Therapeutic targeting of Notch signaling and immune checkpoint blockade in a spontaneous, genetically heterogeneous mouse model of T-cell acute lymphoblastic leukemia

Key Points CD45-driven expression of Cre generates the first mouse model that allows specific and exclusive deletion of Pten in hematopoietic cells. Pten deletion in CD45-expressing cells causes T-cell acute lymphoblastic leukemia, but no other hematologic malignancies.

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Notch Signaling Represses Mir-223 in T-Cell Acute Lymphoblastic Leukemia

Blood ◽

10.1182/blood.v118.21.4630.4630 ◽

2011 ◽

Vol 118 (21) ◽

pp. 4630-4630

Author(s):

Samuel D Gusscott ◽

Florian Kuchenbauer ◽

Andrew P Weng

Keyword(s):

Acute Lymphoblastic Leukemia ◽

T Cell ◽

Cell Growth ◽

Notch Signaling ◽

Cell Lines ◽

Lymphoblastic Leukemia ◽

Gamma Secretase ◽

Protein Levels ◽

Cell Acute Lymphoblastic Leukemia ◽

T Cell Leukemogenesis

Abstract Abstract 4630 T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive cancer of immature T cells that often shows aberrant activation of the Notch1 signaling pathway. Several studies have utilized mRNA expression profiling to identify downstream mediators of oncogenic Notch signaling in this context. Since microRNAs (miRNAs) have in recent years been shown to play important roles in hematological maliganancy, we performed a microarray-based screen for Notch-dependent miRNA expression in T-ALL. Jurkat and P12-Ichikawa cell lines were treated with gamma-secretase inhibitor to block Notch signaling vs. DMSO control for 4 days and profiled using Exigon miRCURY LNA miRNA microarrays. Surprisingly few miRNAs were found to be regulated by this approach; however, one of the hits, miR-223, showed consistent upregulation after gamma-secretase treatment in Jurkat cells and 5 additional human T-ALL cell lines assessed by miRNA qPCR. This observation was unique to human T-ALL as murine models of T-ALL showed no evidence for Notch-dependent miR-223 expression. Given that canonical Notch signaling results in transcriptional activation, our observation that Notch signaling is associated with reduced miR-223 expression suggests an intermediary repressor may be involved. miR-223 has been reported to play an important role in normal granulopoiesis, to be expressed relatively highly in T-ALL with myeloid-like gene features, and most recently to accelerate Notch-mediated T-cell leukemogenesis. To explore potential functional consequences for Notch-dependent miR-223 repression in T-ALL, candidate miR-223 targets identified by TargetScan software were analyzed with Ingenuity Pathway Analysis software, which indicated IGF-1, insulin receptor, PTEN, and ERK5 signaling pathways as the top hits. We recently reported IGF1R signaling to be important for growth and viability of bulk T-ALL cells as well as for leukemia-initiating cell activity. Additionally, we reported that Notch signaling directly upregulates IGF1R transcription by binding to an intronic enhancer which is present between exons 21/22 in the human, but not mouse IGF1R locus. As miR-223 has previously been reported to target IGF1R mRNA and reduce its translation, we hypothesized that Notch signaling may also upregulate net IGF1R protein expression by repressing miR-223. To test this hypothesis, we transduced several human T-ALL cell lines with miR-223 retrovirus and observed a modest decrease in total IGF1R protein levels by western blot; however, no significant change was observed in surface IGF1R levels as assessed by flow cytometry. Addtionally, knockdown of miR-223 by lentiviral expression miR-223 target sequences (miR-223 “sponge”) resulted in modestly increased total IGF1R protein levels, but again showed no demonstrable effect on surface IGF1R levels. Of note, we also observed no apparent effect of either overexpression or knockdown of miR-223 on bulk cell growth or viability. We interpret these findings to suggest that Notch signaling does not have major effects on the miR transcriptome, and that up- or down-modulation of miR-223 in established T-ALL cells does not have significant effects on overall cell growth/viability. Further studies will be required to determine if miR-223 may act in concert with other Notch target genes to modulate cell physiology. Disclosures: No relevant conflicts of interest to declare.

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Deletion of Atm in the Hematopoetic Stem Cell As a Mouse Model for Human T Cell Acute Lymphoblastic Leukemia/Lymphoma.

Blood ◽

10.1182/blood.v120.21.2418.2418 ◽

2012 ◽

Vol 120 (21) ◽

pp. 2418-2418

Author(s):

Lori A. Ehrlich ◽

Katherine S. Yang-Iott ◽

Amy DeMicco ◽

Craig H. Bassing

Keyword(s):

T Cells ◽

Acute Lymphoblastic Leukemia ◽

T Cell ◽

Mouse Model ◽

Genomic Instability ◽

Lymphoblastic Leukemia ◽

Drug Combinations ◽

Thymic Epithelial Cells ◽

Cell Acute Lymphoblastic Leukemia

Abstract Abstract 2418 Acute lymphoblastic leukemia (ALL) is diagnosed in approximately 2500 children per year. Although high cure rates have been achieved for ALL, these cancers account for the highest number of non-brain tumor cancer-related deaths in children. T cell acute lymphoblastic leukemia (T-ALL) is an aggressive malignancy of immature TCRβ−CD4+/CD8+ T-cells that represents ∼15% of pediatric ALL diagnoses, comprises most of the therapy-resistant ALL tumors, and exhibits a high frequency of relapse. The Ataxia Telangiectasia mutated (ATM) protein kinase activates the cellular response to DNA double strand breaks (DSBs) to coordinate DNA repair with cell survival, proliferation, and differentiation. Somatic inactivating ATM mutations occur in 10–20% of T-ALL and T cell lymphoblastic lymphoma (T-LL) tumors and are associated with resistance to genotoxic chemotherapy drugs and therapy relapse, likely driven by increased genomic instability in cells lacking functional ATM. The impaired DSB response of ATM-deficient cells can be exploited to design combinations of genotoxic drugs that specifically kill these cells in vitro. However, the in vivo potential of such drug combinations to treat T-ALL have not been reported. We sought to develop a pre-clinical mouse model that could be used to test effectiveness of such drug combinations to treat T-ALLs and T-LLs with somatic ATM inactivation. Although germline ATM-deficient (Atm−/−) mice succumb by six months of age to immature CD4+/CD8+ T-cell lymphomas containing genomic instability analogous to human T-ALL tumors, we sought a more physiologic model that would avoid potential complications due to ATM-deficiency in thymic epithelial cells. Thus, we generated and characterized VavCre:Atmflox/flox mice with conditional Atm inactivation restricted to hematopoietic cell lineages. These mice contain reduced numbers of TCRβ−CD4+/CD8+, TCRβ+CD4+/CD8−, and TCRβ+CD4−/CD8+ thymocytes and of TCRβ+CD4+ and TCRb+CD8+ splenic T-cells, mirroring the phenotype of Atm−/− mice. We have found that VavCre:Atmflox/flox mice succumb at an average of 95 days (range 53–183 days) to clonal TCRβ−CD4+/CD8+ or TCRβ+CD4−/CD8+ thymic lymphomas. Evaluation of the bone marrow in a subset of these mice indicates that the lymphoma has disseminated and are classified as leukemia. Our initial cytogenetic analyses of these tumors indicate that they contain both clonal translocations involving chromosome 12 and/or chromosome 14 and deletion of one allelic copy of the haploinsufficient Bcl11b tumor suppressor gene. Hemizygous BCL11B inactivation occurs in ∼20% of human T-ALL tumors, indicating the clinical relevance of VavCre:Atmflox/flox mice as a model for human T-ALL. Our ongoing studies include complete cytogenetic and molecular characterization of VavCre:Atmflox/flox tumors and in vivo testing of chemotherapeutics targeting the Atm pathway in this mouse model of T-ALL/T-LL. Disclosures: No relevant conflicts of interest to declare.

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Investigation of deregulated genes of Notch signaling pathway in human T cell acute lymphoblastic leukemia cell lines and clinical samples

Molecular Biology Reports ◽

10.1007/s11033-013-2654-8 ◽

2013 ◽

Vol 40 (10) ◽

pp. 5531-5540 ◽

Cited By ~ 10

Author(s):

Mahdi Paryan ◽

Samira Mohammadi-Yeganeh ◽

Siamak Mirab Samiee ◽

Masoud Soleimani ◽

Ehsan Arefian ◽

...

Keyword(s):

Acute Lymphoblastic Leukemia ◽

T Cell ◽

Notch Signaling ◽

Lymphoblastic Leukemia ◽

Leukemia Cell ◽

Notch Signaling Pathway ◽

Clinical Samples ◽

Human T Cell ◽

Leukemia Cell Lines ◽

Cell Acute Lymphoblastic Leukemia

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Identification of a novel chalcone derivative that inhibits Notch signaling in T-cell acute lymphoblastic leukemia

Scientific Reports ◽

10.1038/s41598-017-02316-9 ◽

2017 ◽

Vol 7 (1) ◽

Cited By ~ 27

Author(s):

Mattia Mori ◽

Luca Tottone ◽

Deborah Quaglio ◽

Nadezda Zhdanovskaya ◽

Cinzia Ingallina ◽

...

Keyword(s):

Acute Lymphoblastic Leukemia ◽

T Cell ◽

Notch Signaling ◽

Lymphoblastic Leukemia ◽

Chalcone Derivative ◽

Cell Acute Lymphoblastic Leukemia

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Growth Arrest-Specific 2 Promotes the Growth of T-Cell Acute Lymphoblastic Leukemia Cells Via Its Regulation of CXCR4

Blood ◽

10.1182/blood-2018-99-114354 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 4081-4081

Author(s):

Wenjuan Ma ◽

Yan Wan ◽

Haixia Zhou ◽

Li Zhu ◽

Yun Zhao

Keyword(s):

Acute Lymphoblastic Leukemia ◽

T Cell ◽

Protein Expression ◽

Notch Signaling ◽

Lymphoblastic Leukemia ◽

Growth Arrest ◽

Jurkat Cells ◽

Control Group ◽

Migration Ability ◽

Cell Acute Lymphoblastic Leukemia

Abstract Growth arrest-specific 2 (GAS2) has multiple functions including the regulation of cell morphology, cell cycle, apoptosis and calpain activity.GAS2 has a dual function in cancer cells, however its expression and underlying mechanism in human T-cell acute lymphoblastic leukemia (T-ALL) remain unclear. In the present study, qRT-PCR analysis showed that GAS2 has significantly higher expression (155.5-fold, P=0.0048) in CD3+ cells from T-ALL patients (n=25) than healthy donors (n=13). GAS2 was present in Jurkat cells, while absent in MOLT-4 or HPB-ALL cells. A tiny CpG island of GAS2 was almost fully methylated in both MOLT-4 (100%) and HPB-ALL cells (80%), while 40% methylation in Jurkat cells; suggesting that DNA methylation played a subtle role in regulating GAS2 expression. Two independent shRNA sequences were delivered into Jurkat cells with lentiviral vector. GAS2 silencing inhibited the growth and colony-forming cell (CFC) production significantly. Conversely, GAS2 overexpression enhanced the growth and CFC production of both MOLT-4 and HPB-ALL cells. In addition, GAS2 overexpression promoted HPB-ALL cell induced leukemia in a xenoengraftment model (5 mice in each control group). In GAS2 expressed group, the disease latency was shortened, the splenomegaly was more severe than control group (0.35±0.04g vs. 0.27±0.05g), and more leukemic cells were present in bone marrow (85±3% vs. 45±7%). To obtain the molecular insights of how GAS2 acts, RNA-seq data comparing GAS2 silenced Jurkat cells with control cells were generated. Several Notch signaling molecules were inhibited, including NOTCH1, HES1 and HES4. Despite the differential expression of these transcripts was validated in Jurkat cells, GAS2 overexpression did not elevated the expression of these transcripts in MOLT-4 or HPB-ALL cells, suggesting GAS2 did not have a consistent impact on Notch signaling. However, we found that GAS2 silencing reduced CXCR4 protein expression in Jurkat cells and GAS2 overexpression enhanced CXCR4 protein expression in MOLT-4 cells, while CXCR4 transcript was not altered upon GAS2 manipulation. Consequently, GAS2 silencing significantly reduced migration ability of Jurkat cells and GAS2 overexpression enhanced migration ability of MOLT-4 cells. Overexpression of CXCR4 "rescued" the inhibited CFC production and migration upon GAS2 silencing. A truncated GAS2 (Δ171-313) coined as GAS2DN (dominant negative form of GAS2) has been known to inhibit normal function of GAS2. Herein, we showed that GAS2DN inhibited the growth of Jurkat cells and the expression of CXCR4. To delineate the role of calpain1 and calpain2 in GAS2 function, shRNA sequences against calpain1 and calpain2 was delivered into GAS2DN expressed Jurkat cells respectively, the results showed that calpain2 but not calpain1 silencing was able to enhance the cell growth and CXCR4 expression. Taken together, the present study has demonstrated that GAS2 is aberrantly expressed in human T-ALL cells, which promotes the growth of T-ALL cells partially via its post-transcriptional regulation of CXCR4 depending on calpain2 activity. These data provide new insights of the pathogenesis of T-ALL and possibly new clues to improve the management of the disease. Disclosures No relevant conflicts of interest to declare.

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