Although the treatment of acute myeloid leukemia (AML) has improved significantly, more than half of all patients develop disease that is refractory to intensive chemotherapy1,2. Functional genomics approaches offer a means to discover specific molecules mediating aberrant growth and survival of cancer cells3–8. Thus, using a loss-of-function RNA interference genomic screen, we identified aberrant expression of the hepatocyte growth factor (HGF) as a critical factor in AML pathogenesis. We found HGF expression leading to autocrine activation of its receptor tyrosine kinase, MET, in nearly half of the AML cell lines and clinical samples studied. Genetic depletion of HGF or MET potently inhibited the growth and survival of HGF-expressing AML cells. However, leukemic cells treated with the specific MET kinase inhibitor crizotinib developed resistance due to compensatory upregulation of HGF expression, leading to restoration of MET signaling. In cases of AML where MET is coactivated with other tyrosine kinases, such as fibroblast growth factor receptor 1 (FGFR1)9, concomitant inhibition of FGFR1 and MET blocked compensatory HGF upregulation, resulting in sustained logarithmic cell kill both in vitro and in xenograft models in vivo. Our results demonstrate widespread dependence of AML cells on autocrine activation of MET, as well as the importance of compensatory upregulation of HGF expression in maintaining leukemogenic signaling by this receptor. We anticipate that these findings will lead to the design of additional strategies to block adaptive cellular responses that drive compensatory ligand expression as an essential component of the targeted inhibition of oncogenic receptors in human cancers.

Summary

The molecular events underlying the progression of T-lymphoblastic lymphoma (T-LBL) to acute T-lymphoblastic leukemia (T-ALL) remain elusive. In our zebrafish model, concomitant overexpression of bcl-2 with Myc accelerated T-LBL onset while inhibiting progression to T-ALL. The T-LBL cells failed to invade the vasculature and showed evidence of increased homotypic cell-cell adhesion and autophagy. Further analysis using clinical biopsy specimens revealed autophagy and increased levels of BCL2, S1P1 and ICAM1 in human T-LBL compared to T-ALL. Inhibition of S1P1 signaling in T-LBL cells led to decreased homotypic adhesion in vitro and increased tumor cell intravasation in vivo. Thus, blockade of intravasation and hematologic dissemination in T-LBL is due to elevated S1P1 signaling, increased expression of ICAM1 and augmented homotypic cell-cell adhesion.

miR-451 represses expression of Myc and acts as a tumor suppressor in murine and human T cell acute lymphoblastic leukemia.

The NOTCH1 signaling pathway is a critical determinant of cell fate decisions and drives oncogenesis through mechanisms that are incompletely understood. Using an established mouse model of T cell acute lymphoblastic leukemia (T-ALL), here we report that induction of intracellular Notch1 (ICN1) leads to repression of miR-451 and miR-709. ICN1 decreases expression of these miRNAs by inducing degradation of the E2a tumor suppressor, which transcriptionally activates the genes encoding miR-451 and miR-709. Both miR-451 and miR-709 directly repress Myc expression. In addition, miR-709 directly represses expression of the Akt and Ras-GRF1 oncogenes. We also show that repression of miR-451 and miR-709 expression is required for initiation and maintenance of mouse T-ALL. miR-451 but not miR-709 is conserved in humans, and human T-ALLs with activating NOTCH1 mutations have decreased miR-451 and increased MYC levels compared with T-ALLs with wild-type NOTCH1. Thus, miR-451 and miR-709 function as potent suppressors of oncogenesis in NOTCH1-induced mouse T-ALL, and miR-451 influences MYC expression in human T-ALL bearing NOTCH1 mutations.

Purpose

To identify children with T-cell acute lymphoblastic leukemia (T-ALL) at high risk of induction chemotherapy failure by using DNA copy number analysis of leukemic cells collected at diagnosis.

Patients and Methods

Array comparative genomic hybridization (CGH) was performed on genomic DNA extracted from diagnostic lymphoblasts from 47 children with T-ALL treated on Children's Oncology Group Study P9404 or Dana-Farber Cancer Institute Protocol 00-01. These samples represented nine patients who did not achieve an initial complete remission, 13 who relapsed, and 25 who became long-term, event-free survivors. The findings were confirmed in an independent cohort of patients by quantitative DNA polymerase chain reaction (DNA-PCR), an assay that is well suited for clinical application.

Results

Analysis of the CGH findings in patients in whom induction chemotherapy failed compared with those in whom induction chemotherapy was successful identified the absence of biallelic TCRγ locus deletion (ABD), a characteristic of early thymocyte precursors before V(D)J recombination, as the most robust predictor of induction failure (P < .001). This feature was also associated with markedly inferior event-free (P = .002) and overall survival (P < .001) rates: 25% versus 58% and 25% versus 72%, respectively. Using a rapid and inexpensive quantitative DNA-PCR assay, we validated ABD as a predictor of a poor response to induction chemotherapy in an independent series of patients.

Conclusion

Lymphoblasts from children with T-ALL should be evaluated at diagnosis for deletion within the TCRγ locus. Patients lacking biallelic deletion, which confers a high probability of induction failure with contemporary therapy, should be assigned to alternative therapy in the context of a prospective clinical trial.

SUMMARY

The tyrosine phosphatase SHP2 (PTPN11) regulates cellular proliferation, survival, migration and differentiation during development. Germline mutations in PTPN11 cause Noonan and LEOPARD syndromes, which have overlapping clinical features. Paradoxically, Noonan syndrome mutations increase SHP2 phosphatase activity, while LEOPARD syndrome mutants are catalytically impaired, raising the possibility that SHP2 has phosphatase-independent roles. By comparing shp2-deficient zebrafish embryos with those injected with mRNA encoding LEOPARD syndrome point mutations, we identify a phosphatase- and Erk-dependent role for Shp2 in neural crest specification and migration. We also identify an unexpected phosphatase-and Erk-independent function, mediated through its SH2 domains, which is evolutionarily conserved and prevents p53-mediated apoptosis in the brain and neural crest. Our results indicate that previously enigmatic aspects of LEOPARD syndrome pathogenesis can be explained by the combined effects of loss of Shp2 catalytic function and retention of an SH2 domain-mediated role that is essential for neural crest cell survival.

Ataxia telangiectasia mutated (ATM) deficiency predisposes humans and mice to T lineage lymphomas with recurrent chromosome 14 translocations involving the T cell receptor α/δ (Tcra/d) locus. Such translocations have been thought to result from aberrant repair of DNA double-strand breaks (DSBs) during Tcra locus V(D)J recombination, and to require the Tcra enhancer (Eα) for Tcra rearrangement or expression of the translocated oncogene. We now show that, in addition to the known chromosome 14 translocation, ATM-deficient mouse thymic lymphomas routinely contain a centromeric fragment of chromosome 14 that spans up to the 5′ boundary of the Tcra/d locus, at which position a 500-kb or larger region centromeric to Tcra/d is routinely amplified. In addition, they routinely contain a large deletion of the telomeric end of one copy of chromosome 12. In contrast to prior expectations, the recurrent translocations and amplifications involve V(D)J recombination–initiated breaks in the Tcrd locus, as opposed to the Tcra locus, and arise independently of the Eα. Overall, our studies reveal previously unexpected mechanisms that contribute to the oncogenic transformation of ATM-deficient T lineage cells.

A growing body of evidence indicates that early mitotic inhibitor 1 (Emi1) is essential for genomic stability, but how this function relates to embryonic development and cancer pathogenesis remains unclear. We have identified a zebrafish mutant line in which deficient emi1 gene expression results in multilineage hematopoietic defects and widespread developmental defects that are p53 independent. Cell cycle analyses of Emi1-depleted zebrafish or human cells showed chromosomal rereplication, and metaphase preparations from mutant zebrafish embryos revealed rereplicated, unsegregated chromosomes and polyploidy. Furthermore, EMI1-depleted mammalian cells relied on topoisomerase IIα-dependent mitotic decatenation to progress through metaphase. Interestingly, the loss of a single emi1 allele in the absence of p53 enhanced the susceptibility of adult fish to neural sheath tumorigenesis. Our results cast Emi1 as a critical regulator of genomic fidelity during embryogenesis and suggest that the factor may act as a tumor suppressor.

SUMMARY

Evasion of DNA damage-induced cell death, via mutation of the p53 tumor suppressor or overexpression of prosurvival Bcl-2 family proteins, is a key step toward malignant transformation and therapeutic resistance. We report that depletion or acute inhibition of checkpoint kinase 1 (Chk1) is sufficient to restore γ-radiation-induced apoptosis in p53 mutant zebrafish embryos. Surprisingly, caspase-3 is not activated prior to DNA fragmentation, in contrast to classical intrinsic or extrinsic apoptosis. Rather, an alternative apoptotic program is engaged that cell autonomously requires atm (ataxia telangiectasia mutated), atr (ATM and Rad3-related) and caspase-2, and is not affected by p53 loss or overexpression of bcl-2/xl. Similarly, Chk1 inhibitor-treated human tumor cells hyperactivate ATM, ATR, and caspase-2 after γ-radiation and trigger a caspase-2-dependent apoptotic program that bypasses p53 deficiency and excess Bcl-2. The evolutionarily conserved “Chk1-suppressed” pathway defines a novel apoptotic process, whose responsiveness to Chk1 inhibitors and insensitivity to p53 and BCL2 alterations have important implications for cancer therapy.

Neuroblastoma, an embryonal tumor of the peripheral sympathetic nervous system, accounts for approximately 15% of all deaths due to childhood cancer1. High-risk neuroblastomas, prevalent in the majority of patients, are rapidly progressive; even with intensive myeloablative chemotherapy, relapse is common and almost uniformly fatal2,3. Here we report the detection of previously unknown mutations in the ALK gene, which encodes a receptor tyrosine kinase, in 8% of primary neuroblastomas. Five non-synonymous sequence variations were identified in the kinase domain of ALK, of which three were somatic and two were germline. The most frequent mutation, F1174L, was also identified in three different neuroblastoma cell lines. ALK cDNAs encoding the F1174L and R1275Q variants, but not the wild-type ALK cDNA, transformed IL-3-dependent murine hematopoietic Ba/F3 cells to cytokine-independent growth. Ba/F3 cells expressing these mutations were sensitive to a small-molecule inhibitor of ALK, TAE6844. Furthermore, two human neuroblastoma cell lines harboring the F1174L mutation were sensitive to the inhibitor. Cytotoxicity was associated with increased levels of apoptosis as measured by TUNEL-labeling. shRNA-mediated knockdown of ALK expression in neuroblastoma cell lines with the F1174L mutation also resulted in apoptosis and impaired cell proliferation. Thus, activating alleles of the ALK receptor tyrosine kinase are present in primary neuroblastoma tumors and in established neuroblastoma cell lines, and confer sensitivity to ALK inhibition with small molecules, providing a molecular rationale for targeted therapy of this disease.

RelA-associated inhibitor (RAI) is an inhibitor of nuclear factor κB (NF-κB) newly identified by yeast two-hybrid screen as an interacting protein of the p65 (RelA) subunit. In this study, we attempted to examine the effect of RAI on transcription and replication of human immunodeficiency virus type 1 (HIV-1). We found that RAI inhibited gene expression from the HIV-1 long terminal repeat (LTR) even at the basal level. Upon in vitro DNA-binding reactions, RAI could directly block the DNA-binding of p65 subunit of NF-κB but not that of the p50 subunit or AP1. We found that RAI could also inhibit the DNA-binding of Sp1 and thus inhibit the basal HIV-1 promoter activity. We further examined the effects of RAI on Sp1 and found that RAI colocalizes with Sp1 in the nucleus and interacts with Sp1 in vitro and in vivo. Moreover, we found that RAI efficiently blocked the HIV-1 replication when cotransfected with a full-length HIV-1 clone. These findings indicate that RAI acts as an efficient inhibitor of HIV-1 gene expression in which both NF-κB and Sp1 play major roles.