The indisputable role of epigenetics in cancer and the fact that epigenetic alterations can be reversed have favoured development of epigenetic drugs. In this study, we design and synthesize potent novel, selective and reversible chemical probes that simultaneously inhibit the G9a and DNMTs methyltransferase activity. In vitro treatment of haematological neoplasia (acute myeloid leukaemia-AML, acute lymphoblastic leukaemia-ALL and diffuse large B-cell lymphoma-DLBCL) with the lead compound CM-272, inhibits cell proliferation and promotes apoptosis, inducing interferon-stimulated genes and immunogenic cell death. CM-272 significantly prolongs survival of AML, ALL and DLBCL xenogeneic models. Our results represent the discovery of first-in-class dual inhibitors of G9a/DNMTs and establish this chemical series as a promising therapeutic tool for unmet needs in haematological tumours.

Key Points• Acquired selective mutations in Bcl2 and BAX conferred resistance to in experimental models of lymphoma.• Monitoring the potential development of such mutations in patients treated with ABT-199 is advised.Acquired resistance to targeted drugs is emerging as an obstacle to successful cancer treatment. Recently, a BCL2-selective BH3 mimetic termed ABT-199 showed promising therapeutic results in BCL2-dependent tumors. Based on its high affinity for BCL2, we studied potential mechanisms conferring resistance upon ABT-199 therapy, aiming to anticipate its occurrence in the clinic. Two models of resistant lymphomas were established by continuous ABT-199 exposure. In resistant Bcl2-expressing mouse lymphoma cells, 2 missense mutations within the Bcl2 BH3 domain were identified. Both F101C and F101L mutations impeded binding to the BH3 domain, therefore suppressing mitochondrial apoptosis. In resistant human lymphoma cells, a missense mutation in the C-terminal transmembrane domain of proapoptotic BAX (G179E) was found, which abrogated BAX anchoring to mitochondria and blocked ABT-199-induced apoptosis both in vitro and in vivo. Importantly, G179E BAX mutation also induced partial cross-resistance to other antineoplastic drugs. Our study reveals the acquisition of mutations in BCL2 family proteins as a novel mechanism of apoptosis resistance in cancer. These results anticipate the potential development of such mutations in patients treated with , providing a basis to preventing their occurrence and to designing drugs able to circumvent the acquired resistance. (Blood. 2014;123(26):4111-4119) IntroductionThe BCL2 family of proteins, which comprise prosurvival members such as BCL2, BCL-XL, BCL-W, MCL1, and BFL1, proapoptotic BH3-only proteins such as BIM and BAD, and the proapoptotic final effectors BAK and BAX, are critical regulators of the mitochondrial apoptotic pathway. [1][2][3] The development of drugs that inhibit the interaction between antiapoptotic BCL2 family members and BH3-only proteins is having a major impact on cancer therapy. [4][5][6][7] Among them, the BH3 mimetic ABT-737 was highly effective by inducing apoptosis through BCL2, BCL-XL, and BCL-W targeting in solid tumors and hematologic malignancies overexpressing BCL2. 8 Its efficacy, however, was largely diminished in tumors expressing high levels of MCL1, BFL1 and BCL-XL, or in cancer cells lacking BAK and BAX. [9][10][11] Despite the promising preclinical therapeutic efficacy of ABT-263, which is the orally available analog of ABT-737, phase II clinical trials showed a rapid, dose-dependent thrombocytopenia due to the inhibition of BCL-XL, limiting the ability to achieve drug concentrations at an efficacious range in cancer patients.12-14 By reengineering of ABT-263, a potent and high-affinity BCL2-selective BH3 mimetic with low avidity for BCL-XL, termed ABT-199, has been developed. 15 In vitro, ABT-199 inhibited the growth of BCL2-expressing leukemia, lymphoma, and myeloma cell lines more effectively than ABT-737. 15 Additionally, a...

Pediatric high-grade glioma (pHGG) and diffuse intrinsic pontine gliomas (DIPGs) are aggressive pediatric brain tumors in desperate need of a curative treatment. Oncolytic virotherapy is emerging as a solid therapeutic approach. Delta-24-RGD is a replication competent adenovirus engineered to replicate in tumor cells with an aberrant RB pathway. This virus has proven to be safe and effective in adult gliomas. Here we report that the administration of Delta-24-RGD is safe in mice and results in a significant increase in survival in immunodeficient and immunocompetent models of pHGG and DIPGs. Our results show that the Delta-24-RGD antiglioma effect is mediated by the oncolytic effect and the immune response elicited against the tumor. Altogether, our data highlight the potential of this virus as treatment for patients with these tumors. Of clinical significance, these data have led to the start of a phase I/II clinical trial at our institution for newly diagnosed DIPG (NCT03178032).

IntroductionTumors that express estrogen receptor alpha (ERα+) comprise 75% of breast cancers in women. While treatments directed against this receptor have successfully lowered mortality rates, many primary tumors initially or later exhibit resistance. The paucity of murine models of this "luminal" tumor subtype has hindered studies of factors that promote their pathogenesis and modulate responsiveness to estrogen-directed therapeutics. Since epidemiologic studies closely link prolactin and the development of ERα+ tumors in women, we examined characteristics of the aggressive ERα+ and ERα- carcinomas which develop in response to mammary prolactin in a murine transgenic model (neu-related lipocalin- prolactin (NRL-PRL)). To evaluate their relationship to clinical tumors, we determined phenotypic relationships among these carcinomas, other murine models of breast cancer, and features of luminal tumors in women.MethodsWe examined a panel of prolactin-induced tumors for characteristics relevant to clinical tumors: histotype, ERα/progesterone receptor (PR) expression and estrogen responsiveness, Activating Protein 1 (AP-1) components, and phosphorylation of signal transducer and activator of transcription 5 (Stat5), extracellular signal regulated kinase (ERK) 1/2 and AKT. We compared levels of transcripts in the ERα-associated "luminal" signature that defines this subtype of tumors in women and transcripts enriched in various mammary epithelial lineages to other well-studied genetically modified murine models of breast cancer. Finally, we used microarray analyses to compare prolactin-induced ERα+ and ERα- tumors, and examined responsiveness to estrogen and the anti-estrogen, Faslodex, in vivo.ResultsProlactin-induced carcinomas were markedly diverse with respect to histotype, ERα/PR expression, and activated signaling cascades. They constituted a heterogeneous, but distinct group of murine mammary tumors, with molecular features of the luminal subtype of human breast cancer. In contrast to morphologically normal and hyperplastic structures in NRL-PRL females, carcinomas were insensitive to ERα-mediated signals. These tumors were distinct from mouse mammary tumor virus (MMTV)-neu tumors, and contained elevated transcripts for factors associated with luminal/alveolar expansion and differentiation, suggesting that they arose from physiologic targets of prolactin. These features were shared by ERα+ and ERα- tumors, suggesting a common origin, although the former exhibited transcript profiles reflecting greater differentiation.ConclusionsOur studies demonstrate that prolactin can promote diverse carcinomas in mice, many of which resemble luminal breast cancers, providing a novel experimental model to examine the pathogenesis, progression and treatment responsiveness of this tumor subtype.

Using knowledge- and structure-based approaches, we designed and synthesized reversible chemical probes that simultaneously inhibit the activity of two epigenetic targets, histone 3 lysine 9 methyltransferase (G9a) and DNA methyltransferases (DNMT), at nanomolar ranges. Enzymatic competition assays confirmed our design strategy: substrate competitive inhibitors. Next, an initial exploration around our hit 11 was pursued to identify an adequate tool compound for in vivo testing. In vitro treatment of different hematological neoplasia cell lines led to the identification of molecules with clear antiproliferative efficacies (GI values in the nanomolar range). On the basis of epigenetic functional cellular responses (levels of lysine 9 methylation and 5-methylcytosine), an acceptable therapeutic window (around 1 log unit) and a suitable pharmacokinetic profile, 12 was selected for in vivo proof-of-concept ( Nat. Commun. 2017 , 8 , 15424 ). Herein, 12 achieved a significant in vivo efficacy: 70% overall tumor growth inhibition of a human acute myeloid leukemia (AML) xenograft in a mouse model.

Epidemiologic studies have demonstrated that increased prolactin (PRL) exposure raises the risk of invasive estrogen receptor a (ERa)-positive breast cancer in women. However, the mechanism(s) whereby this occurs and the interactions with estrogen itself in this disease remain poorly understood. In order to investigate the role of ovarian hormones in the disease process, we employed a transgenic model neu-related lipocalin (NRL)-PRL in which transgenic PRL is directed to mammary epithelial cells by the PRL-and estrogen-insensitive NRL promoter, mimicking the endogenous PRL expression within the breast observed in women. This high local exposure leads to mammary lesion development and eventually carcinomas. Ovariectomy (ovx), shortly after puberty, did not alter the incidence or latency of PRL-induced mammary carcinomas, consistent with the independence of PRL from circulating estrogens as a risk factor for invasive breast cancer in women. However, chronic estrogen administration to ovx NRL-PRL females decreased the latency of both ERa-positive and -negative tumors. We identified multiple mechanisms that may underlie this observation. Elevated estrogen exposure cooperated with PRL to increase epithelial proliferation and myoepithelial abnormalities, increasing the incidence of preneoplastic lesions. Critical components of the extracellular matrix secreted by the myoepithelium were reduced with age, and transgenic PRL raised transcripts for tenascin-C and maspin, both associated with tumor progression and poor prognosis in subclasses of clinical breast tumors. Mammary pERK1/2 and pAkt, but not phosphorylated Stat5, were markedly elevated by local PRL. Together, these findings indicate that PRL employs multiple mechanisms to promote mammary tumorigenesis.

The effect of pre-existent hepatic NO synthesis on liver injury induced by lipopolysaccharide was studied in animals carrying a nitric oxide synthase-2 (NOS-2) transgene under the control of the phosphoenolpyruvate carboxykinase (PEPCK) promoter. These animals expressed NOS-2 in liver cells under fasting conditions. Lipopolysaccharide-induced liver injury in D-galactosamine-conditioned mice, which enhanced notably the effect of the endotoxin on the liver, was impaired in animals expressing NOS-2. This protection against inflammatory liver damage was dependent on NO synthesis and was caused by an inhibition of nuclear factor kB (NF-kB) activity and an impairment of the synthesis of the proinflammatory cytokines tumor necrosis factor a and interleukin 1b. These data indicate that intrahepatic synthesis of NO protects liver by inhibiting the release of cascades of proinflammatory mediators and suggest a beneficial role for local delivery of NO in the control of liver injury.

For millions of years, endogenous retroelements have remained transcriptionally silent within mammalian genomes by epigenetic mechanisms. Modern anticancer therapies targeting the epigenetic machinery awaken retroelement expression, inducing antiviral responses that eliminate tumors through mechanisms not completely understood. Here, we find that massive binding of epigenetically activated retroelements by RIG-I and MDA5 viral sensors promotes ATP hydrolysis and depletes intracellular energy, driving tumor killing independently of immune signaling. Energy depletion boosts compensatory ATP production by switching glycolysis to mitochondrial oxidative phosphorylation, thereby reversing the Warburg effect. However, hyperfunctional succinate dehydrogenase in mitochondrial electron transport chain generates excessive oxidative stress that unleashes RIP1-mediated necroptosis. To maintain ATP generation, hyperactive mitochondrial membrane blocks intrinsic apoptosis by increasing BCL2 dependency. Accordingly, drugs targeting BCL2 family proteins and epigenetic inhibitors yield synergistic responses in multiple cancer types. Thus, epigenetic therapy kills cancer cells by rewiring mitochondrial metabolism upon retroelement activation, which primes mitochondria to apoptosis by BH3-mimetics. Significance: The state of viral mimicry induced by epigenetic therapies in cancer cells remodels mitochondrial metabolism and drives caspase-independent tumor cell death, which sensitizes to BCL2 inhibitor drugs. This novel mechanism underlies clinical efficacy of hypomethylating agents and venetoclax in acute myeloid leukemia, suggesting similar combination therapies for other incurable cancers. This article is highlighted in the In This Issue feature, p. 995