The oncolytic peptide LTX-315 triggers immunogenic cell death

Published version. Source at http://dx.doi.org/10.1038/cddis.2016.47 LTX-315 is a cationic amphilytic peptide that preferentially permeabilizes mitochondrial membranes, thereby causing partially BAX/ BAK1-regulated, caspase-independent necrosis. Based on the observation that intratumorally injected LTX-315 stimulates a strong T lymphocyte-mediated anticancer immune response, we investigated whether LTX-315 may elicit the hallmarks of immunogenic cell death (ICD), namely (i) exposure of calreticulin on the plasma membrane surface, (ii) release of ATP into the extracellular space, (iii) exodus of HMGB1 from the nucleus, and (iv) induction of a type-1 interferon response. Using a panel of biosensor cell lines and robotized fluorescence microscopy coupled to automatic image analysis, we observed that LTX-315 induces all known ICD characteristics. This conclusion was validated by several independent methods including immunofluorescence stainings (for calreticulin), bioluminescence assays (for ATP), immunoassays (for HMGB1), and RT-PCRs (for type-1 interferon induction). When injected into established cancers, LTX-315 caused a transiently hemorrhagic focal necrosis that was accompanied by massive release of HMGB1 (from close-to-all cancer cells), as well as caspase-3 activation in a fraction of the cells. LTX-315 was at least as efficient as the positive control, the anthracycline mitoxantrone (MTX), in inducing local inflammation with infiltration by myeloid cells and T lymphocytes. Collectively, these results support the idea that LTX-315 can induce ICD, hence explaining its capacity to mediate immune-dependent therapeutic effects

Short intron-derived ncRNAs

International audienceIntrons represent almost half of the human genome, although they are eliminated from transcripts through RNA splicing. Yet, different classes of non-canonical miRNAs have been proposed to originate directly from intron splicing. Here, we considered the alternative splicing of introns as an interesting source of miRNAs, compatible with a developmental switch. We report computational prediction of new Short Intron-Derived ncRNAs (SID), defined as precursors of smaller ncRNAs like miRNAs and snoR-NAs produced directly by splicing, and tested their dependence on each key factor in canonical or alternative miRNAs biogenesis (Drosha, DGCR8, DBR1, snRNP70, U2AF65, PRP8, Dicer, Ago2). We found that about half of predicted SID rely on debranch-ing of the excised intron-lariat by the enzyme DBR1, as proposed for mirtrons. However, we identified new classes of SID for which miRNAs biogenesis may rely on intermingling between canonical and alternative pathways. We validated selected SID as putative miR-NAs precursors and identified new endogenous miR-NAs produced by non-canonical pathways, including one hosted in the first intron of SRA (Steroid Receptor RNA activator). Consistent with increased SRA intron retention during myogenic differentiation, release of SRA intron and its associated mature miRNA decreased in cells from healthy subjects but not from myotonic dystrophy patients with splicing defects

A fluorescent biosensor-based platform for the discovery of immunogenic cancer cell death inducers

Systemic anticancer immunity can be reinstated via the induction of immunogenic cell death (ICD) in malignant cells. Thus, certain classes of cytotoxic compounds, for example, anthracyclines, oxaliplatin and taxanes are endowed with the capacity to act on cancer cells to ignite premortem stress pathways that lead to the surface exposure of calreticulin (CALR) and the cellular release of adenosine triphosphate, annexin A1, high mobility group B1 and type-1 interferons. Altogether, these alterations constitute the hallmarks of ICD. Here we report the design of a discovery pipeline for the identification of novel ICD inducers by means of a phenotypic screening platform. The use of fluorescent biosensors as proxies for the manifestation of ICD hallmarks has enabled the exploration of large collections of chemical compounds by automatized screening routines. Imaging-based assessment and phenotypic selection led to the identification of potential ICD inducers that could be validated further in vitro and in vivo, confirming that bona fide ICD inducers possess the capacity to induce immunological long-term memory and to confer resistance against rechallenge with syngeneic tumors. Machine learning algorithms analyzing the physicochemical properties of ICD inducers can assist in the preselection of compounds with potential ICD-stimulatory properties, further accelerating the screening efforts designed to develop new immunotherapeutic agents

The oncolytic peptide LTX-315 kills cancer cells through Bax/Bak-regulated mitochondrial membrane permeabilization

LTX-315 has been developed as an amphipathic cationic peptide that kills cancer cells. Here, we investigated the putative involvement of mitochondria in the cytotoxic action of LTX-315. Subcellular fractionation of LTX-315-treated cells, followed by mass spectrometric quantification, revealed that the agent was enriched in mitochondria. LTX-315 caused an immediate arrest of mitochondrial respiration without any major uncoupling effect. Accordingly, LTX-315 disrupted the mitochondrial network, dissipated the mitochondrial inner transmembrane potential, and caused the release of mitochondrial intermembrane proteins into the cytosol. LTX-315 was relatively inefficient in stimulating mitophagy. Cells lacking the two pro-apoptotic multidomain proteins from the BCL-2 family, BAX and BAK, were less susceptible to LTX-315-mediated killing. Moreover, cells engineered to lose their mitochondria (by transfection with Parkin combined with treatment with a protonophore causing mitophagy) were relatively resistant against LTX-315, underscoring the importance of this organelle for LTX-315-mediated cytotoxicity. Altogether, these results support the notion that LTX-315 kills cancer cells by virtue of its capacity to permeabilize mitochondrial membranes

Lurbinectedin synergizes with immune checkpoint blockade to generate anticancer immunity

Systemic treatment with the active transcription inhibitor lurbinectedin aims at inducing tumor cell death in hyperproliferative neoplasms. Here we show that cell death induced by lurbinectedin reinstates and enhances systemic anticancer immune responses. Lurbinectedin treatment showed traits of immunogenic cell death, including the exposure of calreticulin, the release of ATP, the exodus of high mobility group box 1 (HMGB1) and type 1 interferon responses in vitro. Lurbinectedin treated cells induced antitumor immunity when injected into immunocompetent animals and treatment of transplanted fibrosarcomas reduced tumor growth in immunocompetent yet not in immunodeficient hosts. Anticancer effects resulting from lurbinectedin treatment were boosted in combination with PD-1 and CTLA-4 double immune checkpoint blockade (ICB), and lurbinectedin combined with double ICB exhibited strong antineoplastic effects. Cured animals exhibited long term immune memory effects that rendered them resistant to rechallenge with syngeneic tumors underlining the potency of combination therapy with lurbinectedin

3,4-dimethoxychalcone induces autophagy and reduces neointimal hyperplasia and aortic lesions in mouse models of atherosclerosis

Abstract Autophagy inducers can prevent cardiovascular aging and age-associated diseases including atherosclerosis. Therefore, we hypothesized that autophagy-inducing compounds that act on atherosclerosis-relevant cells might have a protective role in the development of atherosclerosis. Here we identified 3,4-dimethoxychalcone (3,4-DC) as an inducer of autophagy in several cell lines from endothelial, myocardial and myeloid/macrophagic origin, as demonstrated by the aggregation of the autophagosome marker GFP-LC3 in the cytoplasm of cells, as well as the downregulation of its nuclear pool indicative of autophagic flux. In this respect, 3,4-DC showed a broader autophagy-inducing activity than another chalcone (4,4- dimethoxychalcone), spermidine and triethylene tetramine. Thus, we characterized the potential antiatherogenic activity of 3,4-DC in two different mouse models, namely, (i) neointima formation with smooth muscle expansion of vein segments grafted to the carotid artery and (ii) genetically predisposed ApoE −/− mice fed an atherogenic diet. In the vein graft model, local application of 3,4-DC was able to maintain the lumen of vessels and to reduce neointima lesions. In the diet-induced model, intraperitoneal injections of 3,4-DC significantly reduced the number of atherosclerotic lesions in the aorta. In conclusion, 3,4-DC stands out as an autophagy inducer with potent antiatherogenic activity

IGF1 receptor inhibition amplifies the effects of cancer drugs by autophagy and immune-dependent mechanisms−

Background Pharmacological autophagy enhancement constitutes a preclinically validated strategy for preventing or treating most major age-associated diseases. Driven by this consideration, we performed a high-content/high-throughput screen on 65 000 distinct compounds on a robotized fluorescence microscopy platform to identify novel autophagy inducers.Results Here, we report the discovery of picropodophyllin (PPP) as a potent inducer of autophagic flux that acts on-target, as an inhibitor of the tyrosine kinase activity of the insulin-like growth factor-1 receptor (IGF1R). Thus, PPP lost its autophagy-stimulatory activity in cells engineered to lack IGF1R or to express a constitutively active AKT serine/threonine kinase 1 (AKT1) mutant. When administered to cancer-bearing mice, PPP improved the therapeutic efficacy of chemoimmunotherapy with a combination of immunogenic cytotoxicants and programmed cell death 1 (PDCD1, better known as PD-1) blockade. These PPP effects were lost when tumors were rendered PPP-insensitive or autophagy-incompetent. In combination with chemotherapy, PPP enhanced the infiltration of tumors by cytotoxic T lymphocytes, while reducing regulatory T cells. In human triple-negative breast cancer patients, the activating phosphorylation of IGF1R correlated with inhibited autophagy, an unfavorable local immune profile, and poor prognosis.Conclusion Altogether, these results suggest that IGF1R may constitute a novel and druggable therapeutic target for the treatment of cancer in conjunction with chemoimmunotherapies

Control of lysosomal-mediated cell death by the pH-dependent calcium channel RECS1

Programmed cell death is regulated by the balance between activating and inhibitory signals. Here, we have identified RECS1 (responsive to centrifugal force and shear stress 1) [also known as TMBIM1 (transmembrane BAX inhibitor motif containing 1)] as a proapoptotic member of the TMBIM family. In contrast to other proteins of the TMBIM family, RECS1 expression induces cell death through the canonical mitochondrial apoptosis pathway. Unbiased screening indicated that RECS1 sensitizes cells to lysosomal perturbations. RECS1 localizes to lysosomes, where it regulates their acidification and calcium content, triggering lysosomal membrane permeabilization. Structural modeling and electrophysiological studies indicated that RECS1 is a pH-regulated calcium channel, an activity that is essential to trigger cell death. RECS1 also sensitizes whole animals to stress in vivo in Drosophila melanogaster and zebrafish models. Our results unveil an unanticipated function for RECS1 as a proapoptotic component of the TMBIM family that ignites cell death programs at lysosomes.Peer reviewe