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Summary Background l -asparaginase is a universal component of treatment for childhood acute lymphoblastic leukaemia, and is usually administered intramuscularly. Pegylated Escherichia coli asparaginase (PEG-asparaginase) has a longer half-life and is potentially less immunogenic than the native Escherichia coli ( E coli ) preparation, and can be more feasibly administered intravenously. The aim of the Dana-Farber Cancer Institute Acute Lymphoblastic Leukaemia Consortium Protocol 05-001 (DFCI 05-001) was to compare the relative toxicity and efficacy of intravenous PEG-asparaginase and intramuscular native E coli l -asparaginase in children with newly diagnosed acute lymphoblastic leukaemia. Methods DFCI 05-001 enrolled patients aged 1–18 years with newly diagnosed acute lymphoblastic leukaemia from 11 consortium sites in the USA and Canada. Patients were assigned to an initial risk group on the basis of their baseline characteristics and then underwent 32 days of induction therapy. Those who achieved complete remission after induction therapy were assigned to a final risk group and were eligible to participate in a randomised comparison of intravenous PEG-asparaginase (15 doses of 2500 IU/m2 every 2 weeks) or intramuscular native E coli l -asparaginase (30 doses of 25 000 IU/m2 weekly), beginning at week 7 after study entry. Randomisation (1:1) was unmasked, and was done by a statistician-generated allocation sequence using a permuted blocks algorithm (block size of 4), stratified by final risk group. The primary endpoint of the randomised comparison was the overall frequency of asparaginase-related toxicities (defined as allergy, pancreatitis, and thrombotic or bleeding complications). Predefined secondary endpoints were disease-free survival, serum asparaginase activity, and quality of life during therapy as assessed by PedsQL surveys. All analyses were done by intention to treat. This study is registered with ClinicalTrials.gov , number NCT00400946. Findings Between April 22, 2005, and Feb 12, 2010, 551 eligible patients were enrolled. 526 patients achieved complete remission after induction, of whom 463 were randomly assigned to receive intramuscular native E coli l -asparaginase (n=231) or intravenous PEG-asparaginase (n=232). The two treatment groups did not differ significantly in the overall frequency of asparaginase-related toxicities (65 [28%] of 232 patients in the intravenous PEG-asparaginase group vs 59 [26%] of 231 patients in the intramuscular native E coli l -asparaginase group, p=0·60), or in the individual frequency of allergy (p=0·36), pancreatitis (p=0·55), or thrombotic or bleeding complications (p=0·26). Median follow-up was 6·0 years (IQR 5·0–7·1). 5-year disease-free survival was 90% (95% CI 86–94) for patients assigned to intravenous PEG-asparaginase and 89% (85–93) for those assigned to intramuscular native E coli l -asparaginase (p=0·58). The median nadir serum asparaginase activity was significantly higher in patients who received intravenous PEG-asparaginase than in those who received intramuscular native E coli l -asparaginase. Significantly more anxiety was reported by both patients and parent-proxy in the intramuscular native E coli l -asparaginase group than in the intravenous PEG-asparaginase group. Scores for other domains were similar between the groups. The most common grade 3 or worse adverse events were bacterial or fungal infections (47 [20%] of 232 in the intravenous PEG-asparaginase group vs 51 [22%] of 231 patients in the intramuscular E coli l -asparaginase group) and asparaginase-related allergic reactions (14 [6%] vs 6 [3%]). Interpretation Intravenous PEG-asparaginase was not more toxic than, was similarly efficacious to, and was associated with decreased anxiety compared with intramuscular native E coli l -asparaginase, supporting its use as the front-line asparaginase preparation in children with newly diagnosed acute lymphoblastic leukaemia. Funding National Cancer Institute and Enzon Pharmaceuticals.

The metastatic spread of epithelial cancer cells from the primary tumor to distant organs mimics the cell migrations that occur during embryogenesis. Using gene expression profiling, we have found that the FOXC2 transcription factor, which is involved in specifying mesenchymal cell fate during embryogenesis, is associated with the metastatic capabilities of cancer cells. FOXC2 expression is required for the ability of murine mammary carcinoma cells to metastasize to the lung, and overexpression of FOXC2 enhances the metastatic ability of mouse mammary carcinoma cells. We show that FOXC2 expression is induced in cells undergoing epithelial-mesenchymal transitions (EMTs) triggered by a number of signals, including TGF-β1 and several EMT-inducing transcription factors, such as Snail, Twist, and Goosecoid. FOXC2 specifically promotes mesenchymal differentiation during an EMT and may serve as a key mediator to orchestrate the mesenchymal component of the EMT program. Expression of FOXC2 is significantly correlated with the highly aggressive basal-like subtype of human breast cancers. These observations indicate that FOXC2 plays a central role in promoting invasion and metastasis and that it may prove to be a highly specific molecular marker for human basal-like breast cancers.

MicroRNAs are small RNA species involved in biological control at multiple levels. Using genetic deletion and transgenic approaches, we show that the evolutionarily conserved microRNA-155 (miR-155) has an important role in the mammalian immune system, specifically in regulating T helper cell differentiation and the germinal center reaction to produce an optimal T cell-dependent antibody response. miR-155 exerts this control, at least in part, by regulating cytokine production. These results also suggest that individual microRNAs can exert critical control over mammalian differentiation processes in vivo.

Classical Hodgkin lymphomas (cHLs) contain small numbers of neoplastic Reed-Sternberg (RS) cells within an extensive inflammatory infiltrate that includes abundant T helper (Th)-2 and T regulatory (Treg) cells. The skewed nature of the T cell infiltrate and the lack of an effective host antitumor immune response suggest that RS cells use potent mechanisms to evade immune attack. In a screen for T cell-inhibitory molecules in cHL, we found that RS cells selectively overexpressed the immunoregulatory glycan-binding protein, galectin-1 (Gal1), through an AP1-dependent enhancer. In cocultures of activated T cells and Hodgkin cell lines, RNAi-mediated blockade of RS cell Gal1 increased T cell viability and restored the Th1/Th2 balance. In contrast, Gal1 treatment of activated T cells favored the secretion of Th2 cytokines and the expansion of CD4⁺CD25high FOXP3⁺ Treg cells. These data directly implicate RS cell Gal1 in the development and maintenance of an immunosuppressive Th2/Treg-skewed microenvironment in cHL and provide the molecular basis for selective Gal1 expression in RS cells. Thus, Gal1 represents a potential therapeutic target for restoring immune surveillance in cHL.

TP53 is the most frequently mutated tumor suppressor gene in human cancer, with nearly 50% of all tumors exhibiting a loss-of-function mutation. To further elucidate the genetic pathways involving TP53 and cancer, we have exploited the zebrafish, a powerful vertebrate model system that is amenable to whole-genome forward-genetic analysis and synthetic-lethal screens. Zebrafish lines harboring missense mutations in the tp53 DNA-binding domain were identified by using a target-selected mutagenesis strategy. Homozygous mutant fish from two of these lines were viable and exhibited mutations similar to those found in human cancers (tp53N168 Kand tp53M214 K). Although homozygous tp53N168 Kmutants were temperature-sensitive and suppressed radiation-induced apoptosis only at 37°C, cells in the tp53M214 Kembryos failed to undergo apoptosis in response to γ radiation at both 28 and 37°C. Unlike wild-type control embryos, irradiated tp53M214 Kembryos also failed to up-regulate p21 and did not arrest at the G1/ S checkpoint. Beginning at 8.5 months of age, 28% of tp53M214 Kmutant fish developed malignant peripheral nerve sheath tumors. In addition to providing a model for studying the molecular pathogenic pathways of malignant peripheral nerve sheath tumors, these mutant zebrafish lines provide a unique platform for modifier screens to identify genetic mutations or small molecules that affect tp53-related pathways, including apoptosis, cell-cycle delay, and tumor suppression.

We have created a stable transgenic rag2-EGFP-mMyc zebrafish line that develops GFP-labeled T cell acute lymphoblastic leukemia (T-ALL), allowing visualization of the onset and spread of this disease. Here, we show that leukemias from this transgenic line are highly penetrant and render animals moribund by 80.7 ± 17.6 days of life (±1 SD, range = 50-158 days). These T cell leukemias are clonally aneuploid, can be transplanted into irradiated recipient fish, and express the zebrafish orthologues of the human T-ALL oncogenes tal1/scl and lmo2, thus providing an animal model for the most prevalent molecular subgroup of human T-ALL. Because T-ALL develops very rapidly in rag2-EGFP-mMyc transgenic fish (in which \"mMyc\" represents mouse c-Myc), this line can only be maintained by in vitro fertilization. Thus, we have created a conditional transgene in which the EGFP-mMyc oncogene is preceded by a loxed dsRED2 gene and have generated stable rag2-loxP-dsRED2-loxP-EGFP-mMyc transgenic zebrafish lines, which have red fluorescent thymocytes and do not develop leukemia. Transgenic progeny from one of these lines can be induced to develop T-ALL by injecting Cre RNA into one-cell-stage embryos, demonstrating the utility of the Cre/lox system in the zebrafish and providing an essential step in preparing this model for chemical and genetic screens designed to identify modifiers of Myc-induced T-ALL.

Transgenic zebrafish that express GFP under control of the T cell-specific tyrosine kinase (lck) promoter were used to analyze critical aspects of the immune system, including patterns of T cell development and T cell homing after transplant. GFP-labeled T cells could be ablated in larvae by either irradiation or dexamethasone added to the water, illustrating that T cells have evolutionarily conserved responses to chemical and radiation ablation. In transplant experiments, thymocytes from lck-GFP fish repopulated the thymus of irradiated wild-type fish only transiently, suggesting that the thymus contains only short-term thymic repopulating cells. By contrast, whole kidney marrow permanently reconstituted the T lymphoid compartment of irradiated wild-type fish, suggesting that long-term thymic repopulating cells reside in the kidney.

RAS family members are among the most frequently mutated oncogenes in human cancers. Given the utility of zebrafish in both chemical and genetic screens, developing RAS-induced cancer models will make large-scale screens possible to understand further the molecular mechanisms underlying malignancy. We developed a heat shock-inducible Cre/Lox-mediated transgenic approach in which activated human kRASG12D can be conditionally induced within transgenic animals by heat shock treatment. Specifically, double transgenic fish Tg(B-actin-LoxP-EGFP-LoxP-kRASG12D; hsp70-Cre) developed four types of tumors and hyperplasia after heat shock of whole zebrafish embryos, including rhabdomyosarcoma, myeloproliferative disorder, intestinal hyperplasia, and malignant peripheral nerve sheath tumor. Using ex vivo heat shock and transplantation of whole kidney marrow cells from double transgenic animals, we were able to generate specifically kRASG12D-induced myeloproliferative disorder in recipient fish. This heat shock-inducible recombination approach allowed for the generation of multiple types of RAS-induced tumors and hyperplasia without characterizing tissue-specific promoters. Moreover, these tumors and hyperplasia closely resemble human diseases at both the morphologic and molecular levels.

Targeting tumour-infiltrating suppressive myeloid cells with a selective PI3Kγ inhibitor overcomes resistance to checkpoint blockade therapy in various mouse myeloid-rich tumour models. Causes of checkpoint blockade resistance Therapeutic blockade of immune checkpoints with antibodies against CTLA-4 and PD-1 has proved effective against some cancer types, but clinical benefit has been limited to a subset of patients. Here Olivier De Henau et al . show that resistance to checkpoint blockade is associated with a high level of infiltration by suppressive myeloid cells in various mouse tumour models. In addition, targeting the myeloid-derived suppressor cells with a selective inhibitor of the γ isoform of phosphoinositide 3-kinase (PI3Kγ) increases sensitivity to checkpoint blockade therapy in a melanoma mouse model. Recent clinical trials using immunotherapy have demonstrated its potential to control cancer by disinhibiting the immune system. Immune checkpoint blocking (ICB) antibodies against cytotoxic-T-lymphocyte-associated protein 4 or programmed cell death protein 1/programmed death-ligand 1 have displayed durable clinical responses in various cancers 1 . Although these new immunotherapies have had a notable effect on cancer treatment, multiple mechanisms of immune resistance exist in tumours. Among the key mechanisms, myeloid cells have a major role in limiting effective tumour immunity 2 , 3 , 4 . Growing evidence suggests that high infiltration of immune-suppressive myeloid cells correlates with poor prognosis and ICB resistance 5 , 6 . These observations suggest a need for a precision medicine approach in which the design of the immunotherapeutic combination is modified on the basis of the tumour immune landscape to overcome such resistance mechanisms. Here we employ a pre-clinical mouse model system and show that resistance to ICB is directly mediated by the suppressive activity of infiltrating myeloid cells in various tumours. Furthermore, selective pharmacologic targeting of the gamma isoform of phosphoinositide 3-kinase (PI3Kγ), highly expressed in myeloid cells, restores sensitivity to ICB. We demonstrate that targeting PI3Kγ with a selective inhibitor, currently being evaluated in a phase 1 clinical trial (NCT02637531), can reshape the tumour immune microenvironment and promote cytotoxic-T-cell-mediated tumour regression without targeting cancer cells directly. Our results introduce opportunities for new combination strategies using a selective small molecule PI3Kγ inhibitor, such as IPI-549, to overcome resistance to ICB in patients with high levels of suppressive myeloid cell infiltration in tumours.

The mammalian protein 53BP1 is activated in many cell types in response to genotoxic stress, including DNA double-strand breaks (DSBs). We now examine potential functions for 53BP1 in the specific genomic alterations that occur in B lymphocytes. Although 53BP1 was dispensable for V(D)J recombination and somatic hypermutation (SHM), the processes by which immunoglobulin (Ig) variable region exons are assembled and mutated, it was required for Igh class-switch recombination (CSR), the recombination and deletion process by which Igh constant region genes are exchanged. When stimulated to undergo CSR, 53BP1-deficient cells exhibited no defect in C H germline transcription or AID expression, however these cells had a profound decrease in switch junctions. The current findings, in combination with the known 53BP1 functions and how it is activated, implicate the DNA damage response to DSBs in the joining phase of class-switch recombination.