Frida Ponthan

Ras pathway mutations are prevalent in relapsed childhood acute lymphoblastic leukemia and confer sensitivity to MEK inhibition

For most children who relapse with acute lymphoblastic leukemia (ALL), the prognosis is poor, and there is a need for novel therapies to improve outcome. We screened samples from children with B-lineage ALL entered into the ALL-REZ BFM 2002 clinical trial (www.clinicaltrials.gov, #NCT00114348) for somatic mutations activating the Ras pathway (KRAS, NRAS, FLT3, and PTPN11) and showed mutation to be highly prevalent (76 from 206). Clinically, they were associated with high-risk features including early relapse, central nervous system (CNS) involvement, and specifically for NRAS/KRAS mutations, chemoresistance. KRAS mutations were associated with a reduced overall survival. Mutation screening of the matched diagnostic samples found many to be wild type (WT); however, by using more sensitive allelic-specific assays, low-level mutated subpopulations were found in many cases, suggesting that they survived up-front therapy and subsequently emerged at relapse. Preclinical evaluation of the mitogen-activated protein kinase kinase 1/2 inhibitor selumetinib (AZD6244, ARRY-142886) showed significant differential sensitivity in Ras pathway-mutated ALL compared with WT cells both in vitro and in an orthotopic xenograft model engrafted with primary ALL; in the latter, reduced RAS-mutated CNS leukemia. Given these data, clinical evaluation of selumetinib may be warranted for Ras pathway-mutated relapsed ALL.

Expression of enzymes and receptors of the leukotriene pathway in human neuroblastoma promotes tumor survival and provides a target for therapy

The metabolism of arachidonic acid by the cyclooxygenase (COX) or lipoxygenase (LO) pathways generates eicosanoids that have been implicated in the pathogenesis of a variety of human diseases, including cancer. In this study, we examined the expression and significance of components within the 5‐LO pathway in human neuroblastoma, an embryonal tumor of the sympathetic nervous system. High expression of 5‐LO, 5‐LO‐activating protein (FLAP), leukotriene A4 hydrolase, leukotriene C4 synthase, and leukotriene receptors was detected in a majority of primary neuro‐blastoma tumors and all cell lines investigated. Expression of 5‐LO and FLAP was evident in tumor cells but not in nonmalignant adrenal medulla where neuroblastomas typically arise. Moreover, neuroblastoma cells produce leukotrienes, and stimulation of neuroblastoma cells with leukotrienes increased neuroblastoma cell viability. Inhibitors of 5‐LO (AA‐861), FLAP (MK‐886), or the leukotriene receptor antagonist montelukast inhibited neuroblastoma cell growth by induction of G1‐cell cycle arrest and apoptosis. Similarly, specific 5‐LO and leukotriene receptor silencing by small interfering RNA decreased neuroblastoma cell growth. These findings provide new insights into the pathobiology of neuroblastoma, and the use of leukotriene pathway inhibitors as a novel adjuvant therapy for children with neuroblastoma warrants further consideration.—Sveinbjörnsson, B., Rasmuson, A., Baryawno, N., Wan, M., Ingvild Pettersen, I., Frida Ponthan, F., Orrego, A., Haeggström, J. Z., Johnsen, J. I., Kogner, P. Expression of enzymes and receptors of the leukotriene pathway in human neuroblastoma promotes tumor survival and provides a target for therapy. FASEB J. 22, 3525–3536 (2008)

A Drosophila model identifies calpains as modulators of the human leukemogenic fusion protein AML1-ETO

The t(8:21)(q22;q22) translocation is 1 of the most common chromosomal abnormalities linked to acute myeloid leukemia (AML). AML1-ETO, the product of this translocation, fuses the N-terminal portion of the RUNX transcription factor AML1 (also known as RUNX1), including its DNA-binding domain, to the almost entire transcriptional corepressor ETO (also known as MTG8 or RUNX1T1). This fusion protein acts primarily by interfering with endogenous AML1 function during myeloid differentiation, although relatively few genes are known that participate with AML1-ETO during leukemia progression. Here, we assessed the consequences of expressing this chimera in Drosophila blood cells. Reminiscent of what is observed in AML, AML1-ETO specifically inhibited the differentiation of the blood cell lineage whose development depends on the RUNX factor Lozenge (LZ) and induced increased numbers of LZ+ progenitors. Using an in vivo RNAi-based screen for suppressors of AML1-ETO, we identified calpainB as required for AML1-ETO-induced blood cell disorders in Drosophila. Remarkably, calpain inhibition triggered AML1-ETO degradation and impaired the clonogenic potential of the human t(8;21) leukemic blood cell line Kasumi-1. Therefore Drosophila provides a promising genetically tractable model to investigate the conserved basis of leukemogenesis and to open avenues in AML therapy.

Omega-3 fatty acid supplementation delays the progression of neuroblastoma in vivo

Epidemiological and preclinical studies have revealed that omega‐3 fatty acids have anticancer properties. We have previously shown that the omega‐3 fatty acid docosahexaenoic acid (DHA) induces apoptosis of neuroblastoma cells in vitro by mechanisms involving intracellular peroxidation of DHA by means of 15‐lipoxygenase or autoxidation. In our study, the effects of DHA supplementation on neuroblastoma tumor growth in vivo were investigated using two complementary approaches. For the purpose of prevention, DHA as a dietary supplement was fed to athymic rats before the rats were xenografted with human neuroblastoma cells. For therapeutic purposes, athymic rats with established neuroblastoma xenografts were given DHA daily by gavage and tumor growth was monitored. DHA levels in plasma and tumor tissue were analyzed by gas liquid chromatography. DHA delayed neuroblastoma xenograft development and inhibited the growth of established neuroblastoma xenografts in athymic rats. A revised version of the Pediatric Preclinical Testing Program evaluation scheme used as a measurement of treatment response showed that untreated control animals developed progressive disease, whereas treatment with DHA resulted in stable disease or partial response, depending on the DHA concentration. In conclusion, prophylactic treatment with DHA delayed neuroblastoma development, suggesting that DHA could be a potential agent in the treatment of minimal residual disease and should be considered for prevention in selected cases. Treatment results on established aggressive neuroblastoma tumors suggest further studies aiming at a clinical application in children with high‐risk neuroblastoma.

Lentiviral marking of patient-derived acute lymphoblastic leukaemic cells allows in vivo tracking of disease progression

Lentiviral marking of patient-derived acute lymphoblastic leukaemic cells allows in vivo tracking of disease progression

Cell Survival Signalling through PPARδ and Arachidonic Acid Metabolites in Neuroblastoma

Retinoic acid (RA) has paradoxical effects on cancer cells: promoting cell death, differentiation and cell cycle arrest, or cell survival and proliferation. Arachidonic acid (AA) release occurs in response to RA treatment and, therefore, AA and its downstream metabolites may be involved in cell survival signalling. To test this, we inhibited phospholipase A2-mediated AA release, cyclooxygenases and lipoxygenases with small-molecule inhibitors to determine if this would sensitise cells to cell death after RA treatment. The data suggest that, in response to RA, phospholipase A2-mediated release of AA and subsequent metabolism by lipoxygenases is important for cell survival. Evidence from gene expression reporter assays and PPARδ knockdown suggests that lipoxygenase metabolites activate PPARδ. The involvement of PPARδ in cell survival is supported by results of experiments with the PPARδ inhibitor GSK0660 and siRNA-mediated knockdown. Quantitative reverse transcriptase PCR studies demonstrated that inhibition of 5-lipoxygenase after RA treatment resulted in a strong up-regulation of mRNA for PPARδ2, a putative inhibitory PPARδ isoform. Over-expression of PPARδ2 using a tetracycline-inducible system in neuroblastoma cells reduced proliferation and induced cell death. These data provide evidence linking lipoxygenases and PPARδ in a cell survival-signalling mechanism and suggest new drug-development targets for malignant and hyper-proliferative diseases.

Gene Silencing by RNAi in Mammalian Cells

This unit provides information how to use short interfering RNA (siRNA) for sequence‐specific gene silencing in mammalian cells. Several methods for siRNA generation and optimization, as well as recommendations for cell transfection and transduction, are presented.

MLL-AF4 Spreading Identifies Binding Sites that Are Distinct from Super-Enhancers and that Govern Sensitivity to DOT1L Inhibition in Leukemia

Summary Understanding the underlying molecular mechanisms of defined cancers is crucial for effective personalized therapies. Translocations of the mixed-lineage leukemia (MLL) gene produce fusion proteins such as MLL-AF4 that disrupt epigenetic pathways and cause poor-prognosis leukemias. Here, we find that at a subset of gene targets, MLL-AF4 binding spreads into the gene body and is associated with the spreading of Menin binding, increased transcription, increased H3K79 methylation (H3K79me2/3), a disruption of normal H3K36me3 patterns, and unmethylated CpG regions in the gene body. Compared to other H3K79me2/3 marked genes, MLL-AF4 spreading gene expression is downregulated by inhibitors of the H3K79 methyltransferase DOT1L. This sensitivity mediates synergistic interactions with additional targeted drug treatments. Therefore, epigenetic spreading and enhanced susceptibility to epidrugs provides a potential marker for better understanding combination therapies in humans.

Abundant and equipotent founder cells establish and maintain acute lymphoblastic leukaemia

High frequencies of blasts in primary acute lymphoblastic leukaemia (ALL) samples have the potential to induce leukaemia and to engraft mice. However, it is unclear how individual ALL cells each contribute to drive leukaemic development in a bulk transplant and the extent to which these blasts vary functionally. We used cellular barcoding as a fate mapping tool to track primograft ALL blasts in vivo. Our results show that high numbers of ALL founder cells contribute at similar frequencies to leukaemic propagation over serial transplants, without any clear evidence of clonal succession. These founder cells also exhibit equal capacity to home and engraft to different organs, although stochastic processes may alter the composition in restrictive niches. Our findings enhance the stochastic stem cell model of ALL by demonstrating equal functional abilities of singular ALL blasts and show that successful treatment strategies must eradicate the entire leukaemic cell population.

COX2 expression in neuroblastoma increases tumorigenicity but does not affect cell death in response to the COX2 inhibitor celecoxib

COX2 is an inducible cyclooxygenase implicated in the metastasis and migration of tumour cells. In neuroblastoma, COX2 expression has been detected in both cell lines and tumours. The treatment of neuroblastoma cells in vitro with celecoxib, a COX2 inhibitor, induces apoptosis. The aim of this study was to investigate the role of COX2 in neuroblastoma tumour biology by creating a cell line in which COX2 could be conditionally expressed. Xenograft studies showed that the conditional expression of COX2 enhanced tumour growth and malignancy. Elevated COX2 expression enhanced the proliferation and migration of neuroblastoma cells in vitro. However, elevated COX2 expression or variation between cell lines did not affect sensitivity to the COX2 inhibitor celecoxib, indicating that celecoxib does not promote cell death through COX2 inhibition. These data show that increased COX2 expression alone can enhance the tumorigenic properties of neuroblastoma cells; however, high levels of COX2 may not be a valid biomarker of sensitivity to non-steroidal anti-inflammatory drugs such as celecoxib.