scholarly journals Autophagy Collaborates with Ubiquitination to Down-Regulate Oncoprotein E2A/Pbx1 in B Cell Acute Lymphoblastic Leukemia

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 5276-5276 ◽  
Author(s):  
Suping Zhang ◽  
Lin Song ◽  
Na Yuan ◽  
Weiwei Lin ◽  
Yan Cao ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Mouse Model ◽  
B Cell ◽  
Western Blotting ◽  
Degradation Mechanism ◽  
Lymphoblastic Leukemia ◽  
Leukemia Cells ◽  
Hematopoietic Stem ◽  
Treatment Groups ◽  
Cell Acute Lymphoblastic Leukemia

Abstract Background: B cell acute lymphoblastic leukemia (B-ALL) accounts for the most cancer incidences in children. The t(1;19) translocation leukemia accounts for a quarter of pre-B ALL and up to 5% of all ALL patients, in which the transcriptional activator E2A and homeobox pre-B-cell leukemia transcription factor 1 (PBX1) fuses, resulting in expression of the chimeric transcription factor E2A/PBX1. E2A/PBX1 has been proved to be an oncogene and could induce malignant transformation. Methods: (1) Childhood B-ALL patients were collected and the stem/progenitor cells (CD34+CD38-) and leukemia cells (CD19+) were sorted with BD FACS Aria III. The autophagy level in these cells was measured by real-time Q-PCR, including gene expression of Beclin1, Atg7, Atg5, LC3 and p62. Normal bone marrow cells from healthy donors were used as control. (2) E2A/PBX1 fusion gene positive pre-B ALL 697 cells were used to establish leukemia mouse model and the autophagy activity in the mice was enhanced by administration of rapamycin. Mice were sacrificed three weeks post treatment, leukemia phenotype was then identified and E2A/PBX1 oncoprotein of liver was detected by western blotting. (3) Autophagy and ubiquitination were manipulated with inhibitors or starvation in 697 cells and the degradation mechanism of E2A/PBX1 was explored. Co-localization of E2A/PBX1-LC3 and E2A/PBX1-Ub was observed by confocal microscopy and quantified by Amnis image flow cytometry. Results: (1) B-ALL primary cells from childhood patients show down-regulated level of autophagy. (2) The NOD-SCID mouse model study shows that activating autophagy of mice by rapamycin improved the survival of leukemia animals, prevented leukemiagenesis by inhibition on the transplanted leukemia cells (examined by blood cell counting, liver HE staining and expression of CD 45, 10, 19 from transplanted human 697 cells by flow cytometry), promoted the degradation of oncoprotein E2A/PBX1 (by Western blotting)), and more importantly, restored hematopoietic stem cells (LSKCD34- cell number detected by flow cytometry). (3) The ALL 697 cell line study shows that activation of autophagy by rapamycin and starvation could down-regulate E2A/PBX1 expression detected by flow cytometry and western blotting. The confocal microscopic results show co-localization of E2A/PBX1 with autophagy marker GFP-LC3 in both rapamycin and starvation treatment groups. To confirm the degradation mechanism, autophagy inhibitor (3-MA or Baf-A1) and ubiquitin-proteasome inhibitor (MG132) were used to treat 697 cells. The results show that inhibition of autophagy in the early stage by 3-MA fails to degrade E2A/PBX1 in 697 cells, but ubiquitination also contributes to the degradation of E2A/PBX1. Quantitative analysis shows increased co-localization percentage of E2A/PBX1-LC3 in rapamycin and starvation treatment groups and increased co-localization of E2A/PBX1 with Ubiquitin in starvation group; but MG132 treatment inhibited the co-localization of E2A/PBX1-Ub induced by starvation, indicating a collaborative role between autophagy and ubiquitination in the degradation of E2A/PBX1. Conclusions: B-ALL primary cells from patients show low autophagy activity; Autophagy activation fights against B-ALL by inhibition on transplanted leukemia cells, degradation of oncoprotein E2A/Pbx1 and restoration of hematopoietic stem cells in the NOD-SCID B-ALL mouse model; autophagy collaborates with ubiquitination in the degradation of E2A/PBX1 in the 697 cells, thereby proposing a novel strategy for targeted therapy on childhood B-ALL. Disclosures No relevant conflicts of interest to declare.

scholarly journals Efficacy of an Anti-CD22 Antibody-Monomethyl Auristatin E Conjugate in a Preclinical Xenograft Model of Precursor B Cell Acute Lymphoblastic Leukemia

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 971-971
Author(s):  
Sakiko Yoshida ◽  
Emily Tuscano ◽  
Connie Duong ◽  
Joseph Tuscano ◽  
Noriko Satake
Keyword(s):  
Acute Lymphoblastic Leukemia ◽  
B Cell ◽  
Childhood Cancer ◽  
Late Effects ◽  
Lymphoblastic Leukemia ◽  
Wilcoxon Test ◽  
Leukemia Cells ◽  
Treatment Groups ◽  
Cell Acute Lymphoblastic Leukemia ◽  
Target Effects

Abstract Precursor B cell acute lymphoblastic leukemia (preB ALL) is the most common childhood cancer, as well as the leading cause of childhood cancer-related mortality. Despite overall progress in treatment, certain types of patients with preB ALL have a dismal prognosis with an overall survival of 30%. In addition, current approaches predispose these young patients to late effects, including secondary malignancies. Therefore, more efficacious and less toxic approaches are needed. Targeted therapy for leukemia has the potential to reduce off-target effects, thus minimizing toxicity and late effects and improving efficacy. Monoclonal antibodies (mAbs) have proven utility in leukemia therapy based on their ability to specifically target the leukemic clone and minimize off target effects. However, mAbs are generally not adequate as single agents because of limited efficacy. Antibody drug conjugates (ADCs) provide a method to deliver a potent toxin to the interior of antigen-positive tumor cells. CD22 is an ideal target for ADC-mediated therapeutics for B-cell malignancies because 1) there is high CD22 expression (more than 90%) in B-cell type ALL and 2) CD22 undergoes rapid internalization upon mAb binding. In this study, we evaluated the anti-CD22 (aCD22) mAb as a vehicle for the targeted delivery of Monomethyl Auristatin E (mMAE), a derivative of the cytotoxic tubulin modifier auristatin E. Figure 1 Treatments and outcome of the animals Figure 1. Treatments and outcome of the animals First, we assessed the in vitro cytotoxicities of the aCD22 mAb-mMAE in preB ALL cell lines Reh and JM1. MTS assay showed that IC50 doses of aCD22 mAb-mMAE were 0.7nM and 1nM in Reh and JM1, respectively. Next, we assessed in vivo therapeutic efficacy of the aCD22 mAb-mMAE in a pre-clinical xenograft animal model of preB ALL, using a primary leukemia sample which was confirmed to be CD22 positive. Age matched female NOD/SCID/IL2Rg-/- (NSG) mice were randomly assigned to 4 treatment groups (n=8 per group): 1) PBS, 2) free mMAE (0.165mg/kg), 3) free aCD22 mAb (7.335mg/kg), and 4) aCD22 mAb-mMAE conjugate (7.5mg/kg). The dose of free aCD22 mAb and free mMAE was equivalent to those of each component in the aCD22 mAb-mMAE conjugate. Five million leukemia cells were inoculated per mouse via intra-bone marrow injection. Twenty four hours after leukemia inoculation, animals started receiving weekly iv treatments for 3 weeks. When compared to controls (PBS, free aCD22 mAb or mMAE treatments), the treatment with the aCD22 mAb-mMAE conjugate increased the median survival time of the mice by two fold (Figure. PBS vs. aCD22 mAb-mMAE p<0.005, free mMAE vs. aCD22 mAb-mMAE p<0.05, free aCD22 mAb vs. aCD22 mAb-mMAE p<0.05, by Gehan-Breslow-Wilcoxon test). Leukemia-related death was confirmed by necropsy. Harvested leukemia cells were assessed by flow cytometry and found to be HLA and CD22 positive. During treatment, the mice in all the treatment groups remained healthy and active, and did not lose weight. Toxicity was assessed with weekly CBC and chemistry panels which revealed no significant toxicity. In conclusion, we demonstrated that aCD22 mAb-mMAE is efficacious in a preclinical preB ALL xenograft mouse model. Disclosures No relevant conflicts of interest to declare.

B-Cell Acute Lymphoblastic Leukemia Arising in Patients with a Preexisting Diagnosis of Multiple Myeloma Is a Novel Cancer with High Incidence of TP53 Mutations

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 20-20
Author(s):  
Monique Chavez ◽  
Erica Barnell ◽  
Malachi Griffith ◽  
Zachary Skidmore ◽  
Obi Griffith ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Acute Lymphoblastic Leukemia ◽  
B Cells ◽  
B Cell ◽  
Mechanism Of Action ◽  
Lymphoblastic Leukemia ◽  
Treatment Options ◽  
Hematopoietic Stem ◽  
Tp53 Mutations ◽  
Cell Acute Lymphoblastic Leukemia

Multiple Myeloma (MM) is a malignancy of plasma cells that affects over 30,000 Americans every year. Despite advances in the treatment of the disease, approximately 12,000 American patients will still die of MM in 2019. One of the mainstays of treatment for MM is the immunomodulatory and antiangiogenic drug lenalidomide; which is used in induction therapy, maintenance therapy and treatment of relapsed disease. Although not fully elucidated, lenalidomide's mechanism of action in MM involves the drug binding to Cerebelon (CBN) and leads to the subsequent degradation of the Ikaros (IKZF1) and Aiolos (IKZF3) transcription factors (TF). These TFs play important regulatory roles in lymphocyte development. Despite lenalidomide's importance in MM treatment, several groups have reported that MM patients treated with lenalidomide rarely go on to develop B-cell acute lymphoblastic leukemia (B-ALL). The genetics and clonal relationship between the MM and subsequent B-ALL have not been previously defined. Importantly, it is not clear if the MM and B-ALL arise from the same founding clone that has been under selective pressure during lenalidomide treatment. As deletions in IKZF1 are common in B-ALL, one could hypothesize that lenalidomide's mechanism of action mimics this alteration and contributes to leukemogenesis. We sequenced the tumors from a cohort of seven patients with MM treated with lenalidomide who later developed B-ALL. These data did not show any mutational overlap between the MM and ALL samples-the tumors arose from different founding clones in each case. However, several genes were recurrently mutated in the B-ALL samples across the seven patients. These genes included TP53, ZFP36L2, KIR3DL2, RNASE-L, and TERT. Strikingly, five of the seven patients had a TP53 mutations in the B-ALL sample that was not present in the matched MM sample. The frequency of TP53 mutations in our cohort was much higher than that reported in adult de novo B-ALL patients which can range between 4.1-6.4% (Hernández-Rivas et al. 2017 and Foa et al. 2013). Utilizing CRISPR-Cas9 gene editing, we disrupted the Zfp36l2 or Actb in murine hematopoietic stem cells (HSCs) of mice with or without loss of Trp53. We performed our first transplantation experiment in which the cohorts of mice have loss of Trp53 alone, loss of Zfp36l2 alone, loss of both Trp53 and Zfp36l2, or a control knockout (KO) of Actb. To characterize the disruption of Zfp36l2 alone and in combination with Trp53 we analyzed the hematopoietic stem and progenitor cell compartments in the bone marrow of the above transplanted mice. In mice with a loss of Zfp36l2 there is a decrease in Lin- Sca-1+ c-Kit+ (LSK), short term-HSC (ST-HSC), and multipotent progenitors (MPP). This decrease was not observed in the mice with a loss of both Trp53 and Zfp36l2, where instead we noted an increase in monocyte progenitors (MP), granulocytes-macrophage progenitors (GMP), and common myeloid progenitors (CMP) cells. In this Trp53 Zfp36l2 double loss model we also noted a decrease in B220+ B-cells that was not seen in the Zfp36l2 alone. In this cohort of Trp53 Zfp36l2 loss, we characterized B-cell development through hardy fraction flow cytometry, and identified a decrease in fractions A and B/C (pre-pro and pro-B-cells, respectively) as compared to Zfp36l2 or Actb alone. As lenalidomide does not bind to Cbn in mice, we used the human B-ALL NALM6 cell line to test if treatment with lenalidomide will lead to a selective growth advantage of cells with the same genes knocked out versus wild-type control cells grown in the same culture. We hypothesize that lenalidomide treatment selectively enriched for pre-existing mutated cell clones that evolved into the B-ALL. Preliminary data in NALM6 cells with a loss of TP53 demonstrate a slight increase in cell number at day 7 compared to a RELA control. These experiments will be repeated with concurrent ZFP36L2 and TP53 mutations as well as ZFP36L2 alone. Treatment-related disease is a key consideration when deciding between different treatment options, and this project aims to understand the relationship between MM treatment and B-ALL occurrence. It may be possible to identify MM patients who are at-risk for B-ALL. For example, MM patients who harbor low-level TP53 mutations prior to lenalidomide treatment could be offered alternative treatment options. Disclosures Barnell: Geneoscopy Inc: Current Employment, Current equity holder in private company, Membership on an entity's Board of Directors or advisory committees. Wartman:Novartis: Consultancy; Incyte: Consultancy.

scholarly journals Aberrant GATA2 Activation in Pediatric B-Cell Acute Lymphoblastic Leukemia

2022 ◽  
Vol 9 ◽  
Author(s):  
Han Wang ◽  
Bowen Cui ◽  
Huiying Sun ◽  
Fang Zhang ◽  
Jianan Rao ◽  
...  
Keyword(s):  
Acute Lymphoblastic Leukemia ◽  
B Cell ◽  
Transcriptome Sequencing ◽  
Lymphoblastic Leukemia ◽  
Myeloid Differentiation ◽  
Sequencing Data ◽  
Hematopoietic Progenitors ◽  
Hematopoietic Stem ◽  
Topologically Associating Domains ◽  
Cell Acute Lymphoblastic Leukemia

GATA2 is a transcription factor that is critical for the generation and survival of hematopoietic stem cells (HSCs). It also plays an important role in the regulation of myeloid differentiation. Accordingly, GATA2 expression is restricted to HSCs and hematopoietic progenitors as well as early erythroid cells and megakaryocytic cells. Here we identified aberrant GATA2 expression in B-cell acute lymphoblastic leukemia (B-ALL) by analyzing transcriptome sequencing data obtained from St. Jude Cloud. Differentially expressed genes upon GATA2 activation showed significantly myeloid-like transcription signature. Further analysis identified several tumor-associated genes as targets of GATA2 activation including BAG3 and EPOR. In addition, the correlation between KMT2A-USP2 fusion and GATA2 activation not only indicates a potential trans-activating mechanism of GATA2 but also suggests that GATA2 is a target of KMT2A-USP2. Furthermore, by integrating whole-genome and transcriptome sequencing data, we showed that GATA2 is also cis activated. A somatic focal deletion located in the GATA2 neighborhood that disrupts the boundaries of topologically associating domains was identified in one B-ALL patient with GATA2 activation. These evidences support the hypothesis that GATA2 could be involved in leukemogenesis of B-ALL and can be transcriptionally activated through multiple mechanisms. The findings of aberrant activation of GATA2 and its molecular function extend our understanding of transcriptional factor dysregulation in B-ALL.

scholarly journals Lymphoblast purine pathway enzymes in B-cell acute lymphoblastic leukemia

Blood ◽  
1981 ◽  
Vol 58 (2) ◽  
pp. 330-332 ◽  
Author(s):  
GH Reaman ◽  
J Blatt ◽  
DG Poplack
Keyword(s):  
Acute Lymphoblastic Leukemia ◽  
B Cell ◽  
Metabolic Pathway ◽  
Purine Nucleoside Phosphorylase ◽  
Lymphoblastic Leukemia ◽  
Leukemia Cells ◽  
Malignant Cells ◽  
B Cell Malignancies ◽  
Cell Acute Lymphoblastic Leukemia ◽  
Purine Pathway

Abstract Activities of enzymes of the purine metabolic pathway, adenosine deaminase (ADA), purine nucleoside phosphorylase (PNP), and 5′- nucleotidase (5′-N), were investigated in the lymphoblasts of a patient with B-cell acute lymphoblastic leukemia. These lymphoblasts exhibited increased ADA activity and diminished activities of both PNP and 5′N' as compared to normal lymphocytes as well as non-T, non-B leukemia cells. This enzymatic pattern is identical to that which has been described in T-cell leukemic lymphoblasts and differs from that which has been observed in the malignant cells of undifferentiated B-cell lymphomas. These data suggest that there is biochemical heterogeneity within the spectrum of B-cell malignancies. Furthermore, inhibitors of ADA may be of use in those B-cell lymphoid neoplasms that exhibit increased ADA activity.

scholarly journals The hematopoietic stem cell marker VNN2 is associated with chemoresistance in pediatric B-cell precursor ALL

2020 ◽  
Vol 4 (17) ◽  
pp. 4052-4064
Author(s):  
Beat Bornhauser ◽  
Gunnar Cario ◽  
Anna Rinaldi ◽  
Thomas Risch ◽  
Virginia Rodriguez Martinez ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Cell Surface ◽  
B Cell ◽  
Residual Disease ◽  
Lymphoblastic Leukemia ◽  
Quantitative Polymerase Chain Reaction ◽  
Surface Protein ◽  
Stem Cell Marker ◽  
Cell Precursor ◽  
Hematopoietic Stem

Abstract Most relapses of acute lymphoblastic leukemia (ALL) occur in patients with a medium risk (MR) for relapse on the Associazione Italiana di Ematologia e Oncologia Pediatrica and Berlin-Frankfurt-Münster (AIEOP-BFM) ALL protocol, based on persistence of minimal residual disease (MRD). New insights into biological features that are associated with MRD are needed. Here, we identify the glycosylphosphatidylinositol-anchored cell surface protein vanin-2 (VNN2; GPI-80) by charting the cell surface proteome of MRD very high-risk (HR) B-cell precursor (BCP) ALL using a chemoproteomics strategy. The correlation between VNN2 transcript and surface protein expression enabled a retrospective analysis (ALL-BFM 2000; N = 770 cases) using quantitative polymerase chain reaction to confirm the association of VNN2 with MRD and independent prediction of worse outcome. Using flow cytometry, we detected VNN2 expression in 2 waves, in human adult bone marrow stem and progenitor cells and in the mature myeloid compartment, in line with proposed roles for fetal hematopoietic stem cells and inflammation. Prospective validation by flow cytometry in the ongoing clinical trial (AIEOP-BFM 2009) identified 10% (103/1069) of VNN2+ BCP ALL patients at first diagnosis, primarily in the MRD MR (48/103, 47%) and HR (37/103, 36%) groups, across various cytogenetic subtypes. We also detected frequent mutations in epigenetic regulators in VNN2+ ALLs, including histone H3 methyltransferases MLL2, SETD2, and EZH2 and demethylase KDM6A. Inactivation of the VNN2 gene did not impair leukemia repopulation capacity in xenografts. Taken together, VNN2 marks a cellular state of increased resistance to chemotherapy that warrants further investigations. Therefore, this marker should be included in diagnostic flow cytometry panels.

scholarly journals Reprogramming of B cell acute lymphoblastic leukemia cells: Do we need to shoot a moving target?

2015 ◽  
Vol 112 (27) ◽  
pp. E3455-E3455 ◽  
Author(s):  
Karel Fišer ◽  
Lucie Slámová ◽  
Jean-Pierre Bourquin ◽  
Jan Trka ◽  
Jan Starý ◽  
...  
Keyword(s):  
Acute Lymphoblastic Leukemia ◽  
B Cell ◽  
Lymphoblastic Leukemia ◽  
Leukemia Cells ◽  
Moving Target ◽  
Cell Acute Lymphoblastic Leukemia

scholarly journals IKZF1deletions Coupled with CD20 Expression Represents a Novel High-Risk Subtype in Adult B-Cell Progenitor Acute Lymphoblastic Leukemia

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 4474-4474
Author(s):  
Bingqing Tang ◽  
Zhixiang Wang ◽  
Dainan Lin ◽  
Xianjun He ◽  
Zihong Cai ◽  
...  
Keyword(s):  
Acute Lymphoblastic Leukemia ◽  
High Risk ◽  
B Cell ◽  
Validation Cohort ◽  
Conflicts Of Interest ◽  
Lymphoblastic Leukemia ◽  
Hematopoietic Stem ◽  
Poor Outcome ◽  
Cd20 Expression ◽  
Cell Acute Lymphoblastic Leukemia

Abstract Genetic deletions of IKZF1 are associated with poor prognosis in B-cell acute lymphoblastic leukemia (B-ALL). Here we investigated the effect of IKZF1 deletions (IKZF1 del) plus with immunotype in adult B-ALL in PDT-ALL-2016 cohort. This cohort study involved 161 patients with B-ALL from 2016 to 2019, with detailed information about IKZF1 del and CD20 expression. Validation cohort consists N= patients from TARGET cohort. IKZF1 del was detected in 36.0% of patients with 3-year event-free survival (EFS) of 37.2±6.7% and overall survival (OS) of 51.1±7.3%, compared to IKZF1 wild-type (IKZF1 wt) with EFS 55.4±5.1% (P&lt;0.01) and OS 74.6±4.5% (P&lt;0.05), respectively. CD20 expression was also associated with inferior EFS than CD20-negative group (P&lt;0.05). Furthermore, IKZF1 del coupled with CD20 expression, termed as IKZF1 del/CD20+, comprised 12.4% of patients with 3-year EFS of 25.0±9.7% compared with IKZF1 wt (P&lt;0.05 ) and IKZF1 del/CD20- (P&lt;0.05 ) groups, respectively. Multivariable analyses demonstrated independence of IKZF1 del/CD20+ with highest hazard ratio for EFS and OS. Furthermore, the prognostic strength of IKZF1 del/CD20+ was confirmed in TARGET validation cohort. Eighty-one patients received allogeneic hematopoietic stem cell transplantation (allo-HSCT). Notably, neither IKZF1 del(P=0.6288), CD20 (P=0.0705) or IKZF1 del/CD20 (P=0.3410) groups were identified as poor outcome in allo-HSCT cohort. Collectively, our data demonstrate that IKZF1 del/CD20+ represents a very high-risk subtype in adult B-ALL; and particularly, allo-HSCT could overcome the poor outcome of IKZF1 del and IKZF1 del/CD20+. Disclosures No relevant conflicts of interest to declare.

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