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Systematic exploration of cancer cell vulnerabilities can inform the development of novel cancer therapeutics. Here, through analysis of genome-scale loss-of-function datasets, we identify adenosine deaminase acting on RNA (ADAR or ADAR1) as an essential gene for the survival of a subset of cancer cell lines. ADAR1-dependent cell lines display increased expression of interferon-stimulated genes. Activation of type I interferon signaling in the context of ADAR1 deficiency can induce cell lethality in non-ADAR1-dependent cell lines. ADAR deletion causes activation of the double-stranded RNA sensor, protein kinase R (PKR). Disruption of PKR signaling, through inactivation of PKR or overexpression of either a wildtype or catalytically inactive mutant version of the p150 isoform of ADAR1, partially rescues cell lethality after ADAR1 loss, suggesting that both catalytic and non-enzymatic functions of ADAR1 may contribute to preventing PKR-mediated cell lethality. Together, these data nominate ADAR1 as a potential therapeutic target in a subset of cancers., Specific cancer cell vulnerabilities can provide an opportunity for the development of novel cancer therapeutics. In this study the authors demonstrate that targeting ADAR1 represents a potential therapeutic vulnerability in cancers with activated interferon response signatures.

High-risk neuroblastoma remains lethal in about 50% of patients despite multimodal treatment. Recent attempts to identify molecular targets for specific therapies have shown that Neuroblastoma RAS (NRAS) is significantly mutated in a small number of patients. However, few inhibitors for the potential treatment for NRAS mutant neuroblastoma have been investigated so far. In this in-vitro study, we show that MEK inhibitors AZD6244, MEK162 and PD0325901 block cell growth in NRAS mutant neuroblastoma cell lines but not in NRAS wild-type cell lines. Several studies show that mutant NRAS leads to PI3K pathway activation and combined inhibitors of PI3K/mTOR effectively block cell growth. However, we observed the combination of MEK inhibitors with PI3K or AKT inhibitors did not show synergestic effects on cell growth. Thus, we tested single mTOR inhibitors Everolimus and AZD8055. Interestingly, Everolimus and AZD8055 alone were sufficient to block cell growth in NRAS mutant cell lines but not in wild-type cell lines. We found that Everolimus alone induced apoptosis in NRAS mutant neuroblastoma. Furthermore, the combination of mTOR and MEK inhibitors resulted in synergistic growth inhibition. Taken together, our results show that NRAS mutant neuroblastoma can be targeted by clinically available Everolimus alone or in combination with MEK inhibitors which could impact future clinical studies.

Aberrant signaling of the nuclear facotr (NF-κB) pathway has been identified as a mediator of survival and apoptosis resistance in leukemias and lymphomas. Here, we report that cell death of cutaneous T-cell lymphoma cell lines induced by inhibition of the NF-κB pathway is independent of caspases or classic death receptors. We found that free intracellular iron and reactive oxygen species (ROS) are the main mediators of this cell death. Antioxidants such as N-Acetyl-l-cysteine and glutathione or the iron chelator desferrioxamine effectively block cell death in cutaneous T-cell lymphoma cell lines or primary T cells from Sézary patients. We show that inhibition of constitutively active NF-κB causes down-regulation of ferritin heavy chain (FHC) that leads to an increase of free intracellular iron, which, in turn, induces massive generation of ROS. Furthermore, direct down-regulation of FHC by siRNA caused a ROS-dependent cell death. Finally, high concentrations of ROS induce cell death of malignant T cells. In contrast, T cells isolated from healthy donors do not display down-regulation of FHC and, therefore, do not show an increase in iron and cell death upon NF-κB inhibition. In addition, in a murine T-cell lymphoma model, we show that inhibition of NF-κB and subsequent down-regulation of FHC significantly delays tumor growth in vivo. Thus, our results promote FHC as a potential target for effective therapy in lymphomas with aberrant NF-κB signaling. [Cancer Res 2009;69(6):2365–74]

Reactive oxygen species (ROS) play a key role in regulation of activation-induced T-cell death (AICD) by induction of CD95L expression. However, the molecular source and the signaling steps necessary for ROS production are largely unknown. Here, we show that the proximal T-cell receptor-signaling machinery, including ZAP70 (zeta chain-associated protein kinase 70), LAT (linker of activated T cells), SLP76 (SH2 domain-containing leukocyte protein of 76 kDa), PLCgamma1 (phospholipase Cgamma1), and PKCtheta (protein kinase Ctheta), are crucial for ROS production. PKCtheta is translocated to the mitochondria. By using cells depleted of mitochondrial DNA, we identified the mitochondria as the source of activation-induced ROS. Inhibition of mitochondrial electron transport complex I assembly by small interfering RNA (siRNA)-mediated knockdown of the chaperone NDUFAF1 resulted in a block of ROS production. Complex I-derived ROS are converted into a hydrogen peroxide signal by the mitochondrial superoxide dismutase. This signal is essential for CD95L expression, as inhibition of complex I assembly by NDUFAF1-specific siRNA prevents AICD. Similar results were obtained when metformin, an antidiabetic drug and mild complex I inhibitor, was used. Thus, we demonstrate for the first time that PKCtheta-dependent ROS generation by mitochondrial complex I is essential for AICD.

Targeting of oncogenic driver mutations with small-molecule inhibitors resulted in powerful treatment options for cancer patients in recent years. The RAS (rat sarcoma) pathway is among the most frequently mutated pathways in human cancer. Whereas targeting mutant Kirsten RAS (KRAS) remains difficult, mutant B rapidly accelerated fibrosarcoma (BRAF) kinase is an established drug target in cancer. Now data show that neuroblastoma RAS (NRAS) and even Harvey RAS (HRAS) mutations could be predictive markers for treatment with mitogen-activated protein kinase (MEK) inhibitors. This review discusses recent preclinical and clinical studies of MEK inhibitors in BRAF and RAS mutant cancer.

Biosimilars are increasingly being licensed as equipotent drugs, although efficacy and safety data are not available for all clinical indications. Accordingly, the efficacy of the biosimilar filgrastim Zarzio® combined with vinorelbine for chemo-mobilization of CD34+ hematopoietic progenitor cells (HPC) in patients with multiple myeloma has not been evaluated yet. We compared the efficacy of vinorelbine combined with this biosimilar filgrastim for HPC mobilization to vinorelbine plus original filgrastim (Neupogen®). Overall, 105 multiple myeloma patients received vinorelbine 35 mg/m

HRAS is a frequently mutated oncogene in cancer. However, mutant HRAS as drug target has not been investigated so far. Here, we show that mutant HRAS hyperactivates the RAS and the mTOR pathway in various cancer cell lines including lung, bladder and esophageal cancer. HRAS mutation sensitized toward growth inhibition by the MEK inhibitors AZD6244, MEK162 and PD0325901. Further, we found that MEK inhibitors induce apoptosis in mutant HRAS cell lines but not in cell lines lacking RAS mutations. In addition, knockdown of HRAS by siRNA blocked cell growth in mutant HRAS cell lines. Inhibition of the PI3K pathway alone or in combination with MEK inhibitors did not alter signaling nor had an impact on viability. However, inhibition of mTOR or combined inhibition of MEK and mTOR reduced cell growth in a synergistic manner. Finally, Ba/F3 cells transformed with mutant HRAS isoforms Q61L, Q61R and G12V demonstrated equal sensitivity towards MEK and mTOR inhibition. Our results show that HRAS mutations in cancer activate the RAS and mTOR pathways which might serve as a therapeutic option for patients with HRAS mutant tumors.

Cutaneous T-cell lymphomas (CTCLs) are malignancies of skin-homing lymphoid cells, which have so far not been investigated thoroughly for common oncogenic mutations. We screened 90 biopsy specimens from CTCL patients (41 mycosis fungoides, 36 Sézary syndrome, and 13 non–mycosis fungoides/Sézary syndrome CTCL) for somatic mutations using OncoMap technology. We detected oncogenic mutations for the RAS pathway in 4 of 90 samples. One mycosis fungoides and one pleomorphic CTCL harbored a KRASG13D mutation; one Sézary syndrome and one CD30+ CTCL harbored a NRASQ61K amino acid change. All mutations were found in stage IV patients (4 of 42) who showed significantly decreased overall survival compared with stage IV patients without mutations (P = .04). In addition, we detected a NRASQ61K mutation in the CTCL cell line Hut78. Knockdown of NRAS by siRNA induced apoptosis in mutant Hut78 cells but not in CTCL cell lines lacking RAS mutations. The NRASQ61K mutation sensitized Hut78 cells toward growth inhibition by the MEK inhibitors U0126, AZD6244, and PD0325901. Furthermore, we found that MEK inhibitors exclusively induce apoptosis in Hut78 cells. Taken together, we conclude that RAS mutations are rare events at a late stage of CTCL, and our preclinical results suggest that such late-stage patients profit from MEK inhibitors.

NF-κB is a crucial transcription factor regulating apoptosis sensitivity and resistance. It has been shown that inhibition of NF-κB in T lymphocytes leads to sensitization towards apoptosis. The underlying molecular mechanism is not entirely understood. Therefore, we investigated T cell receptor (TCR) stimulated apoptosis in T cells in which NF-κB activity is blocked by an inhibitor or IκBα overexpression. We show that enhanced apoptosis upon TCR stimulation is caspase- and JNK-dependent, but independent of the CD95/CD95L system. Generation of reactive oxygen species (ROS) induced sustained JNK phosphorylation by inactivation of MAP kinase phosphatase 7 (MKP7). Sustained JNK activation causes upregulation of the pro-apototic protein BIM. Thus, inhibition of NF-κB causes a switch from classical activation-induced cell death (AICD) to CD95L-independent apoptosis.

Background Genome-wide CRISPR-Cas9 dropout screens can identify genes whose knockout affects cell viability. Recent CRISPR screens detected thousands of essential genes required for cellular survival and key cellular processes; however discovering novel lineage-specific genetic dependencies from the many hits still remains a challenge. Results To assess whether CRISPR-Cas9 dropout screens can help identify cancer dependencies, we screened two human cancer cell lines carrying known and distinct oncogenic mutations using a genome-wide sgRNA library. We found that the gRNA targeting the driver mutation EGFR was one of the highest-ranking candidates in the EGFR-mutant HCC-827 lung adenocarcinoma cell line. Likewise, sgRNAs for NRAS and MAP2K1 (MEK1), a downstream kinase of mutant NRAS, were identified among the top hits in the NRAS-mutant neuroblastoma cell line CHP-212. Depletion of these genes targeted by the sgRNAs strongly correlated with the sensitivity to specific kinase inhibitors of the EGFR or RAS pathway in cell viability assays. In addition, we describe other dependencies such as TBK1 in HCC-827 cells and TRIB2 in CHP-212 cells which merit further investigation. Conclusions We show that genome-wide CRISPR dropout screens are suitable for the identification of oncogenic drivers and other essential genes. Electronic supplementary material The online version of this article (doi:10.1186/s12864-016-3042-2) contains supplementary material, which is available to authorized users.