNA表达水平，Western blot法检测c-MYC、CDK6、Bcl-2蛋白的表达。结果GSK525762A对14例普通型B-ALL患者原代白血病细胞均有抑制增殖作用，且呈剂量依赖性，其中位IC50值为256.25（90.64~1 378.39）nmol/L。500、1 000、2 500 nmol/L GSK525762A作用后中位细胞凋亡率分别为45.17%（9.38%~70.91%）、66.02%（24.36%~96.34%）、89.29%（39.29%~99.37%）。GSK525762A对Ph+与Ph−B-ALL细胞具有类似的作用，但对Ph+ B-ALL细胞抑制增殖和诱导凋亡作用均弱于Ph−B-ALL细胞。1 000 nmol/L GSK525762A处理白血病细胞24、48 h后，c-MYC、CDK6和Bcl-2 mRNA表达水平下降，其中对Ph+和Ph−B-ALL细胞c-MYC、CDK6 mRNA的下调作用差异无统计学意义，而对Ph+ B-ALL细胞Bcl-2 mRNA表达水平的下调弱于对Ph−B-ALL细胞；GSK525762A处理后白血病细胞c-MYC、CDK6、Bcl-2蛋白表达水平均下调。结论GSK525762A可抑制普通型B-ALL原代细胞的增殖，并促进其凋亡，且对Ph+ ALL细胞在体外具有一定作用。该作用可能通过下调c-MYC、CDK6和Bcl-2而实现。

IKZF1 Status As an Independent Prognostic Marker in Adult Common B-Cell Acute Lymphoblastic Leukemia

Previous studies have suggested that IKZF1 gene deletions are associated with an adverse prognosis in acute lymphoblastic leukemia (ALL). However, the prognostic value of IKZF1 deletions in adult common B-cell ALL (Com-B ALL) has not been well-defined, especially in the context of different post-remission therapies. The objectives of this study were to evaluate the prognostic role of IKZF1 deletions in adult Com-B ALL.Overall, 162 untreated adult Com-B ALL patients were recruited between April 2006 and April 2013. Patients received one or two cycles of induction therapy and, if in remission, were allowed to select either ongoing systemic chemotherapy or hematopoietic stem cell transplantation (HSCT). BCR-ABL positive patients were additionally treated with tyrosine kinase inhibitors (TKIs). Deletions in the IKZF1 gene were detected using multiplex RQ-PCR, multiplex fluorescent PCR, multiplex ligation-dependent probe amplification (MLPA) and sequence analysis. High-risk (HR) was defined as having any of the following factors: central nervous system involvement at diagnosis; age ¡Ý50 years; WBC count ¡Ý30x109/L; hypodiploidy; mixed lineage leukemia (MLL) gene rearrangements; BCR-ABL rearrangements; t(1;19) translocations; or a complicated karyotype (¡Ý5 chromosomal abnormalities); failure to achieve remission after two cycles of induction therapy. The study end points included relapse-free survival (RFS), disease-free survival (DFS) and overall survival (OS), which was analyzed using the Kaplan-Meier method. The cumulative incidence of relapse (CIR) was estimated, adjusting for competing risks of death, and compared by Gray’s test.IKZF1 deletions were detected in 79 (48.8%) out of 162 adult Com-B ALL patients. Patients with an IKZF1 deletion (n=79) had a significantly inferior prognosis than those with wild-type IKZF1 (n=83) (Fig.1). The prognosis of patients with IKZF1 deletions with or without the BCR-ABL rearrangements was significantly inferior compared to those of patients with neither IKZF1 deletions nor BCR-ABL rearrangements. The power of IKZF1 deletion status as a prognostic factor remained in both non-HR group (5-yr OS: 52.5%¡À13.1% for IKZF1 deletions (n=25) vs. 72.2%¡À8.8% for wild-type IKZF1 (n=42), P=0.048) and HR group (5-yr OS: 36.7%¡À8.2% for IKZF1 deletions (n=54) vs. 69.3%¡À8.3% for wild-type IKZF1 (n=41), P=0.015). Among patients who received chemotherapy as post-remission therapy (n=52), IKZF1 deletions were associated with an unfavorable OS (P=0.003). In this group, the median OS time for IKZF1-deletion patients (n=28) was 10.0¡À1.0 months, while the median OS time for IKZF1-wild-type carriers (n=24) was not achieved. Among those that received HSCT as post-remission therapy (n=94), IKZF1 status did not affect the prognosis.For patients with IKZF1 deletions, HSCT (n=40) was significantly superior to chemotherapy (n=28) as a post-remission therapy. In the wild-type IKZF1 HR group, patients that received HSCT (n=24) achieved a longer DFS and RFS while the OS did not significantly differ from that of patients who received chemotherapy alone (n=12). In the wild-type IKZF1 non-HR group, there was no significant difference between patients that received HSCT (n=30) and those who received chemotherapy (n=12) as a post-remission therapy. Comparing survival curves of HSCT to chemotherapy are shown in Fig.2. Multivariate analysis confirmed the negative impact of IKZF1 deletions on OS (RR 2.3; 95% CI, 1.3-4.3; P=0.007), DFS (RR 1.8; 95% CI, 1.1-3.0; P=0.029), RFS (RR 2.1; 95% CI, 1.3-3.9; P=0.018) and favorable impact of HSCT on OS (RR 0.2; 95% CI, 1.0-0.5; P=0.000), DFS (RR 0.2; 95% CI, 0.1-0.4; P=0.000), RFS (RR 0.2; 95% CI, 0.1-0.4; P=0.000).These results indicated that IKZF1 status served as an independent negative prognostic marker in adult Com-B ALL and surpassed conventional HR features in the era of TKIs. Patients may benefit from therapy stratified by IKZF1 deletions and conventional HR features. [Display omitted] [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Individualized leukemia cell-population profiles in common B-cell acute lymphoblastic leukemia patients

Immunophenotype is critical for diagnosing common B-cell acute lymphoblastic leukemia (common ALL) and detecting minimal residual disease. We developed a protocol to explore the immunophenotypic profiles of common ALL based on the expression levels of the antigens associated with B lymphoid development, including IL-7Rα (CD127), cytoplasmic CD79a (cCD79a), CD19, VpreB (CD179a), and sIgm, which are successive and essential for progression of B cells along their developmental pathway. Analysis of the immunophenotypes of 48 common ALL cases showed that the immunophenotypic patterns were highly heterogeneous, with the leukemic cell population differing from case to case. Through the comprehensive analysis of immunophenotypic patterns, the profiles of patient-specific composite leukemia cell populations could provide detailed information helpful for the diagnosis, therapeutic monitoring, and individualized therapies for common ALL.

High Pro-Apoptotic Effects of 17-Allylamino-17-Demethoxy Geldanamycin (17-AAG) In Philadelphia Acute Lymphoblastic Leukemia (Ph+ ALL)

AbstractAbstract 3226Heat Shock Protein 90 (HSP 90), a 90 Kda molecular weight protein, is one of the most abundant cytosolic chaperone, acting in protein folding, refolding and degradation and of particular importance in cell survival. HSP 90 is overexpressed in acute leukemia cells and this high expression is related with a poor prognosis. This ATP-dependent chaperone could be an interesting potential drug target. 17-AAG, a Geldanamycin derivates, is a HSP 90 inhibitor with a promising antitumor activity.Very few reports have evaluated the effects of 17-AAG in acute lymphoblastic leukemia (ALL). The aim of this work is to study the cytotoxicity of 17-AAG and to compare Philadelphia positive (Ph+) ALL to common B-cell ALL.Two human leukemia cell lines, Reh (common B-cell ALL) and SUPB15 (Ph+ ALL) were used in this study. We identified 63 consecutive patients treated in our institution for ALL (44 common B-cell ALL and 19 Ph+ ALL) and leukemic samples were collected at diagnosis from bone marrow aspiration after patient's consent.The expression of HSP 90, pro-apoptotic BAX protein, anti-apoptotic bcl-2 and bcl-xl proteins was studied by flow cytometry. Cell lines and ALL patient's cells were cultured in RPMI 1640 and exposed to various concentrations of 17-AAG. Apoptosis was evaluated by an Annexin V and an activated caspase-3 staining by flow cytometry. Results were expressed as percentage of positive cells.Pro-apoptotic effect of 17-AAG in Ph+ ALL cells was superior when compared to common B-cells with a 100% mortality rate after exposure to [10μM] 17-AAG for 24 hours for SUPB15 cell line whereas 24% of cell surviving was noted for Reh cell line. Similar results were observed with patient's leukemic samples (as shown in figure). The susceptibility of Ph+ ALL cells to 17-AAG was confirmed by the assessment of the IC50, estimated at 1.1 μM for SUPB15 cells versus 2.9 μM for ReH cells. [Display omitted] As assessed by Annexin V binding and activated caspase-3 staining, 17-AAG induced apoptosis in ALL cells. As regard Ph+ ALL, the median percentage of Annexin-V positive cells and caspase-3 positive cells after exposure to [5μM] 17 AAG for 24 hours was 54% and 57% respectively and increased up to 99% and 99,5% after exposure for 48 hours. Spontaneous apoptosis in control cell culture was measured in 1% at 24 hours and 3% at 48 hours.The pattern of expression of HSP90 and apoptotic proteins was different between common B-cell ALL and Ph+ ALL cells. The percentage of HSP90 positive cells was 62% for Reh cell line whereas it reached 100% for SUP B15 cell line. As shown in the Table, the percentage of HSP90-positive cells and anti-apoptotic bcl-2 and bcl-xl proteins expression were higher for Ph+ ALL samples when compared to common B-cell ALL samples.HSP90Bcl-2Bcl-xlBaxCommon B-cell ALL samples n=4432.5% (±19.8)33% (± 21.4)28.5% (±19.5)33% (±17)Ph+ ALL samples n = 1979% (±7.5)82% (±8.4)84% (±7.8)12% (±4.3)When ALL cells were exposed in culture to various concentrations of 17-AAG, there was no change in HSP90 expression but we observed a down-regulation in bcl-2 and bcl-xl expression and an up-regulation in bax expression.In summary, we showed that HSP90 and anti-apoptotic proteins were expressed at a higher level on Ph+ ALL cells when compared to common B-cell ALL. High percentage of HSP90-positive cells was associated with high sensitivity to 17-AAG. 17-AAG is a new targeted therapy that induces the apoptotic death of leukemic cells via a caspase-3 dependant way. It would be interesting to test its antileukemic activity in combination with chemotherapeutic agents to study additional or synergistic effects. Despite therapeutic improvement with the development of tyrosine kinase inhibitors (TKI) in the treatment of Ph+ ALL, relapse remains a major problem. Considering that Bcr-Abl constitutes HSP 90 substrates and depends on this chaperone for its maturation and conformational maintenance, 17-AAG could be of particular interest for Ph+ ALL disease, in combination with TKI. We can hypothesise that this drug could restore the sensitivity to TKI treatment for patients with Bcr-Abl mutation. Disclosures:No relevant conflicts of interest to declare.

Keep the Sprays Away?: Home Pesticides Linked to Childhood Cancers

Vol. 115, No. 12 EnvironewsOpen AccessKeep the Sprays Away?: Home Pesticides Linked to Childhood Cancers Tina Adler Tina Adler Search for more papers by this author Published:1 December 2007https://doi.org/10.1289/ehp.115-a594aAboutSectionsPDF ToolsDownload CitationsTrack Citations ShareShare onFacebookTwitterLinked InReddit Previous studies have suggested a link between pesticide use in the home and childhood hematopoietic tumors, the most common type of childhood cancer. A new epidemiologic study of French children diagnosed with leukemia or lymphoma in 2003 or 2004 suggests that a child has about twice the risk of developing acute leukemia (AL) or non-Hodgkin lymphoma (NHL) if his or her mother used insecticides in the home while pregnant [EHP 115:1787–1793; Rudant et al.].The researchers interviewed 1,060 children diagnosed within the prior 6 months and 1,681 control children. When analyzing the data on the children, the team controlled for other factors that may alter a child’s risk of getting cancer, including family cancer history and whether the child was breastfed. The children with cancer were part of the French National Registry of Childhood Blood Malignancies, begun in 1990, which documents all children in the country under age 15 year who have had hematopoietic tumors.The researchers asked the children’s mothers about their use while pregnant of pesticides in their homes, on pets, and in the garden. They also asked about the father’s use of pesticides while the mother was pregnant and after the child’s birth. Just over 50% of the parents who had a child with AL or NHL had used pesticides at least once during the pregnancy, as did just under 40% of the parents of the control group. Children had 2.1 and 1.8 times the risk of developing AL or NHL, respectively, with maternal use of pesticides during pregnancy.Mothers’ use of insecticides during pregnancy was significantly associated with childhood AL, NHL, and mixed-cell Hodgkin lymphomas (HLs). Use of pesticides by fathers was also related to AL and NHL. The association was stronger for common B-cell acute lymphoblastic leukemia (ALL) and acute myeloblastic leukemia than for T-cell ALL or mature B-cell ALL. It was also stronger for Burkitt lymphoma than for the other NHLs. Of the HLs, the study linked only the mixed-cell subtype to pesticide use. The strength of the association between pesticide use in the home and cancer did not change as the children grew older.This is the first study to tease out the different types of hematopoietic cancers as they relate to pesticide use in the home. Other studies have found a link between parents’ occupational exposure to pesticides and childhood cancers, but few of the parents in the French study were exposed to pesticides at work or through farming. Whether a family was rural or urban didn’t alter a child’s risk of developing cancer.The two types of lymphoma associated with maternal pesticide use during pregnancy have also both been linked to the Epstein-Barr virus, which may suggest a link between pesticide exposure and susceptibility to a viral lymphoma. According to the authors, the consistency of the findings suggests pregnant women may want to avoid pesticide use.FiguresReferencesRelatedDetails Vol. 115, No. 12 December 2007Metrics About Article Metrics Publication History Originally published1 December 2007Published in print1 December 2007 Financial disclosuresPDF download License information EHP is an open-access journal published with support from the National Institute of Environmental Health Sciences, National Institutes of Health. All content is public domain unless otherwise noted. Note to readers with disabilities EHP strives to ensure that all journal content is accessible to all readers. However, some figures and Supplemental Material published in EHP articles may not conform to 508 standards due to the complexity of the information being presented. If you need assistance accessing journal content, please contact [email protected]. Our staff will work with you to assess and meet your accessibility needs within 3 working days.

RUNX1 amplification in lineage conversion of childhood B-cell acute lymphoblastic leukemia to acute myelogenous leukemia

Amplification of RUNX1 (alias AML1) is a recurrent karyotypic abnormality in childhood acute lymphoblastic leukemia (ALL) that is generally associated with a poor outcome. It does not occur with other primary chromosomal abnormalities in acute ALL. AML1 amplification in acute myelogenous leukemia (AML) is a rare secondary event described mainly in therapy-related cases. AML1 amplification was found in a 13-year-old patient with AML M4/M5 leukemia that occurred 5 years after she had been diagnosed with common B-cell ALL. Conventional cytogenetic, fluorescent in situ hybridization (FISH), and polymerase chain reaction methods revealed no other chromosomal change expected to occur in a disease that we assumed to be a secondary leukemia. Due to the lack of cytogenetic data from the diagnostic sample, we developed a new approach to analyze the archived bone marrow smear, which had been stained previously with May-Grünwald-Geimsa by the FISH method. This analysis confirmed that in addition to t(12;21), AML1 amplification and overexpression existed already at the time the diagnosis was made. The chromosomal changes, however, were found in different clones of bone marrow cells. While the first course of chemotherapy successfully eradicated the cell line with the t(12;21), the second cell line with AML1 amplification remained latent during the time of complete remission and reappeared with a different immunophenotype.

Dicentric Translocation (9;12) Presenting as Refractory Philadelphia Chromosome-Positive Acute B-Cell Lymphoblastic Leukemia

A 66-year-old Caucasian woman presented with a Philadelphia chromosome positive common B-cell acute lymphoblastic leukemia and a dic(9;12) involving the der(9)t(9;22), a rearrangement so far not observed in acute lymphoblastic leukemia. The patient was treated for the acute lymphoblastic leukemia, but showed refractory disease and died 6 months after initial diagnosis. This case suggests that, in the combination of t(9;22) and dic(9;12), the known poor prognostic feature of t(9;22) in acute lymphoblastic leukemia may outweigh the favorable outcome reported in patients with dic(9;12).