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P66(super Shc) is a genetic determinant of life span in mammals, which regulates reactive oxygen species (ROS) metabolism and apoptosis. Study is conducted to show that P66(super Shc) is a redox enzyme that generates mitochondrial ROS as signaling molecules for apoptosis.

Combined inhibition of oxidative phosphorylation (OXPHOS) and glycolysis has been shown to activate a PP2A-dependent signaling pathway, leading to tumor cell death. Here, we analyze highly selective mitochondrial complex I or III inhibitors in vitro and in vivo to elucidate the molecular mechanisms leading to cell death following OXPHOS inhibition. We show that IACS-010759 treatment (complex I inhibitor) induces a reactive oxygen species (ROS)-dependent dissociation of CIP2A from PP2A, leading to its destabilization and degradation through chaperone-mediated autophagy. Mitochondrial complex III inhibition has analogous effects. We establish that activation of the PP2A holoenzyme containing B56δ regulatory subunit selectively mediates tumor cell death, while the arrest in proliferation that is observed upon IACS-010759 treatment does not depend on the PP2A-B56δ complex. These studies provide a molecular characterization of the events subsequent to the alteration of critical bioenergetic pathways and help to refine clinical studies aimed to exploit metabolic vulnerabilities of tumor cells. [Display omitted] • Complex I is a key regulator of CIP2A, a PP2A inhibitor with protumor activity • Increased mROS mediates the dissociation of CIP2A from PP2A and its degradation • Hypoglycemia and low dosages of mROS inducers can be exploited as cancer therapy Cazzoli et al. show that reactive oxygen species production, under low-glucose conditions in combination with low doses of mitochondrial drug inhibitor, enhances CIP2A degradation. CIP2A is an endogenous PP2A inhibitor with a strong prognostic and therapeutic value; more invasive and lethal tumors show increased levels of CIP2A protein. [ABSTRACT FROM AUTHOR]

After years of limited progress in the treatment of neuroendocrine neoplasms (NENs), an increasing number of therapeutic targets have recently emerged as potential tools to improve disease outcome. The mammalian target of rapamycin (mTOR) pathway and vascular endothelial growth factor (VEGF) signalling are implicated in the regulation of cell growth, proliferation, neo-angiogenesis and tumour cell spread. Their combined blockade, in a simultaneous or sequential strategy, represents an intriguing biological rationale to overcome the onset of resistance mechanisms. However, is becoming increasingly imperative to find the optimal sequential strategy according to the best toxicity profile, and also to identify predictive biomarkers. We will provide an overview of the pre-clinical and clinical data relating to mTOR pathway/VEGF signalling as a potential targets of treatment in NENs. [ABSTRACT FROM AUTHOR]

Abstract: Epigenetic alterations complement genetic mutations as a molecular mechanism leading to cell transformation, and maintenance of the cancer phenotype. Of note, they are reversible by pharmacological manipulation of the enzymes responsible for chromatin modification: indeed, epigenetic drugs (histone deacetylase inhibitors and DNA demethylating agents) are currently on the market, inducing proliferative arrest and death of tumor cells. These drugs, however, have been effective only in a few tumor types: the lack of consistent clinical results is mainly due to their use in a poorly targeted approach, since the epigenetic alterations present in cancer cells are mostly unknown. In a few cases (notably, leukemias expressing RAR and MLL fusion proteins), the molecular mechanisms underlying tumor-selective and tumor-specific epigenetic alterations have started to be deciphered. These studies are revealing a dazzling complexity in the mechanisms leading to alterations of the epigenome, and the need of combination therapies targeting different chromatin modifiers to reach an effective reversion of epigenetic alterations. [Copyright &y& Elsevier]

Abstract: DNA methylation, post-translational modifications of histones and high order organization of chromatin in cell nuclei are the components of the epigenome. Epigenetic regulation of gene expression is specific for each cell type, within different tissues, according to stages of development and (in the adult organism) of differentiation. Almost invariably, this regulation is altered in disease states, including cancer. The complete understanding of the identity of the epigenome of cancer has been so far hampered, due to the technical limitations and costs of the genome-wide analyses required. The recent development of next generation sequencing (NGS) technologies, however, holds the promise of fast, reliable and cost-effective analyses. Here we review the main approaches employed thus far to identify altered epigenetic patterns in cancer cells, and analyse how they are predicted to evolve in the scenario of the ultra high-throughput (UHT) screenings. [Copyright &y& Elsevier]

Acute promyelocytic leukemia (APL) is caused by chromosomal translocations that involve the retinoic acid receptor α (RAR) and several other genes to yield X-RAR fusion proteins. Unlike wild-type RARs, which require heterodimerization with the retinoid X receptor (RXR) for their function as DNA-binding transcriptional regulators, X-RAR fusion proteins bind DNA and deregulate transcription as homo-oligomers. In this issue of Cancer Cell, however, Zeisig et al. and Zhu et al. show that RXR recruitment is a critical determinant for the transforming potential of oligomeric X-RAR fusion proteins and explore the possibility for targeted interventions in APL with either RAR or RXR ligands. [Copyright &y& Elsevier]

Among the functional subpopulations that coexist within the tumor, ‘cancer stem cells’ are characterized by increased self-renewal and the ability to derive all of the other subpopulations of tumor cells (‘bulk’). The functional heterogeneity among cancer stem cells and bulk cells must reflect distinct cellular epigenetic landscapes, but — due to the difficulty to isolate bona fide cancer stem cells with a high degree of purity — those different epigenetic landscapes, and the molecular mechanisms underlying them, remain largely unknown. Cues of intratumor phenotypic plasticity complicate the interpretation of the cancer stem cell phenotype: we contend that, however, the concept of cancer stem cell has crucial therapeutic implication, and remains a key target for the exploration of the cancer epigenome. [ABSTRACT FROM AUTHOR]

Recent evidence highlights patients' sex relevance in antitumor immune response through a complex interaction—among hormones, genes, behaviors, and the microbiome—that affects both innate and adaptive immune functions, as well as immune evasion mechanisms. These complex interactions ultimately influence the efficacy and toxicity of immune checkpoint inhibitors in solid tumors. [ABSTRACT FROM AUTHOR]

Abstract: Absent or altered differentiation is one of the major features of cancer cells. Histone deacetylases (HDACs) play a central role in the epigenetic regulation of gene expression. Aberrant activity of HDACs has been documented in several types of cancers, leading to the development of HDAC inhibitors (HDACi) as anti-tumor drugs. In vitro and in vivo experimental evidences show that HDACi are able to resume the process of maturation in undifferentiated cancer cells, justifying their introduction as differentiating agents in several clinical trials. Modulation of cell fate by HDACi is observed at several levels, including the stem cell compartment: HDACi can act both on cancer stem cells, and with the rest of the tumor cell mass, leading to complex biological outputs. As a note of caution, when used as single agent, HDACi show only a moderate and limited biological response, which is augmented in combinatorial therapies with drugs designed against other epigenetic targets. The optimal employment of these molecules may be therefore in combination with other epigenetic drugs acting against the set of enzymes responsible for the set-up and maintenance of epigenetic information. [Copyright &y& Elsevier]

• Epigenetics, and in particular, chromatin modifiers. • Cancer metabolism. • Cancer stem cells. • Tumor cell signaling. • The immune system. During the Tenth Edition of the Annual Congress on "Anticancer Innovative Therapy" [Milan, 23/24 January 2020], experts in the fields of immuno-oncology, epigenetics, tumor cell signaling, and cancer metabolism shared their latest knowledge on the roles of i] epigenetics, and in particular, chromatin modifiers, ii] cancer metabolism, iii] cancer stem cells [CSCs], iv] tumor cell signaling, and iv] the immune system. The novel therapeutic approaches presented included epigenetic drugs, cell cycle inhibitors combined with ICB, antibiotics and other off-label drugs, small-molecules active against CSCs, liposome-delivered miRNAs, tumor-specific CAR-T cells, and T-cell–based immunotherapy. Moreover, important evidence on possible mechanisms of resistance to these innovative therapies were also discussed, in particular with respect to resistance to ICB. Overall, this conference provided scientists and clinicians with a broad overview of future challenges and hopes to improve cancer treatment reasonably in the medium-short term. [ABSTRACT FROM AUTHOR]

The Food and Drug Administration has lately approved atezolizumab, anti-programmed death ligand 1 (PD-L1), to be used together with nanoparticle albumin-bound (nab) paclitaxel in treating patients with triple negative breast cancer (BC) expressing PD-L1. Nonetheless, immune checkpoint inhibitors (ICIs) are still challenged by the resistance and immune-related adverse effects evident in a considerable subset of treated patients without conclusive comprehension of the underlying molecular basis, biomarkers and tolerable therapeutic regimens capable of unleashing the anti-tumour immune responses. Stepping back to preclinical models is thus inevitable to address these inquiries. Herein, we comprehensively review diverse preclinical models of BC exploited in investigating ICIs underscoring their pros and cons as well as the learnt and awaited lessons to allow full exploitation of ICIs in BC therapy. • Triple-negative breast cancer patients are more likely to benefit from immune checkpoint inhibitors. • Preclinical models are indispensable for identifying biomarker and safe combo regimens. • Optimised preclinical models recapitulating human breast cancer heterogeneity are urged. • Preclinical integration of pharmacokinetic/pharmacodynamic modelling and scRNA profiling is fundamental. [ABSTRACT FROM AUTHOR]

Abstract Olaparib is a PARP inhibitor (PARPi). For patients bearing BRCA1 or BRCA2 mutations, olaparib is approved to treat ovarian cancer and in clinical trials to treat breast and pancreatic cancers. In BRCA2-defective patients, PARPi inhibits DNA single-strand break repair, while BRCA2 mutations hamper double-strand break repair. Recently, we identified a series of triazole derivatives that mimic BRCA2 mutations by disrupting the Rad51-BRCA2 interaction and thus double-strand break repair. Here, we have computationally designed, synthesized, and tested over 40 novel derivatives. Additionally, we designed and conducted novel biological assays to characterize how they disrupt the Rad51-BRCA2 interaction and inhibit double-strand break repair. These compounds synergized with olaparib to target pancreatic cancer cells with functional BRCA2. This supports the idea that small organic molecules can mimic genetic mutations to improve the profile of anticancer drugs for precision medicine. Moreover, this paradigm could be exploited in other genetic pathways to discover innovative anticancer targets and drug candidates. Graphical abstract Image 1 Highlights • Olaparib is a PARPi approved to treat ovarian cancer in BRCA2 defective patients. • BRCA2 mutations can be mimicked by disrupting Rad51-BRCA2 interaction. • A virtual screening allowed the identification of a triazole as small molecule PPI inhibitors. • Over 40 novel derivatives have computationally designed, synthesized, and tested. • 26 significantly increase the therapeutic power of olaparib in pancreatic cancer cells. [ABSTRACT FROM AUTHOR]

In light of the emergence of resistance against the currently available EGFR inhibitors, our study focuses on tackling this problem through the development of dual EGFR/HER2 inhibitors with improved enzymatic affinities. Guided by the binding mode of the marketed dual EGFR/HER2 inhibitor, Lapatinib, we proposed the design of dual EGFR/HER2 inhibitors based on the 6-phenylthieno[2,3- d ]pyrimidine as a core scaffold and hinge binder. After two cycles of screening aiming to identify the optimum aniline headgroup and solubilizing group, we eventually identified 27b as a dual EGFR/HER2 inhibitor with IC 50 values of 91.7 nM and 1.2 μM, respectively. Notably, 27b dramatically reduced the viability of various patient-derived cancer cells preferentially overexpressing EGFR/HER2 (A431, MDA-MBA-361 and SKBr3 with IC 50 values of 1.45, 3.5 and 4.83 μM, respectively). Additionally, 27b efficiently thwarted the proliferation of lapatinib-resistant human non-small lung carcinoma (NCI-H1975) cells, harboring T790 M mutation, with IC 50 of 4.2 μM. Consistently, 27b significantly blocked EGF-induced EGFR activation and inactivated its downstream AKT/mTOR/S6 signalling pathway triggering apoptotic cell death in NCI-H1975 cells. The present study presents a promising candidate for further design and development of novel EGFR/HER2 inhibitors capable of overcoming EGFR TKIs resistance. [ABSTRACT FROM AUTHOR]

[Display omitted] Targeting endoplasmic reticulum (ER) stress presents a promising strategy in cancer therapy. We previously reported a series of 1,2,4-oxadiazole derivatives that induced the degradation of EGFR and c-Met which are implicated in tumorigenesis. Based on our previous SAR studies, herein, we report the discovery of EMD37, a novel 1,2,4-oxadiazole derivative, which demonstrated potent anticancer activity against NCI-60 cancer cell lines panel compared to its parent/lead compounds. Anti-proliferative assays revealed preferential cytotoxicity of EMD37 on cancer cells compared to normal cells. Delving deeper, we exploited unbiased genome-wide transcriptome profiling of EMD37-treated cancer cells. Gene Ontology and gene set enrichment analyses revealed that EMD37 promoted ER stress and unfolded protein response (UPR) machinery which was confirmed using RT-qPCR. Mining drug signature databases also confirmed the enrichment of the signature of canonical UPR inducers. Knocking down ER stress transcription factors compromised at least partly the anticancer activity of EMD37. Immunoblot analysis showed that EMD37 induced the accumulation of polyubiquitinated proteins and inhibited mTOR signaling. EMD37 induced G2/M cell cycle arrest and apoptosis of human cancer cells. Inhibiting apoptosis evidently abrogated the anticancer efficacy of EMD37. Altogether, this study introduces EMD37 as a novel ER inducer which warrants further investigation as a potentially relevant anti-cancer therapy. [ABSTRACT FROM AUTHOR]

In the last decades, inhibitors of histone deacetylases (HDAC) have become an important class of anti-cancer agents. In a previous study we described the synthesis of spiro[chromane-2,4′-piperidine]hydroxamic acid derivatives able to inhibit histone deacetylase enzymes. Herein, we present our exploration for new derivatives by replacing the piperidine moiety with various cycloamines. The goal was to obtain highly potent compounds with a good in vitro ADME profile. In addition, molecular modeling studies unravelled the binding mode of these inhibitors. [ABSTRACT FROM AUTHOR]

LSD1 is a histone lysine demethylase proposed as therapeutic target in cancer. Chemical modifications applied at C2, C4 and/or C7 positions of the quinazoline core of the previously reported dual LSD1/G9a inhibitor 1 led to a series of non-covalent, highly active, and selective LSD1 inhibitors (2 – 4 and 6 – 30) and to the dual LSD1/G9a inhibitor 5 that was more potent than 1 against LSD1. In THP-1 and MV4-11 leukemic cells, the most potent compounds (7 , 8 , and 29) showed antiproliferative effects at sub-micromolar level without significant toxicity at 1 μM in non-cancer AHH-1 cells. In MV4-11 cells, the new derivatives increased the levels of the LSD1 histone mark H3K4me2 and induced the re-expression of the CD86 gene silenced by LSD1, thereby confirming the inhibition of LSD1 at cellular level. In breast MDA-MB-231 as well as in rhabdomyosarcoma RD and RH30 cells, taken as examples of solid tumors, the same compounds displayed cell growth arrest in the same IC 50 range, highlighting a crucial anticancer role for LSD1 inhibition and suggesting no added value for the simultaneous G9a inhibition in these tumor cell lines. [Display omitted] • Novel potent and selective non-covalent LSD1 inhibitors are presented. • The crystal structure of LSD1 inhibitor 7 in complex with LSD1-CoREST unveils an unusual binding mode. • The most potent LSD1 inhibitors (7 , 8 and 29) display sub-micromolar antiproliferative effects in leukemia cells. • The same compounds inhibit breast cancer and rhabdomyosarcoma cell proliferation. • Functional assays confirm the inhibition of LSD1 at cellular level in both solid and hematological cancer cell lines. [ABSTRACT FROM AUTHOR]

The pure enantiomers of the N -(2-, 3-, and 4-(2-aminocyclopropyl)phenyl)benzamides hydrochlorides 11a – j were prepared and tested against LSD1 and MAO enzymes. The evaluation of the regioisomers 11a – j highlighted a net increase of the anti-LSD1 potency by shifting the benzamide moiety from ortho to meta and mainly to para position of tranylcypromine phenyl ring, independently from their trans or cis stereochemistry. In particular, the para -substituted 11a,b ( trans ) and 11g,h ( cis ) compounds displayed LSD1 and MAO-A inhibition at low nanomolar levels, while were less potent against MAO-B. The meta analogs 11c,d ( trans ) and 11i,j ( cis ) were in general less potent, but more efficient against MAO-A than against LSD1. In cellular assays, all the para and meta enantiomers were able to inhibit LSD1 by inducing Gfi-1b and ITGAM gene expression, with 11b , c and 11g – i giving the highest effects. Moreover, 11b and 11g,h strongly inhibited the clonogenic potential of murine promyelocytic blasts. [ABSTRACT FROM AUTHOR]

Histone demethylase KDM1A (also known as LSD1) has become an attractive therapeutic target for the treatment of cancer as well as other disorders such as viral infections. We report on the synthesis of compounds derived from the expansion of tranylcypromine as a chemical scaffold for the design of novel demethylase inhibitors. These compounds, which are substituted on the cyclopropyl core moiety, were evaluated for their ability to inhibit KDM1A in vitro as well as to function in cells by modulating the expression of Gfi-1b, a well recognized KDM1A target gene. The molecules were all found to covalently inhibit KDM1A and to become increasingly selective against human monoamine oxidases MAO A and MAO B through the introduction of bulkier substituents on the cyclopropylamine ring. Structural and biochemical analysis of selected trans isomers showed that the two stereoisomers are endowed with similar inhibitory activities against KDM1A, but form different covalent adducts with the FAD co-enzyme. [ABSTRACT FROM AUTHOR]

[Display omitted] • A benzimidazole derivative with nanomolar activity against FLT3 and TrKA kinase. • 4ACP was identified via scaffold hopping & structural simplification of quizartinib. • 4ACP prompted apoptotic and necrotic cell death and G0/G1 cell cycle arrest. • 4ACP exhibited selective antiproliferative profile and low toxicity in normal cells. • 4ACP represents a novel FLT3/TrKA dual kinase inhibitor for targeted therapy of AML. FMS-like tyrosine kinase 3 (FLT3) enzyme overexpression and mutations are the most common molecular abnormalities associated with acute myeloid leukemia (AML). In addition, recent studies investigated the role of tropomyosin receptor kinase A (TrKA) enzyme fusions in promoting AML growth and survival. Based on these premises, targeting both kinases using dual inhibitors would constitute a promising therapeutic approach to target resistant AML. Guided by ligand-based design and structure simplification of the FLT3 inhibitor, quizartinib, we developed a benzimidazole-based small molecule, 4ACP , that exhibited nanomolar activity against wild-type FLT3, FLT3-Internal tandem duplications (FLT3-ITD), and FLT3-D835Y (FLT3-TKD) mutation (IC 50 = 43.8, 97.2, and 92.5 nM respectively). Additionally, 4ACP demonstrated potent activity against colon cancer KM12 cell line (IC 50 = 358 nM) and subsequent mechanistic deconvolution identified TrKA enzyme as a second plausible target (IC 50 = 23.6 nM) for our compound. 4ACP manifested preferential antiproliferative activity against FLT3-ITD positive AML cell lines (MV4-11 IC 50 = 38.8 ± 10.7 nM and MOLM-13 IC 50 = 54.9 ± 4.1 nM), while lacking activity against FLT3-ITD negative AML cell lines. Western blot analysis confirmed 4ACP ability to downregulate ERK1/2 and mTOR signaling downstream of FLT3-ITD in AML cells. Furthermore, 4ACP prompted apoptotic and necrotic cell death and G0/G1 cell cycle arrest as indicated by cell cycle analysis. 4ACP did not show cytotoxic effects on normal BNL and H9c2 cells and demonstrated decreased activity against c-Kit enzyme, hence, indicating lower probability of synthetic lethal toxicity and a relatively safer profile. In light of these data, 4ACP represents a novel FLT3/TrKA dual kinase inhibitor for targeted therapy of AML. [ABSTRACT FROM AUTHOR]

Abstract: Histone Deacetylases (HDACs) have become important targets for the treatment of cancer and other diseases. In previous studies we described the development of novel spirocyclic HDAC inhibitors based on the combination of privileged structures with hydroxamic acid moieties as zinc binding group. Herein, we report further explorations, which resulted in the discovery of a new class of spiro[2H-(1,3)-benzoxazine-2,4′-piperidine] derivatives. Several compounds showed good potency of around 100 nM and less in the HDAC inhibition assays, submicromolar IC50 values when tested against tumour cell lines and a remarkable stability in human and mouse microsomes. Two representative examples exhibited a good pharmacokinetic profile with an oral bioavailability equal or higher than 35% and one of them studied in an HCT116 murine xenograft model showing a robust tumour growth inhibition. In addition, the two benzoxazines were found to have a minor affinity for the hERG potassium channel compared to their corresponding ketone analogues. [Copyright &y& Elsevier]

Abstract: Although epigenetic drugs have been approved for use in selected malignancies, there is significant need for a better understanding of their mechanism of action. Here, we study the action of a clinically approved DNA-methyltransferase inhibitor – decitabine (DAC) – in acute myeloid leukemia (AML) cells. At low doses, DAC treatment induced apoptosis of NB4 Acute Promyelocytic Leukemia (APL) cells, which was associated with the activation of the extrinsic apoptotic pathway. Expression studies of the members of the Death Receptor family demonstrated that DAC induces the expression of TNF-related apoptosis-inducing ligand (TRAIL). Upregulation of TRAIL, upon DAC treatment, was associated with specific epigenetic modifications induced by DAC in the proximity of the TRAIL promoter, as demonstrated by DNA demethylation, increased DNaseI sensitivity and histone acetylation of a non-CpG island, CpG-rich region located 2kb upstream to the transcription start site. Luciferase assay experiments showed that this region behave as a DNA methylation sensitive transcriptional regulatory element. The CpG regulatory element was also found methylated in samples derived from APL patients. These findings have been confirmed in the non-APL, AML Kasumi cell line, suggesting that this regulatory mechanism may be extended to other AMLs. Our study suggests that DNA methylation is a regulatory mechanism relevant for silencing of the TRAIL apoptotic pathway in leukemic cells, and further elucidates the mechanism by which epigenetic drugs mediate their anti-leukemic effects. [Copyright &y& Elsevier]

Summary: It is well established that epigenetic modulation of genome accessibility in chromatin occurs during biological processes. Here we describe a method based on restriction enzymes and next-generation sequencing for identifying accessible DNA elements using a small amount of starting material, and use it to examine myeloid differentiation of primary human CD34+ cells. The accessibility of several classes of cis-regulatory elements was a predictive marker of in vivo DNA binding by transcription factors, and was associated with distinct patterns of histone posttranslational modifications. We also mapped large chromosomal domains with differential accessibility in progenitors and maturing cells. Accessibility became restricted during differentiation, correlating with a decreased number of expressed genes and loss of regulatory potential. Our data suggest that a permissive chromatin structure in multipotent cells is progressively and selectively closed during differentiation, and illustrate the use of our method for the identification of functional cis-regulatory elements. [Copyright &y& Elsevier]

Reactive oxygen species (ROS) and insulin signaling in the adipose tissue are critical determinants of aging and age-associated diseases. It is not clear, however, if they represent independent factors or they are mechanistically linked. We investigated the effects of ROS on insulin signaling using as model system the p66Shc-null mice. p66Shc is a redox enzyme that generates mitochondrial ROS and promotes aging in mammals. We report that insulin activates the redox enzyme activity of p66Shc specifically in adipocytes and that p66Shc-generated ROS regulate insulin signaling through multiple mechanisms, including AKT phosphorylation, Foxo localization, and regulation of selected insulin target genes. Deletion of p66Shc resulted in increased mitochondrial uncoupling and reduced triglyceride accumulation in adipocytes and in vivo increased metabolic rate and decreased fat mass and resistance to diet-induced obesity. In addition, p66Shc-/- mice showed impaired thermo-insulation. These findings demonstrate that p66Shc-generated ROS regulate the effect of insulin on the energetic metabolism in mice and suggest that intracellular oxidative stress might accelerate aging by favoring fat deposition and fat-related disorders. [ABSTRACT FROM AUTHOR]

Atg6Beclin 1 mediates autophagy and endosomal trafficking. We investigated how Atg6 influences replication stress. Combining genetic, genomic, metabolomic, and proteomic approaches, we found that the Vps34-Vps15-Atg6Beclin 1-Vps38UVRAG-phosphatydilinositol-3 phosphate (PtdIns(3)P) axis sensitizes cells to replication stress by favoring the degradation of plasma membrane amino acid (AA) transporters via endosomal trafficking and ESCRT proteins, while the PtdIns(3)P phosphatases Ymr1 and Inp53 promote survival to replication stress by reversing this process. An impaired AA uptake triggers activation of Gcn2, which attenuates protein synthesis by phosphorylating eIF2α. Mec1Atr-Rad53Chk1/Chk2 activation during replication stress further hinders translation efficiency by counteracting eIF2α dephosphorylation through Glc7PP1. AA shortage-induced hyperphosphorylation of eIF2α inhibits the synthesis of 65 stress response proteins, thus resulting in cell sensitization to replication stress, while TORC1 promotes cell survival. Our findings reveal an integrated network mediated by endosomal trafficking, translational control pathways, and checkpoint kinases linking AA availability to the response to replication stress. [Display omitted] • Atg6Beclin 1 affects survival to replication stress by controlling protein translation • Endosomal PtIns3P levels regulate vacuolar degradation of amino acid transporters • Opposing roles of Gcn2 and Torc1 in affecting cell survival during replication stress • Rad53Chk1/Chk2 modulates general amino acid control axis during replication stress Ajazi et al. show a link between endosomal PtdIns(3)P levels, essential amino acid uptake, and translational control pathways that affects cell survival during replication stress. The study reveals a connection between the Vps34-Atg6 axis, Gcn2, Torc1, and the Rad53 DDR kinase. [ABSTRACT FROM AUTHOR]

Summary: Epigenetic changes are common alterations in cancer cells. Here, we have investigated the role of Polycomb group proteins in the establishment and maintenance of the aberrant silencing of tumor suppressor genes during transformation induced by the leukemia-associated PML-RARα fusion protein. We show that in leukemic cells knockdown of SUZ12, a key component of Polycomb repressive complex 2 (PRC2), reverts not only histone modification but also induces DNA demethylation of PML-RARα target genes. This results in promoter reactivation and granulocytic differentiation. Importantly, the epigenetic alterations caused by PML-RARα can be reverted by retinoic acid treatment of primary blasts from leukemic patients. Our results demonstrate that the direct targeting of Polycomb group proteins by an oncogene plays a key role during carcinogenesis. [Copyright &y& Elsevier]

Mammalian homologues of Drosophila Seven in Absentia (SIAHs) target for proteasome-mediated degradation several factors involved in cell growth and tumorigenesis. Here we show that SIAH-1/2 binds and targets for proteasome-mediated degradation the putative tumor suppressor and tripartite motif (TRIM) family member PML, leading to the loss of its transcriptional co-activating properties and a reduction in the number of endogenous PML nuclear bodies. Association with PML requires the substrate-binding domain (SBD) of SIAH-1/2 through an interacting surface apparently distinct from those predicted by the structural studies, or shown experimentally to mediate binding to SIAH-associated factors. Within PML, the coiled-coil domain is required for Siah- and proteasome-mediated degradation, and deletions of regions critical for the integrity of this region impair the ability of Siah to trigger PML-RAR degradation. Fusion of the coiled-coil domain to heterologous proteins resulted in the capacity of mSiah-2 to target their degradation. All of the TRIM proteins tested were degraded upon mSiah-2 overexpression. Finally, we show that the fusion protein PML-RAR (that retains the coiled-coil domain), which causes acute promyelocytic leukemias, is also a potential substrate of mSiah-2. As a result of mSiah-2 overexpression and subsequent degradation of the fusion protein, the arrest in hematopoietic differentiation because of expression of PML-RAR is partially rescued. These results identify PML and other TRIMs as new factors post-translationally regulated by SIAH and involve the coiled-coil region of PML and of other SIAH substrates as a novel structural determinant for targeted degradation. [ABSTRACT FROM AUTHOR]

Chicken ovalbumin upstream promoter transcription factor (COUP-TF)-interacting proteins 1 and 2 (CTIP1 and CTIP2) enhance transcriptional repression mediated by COUP-TF II and have been implicated in hematopoietic cell development and malignancies. CTIP1 and CTIP2 are also sequence-specific DNA-binding proteins that repress transcription through direct, COUP-TF-independent binding to a GC-rich response element. CTIP1- and CTIP2-mediated transcriptional repression is insensitive to trichostatin A, an inhibitor of known class I and II histone deacetylases. However, chromatin immunoprecipitation assays revealed that expression of CTIP2 in mammalian cells resulted in deacetylation of histones H3 and/or H4 that were associated with the promoter region of a reporter gene. CTIP2-mediated transcriptional repression, as well as deacetylation of promoter-associated histones H3/H4 in CTIP2-transfected cells, was reversed by nicotinamide, an inhibitor of class III histone deacetylases such as the mammalian homologs of yeast Silent Information Regulator 2 (Sir2). The human homolog of yeast Sir2, SIRT1, was found to interact directly with CTIP2 and was recruited to the promoter template in a CTIP2-dependent manner. Moreover, SIRT1 enhanced the deacetylation of template-associated histones H3/H4 in CTIP2-transfected cells, and stimulated CTIP2-dependent transcriptional repression. Finally, endogenous SIRT1 and CTIP2 co-purified from Jurkat cell nuclear extracts in the context of a large (1-2 mDa) complex. These findings implicate SIRT1 as a histone H3/H4 deacetylase in mammalian cells and in transcriptional repression mediated by CTIP2. [ABSTRACT FROM AUTHOR]

All-trans-retinoic acid (RA) is a potent inhibitor of leukemia cell proliferation and induces differentiation of acute promyelocytic leukemia cells in vitro and in vivo. For RA to induce its biological effects in target cells, binding to specific retinoic acid nuclear receptors is required. The resulting complexes bind to RA-responsive elements (RAREs) in the promoters of RA-inducible genes to initiate gene transcription and to generate protein products that mediate the biological effects of RA. In this report, we provide evidence that a member of the protein kinase C (PKC) family of proteins, PKCδ, is activated during RA treatment of the NB-4 and HL-60 acute myeloid leukemia cell lines as well as the MCF-7 breast cancer cell line. Such RA-dependent phosphorylation was also observed in primary acute promyelocytic leukemia cells and resulted in activation of the kinase domain of PKCδ. In studies aimed at understanding the functional relevance of PKCδ in the induction of RA responses, we found that pharmacological inhibition of PKCδ (but not of other PKC isoforms) diminished RA-dependent gene transcription via RAREs. On the other hand, overexpression of a constitutively active form of the kinase strongly enhanced RA-dependent gene transcription via RAREs. Gel shift assays and chromatin immunoprecipitation studies demonstrated that PKCδ associated with retinoic acid receptor-α and was present in an RA-inducible protein complex that bound to RAREs. Pharmacological inhibition of PKCδ activity abrogated the induction of cell differentiation and growth inhibition of NB-4 blast cells, demonstrating that its function is required for such effects. Altogether, our data provide strong evidence that PKCδ is activated in an RA-dependent manner and plays a critical role in the generation of the biological effects of RA in malignant cells. [ABSTRACT FROM AUTHOR]

: ObjectiveWe studied the in vitro and in vivo hematopoietic potential of human stem cells residing in muscle tissue collected from adults with head and neck cancer.: Materials and MethodsAdherent muscle cells were cultured in F12 medium with 10% fetal bovine serum and transplanted into immunodeficient mice.: ResultsOn day 12 we obtained a median of 500,000 adherent cells per gram muscle sample. Thy-1, endoglin, HER2/neu, and P1H12 were expressed in the majority of cells. CD34, VEGFR2, c-kit, VCAM-1, and CXCR4 were expressed in 0.5–1.5%, 1–5%, 1–15%, 9–15%, and 30% of cells, respectively. Immunodeficient mice transplanted with fresh muscle cells or less than 500,000 cultured cells showed little or no human engraftment. In mice transplanted with more than 500,000 cultured cells, up to 14% human CD45+ hematopoietic cells (including myeloid and lymphoid subsets) were detected by flow cytometry. Engraftment was confirmed by polymerase chain reaction, Southern blotting, and DNA sequencing. Liver, muscle, and spleen evaluated for human DNA were positive in the majority of mice showing hematopoietic engraftment in the bone marrow. In vivo hematopoietic engraftment potential was maintained in cultured CD45− muscle cells transduced with the green fluorescence protein gene.: ConclusionsHuman stem cells residing in muscle tissue can generate multilineage hematopoiesis in immunodeficient mice. Surprisingly, this hematopoietic potential increased in cultured versus fresh cells from muscle tissue. [Copyright &y& Elsevier]

Tumor cells may adapt to metabolic challenges by alternating between glycolysis and oxidative phosphorylation (OXPHOS). To target this metabolic plasticity, we combined intermittent fasting, a clinically feasible approach to reduce glucose availability, with the OXPHOS inhibitor metformin. In mice exposed to 24-h feeding/fasting cycles, metformin impaired tumor growth only when administered during fasting-induced hypoglycemia. Synergistic anti-neoplastic effects of the metformin/hypoglycemia combination were mediated by glycogen synthase kinase 3β (GSK3β) activation downstream of PP2A, leading to a decline in the pro-survival protein MCL-1, and cell death. Mechanistically, specific activation of the PP2A-GSK3β axis was the sum of metformin-induced inhibition of CIP2A, a PP2A suppressor, and of upregulation of the PP2A regulatory subunit B56δ by low glucose, leading to an active PP2A-B56δ complex with high affinity toward GSK3β. • Metformin plus fasting-induced hypoglycemia synergistically reduces tumor growth • PP2A-GSK3β-MCL-1 axis mediates the synergistic cytotoxicity of the combination • Simultaneous CIP2A inhibition and B56δ upregulation dictate combination specificity Elgendy et al. show that metformin administered during the fasting period synergizes with 24-h feeding/fasting cycles to suppress tumor growth. Inhibition of CIP2A by metformin and upregulation of B56δ by low glucose activates PP2A toward GSK3β leading to reduced pro-survival protein MCL-1 and cell death. [ABSTRACT FROM AUTHOR]