Notch1 Gene Mutations Target KRAS G12D-expressing CD8+ Cells and Contribute to Their Leukemogenic Transformation

Acute T-cell lymphoblastic leukemia/lymphoma (T-ALL) is an aggressive hematopoietic malignancy affecting both children and adults. Previous studies of T-ALL mouse models induced by different genetic mutations have provided highly diverse results on the issues of T-cell leukemia/lymphoma-initiating cells (T-LICs) and potential mechanisms contributing to T-LIC transformation. Here, we show that oncogenic Kras (Kras G12D) expressed from its endogenous locus is a potent inducer of T-ALL even in a less sensitized BALB/c background. Notch1 mutations, including exon 34 mutations and recently characterized type 1 and 2 deletions, are detected in 100% of Kras G12D-induced T-ALL tumors. Although these mutations are not detected at the pre-leukemia stage, incremental up-regulation of NOTCH1 surface expression is observed at the pre-leukemia and leukemia stages. As secondary genetic hits in the Kras G12D model, Notch1 mutations target CD8⁺ T-cells but not hematopoietic stem cells to further promote T-ALL progression. Pre-leukemia T-cells without detectable Notch1 mutations do not induce T-ALL in secondary recipient mice compared with T-ALL tumor cells with Notch1 mutations. We found huge variations in T-LIC frequency and immunophenotypes of cells enriched for T-LICs. Unlike Pten deficiency-induced T-ALL, oncogenic Kras-initiated T-ALL is not associated with up-regulation of the Wnt/β-catenin pathway. Our results suggest that up-regulation of NOTCH1 signaling, through either overexpression of surface NOTCH1 or acquired gain-of-function mutations, is involved in both T-ALL initiation and progression. Notch1 mutations and Kras G12D contribute cooperatively to leukemogenic transformation of normal T-cells.     

Frequent epigenetic inactivation of KIBRA,an upstream member of the Salvador/Warts/Hippo (SWH) tumor suppressor network,is associated with specific genetic event in B-cell acute lymphocytic leukemia

The WW-domain containing protein KIBRA has recently been identified as a new member of the Salvador/Warts/Hippo (SWH) pathway in Drosophila and is shown to act as a tumor suppressor gene in Drosophila. This pathway is conserved in humans and members of the pathway have been shown to act as tumor suppressor genes in mammalian systems. We determined the methylation status of the 5′ CpG island associated with the KIBRA gene in human cancers. In a large panel of cancer cell lines representing common epithelial cancers KIBRA was unmethylated. But in pediatric acute lymphocytic leukemia (ALL) cell lines KIBRA showed frequent hypermethylation and silencing of gene expression, which could be reversed by treatment with 5-aza-2′-deoxycytidine. In ALL patient samples KIBRA was methylated in 70% B-ALL but was methylated in <20% T-ALL leukemia (p = 0.0019). In B-ALL KIBRA methylation was associated with ETV6/RUNX1 [t(12;21) (p13;q22)] chromosomal translocation (p = 0.0082) phenotype, suggesting that KIBRA may play an important role in t(12;21) leukemogenesis. In ALL paired samples at diagnosis and remission KIBRA methylation was seen in diagnostic but not in any of the remission samples accompanied by loss of KIBRA expression in disease state compared to patients in remission. Hence KIBRA methylation occurs frequently in B-cell acute lymphocytic leukemia but not in epithelial cancers and is linked to specific genetic event in B-ALL.Key words: KIBRA, methylation, ALL, SWH pathway, ETV6/RUNX1 translocation     

Sulforaphane Induces Cell Cycle Arrest and Apoptosis in Acute Lymphoblastic Leukemia Cells

Acute lymphoblastic leukemia (ALL) is the most common hematological cancer in children. Although risk-adaptive therapy, CNS-directed chemotherapy, and supportive care have improved the survival of ALL patients, disease relapse is still the leading cause of cancer-related death in children. Therefore, new drugs are needed as frontline treatments in high-risk disease and as salvage agents in relapsed ALL. In this study, we report that purified sulforaphane, a natural isothiocyanate found in cruciferous vegetables, has anti-leukemic properties in a broad range of ALL cell lines and primary lymphoblasts from pediatric T-ALL and pre-B ALL patients. The treatment of ALL leukemic cells with sulforaphane resulted in dose-dependent apoptosis and G2/M cell cycle arrest, which was associated with the activation of caspases (3, 8, and 9), inactivation of PARP, p53-independent upregulation of p21^CIP1/WAF1, and inhibition of the Cdc2/Cyclin B1 complex. Interestingly, sulforaphane also inhibited the AKT and mTOR survival pathways in most of the tested cell lines by lowering the levels of both total and phosphorylated proteins. Finally, the administration of sulforaphane to the ALL xenograft models resulted in a reduction of tumor burden, particularly following oral administration, suggesting a potential role as an adjunctive agent to improve the therapeutic response in high-risk ALL patients with activated AKT signaling.     

FLT3 Mutations in Early T-Cell Precursor ALL Characterize a Stem Cell Like Leukemia and Imply the Clinical Use of Tyrosine Kinase Inhibitors

Early T-cell precursor acute lymphoblastic leukemia (ETP-ALL) has been identified as high-risk subgroup of acute T-lymphoblastic leukemia (T-ALL) with a high rate of FLT3-mutations in adults. To unravel the underlying pathomechanisms and the clinical course we assessed molecular alterations and clinical characteristics in a large cohort of ETP-ALL (n = 68) in comparison to non-ETP T-ALL adult patients. Interestingly, we found a high rate of FLT3-mutations in ETP-ALL samples (n = 24, 35%). Furthermore, FLT3 mutated ETP-ALL was characterized by a specific immunophenotype (CD2+/CD5-/CD13+/CD33-), a distinct gene expression pattern (aberrant expression of IGFBP7, WT1, GATA3) and mutational status (absence of NOTCH1 mutations and a low frequency, 21%, of clonal TCR rearrangements). The observed low GATA3 expression and high WT1 expression in combination with lack of NOTCH1 mutations and a low rate of TCR rearrangements point to a leukemic transformation at the pluripotent prothymocyte stage in FLT3 mutated ETP-ALL. The clinical outcome in ETP-ALL patients was poor, but encouraging in those patients with allogeneic stem cell transplantation (3-year OS: 74%). To further explore the efficacy of targeted therapies, we demonstrate that T-ALL cell lines transfected with FLT3 expression constructs were particularly sensitive to tyrosine kinase inhibitors. In conclusion, FLT3 mutated ETP-ALL defines a molecular distinct stem cell like leukemic subtype. These data warrant clinical studies with the implementation of FLT3 inhibitors in addition to early allogeneic stem cell transplantation for this high risk subgroup.     

Targeted cellular ionic calcium chelation by oxalates: Implications for the treatment of tumor cells

Background

The Notch signaling pathway is crucial in T-cell development, Notch1 mutations are frequently present in T-cell acute lymphoblastic leukemia (T-ALL). To investigate the feature of Notch1 mutation and its corresponding expression level in Chinese patients with T-ALL, detection of mutation and the expression level of Notch1 gene was preformed using RT-PCR, sequencing and real-time PCR respectively.

Results

Two Notch1 point mutations (V1578E and L1593P) located on HD-N domain were identified in three cases out of 13 T-ALL patients. The mutation on 4733 position (V1578E) found in two cases was a novel mutation. The overexpression of Notch1 was detected in all samples with T-ALL, moreover, significantly higher expression of Notch1 was detected in the T-ALL with Notch1 mutation group compared with T-ALL with WT Notch1 group (p = 0.0192).

Conclusions

Higher expression of Notch1 was associated with Notch1 mutation, more novel mutation of this gene might be identified in different populations and its contribution to the molecular pathogenesis of T-ALL is needed further research.     

Frequent epigenetic inactivation of KIBRA,an upstream member of the Salvador/Warts/Hippo (SWH) tumor suppressor network,is associated with specific genetic event in B-cell acute lymphocytic leukemia

The WW-domain containing protein KIBRA has recently been identified as a new member of the Salvador/Warts/Hippo (SWH) pathway in Drosophila and is shown to act as a tumor suppressor gene in Drosophila. This pathway is conserved in humans and members of the pathway have been shown to act as tumor suppressor genes in mammalian systems. We determined the methylation status of the 5' CpG island associated with the KIBRA gene in human cancers. In a large panel of cancer cell lines representing common epithelial cancers KIBRA was unmethylated. But in pediatric acute lymphocytic leukemia (ALL) cell lines KIBRA showed frequent hypermethylation and silencing of gene expression, which could be reversed by treatment with 5-aza-2'-deoxycytidine. In ALL patient samples KIBRA was methylated in 70% B-ALL but was methylated in &lt; 20% T-ALL leukemia (p = 0.0019). In B-ALL KIBRA methylation was associated with ETV6/RUNX1 {t(12;21) (p13;q22)} chromosomal translocation (p = 0.0082) phenotype, suggesting that KIBRA may play an important role in t(12;21) leukemogenesis. In ALL paired samples at diagnosis and remission KIBRA methylation was seen in diagnostic but not in any of the remission samples accompanied by loss of KIBRA expression in disease state compared to patients in remission. Hence KIBRA methylation occurs frequently in B-cell acute lymphocytic leukemia but not in epithelial cancers and is linked to specific genetic event in B-ALL.     

Activation of Notch1 promotes development of human CD8 single positive T cells in humanized mice

Notch1 mutations are found in more than 50% of human T cell acute lymphoblastic leukemia (T-ALL) cells. However, the functions of Notch1 for human T cell development and leukemogenesis are not well understood. To examine the role of Notch1, human hematopoietic stem cells (HSCs), which had been transduced with a constitutively active form of Notch1 (ICN1), were transplanted into severely immunodeficient NOD/Shi-scid-IL2rγ^null (NOG) mice. We found that the great majority of the ICN1-expressing hematopoietic cells in the bone marrow expressed surface markers for T cells, such as CD3, CD4, and CD8, and that this T cell development was independent of the thymus. Accordingly, phenotypically mature CD8⁺ single positive (SP) T cells were observed in the spleen. Furthermore, T-ALL developed in one NOG recipient mouse out of 26 that had been secondary transferred with the T cells developed in the first NOG mice. These results indicate that Notch1 signaling in HSCs promotes CD8⁺ SP T cell development, and that T cell leukemogenesis may require additional oncogenic factors other than Notch1 activation.     

c-MYC Copy-Number Gain Is an Independent Prognostic Factor in Patients with Colorectal Cancer

Background

The aim of this study was to determine the incidence and clinicopathological significance of c-MYC gene copy-number (GCN) gain in patients with primary colorectal cancer (CRC).

Methods

The c-MYC GCN was investigated in 367 consecutive CRC patients (cohort 1) by using dual-color silver in situ hybridization. Additionally, to evaluate regional heterogeneity, we examined CRC tissue from 3 sites including the primary cancer, distant metastasis, and lymph-node metastasis in 152 advanced CRC patients (cohort 2). KRAS exons 2 and 3 were investigated for mutations.

Results

In cohort 1, c-MYC gene amplification, defined by a c-MYC:centromere of chromosome 8 ratio ≥ 2.0, was detected in 31 (8.4%) of 367 patients. A c-MYC GCN gain, defined by ≥ 4.0 c-MYC copies/nucleus, was found in 63 (17.2%) patients and was associated with poor prognosis (P = 0.015). Multivariate Cox regression analysis showed that the hazard ratio for c-MYC GCN gain was 2.35 (95% confidence interval, 1.453–3.802; P < 0.001). In a subgroup of stage II-III CRC patients, c-MYC GCN gain was significantly associated with poor prognosis by univariate (P = 0.034) and multivariate (P = 0.040) analyses. c-MYC protein overexpression was observed in 201 (54.8%) out of 367 patients and weakly correlated with c-MYC GCN gain (ρ, 0.211). In cohort 2, the c-MYC genetic status was heterogenous in advanced CRC patients. Discordance between GCN gain in the primary tumor and either distant or lymph-node metastasis was 25.7% and 30.4%, respectively. A similar frequency for c-MYC GCN gain and amplification was observed in CRC patients with both wild-type and mutated KRAS.

Conclusions

c-MYC GCN gain was an independent factor for poor prognosis in consecutive CRC patients and in the stage II-III subgroup. Our findings indicate that the status of c-MYC may be helpful in predicting the patients’ outcome and for managing CRC patients.     

Notches,NF-kBs and the Making of T Cell Leukemia

T cell lymphoblastic leukemia (T-ALL) is an aggressive hematological cancer frequent within pediatric ALL patients. Recent findings suggested that the transmembrane receptor NOTCH1 is the major oncogene for the majority of T-ALL cases. In these cases activating mutations of NOTCH1 are responsible for the transformation of developing T cell progenitors. These observations prompted us to study the mechanisms of Notch1-induced T cell transformation. Using parallel studies in T cell progenitors and established T-ALL lines we have demonstrated that the NF-kB signaling pathway is targeted and induced by Notch1 activation. Our studies suggested that the NF-kB activation by Notch1 can be direct, as Notch1 can bind and activate the promoters of the RELB and NFKB2 factors and indirect, as Notch1 can form a complex with the NF-kB kinase IKK. NF-kB appears to be important for the development of the disease as suppression of the pathway antagonizes T cell transformation both in vitro and in vivo, using animal models of T-ALL. We believe that these findings could be important for the understanding of Notch1 signaling and the therapeutic treatment of T-ALL.     

The role of interleukin-15 polymorphisms in adult acute lymphoblastic leukemia

Background

Interleukin-15 (IL-15) plays important roles in the immune system and in the development of hematopoietic cells. Previous studies revealed that five SNPs in IL-15, rs10519612, rs10519613, rs35964658, rs17007695 and rs17015014, were significantly associated with childhood Acute Lymphoblastic Leukemia (ALL) treatment response. In adult ALL, the expression of IL-15 was also correlated with the immunophenotypes of ALL. Therefore, we hypothesize that SNPs of IL-15 might also be associated with adult ALL.

Methods and Findings

We genotyped the above five SNPs of IL-15 gene by PCR-RFLP assays in adult ALL case-control studies. The current study included 121 adult ALL patients and 263 healthy controls. IL-15 genotypes and haplotypes were determined and the associations with the risk of ALL were analyzed by logistic regression. SNPs rs10519612 and rs17007695 were significantly associated with ALL (P = 0.013 and P = 0.001). We observed a 2-fold and 2.4-fold excess risk of developing ALL for the rs10519612 CC and rs17007695 TC genotype carriers compared with non-carriers, respectively. Haplotype analysis revealed that haplotypes ACAC, CAGT and CCAT were significantly associated with adult B-ALL, while haplotype CCAT conferred susceptibility to T-ALL.

Conclusion

These findings suggest that IL-15 gene polymorphisms are significantly associated with ALL in adult Chinese population.