Molecular portraits of cell cycle checkpoint kinases in cancer evolution, progression, and treatment responsiveness.

Cell cycle dysregulation is prerequisite for cancer formation. However, it is unknown whether the mode of dysregulation affects disease characteristics. Here, we conduct comprehensive analyses of cell cycle checkpoint dysregulation using patient data and experimental investigations. We find that ATM mutation predisposes the diagnosis of primary estrogen receptor (ER)+/human epidermal growth factor (HER)2- cancer in older women. Conversely, CHK2 dysregulation induces formation of metastatic, premenopausal ER+/HER2- breast cancer (P = 0.001) that is treatment-resistant (HR = 6.15, P = 0.01). Lastly, while mutations in ATR alone are rare, ATR/TP53 co-mutation is 12-fold enriched over expected in ER+/HER2- disease (P = 0.002) and associates with metastatic progression (HR = 2.01, P = 0.006). Concordantly, ATR dysregulation induces metastatic phenotypes in TP53 mutant, not wild-type, cells. Overall, we identify mode of cell cycle dysregulation as a distinct event that determines subtype, metastatic potential, and treatment responsiveness, providing rationale for reconsidering diagnostic classification through the lens of the mode of cell cycle dysregulation..

Marine Polyether Phycotoxin Palytoxin Induces Apoptotic Cell Death via Mcl-1 and Bcl-2 Downregulation.

Palytoxin is considered one of the most potent biotoxins. As palytoxin-induced cancer cell death mechanisms remain to be elucidated, we investigated this effect on various leukemia and solid tumor cell lines at low picomolar concentrations. As palytoxin did not affect the viability of peripheral blood mononuclear cells (PBMC) from healthy donors and did not create systemic toxicity in zebrafish, we confirmed excellent differential toxicity. Cell death was characterized by a multi-parametric approach involving the detection of nuclear condensation and caspase activation assays. zVAD-sensitive apoptotic cell death was concomitant with a dose-dependent downregulation of antiapoptotic Bcl-2 family proteins Mcl-1 and Bcl-xL. Proteasome inhibitor MG-132 prevented the proteolysis of Mcl-1, whereas the three major proteasomal enzymatic activities were upregulated by palytoxin. Palytoxin-induced dephosphorylation of Bcl-2 further exacerbated the proapoptotic effect of Mcl-1 and Bcl-xL degradation in a range of leukemia cell lines. As okadaic acid rescued cell death triggered by palytoxin, protein phosphatase (PP)2A was involved in Bcl-2 dephosphorylation and induction of apoptosis by palytoxin. At a translational level, palytoxin abrogated the colony formation capacity of leukemia cell types. Moreover, palytoxin abrogated tumor formation in a zebrafish xenograft assay at concentrations between 10 and 30 pM. Altogether, we provide evidence of the role of palytoxin as a very potent and promising anti-leukemic agent, acting at low picomolar concentrations in cellulo and in vivo.

Endocannabinoid dysfunction in neurological disease: neuro-ocular DAGLA-related syndrome.

The endocannabinoid system is a highly conserved and ubiquitous signalling pathway with broad-ranging effects. Despite critical pathway functions, gene variants have not previously been conclusively linked to human disease. We identified nine children from eight families with heterozygous, de novo truncating variants in the last exon of DAGLA with a neuro-ocular phenotype characterized by developmental delay, ataxia and complex oculomotor abnormality. All children displayed paroxysms of nystagmus or eye deviation accompanied by compensatory head posture and worsened incoordination most frequently after waking. RNA sequencing showed clear expression of the truncated transcript and no differences were found between mutant and wild-type DAGLA activity. Immunofluorescence staining of patient-derived fibroblasts and HEK cells expressing the mutant protein showed distinct perinuclear aggregation not detected in control samples. This report establishes truncating variants in the last DAGLA exon as the cause of a unique paediatric syndrome. Because enzymatic activity was preserved, the observed mislocalization of the truncated protein may account for the observed phenotype. Potential mechanisms include DAGLA haploinsufficiency at the plasma membrane or dominant negative effect. To our knowledge, this is the first report directly linking an endocannabinoid system component with human genetic disease and sets the stage for potential future therapeutic avenues.

Raloxifene Suppresses Tumor Growth and Metastasis in an Orthotopic Model of Castration-Resistant Prostate Cancer.

Androgen receptor (AR)-castrate-resistant prostate cancer (CRPC) is an aggressive form of prostate cancer that does not have clinically approved targeted treatment options. To this end, the cytotoxic potential of raloxifene and the synthetic curcumin derivative 2,6-bis (pyridin-4-ylmethylene)-cyclohexanone (RL91) was examined in AR-(PC3 and DU145) cells and AR+ (LnCaP) CRPC cells. The results showed that both raloxifene and RL91 elicited significant cytotoxicity across three cell lines with the lowest EC50 values in PC3 cells. Additionally, the two drugs were synergistically cytotoxic toward the PC3, DU-145 and LNCaP cell lines. To determine the effect of the drug combination in vivo, an orthotopic model of CRPC was used. Male mice were injected with PC3 prostate cancer cells and then treated with vehicle (5 mL/kg), raloxifene (8.5 mg/kg, po), RL91 (8.5 mg/kg, po) or a combination of raloxifene and RL91 for six weeks. Sham animals were subjected to the surgical procedure but were not implanted with PC3 cells. The results showed that raloxifene decreased tumor size and weight as well as metastasis to renal lymph nodes. However, combination treatment reversed the efficacy of raloxifene as tumor volume and metastasis returned to control levels. The results suggest that raloxifene has tumor suppressive and anti-metastatic effects and has potential for further clinical use in AR-CRPC.

The DNA damage repair landscape in Black women with breast cancer.

BACKGROUND: Estrogen receptor positive (ER+) breast cancer is one of the most commonly diagnosed malignancies in women irrespective of their race or ethnicity. While Black women with ER+ breast cancer are 42% more likely to die of their disease than White women, molecular mechanisms underlying this disparate outcome are understudied. Recent studies identify DNA damage repair (DDR) genes as a new class of endocrine therapy resistance driver that contributes to poor survival among ER+ breast cancer patients. Here, we systematically analyze DDR regulation in the tumors and normal breast of Black women and its impact on survival outcome. METHOD: Mutation and up/downregulation of 104 DDR genes in breast tumor and normal samples from Black patients relative to White counterparts was assessed. For DDR genes that were differently regulated in the tumor samples from Black women in multiple datasets associations with survival outcome were tested. RESULTS: Overall, Black patient tumors upregulate or downregulate RNA levels of a wide array of single strand break repair (SSBR) genes relative to their white counterparts and uniformly upregulate double strand break repair (DSBR) genes. This DSBR upregulation was also detectable in samples of normal breast tissue from Black women. Eight candidate DDR genes were reproducibly differently regulated in tumors from Black women and associated with poor survival. A unique DDR signature comprised of simultaneous upregulation of homologous recombination gene expression and downregulation of SSBR genes was enriched in Black patients. This signature associated with cell cycle dysregulation (p < 0.001), a hallmark of endocrine therapy resistance, and concordantly, with significantly worse survival outcomes in all datasets analyzed (hazard ratio of 9.5, p < 0.001). CONCLUSION: These results constitute the first systematic analysis of DDR regulation in Black women and provide strong rationale for refining biomarker profiles to ensure precision medicine for underserved populations.

HER2 Activation and Endocrine Treatment Resistance in HER2-negative Breast Cancer.

The lethality of estrogen receptor alpha positive (ER+) breast cancer, which is often considered to have better prognosis than other subtypes, is defined by resistance to the standard of care endocrine treatment. Relapse and metastasis are inevitable in almost every patient whose cancer is resistant to endocrine treatment. Therefore, understanding the underlying causes of treatment resistance remains an important biological and clinical focus of research in this area. Growth factor receptor pathway activation, specifically HER2 activation, has been identified as 1 mechanism of endocrine treatment resistance across a range of experimental model systems. However, clinical trials conducted to test whether targeting HER2 benefits patients with endocrine treatment-resistant ER+ breast cancer have consistently and disappointingly shown mixed results. One reason for the failure of these clinical trials could be the complexity of crosstalk between ER, HER2, and other growth factor receptors and the fluidity of HER2 activation in these cells, which makes it challenging to identify stratifiers for this targeted intervention. In the absence of stratifiers that can be assayed at diagnosis to allow prospective tailoring of HER2 inhibition to the right patients, clinical trials will continue to disappoint. To understand stratifiers, it is important that the field invests in key understudied areas of research including characterization of the tumor secretome and receptor activation in response to endocrine treatment, and mapping the ER-HER2 growth factor network in the normal and developing mammary gland. Understanding these mechanisms further is critical to improving outcomes for the hard-to-treat endocrine treatment-resistant ER+ breast cancer cohort.

Mismatch repair deficiency predicts response to HER2 blockade in HER2-negative breast cancer.

Resistance to endocrine treatment occurs in ~30% of ER+ breast cancer patients resulting in ~40,000 deaths/year in the USA. Preclinical studies strongly implicate activation of growth factor receptor, HER2 in endocrine treatment resistance. However, clinical trials of pan-HER inhibitors in ER+/HER2- patients have disappointed, likely due to a lack of predictive biomarkers. Here we demonstrate that loss of mismatch repair activates HER2 after endocrine treatment in ER+/HER2- breast cancer cells by protecting HER2 from protein trafficking. Additionally, HER2 activation is indispensable for endocrine treatment resistance in MutL- cells. Consequently, inhibiting HER2 restores sensitivity to endocrine treatment. Patient data from multiple clinical datasets supports an association between MutL loss, HER2 upregulation, and sensitivity to HER inhibitors in ER+/HER2- patients. These results provide strong rationale for MutL loss as a first-in-class predictive marker of sensitivity to combinatorial treatment with endocrine intervention and HER inhibitors in endocrine treatment-resistant ER+/HER2- breast cancer patients.

The HDAC6 inhibitor 7b induces BCR-ABL ubiquitination and downregulation and synergizes with imatinib to trigger apoptosis in chronic myeloid leukemia.

Despite the discovery of tyrosine kinase inhibitors (TKIs) for the treatment of breakpoint cluster region-Abelson (BCR-ABL)+ cancer types, patients with chronic myeloid leukemia (CML) treated with TKIs develop resistance and severe adverse effects. Combination treatment, especially with a histone deacetylase (HDAC) 6 inhibitor (HDAC6i), appears to be an attractive option to prevent TKI resistance, considering the potential capacity of an HDAC6i to diminish BCR-ABL expression. We first validated the in vivo anti-cancer potential of the compound 7b by significantly reducing the tumor burden of BALB/c mice xenografted with K-562 cells, without notable organ toxicity. Here, we hypothesize that the HDAC6i compound 7b can lead to BCR-ABL downregulation in CML cells and sensitize them to TKI treatment. The results showed that combination treatment with imatinib and 7b resulted in strong synergistic caspase-dependent apoptotic cell death and drastically reduced the proportion of leukemia stem cells, whereas this treatment only moderately affected healthy cells. Ultimately, the combination significantly decreased colony formation in a semisolid methylcellulose medium and tumor mass in xenografted zebrafish compared to each compound alone. Mechanistically, the combination induced BCR-ABL ubiquitination and downregulation followed by disturbance of key proteins in downstream pathways involved in CML proliferation and survival. Taken together, our results suggest that an HDAC6i potentiates the effect of imatinib and could overcome TKI resistance in CML cells.

Novel HDAC inhibitor MAKV-8 and imatinib synergistically kill chronic myeloid leukemia cells via inhibition of BCR-ABL/MYC-signaling: effect on imatinib resistance and stem cells.

BACKGROUND: Chronic myeloid leukemia (CML) pathogenesis is mainly driven by the oncogenic breakpoint cluster region-Abelson murine leukemia viral oncogene homolog 1 (BCR-ABL) fusion protein. Since BCR-ABL displays abnormal constitutive tyrosine kinase activity, therapies using tyrosine kinase inhibitors (TKis) such as imatinib represent a major breakthrough for the outcome of CML patients. Nevertheless, the development of TKi resistance and the persistence of leukemia stem cells (LSCs) remain barriers to cure the disease, justifying the development of novel therapeutic approaches. Since the activity of histone deacetylase (HDAC) is deregulated in numerous cancers including CML, pan-HDAC inhibitors may represent promising therapeutic regimens for the treatment of CML cells in combination with TKi. RESULTS: We assessed the anti-leukemic activity of a novel hydroxamate-based pan-HDAC inhibitor MAKV-8, which complied with the Lipinski's "rule of five," in various CML cells alone or in combination with imatinib. We validated the in vitro HDAC-inhibitory potential of MAKV-8 and demonstrated efficient binding to the ligand-binding pocket of HDAC isoenzymes. In cellulo, MAKV-8 significantly induced target protein acetylation, displayed cytostatic and cytotoxic properties, and triggered concomitant ER stress/protective autophagy leading to canonical caspase-dependent apoptosis. Considering the specific upregulation of selected HDACs in LSCs from CML patients, we investigated the differential toxicity of a co-treatment with MAKV-8 and imatinib in CML versus healthy cells. We also showed that beclin-1 knockdown prevented MAKV-8-imatinib combination-induced apoptosis. Moreover, MAKV-8 and imatinib co-treatment synergistically reduced BCR-ABL-related signaling pathways involved in CML cell growth and survival. Since our results showed that LSCs from CML patients overexpressed c-MYC, importantly MAKV-8-imatinib co-treatment reduced c-MYC levels and the LSC population. In vivo, tumor growth of xenografted K-562 cells in zebrafish was completely abrogated upon combined treatment with MAKV-8 and imatinib. CONCLUSIONS: Collectively, the present findings show that combinations HDAC inhibitor-imatinib are likely to overcome drug resistance in CML pathology.

Tetrahydrobenzimidazole TMQ0153 triggers apoptosis, autophagy and necroptosis crosstalk in chronic myeloid leukemia.

By comparing imatinib-sensitive and -resistant chronic myeloid leukemia (CML) cell models, we investigated the molecular mechanisms by which tetrahydrobenzimidazole derivative TMQ0153 triggered caspase-dependent apoptosis at low concentrations accompanied by loss of mitochondrial membrane potential (MMP) and increase of cytosolic free Ca2+ levels. Interestingly, at higher concentrations, TMQ0153 induced necroptotic cell death with accumulation of ROS, both preventable by N-acetyl-L-cysteine (NAC) pretreatment. At necroptosis-inducing concentrations, we observed increased ROS and decreased ATP and GSH levels, concomitant with protective autophagy induction. Inhibitors such as bafilomycin A1 (baf-A1) and siRNA against beclin 1 abrogated autophagy, sensitized CML cells against TMQ0153 and enhanced necroptotic cell death. Importantly, TMQ153-induced necrosis led to cell surface exposure of calreticulin (CRT) and ERp57 as well as the release of extracellular ATP and high mobility group box (HMGB1) demonstrating the capacity of this compound to release immunogenic cell death (ICD) markers. We validated the anti-cancer potential of TMQ0153 by in vivo inhibition of K562 microtumor formation in zebrafish. Taken together, our findings provide evidence that cellular stress and redox modulation by TMQ0153 concentration-dependently leads to different cell death modalities including controlled necrosis in CML cell models.

Human telomerase reverse transcriptase depletion potentiates the growth-inhibitory activity of imatinib in chronic myeloid leukemia stem cells.

Although tyrosine kinase inhibitors (TKIs) revolutionized the management of chronic myeloid leukemia (CML), resistance against TKIs and leukemia stem cell (LSC) persistence remain a clinical concern. Therefore, new therapeutic strategies combining conventional and novel therapies are urgently needed. Since telomerase is involved in oncogenesis and tumor progression but is silent in most human normal somatic cells, it may be an interesting target for CML therapy by selectively targeting cancer cells while minimizing effects on normal cells. Here, we report that hTERT expression is associated with CML disease progression. We also provide evidence that hTERT-deficient K-562 cells do not display telomere shortening and that telomere length is maintained through the ALT pathway. Furthermore, we show that hTERT depletion exerts a growth-inhibitory effect in K-562 cells and potentiates imatinib through alteration of cell cycle progression leading to a senescence-like phenotype. Finally, we demonstrate that hTERT depletion potentiates the imatinib-induced reduction of the ALDH+-LSC population. Altogether, our results suggest that the combination of telomerase and TKI should be considered as an attractive strategy to treat CML patients to eradicate cancer cells and prevent relapse by targeting LSCs.

Hydroxycoumarin OT-55 kills CML cells alone or in synergy with imatinib or Synribo: Involvement of ER stress and DAMP release.

We synthetized and investigated the anti-leukemic potential of the novel cytostatic bis(4-hydroxycoumarin) derivative OT-55 which complied with the Lipinski's rule of 5 and induced differential toxicity in various chronic myeloid leukemia (CML) cell models. OT-55 triggered ER stress leading to canonical, caspase-dependent apoptosis and release of danger associated molecular patterns. Consequently, OT-55 promoted phagocytosis of OT-55-treated CML cells by both murine and human monocyte-derived macrophages. Moreover, OT-55 inhibited tumor necrosis factor α-induced activation of nuclear factor-кB and produced synergistic effects when used in combination with imatinib to inhibit colony formation in vitro and Bcr-Abl+ patient blast xenograft growth in zebrafish. Furthermore, OT-55 synergized with omacetaxine in imatinib-resistant KBM-5 R cells to inhibit the expression of Mcl-1, triggering apoptosis. In imatinib-resistant K562 R cells, OT-55 triggered necrosis and blocked tumor formation in zebrafish in combination with omacetaxine.

Preclinical Assessment of the Bioactivity of the Anticancer Coumarin OT48 by Spheroids, Colony Formation Assays, and Zebrafish Xenografts.

In vitro and in vivo pre-clinical screening of novel therapeutic agents are an essential tool in cancer drug discovery. Although human cancer cell lines respond to therapeutic compounds in 2D (dimensional) monolayer cell cultures, 3D culture systems were developed to understand the efficacy of drugs in more physiologically relevant models. In recent years, a paradigm shift was observed in pre-clinical research to validate the potency of new molecules in 3D culture systems, more precisely mimicking the tumor microenvironment. These systems characterize the disease state in a more physiologically relevant manner and help to gain better mechanistic insight and understanding of the pharmacological potency of a given molecule. Moreover, with the current trend in improving in vivo cancer models, zebrafish has emerged as an important vertebrate model to assess in vivo tumor formation and study the effect of therapeutic agents. Here, we investigated the therapeutic efficacy of hydroxycoumarin OT48 alone or in combination with BH3 mimetics in lung cancer cell line A549 by using three different 3D culture systems including colony formation assays (CFA), spheroid formation assay (SFA) and in vivo zebrafish xenografts.

Natural scaffolds in anticancer therapy and precision medicine.

The diversity of natural compounds is essential for their mechanism of action. The source, structures and structure activity relationship of natural compounds contributed to the development of new classes of chemotherapy agents for over 40 years. The availability of combinatorial chemistry and high-throughput screening has fueled the challenge to identify novel compounds that mimic nature's chemistry and to predict their macromolecular targets. Combining conventional and targeted therapies helped to successfully overcome drug resistance and prolong disease-free survival. Here, we aim to provide an overview of preclinical investigated natural compounds alone and in combination to further improve personalization of cancer treatment.

Cardiac Glycoside Glucoevatromonoside Induces Cancer Type-Specific Cell Death.

Cardiac glycosides (CGs) are natural compounds used traditionally to treat congestive heart diseases. Recent investigations repositioned CGs as potential anticancer agents. To discover novel cytotoxic CG scaffolds, we selected the cardenolide glucoevatromonoside (GEV) out of 46 CGs for its low nanomolar anti-lung cancer activity. GEV presented reduced toxicity toward non-cancerous cell types (lung MRC-5 and PBMC) and high-affinity binding to the Na+/K+-ATPase α subunit, assessed by computational docking. GEV-induced cell death was caspase-independent, as investigated by a multiparametric approach, and culminates in severe morphological alterations in A549 cells, monitored by transmission electron microscopy, live cell imaging and flow cytometry. This non-canonical cell death was not preceded or accompanied by exacerbation of autophagy. In the presence of GEV, markers of autophagic flux (e.g. LC3I-II conversion) were impacted, even in presence of bafilomycin A1. Cell death induction remained unaffected by calpain, cathepsin, parthanatos, or necroptosis inhibitors. Interestingly, GEV triggered caspase-dependent apoptosis in U937 acute myeloid leukemia cells, witnessing cancer-type specific cell death induction. Differential cell cycle modulation by this CG led to a G2/M arrest, cyclin B1 and p53 downregulation in A549, but not in U937 cells. We further extended the anti-cancer potential of GEV to 3D cell culture using clonogenic and spheroid formation assays and validated our findings in vivo by zebrafish xenografts. Altogether, GEV shows an interesting anticancer profile with the ability to exert cytotoxic effects via induction of different cell death modalities.

The dialkyl resorcinol stemphol disrupts calcium homeostasis to trigger programmed immunogenic necrosis in cancer.

Stemphol (STP) is a novel druggable phytotoxin triggering mixed apoptotic and non-apoptotic necrotic-like cell death in human acute myeloid leukemia (AML). Use of several chemical inhibitors highlighted that STP-induced non-canonical programmed cell death was Ca2+-dependent but independent of caspases, poly (ADP-ribose) polymerase-1, cathepsin, or calpains. Similar to thapsigargin, STP led to increased cytosolic Ca2+ levels and computational docking confirmed binding of STP within the thapsigargin binding pocket of the sarco/endoplasmic reticulum (ER) Ca2+-ATPase (SERCA). Moreover, the inositol 1,4,5-trisphosphate receptor is implicated in STP-modulated cytosolic Ca2+ accumulation leading to ER stress and mitochondrial swelling associated with collapsed cristae as observed by electron microscopy. Confocal fluorescent microscopy allowed identifying mitochondrial Ca2+ overload as initiator of STP-induced cell death insensitive to necrostatin-1 or cycloheximide. Finally, we observed that STP-induced necrosis is dependent of mitochondrial permeability transition pore (mPTP) opening. Importantly, the translational immunogenic potential of STP was validated by HMGB1 release of STP-treated AML patient cells. STP reduced colony and in vivo tumor forming potential and impaired the development of AML patient-derived xenografts in zebrafish.

Tubulin-binding anticancer polysulfides induce cell death via mitotic arrest and autophagic interference in colorectal cancer.

Polysulfanes show chemopreventive effects against gastrointestinal tumors. We identified diallyl tetrasulfide and its derivative, dibenzyl tetrasulfide (DBTTS), to be mitotic inhibitors and apoptosis inducers. Here, we translate their application in colorectal cancer (CRC). MALDI-TOF-MS analysis identified both compounds as reversible tubulin binders, validated by in cellulo α-tubulin degradation. BRAF(V600E)-mutated HT-29 cells were resistant to DBTTS, as evidenced by mitotic arrest for 48 h prior to apoptosis induction compared to KRAS(G12V)-mutated SW480/620 cells, which committed to death earlier. The prolonged mitotic block correlated with autophagy impairment and p62 protein accumulation in HT-29 but not in SW480/620 cells, whereas siRNA-mediated p62 inhibition sensitized HT-29 cells to death. In silico analysis with 484 colorectal cancer patients associated higher p62 expression and reduced autophagic flux with greater overall survival. Accordingly, we hypothesized that DBTTS targets CRC survival/death through autophagy interference in cell types with differential autophagic capacities. We confirmed the therapeutic potential of DBTTS by the inhibition of spheroid and colony formation capacities in CRC cells, as well as in HT-29 zebrafish xenografts in vivo.

Discovery and Characterization of R/S-N-3-Cyanophenyl-N'-(6-tert-butoxycarbonylamino-3,4-dihydro-2,2-dimethyl-2H-1-benzopyran-4-yl)urea, a New Histone Deacetylase Class III Inhibitor Exerting Antiproliferative Activity against Cancer Cell Lines.

A new series of N-aryl-N'-3,4-dihydro-2,2-dimethyl-2H-1-benzopyran-4-yl)ureas bearing an alkoxycarbonylamino group at the 6-position were synthesized and examined as putative anticancer agents targeting sirtuins in glioma cells. On the basis of computational docking combined to in vitro sirtuin 1/2 inhibition assays, we selected compound 18 [R/S-N-3-cyanophenyl-N'-(6-tert-butoxycarbonylamino-3,4-dihydro-2,2-dimethyl-2H-1-benzopyran-4-yl)urea] which displays a potent antiproliferative activity on various glioma cell types, assessed by quantitative videomicroscopy, eventually triggering senescence. The impact on normal glial cells was lower with a selectivity index of >10. Furthermore, human U373 and Hs683 glioblastoma cell lines served to demonstrate the inhibitory activity of 18 against histone deacetylase (HDAC) class III sirtuins 1 and 2 (SIRT1/2) by quantifying acetylation levels of histone and non-histone proteins. The translational potential of 18 was validated by an NCI-60 cell line screen and validation of growth inhibition of drug resistant cancer cell models. Eventually, the anticancer potential of 18 was validated in 3D glioblastoma spheroids and in vivo by zebrafish xenografts. In summary, compound 18 is the first representative of a new class of SIRT inhibitors opening new perspectives in the medicinal chemistry of HDAC inhibitors.

Discovery and characterization of Isofistularin-3, a marine brominated alkaloid, as a new DNA demethylating agent inducing cell cycle arrest and sensitization to TRAIL in cancer cells.

We characterized the brominated alkaloid Isofistularin-3 (Iso-3), from the marine sponge Aplysina aerophoba, as a new DNA methyltransferase (DNMT)1 inhibitor. Docking analysis confirmed our in vitro DNMT inhibition data and revealed binding of Iso-3 within the DNA binding site of DNMT1. Subsequent increased expression of tumor suppressor gene aryl hydrocarbon receptor (AHR) could be correlated to decreased methylation of CpG sites within the essential Sp1 regulatory region of its promoter. Iso-3 induced growth arrest of cancer cells in G0/G1 concomitant with increased p21 and p27 expression and reduced cyclin E1, PCNA and c-myc levels. Reduced proliferation was accompanied by morphological changes typical of autophagy revealed by fluorescent and transmission electron microscopy and validated by LC3I-II conversion. Furthermore, Iso-3 strongly synergized with tumor-necrosis-factor related apoptosis inducing ligand (TRAIL) in RAJI [combination index (CI) = 0.22] and U-937 cells (CI = 0.21) and increased TRAIL-induced apoptosis via a mechanism involving reduction of survivin expression but not of Bcl-2 family proteins nor X-linked inhibitor of apoptosis protein (XIAP). Iso-3 treatment decreased FLIPL expression and triggered activation of endoplasmatic reticulum (ER) stress with increased GRP78 expression, eventually inducing TRAIL receptor death receptor (DR)5 surface expression. Importantly, as a potential candidate for further anticancer drug development, Iso-3 reduced the viability, colony and in vivo tumor forming potential without affecting the viability of PBMCs from healthy donors or zebrafish development.