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Current clinical RAF inhibitors (RAFi) inhibit monomeric BRAF (mBRAF) but are less potent against dimeric BRAF (dBRAF). RAFi equipotent for mBRAF and dBRAF have been developed but are predicted to have lower therapeutic index. Here we identify a third class of RAFi that selectively inhibits dBRAF over mBRAF. Molecular dynamic simulations reveal restriction of the movement of the BRAF αC-helix as the basis of inhibitor selectivity. Combination of inhibitors based on their conformation selectivity (mBRAF- plus dBRAF-selective plus the most potent BRAF–MEK disruptor MEK inhibitor) promoted suppression of tumor growth in BRAFV600E therapy–resistant models. Strikingly, the triple combination showed no toxicities, whereas dBRAF-selective plus MEK inhibitor treatment caused weight loss in mice. Finally, the triple combination achieved durable response and improved clinical well-being in a patient with stage IV colorectal cancer. Thus, exploiting allosteric properties of RAF and MEK inhibitors enables the design of effective and well-tolerated therapies for BRAFV600E tumors.Significance:This work identifies a new class of RAFi that are selective for dBRAF over mBRAF and determines the basis of their selectivity. A rationally designed combination of RAF and MEK inhibitors based on their conformation selectivity achieved increased efficacy and a high therapeutic index when used to target BRAFV600E tumors.See related commentary by Zhang and Bollag, p. 1620.This article is highlighted in the In This Issue feature, p. 1601

Current clinical RAF inhibitors (RAFi) inhibit monomeric BRAF (mBRAF) but are less potent against dimeric BRAF (dBRAF). RAFi equipotent for mBRAF and dBRAF have been developed but are predicted to have lower therapeutic index. Here we identify a third class of RAFi that selectively inhibits dBRAF over mBRAF. Molecular dynamic simulations reveal restriction of the movement of the BRAF αC-helix as the basis of inhibitor selectivity. Combination of inhibitors based on their conformation selectivity (mBRAF- plus dBRAF-selective plus the most potent BRAF–MEK disruptor MEK inhibitor) promoted suppression of tumor growth in BRAFV600E therapy–resistant models. Strikingly, the triple combination showed no toxicities, whereas dBRAF-selective plus MEK inhibitor treatment caused weight loss in mice. Finally, the triple combination achieved durable response and improved clinical well-being in a patient with stage IV colorectal cancer. Thus, exploiting allosteric properties of RAF and MEK inhibitors enables the design of effective and well-tolerated therapies for BRAFV600E tumors. Significance: This work identifies a new class of RAFi that are selective for dBRAF over mBRAF and determines the basis of their selectivity. A rationally designed combination of RAF and MEK inhibitors based on their conformation selectivity achieved increased efficacy and a high therapeutic index when used to target BRAFV600E tumors. See related commentary by Zhang and Bollag, p. 1620. This article is highlighted in the In This Issue feature, p. 1601

Induced pluripotent stem cells (iPSCs) represent a source from which β cells can be derived for diabetes replacement therapy. However, their application may be hindered by immune-mediated responses. Although abrogation of major histocompatibility complex class I (MHC-I) can address this issue, it may trigger natural killer (NK) cells through missing-self recognition mechanisms. By profiling the relevant NK-activating ligands on iPSCs during in vitro differentiation into pancreatic β cells, we find that they express high levels of B7-H3 and CD155. Hypothesizing that such surface ligands could be involved in the amplification of NK-activating signals following missing-self, we generate MHC-I-deprived B7-H3

Frequent deletions impacting the FRA4F fragile site leads to the oncogenic overexpression of the corresponding CCSER1 gene, through the deletion of the TMSB4XP8 pseudogene. The ensuing CCSER1 overexpression elicits mitotic instability, targetable with aurora kinase inhibitors., The oncogenic role of common fragile sites (CFS), focal and pervasive gaps in the cancer genome arising from replicative stress, remains controversial. Exploiting the TCGA dataset, we found that in most CFS the genes residing within the associated focal deletions are down-regulated, including proteins involved in tumour immune recognition. In a subset of CFS, however, the residing genes are surprisingly overexpressed. Within the most frequent CFS in this group, FRA4F, which is deleted in up to 18% of cancer cases and harbours the CCSER1 gene, we identified a region which includes an intronic, antisense pseudogene, TMSB4XP8. TMSB4XP8 focal ablation or transcriptional silencing elicits the overexpression of CCSER1, through a cis-acting mechanism. CCSER1 overexpression increases proliferation and triggers centrosome amplifications, multinuclearity, and aberrant mitoses. Accordingly, FRA4F is associated in patient samples to mitotic genes deregulation and genomic instability. As a result, cells overexpressing CCSER1 become sensitive to the treatment with aurora kinase inhibitors. Our findings point to a novel tumourigenic mechanism where focal deletions increase the expression of a new class of “dormant” oncogenes.

In response to microenvironmental signals, macrophages undergo different activation, including the “classic” proinflammatory phenotype (also called M1), the “alternative” activation induced by the IL-4/IL-13 trigger, and the related but distinct heterogeneous M2 polarization associated with the anti-inflammatory profile. The latter is induced by several stimuli, including IL-10 and TGF-β. Macrophage-polarized activation has profound effects on immune and inflammatory responses and in tumor biology, but information on the underlying molecular pathways is scarce. In the present study, we report that alternative polarization of macrophages requires the transcription factor c-MYC. In macrophages, IL-4 and different stimuli sustaining M2-like polarization induce c-MYC expression and its translocation to the nucleus. c-MYC controls the induction of a subset (45%) of genes associated with alternative activation. ChIP assays indicate that c-MYC directly regulates some genes associated with alternative activation, including SCARB1, ALOX15, and MRC1, whereas others, including CD209, are indirectly regulated by c-MYC. c-MYC up-regulates the IL-4 signaling mediators signal transducer and activator of transcription-6 and peroxisome proliferator–activated receptorγ, is also expressed in tumor-associated macrophages, and its inhibition blocks the expression of protumoral genes including VEGF, MMP9, HIF-1α, and TGF-β. We conclude that c-MYC is a key player in alternative macrophage activation, and is therefore a potential therapeutic target in pathologies related to these cells, including tumors.

There are several diseases whereby the goal of gene therapy is to silence rather than replace a gene function. Paradigmatic examples are diseases caused by a dominant negative mutation or those in which silencing of a host gene confers resistance to a pathogen or compensates the function of the missing gene. Yet, gene silencing can be used to enhance efficacy of cell therapy and for biotechnological applications. Until now, two technologies have been used to silence gene expression, namely RNA interference with short harping RNAs (shRNA) and gene disruption with Artificial Nucleases (ANs). Although some promising pre-clinical and clinical data have been already obtained, the low efficiency of knock-down with shRNA and of biallelic disruption with ANs may limit efficacy of these treatments, especially when residual gene activity can exert a biological function. To overcome this issue, we have developed a novel modality of gene silencing that exploits endogenous epigenetic mechanisms to convey robust and heritable states of repression at the desired target gene. We have generated Artificial Transcriptional Repressors (ATRs), chimeric proteins containing a custom-made DNA binding domain fused to the effector domain of a chromatinmodifying enzyme involved in silencing of Endogenous RetroViruses (ERVs). By performing iterative rounds of selection in human cell lines and primary cells engineered to report for synergistic activity of candidate effector domains, we identified a combination of 3 domains that, when transiently co-assembled on the promoter of the reporter cassette, fully abrogated transgene expression in up to 90% of treated cells. Importantly, silencing was maintained for more than 250 days in cultured cell lines, was resistant to in vitro differentiation or metabolic activation of primary cells, and was confined to the reporter cassette. Silencing was associated with high levels of de novo DNA methylation at the targeted locus and was dependent on this epigenetic mark for its propagation. Finally, transient transfection of 3 ATRs targeted to the promoter region of the Beta-2-microglobulin (B2M) gene resulted in the loss of surface expression of B2M and, consequently, of the MHC-I molecules in up to 80% of treated cells. This phenotype was associated with a switch in the epigenetic and transcriptional state of the constitutively active B2M gene, which became highly decorated with DNA methylation and deprived of RNA PolII and of its transcript. Of note, silencing was resistant to IFN-γ treatment, a potent B2M inducer. Overall, these data provide the first demonstration of efficient and stable silencing of an endogenous gene upon transient delivery of ATRs. This result was made possible by repurposing the machinery involved in silencing of ERVs, which instructs self-sustaining repressive epigenetic states on the gene of interest. While silencing of B2M might be used to generate universally transplantable allogeneic cells, our hit-and-run strategy provides a powerful new alternative to conventional gene silencing for the treatment of several diseases. (LN & AL co-authorship)

Gene silencing holds great promise for the treatment of several diseases and can be exploited to investigate gene function and activity of the regulatory genome. Here, we develop a novel modality of gene silencing that exploits epigenetics to achieve stable and highly efficient repression of target genes. To this end, we generated Artificial Transcriptional Repressors (ATRs), chimeric proteins containing a custom-made DNA binding domain fused to the effector domain of chromatin-modifying enzymes involved in silencing process of Endogenous RetroViruses (ERVs). By performing iterative rounds of selection in cells engineered to report for synergistic activity of candidate effector domains, we identified a combination of 3 domains (namely KRAB, DNMT3A and DNMT3L) that, when transiently co-assembled on the promoter of the reporter cassette, recreate a powerful embryonic-specific repressive complex capable of inducing full and long-term (>150 days) silencing of transgene expression in up to 90% of the cells. The ATR-induced silencing was cell type and locus independent, and resistant to metabolic activation of the cells. Importantly, these findings were holding true also for endogenous genes embedded in their natural chromatin context, as shown for the highly and ubiquitously expressed B2M gene. Here, transient co-delivery of TALE-based ATRs resulted in loss of surface expression of B2M and, consequently, of the MHC-I molecules in up to 80% of the cells. This phenotype was associated with a drastic switch in the epigenetic and transcriptional state of the constitutively active B2M promoter, which become highly decorated with de novo DNA methylation and deprived of RNAP II. Importantly, silencing was sharply confined to the targeted gene and resistant to INF-γ, a potent natural activator of B2M. We further extended these studies by showing that our silencing approach is portable to the CRISPR/dCas9 DNA binding technology. In this setting, comparable levels of B2M silencing (up to 80%) were achieved using either pools or even individual sgRNAs coupled to dCas9-based ATRs. Yet, adoption of this technology allowed performing simultaneous, highly efficient multiplex gene silencing within the same cell, as shown for B2M, IFNAR1 and VEGFA. Finally, we assessed resistance of the silenced gene to activity of potent artificial transcription activators and chromatin remodelers, and found that only targeted DNA demethylation was able to reawaken the silent gene. This allowed performing iterative cycles of silencing and reactivation of the same gene in the same cell population. Overall, these data provide the first demonstration of efficient and stable epigenetic silencing of endogenous genes upon transient delivery of ATRs. This was accomplished by repurposing the ERVs silencing machinery, which instructs self-sustaining repressive epigenetic states to the target gene. While silencing of B2M might be used to generate universally transplantable allogeneic cells, our hit-and-run strategy provides a powerful new alternative to conventional gene silencing for both basic and translational research.

The lysosomal calcium channel TRPML1, whose mutations cause the lysosomal storage disorder (LSD) mucolipidosis type IV (MLIV), contributes to upregulate autophagic genes by inducing the nuclear translocation of the transcription factor EB (TFEB). Here we show that TRPML1 activation also induces autophagic vesicle (AV) biogenesis through the generation of phosphatidylinositol 3-phosphate (PI3P) and the recruitment of essential PI3P-binding proteins to the nascent phagophore in a TFEB-independent manner. Thus, TRPML1 activation of phagophore formation requires the calcium-dependent kinase CaMKKβ and AMPK, which increase the activation of ULK1 and VPS34 autophagic protein complexes. Consistently, cells from MLIV patients show a reduced recruitment of PI3P-binding proteins to the phagophore during autophagy induction, suggesting that altered AV biogenesis is part of the pathological features of this disease. Together, we show that TRPML1 is a multistep regulator of autophagy that may be targeted for therapeutic purposes to treat LSDs and other autophagic disorders., It was known that prolonged TRMPL1 activation induces TFEB translocation and upregulates autophagic gene regulation. Here, the authors show that acute TRMPL1 activation also induces autophagy through VPS34 and by lysosomal calcium release independent of TFEB.