TNFR2 is required for RIP1-dependent cell death in human leukemia.

Despite major advances in the treatment of patients with acute lymphoblastic leukemia in the last decades, refractory and/or relapsed disease remains a clinical challenge, and relapsed leukemia patients have an exceedingly dismal prognosis. Dysregulation of apoptotic cell death pathways is a leading cause of drug resistance; thus, alternative cell death mechanisms, such as necroptosis, represent an appealing target for the treatment of high-risk malignancies. We and other investigators have shown that activation of receptor interacting protein kinase 1 (RIP1)-dependent apoptosis and necroptosis by second mitochondria derived activator of caspase mimetics (SMs) is an attractive antileukemic strategy not currently exploited by standard chemotherapy. However, the underlying molecular mechanisms that determine sensitivity to SMs have remained elusive. We show that tumor necrosis factor receptor 2 (TNFR2) messenger RNA expression correlates with sensitivity to SMs in primary human leukemia. Functional genetic experiments using clustered regularly interspaced short palindromic repeats/Cas9 demonstrate that TNFR2 and TNFR1, but not the ligand TNF-α, are essential for the response to SMs, revealing a ligand-independent interplay between TNFR1 and TNFR2 in the induction of RIP1-dependent cell death. Further potential TNFR ligands, such as lymphotoxins, were not required for SM sensitivity. Instead, TNFR2 promotes the formation of a RIP1/TNFR1-containing death signaling complex that induces RIP1 phosphorylation and RIP1-dependent apoptosis and necroptosis. Our data reveal an alternative paradigm for TNFR2 function in cell death signaling and provide a rationale to develop strategies for the identification of leukemias with vulnerability to RIP1-dependent cell death for tailored therapeutic interventions.

Repurposing anthelmintic agents to eradicate resistant leukemia.

Despite rapid progress in genomic profiling in acute lymphoblastic leukemia (ALL), identification of actionable targets and prediction of response to drugs remains challenging. To identify specific vulnerabilities in ALL, we performed a drug screen using primary human ALL samples cultured in a model of the bone marrow microenvironment combined with high content image analysis. Among the 2487 FDA-approved compounds tested, anthelmintic agents of the class of macrocyclic lactones exhibited potent anti-leukemia activity, similar to the already known anti-leukemia agents currently used in induction chemotherapy. Ex vivo validation in 55 primary ALL samples of both precursor B cell and T-ALL including refractory relapse cases confirmed strong anti-leukemia activity with IC50 values in the low micromolar range. Anthelmintic agents increased intracellular chloride levels in primary leukemia cells, inducing mitochondrial outer membrane depolarization and cell death. Supporting the notion that simultaneously targeting cell death machineries at different angles may enhance the cell death response, combination of anthelmintic agents with the BCL-2 antagonist navitoclax or with the chemotherapeutic agent dexamethasone showed synergistic activity in primary ALL. These data reveal anti-leukemia activity of anthelmintic agents and support exploiting drug repurposing strategies to identify so far unrecognized anti-cancer agents with potential to eradicate even refractory leukemia.

Efficient apoptosis requires feedback amplification of upstream apoptotic signals by effector caspase-3 or -7.

Apoptosis is a complex multi-step process driven by caspase-dependent proteolytic cleavage cascades. Dysregulation of apoptosis promotes tumorigenesis and limits the efficacy of chemotherapy. To assess the complex interactions among caspases during apoptosis, we disrupted caspase-8, -9, -3, -7, or -6 and combinations thereof, using CRISPR-based genome editing in living human leukemia cells. While loss of apical initiator caspase-8 or -9 partially blocked extrinsic or intrinsic apoptosis, respectively, only combined loss of caspase-3 and -7 fully inhibited both apoptotic pathways, with no discernible effect of caspase-6 deficiency alone or in combination. Caspase-3/7 double knockout cells exhibited almost complete inhibition of caspase-8 or -9 activation. Furthermore, deletion of caspase-3 and -7 decreased mitochondrial depolarization and cytochrome c release upon apoptosis activation. Thus, activation of effector caspase-3 or -7 sets off explosive feedback amplification of upstream apoptotic events, which is a key feature of apoptotic signaling essential for efficient apoptotic cell death.

miR-17-92/p38α Dysregulation Enhances Wnt Signaling and Selects Lgr6+ Cancer Stem-like Cells during Lung Adenocarcinoma Progression.

Defining the molecular and cellular roots of lung cancer relapse after initial treatment remains an imperative to improve survival. Here we report that the lung stem cell marker Lgr6 becomes enriched in non-small cell lung cancer (NSCLC) cells during malignant progression. Lgr6(+) NSCLC cells displayed self-renewal and differentiation properties along with a higher tumorigenic potential. Mechanistic investigations suggested that a defective repression of the miR-17-92 gene cluster was responsible for evolution of a selection for outgrowth of Lgr6(+) NSCLC cells. High levels of expression of miR-19 family members were found to target and downregulate levels of p38α kinase, providing a specific survival signal for Lgr6(+) cells as mediated by increased Wnt/ß-catenin activity. Our results identify a specific stem-like cell population in NSCLC with increased malignant potential, the elucidation of which may enable earlier prognosis and possibly the development of more effective targeted treatments. Cancer Res; 76(13); 4012-22. ©2016 AACR.

Combined deletion of Vhl, Trp53 and Kif3a causes cystic and neoplastic renal lesions.

The von Hippel-Lindau (VHL) tumour suppressor gene is bi-allelically inactivated in the majority of cases of clear cell renal cell carcinoma (ccRCC); however, Vhl knockout mouse models do not recapitulate human ccRCC, implying that additional mutations are required for tumour formation. Mutational inactivation of VHL sensitises renal epithelial cells to lose the primary cilium in response to other mutations or extracellular stimuli. Loss of cilia is believed to represent a second hit in VHL mutant cells that causes the development of cystic lesions that, in some cases, can progress to ccRCC. Supporting this idea, genetic ablation of the primary cilium by deletion of the kinesin family member 3A (Kif3a) gene cooperates with loss of Vhl to accelerate cyst formation in mouse kidneys. Additionally, aged Vhl/Trp53 double-mutant mice develop renal cysts and tumours at a relatively low incidence, suggesting that there is a genetic cooperation between VHL and TP53 mutation in the development of ccRCC. Here we generated renal epithelium-specific Kif3a/Trp53 and Vhl/Kif3a/Trp53 mutant mice to investigate whether primary cilium deletion would accelerate the development of cystic precursor lesions or cause their progression to ccRCC. Longitudinal microcomputed tomography (µCT) imaging and histopathological analyses revealed an increased rate of cyst formation, increased proportion of cysts with proliferating cells, higher frequency of atypical cysts as well as the development of neoplasms in Vhl/Kif3a/Trp53 mutant kidneys compared to Kif3a/Trp53 or Vhl/Kif3a mutant kidneys. These findings demonstrate that primary cilium loss, in addition to Vhl and Trp53 losses, promotes the transition towards malignancy and provide further evidence that the primary cilium functions as a tumour suppressor organelle in the kidney. Copyright © 2016 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Induction of long noncoding RNA MALAT1 in hypoxic mice.

Long thought to be "junk DNA", in recent years it has become clear that a substantial fraction of intergenic genomic DNA is actually transcribed, forming long noncoding RNA (lncRNA). Like mRNA, lncRNA can also be spliced, capped, and polyadenylated, affecting a multitude of biological processes. While the molecular mechanisms underlying the function of lncRNAs have just begun to be elucidated, the conditional regulation of lncRNAs remains largely unexplored. In genome-wide studies our group and others recently found hypoxic transcriptional induction of a subset of lncRNAs, whereof nuclear-enriched abundant/autosomal transcript 1 (NEAT1) and metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) appear to be the lncRNAs most ubiquitously and most strongly induced by hypoxia in cultured cells. Hypoxia-inducible factor (HIF)-2 rather than HIF-1 seems to be the preferred transcriptional activator of these lncRNAs. For the first time, we also found strong induction primarily of MALAT1 in organs of mice exposed to inspiratory hypoxia. Most abundant hypoxic levels of MALAT1 lncRNA were found in kidney and testis. In situ hybridization revealed that the hypoxic induction in the kidney was confined to proximal rather than distal tubular epithelial cells. Direct oxygen-dependent regulation of MALAT1 lncRNA was confirmed using isolated primary kidney epithelial cells. In summary, high expression levels and acute, profound hypoxic induction of MALAT1 suggest a hitherto unrecognized role of this lncRNA in renal proximal tubular function.

Regulation of human lung alveolar multipotent cells by a novel p38α MAPK/miR-17-92 axis.

The cellular and molecular mechanisms that control lung homeostasis and regeneration are still poorly understood. It has been proposed that a population of cells exists in the mouse lung with the potential to differentiate into all major lung bronchioalveolar epithelium cell types in homeostasis or in response to virus infection. A new population of E-Cad/Lgr6(+) putative stem cells has been isolated, and indefinitely expanded from human lungs, harbouring both, self-renewal capacity and the potency to differentiate in vitro and in vivo. Recently, a putative population of human lung stem cells has been proposed as being c-Kit(+). Unlike Integrin-α6(+) or c-Kit(+) cells, E-Cad/Lgr6(+) single-cell injections in the kidney capsule produce differentiated bronchioalveolar tissue, while retaining self-renewal, as they can undergo serial transplantations under the kidney capsule or in the lung. In addition, a signalling network involving the p38α pathway, the activation of p53 and the regulation of the miR-17-92 cluster has been identified. Disruption of the proper cross-regulation of this signalling axis might be involved in the promotion of human lung diseases.

Substrate binding and catalysis in carbamate kinase ascertained by crystallographic and site-directed mutagenesis studies: movements and significance of a unique globular subdomain of this key enzyme for fermentative ATP production in bacteria.

Carbamate kinase (CK) makes ATP from ADP and carbamoyl phosphate (CP) in the final step of the microbial fermentative catabolism of arginine, agmatine, and oxalurate/allantoin. Two previously reported CK structures failed to clarify CP binding and catalysis and to reveal the significance of the protruding subdomain (PSD) that hangs over the CK active center as an exclusive and characteristic CK feature. We clarify now these three questions by determining two crystal structures of Enterococcus faecalis CK (one at 1.5 A resolution and containing bound MgADP, and the other at 2.1 A resolution and having in the active center one sulfate and two fixed water molecules that mimic one bound CP molecule) and by mutating active-center residues, determining the consequences of these mutations on enzyme functionality. Superimposition of the present crystal structures reconstructs the filled active center in the ternary complex, immediately suggesting in-line associative phosphoryl group transfer and a mechanism for enzyme catalysis involving N51, K209, K271, D210, and the PSD residue K128. The large respective increases and decreases in K(m)(CP) and k(cat) triggered by the mutations N51A, K128A, K209A, and D210N corroborate the ternary complex active-site architecture and the catalytic mechanism proposed. The extreme negative effects of K128A demonstrate a key role of the PSD in substrate binding and catalysis. The crystal structures reveal large rigid-body movements of the PSD towards the enzyme body that place K128 next to CP and bury the CP site. A mechanism that connects CP site occupation with the PSD approach, involving V206-I207 in the CP site and P162-S163 in the PSD stem, is identified. The effects of the V206A and V206L mutations support this mechanism. It is concluded that the PSD movement allows CK to select against the abundant CP/carbamate analogues acetylphosphate/acetate and bicarbonate, rendering CK highly selective for CP/carbamate.