Dual Targeting of Chromatin Stability By The Curaxin CBL0137 and Histone Deacetylase Inhibitor Panobinostat Shows Significant Preclinical Efficacy in Neuroblastoma.

PURPOSE: We investigated whether targeting chromatin stability through a combination of the curaxin CBL0137 with the histone deacetylase (HDAC) inhibitor, panobinostat, constitutes an effective multimodal treatment for high-risk neuroblastoma. EXPERIMENTAL DESIGN: The effects of the drug combination on cancer growth were examined in vitro and in animal models of MYCN-amplified neuroblastoma. The molecular mechanisms of action were analyzed by multiple techniques including whole transcriptome profiling, immune deconvolution analysis, immunofluorescence, flow cytometry, pulsed-field gel electrophoresis, assays to assess cell growth and apoptosis, and a range of cell-based reporter systems to examine histone eviction, heterochromatin transcription, and chromatin compaction. RESULTS: The combination of CBL0137 and panobinostat enhanced nucleosome destabilization, induced an IFN response, inhibited DNA damage repair, and synergistically suppressed cancer cell growth. Similar synergistic effects were observed when combining CBL0137 with other HDAC inhibitors. The CBL0137/panobinostat combination significantly delayed cancer progression in xenograft models of poor outcome high-risk neuroblastoma. Complete tumor regression was achieved in the transgenic Th-MYCN neuroblastoma model which was accompanied by induction of a type I IFN and immune response. Tumor transplantation experiments further confirmed that the presence of a competent adaptive immune system component allowed the exploitation of the full potential of the drug combination. CONCLUSIONS: The combination of CBL0137 and panobinostat is effective and well-tolerated in preclinical models of aggressive high-risk neuroblastoma, warranting further preclinical and clinical investigation in other pediatric cancers. On the basis of its potential to boost IFN and immune responses in cancer models, the drug combination holds promising potential for addition to immunotherapies.

A Functional Genomic Screen Identifies the Deubiquitinase USP11 as a Novel Transcriptional Regulator of ERα in Breast Cancer.

Approximately 70% of breast cancers express estrogen receptor α (ERα) and depend on this key transcriptional regulator for proliferation and differentiation. While patients with this disease can be treated with targeted antiendocrine agents, drug resistance remains a significant issue, with almost half of patients ultimately relapsing. Elucidating the mechanisms that control ERα function may further our understanding of breast carcinogenesis and reveal new therapeutic opportunities. Here, we investigated the role of deubiquitinases (DUB) in regulating ERα in breast cancer. An RNAi loss-of-function screen in breast cancer cells targeting all DUBs identified USP11 as a regulator of ERα transcriptional activity, which was further validated by assessment of direct transcriptional targets of ERα. USP11 expression was induced by estradiol, an effect that was blocked by tamoxifen and not observed in ERα-negative cells. Mass spectrometry revealed a significant change to the proteome and ubiquitinome in USP11-knockdown (KD) cells in the presence of estradiol. RNA sequencing in LCC1 USP11-KD cells revealed significant suppression of cell-cycle-associated and ERα target genes, phenotypes that were not observed in LCC9 USP11-KD, antiendocrine-resistant cells. In a breast cancer patient cohort coupled with in silico analysis of publicly available cohorts, high expression of USP11 was significantly associated with poor survival in ERα-positive (ERα+) patients. Overall, this study highlights a novel role for USP11 in the regulation of ERα activity, where USP11 may represent a prognostic marker in ERα+ breast cancer. SIGNIFICANCE: A newly identified role for USP11 in ERα transcriptional activity represents a novel mechanism of ERα regulation and a pathway to be exploited for the management of ER-positive breast cancer.

Replication stress induces mitotic death through parallel pathways regulated by WAPL and telomere deprotection.

Mitotic catastrophe is a broad descriptor encompassing unclear mechanisms of cell death. Here we investigate replication stress-driven mitotic catastrophe in human cells and identify that replication stress principally induces mitotic death signalled through two independent pathways. In p53-compromised cells we find that lethal replication stress confers WAPL-dependent centromere cohesion defects that maintain spindle assembly checkpoint-dependent mitotic arrest in the same cell cycle. Mitotic arrest then drives cohesion fatigue and triggers mitotic death through a primary pathway of BAX/BAK-dependent apoptosis. Simultaneously, a secondary mitotic death pathway is engaged through non-canonical telomere deprotection, regulated by TRF2, Aurora B and ATM. Additionally, we find that suppressing mitotic death in replication stressed cells results in distinct cellular outcomes depending upon how cell death is averted. These data demonstrate how replication stress-induced mitotic catastrophe signals cell death with implications for cancer treatment and cancer genome evolution.

Therapeutic Rationale to Target Highly Expressed CDK7 Conferring Poor Outcomes in Triple-Negative Breast Cancer.

Triple-negative breast cancer (TNBC) patients commonly exhibit poor prognosis and high relapse after treatment, but there remains a lack of biomarkers and effective targeted therapies for this disease. Here, we report evidence highlighting the cell-cycle-related kinase CDK7 as a driver and candidate therapeutic target in TNBC. Using publicly available transcriptomic data from a collated set of TNBC patients (n = 383) and the METABRIC TNBC dataset (n = 217), we found CDK7 mRNA levels to be correlated with patient prognosis. High CDK7 protein expression was associated with poor prognosis within the RATHER TNBC cohort (n = 109) and the METABRIC TNBC cohort (n = 203). The highly specific CDK7 kinase inhibitors, BS-181 and THZ1, each downregulated CDK7-mediated phosphorylation of RNA polymerase II, indicative of transcriptional inhibition, with THZ1 exhibiting 500-fold greater potency than BS-181. Mechanistic investigations revealed that the survival of MDA-MB-231 TNBC cells relied heavily on the BCL-2/BCL-XL signaling axes in cells. Accordingly, we found that combining the BCL-2/BCL-XL inhibitors ABT-263/ABT199 with the CDK7 inhibitor THZ1 synergized in producing growth inhibition and apoptosis of human TNBC cells. Collectively, our results highlight elevated CDK7 expression as a candidate biomarker of poor prognosis in TNBC, and they offer a preclinical proof of concept for combining CDK7 and BCL-2/BCL-XL inhibitors as a mechanism-based therapeutic strategy to improve TNBC treatment. Cancer Res; 77(14); 3834-45. ©2017 AACR.

A high through-put screen for small molecules modulating MCM2 phosphorylation identifies Ryuvidine as an inducer of the DNA damage response.

DNA replication is an essential process for cell division and as such it is a process that is directly targeted by several anticancer drugs. CDC7 plays an essential role in the activation of replication origins and has recently been proposed as a novel target for drug discovery. The MCM DNA helicase complex (MCM2-7) is a key target of the CDC7 kinase, and MCM phosphorylation status at specific sites is a reliable biomarker of CDC7 cellular activity. In this work we describe a cell-based assay that utilizes the "In Cell Western Technique" (ICW) to identify compounds that affect cellular CDC7 activity. By screening a library of approved drugs and kinase inhibitors we found several compounds that can affect CDC7-dependent phosphorylation of MCM2 in HeLa cells. Among these, Mitoxantrone, a topoisomerase inhibitor, and Ryuvidine, previously described as a CDK4 inhibitor, cause a reduction in phosphorylated MCM2 levels and a sudden blockade of DNA synthesis that is accompanied by an ATM-dependent checkpoint response. This study sheds light on the previously observed cytotoxity of Ryuvidine, strongly suggesting that it is related to its effect of causing DNA damage.

The anti-cancer activity of a cationic anti-microbial peptide derived from monomers of polyhydroxyalkanoate.

The biodegradable polymer medium chain length polyhydroxyalkanoate (mclPHA), produced by Pseudomonas putida CA-3, was depolymerised and the predominant monomer (R)-3-hydroxydecanoic acid (R10) purified. R10 was conjugated to a d-peptide DP18 and its derivatives. All peptides conjugated with R10 exhibited greater anti-cancer activity compared to the unconjugated peptides. Unconjugated and conjugated peptides were cytocidal for cancer cells. Conjugation of R10 to peptides was essential for enhanced anti-proliferation activity, as unconjugated mixes did not result in enhancement of anti-cancer activity. The conjugation of R10 resulted in more rapid uptake of peptides into HeLa and MiaPaCa cells compared to unconjugated peptide. Both unconjugated and R10 conjugated peptides localized to the mitochondria of HeLa and MiaPaCa cells and induced apoptosis. Peptide conjugated with a terminally hydroxylated decanoic acid (ω-hydroxydecanoic acid) exhibited 3.3 and 6.3 fold higher IC(50) values compared to R10 conjugated peptide indicating a role for the position of the hydroxyl moiety in enhancement of anti-cancer activity. Conjugation of decanoic acid (C10) to peptides resulted in similar or higher IC(50) values compared to R10 conjugates but C10 conjugates did not exhibit any cancer selectivity. Combination studies showed that R10DP18L exhibited synergy with cisplatin, gemcitabine, and taxotere with IC(50) values in the nanomolar range.

Mechanism of cell death mediated by a BF2-chelated tetraaryl-azadipyrromethene photodynamic therapeutic: dissection of the apoptotic pathway in vitro and in vivo.

Photodynamic therapy (PDT) is an established treatment modality for cancer. ADPM06 is an emerging non-porphyrin PDT agent which has been specifically designed for therapeutic application. Recently, we have demonstrated that ADPM06-PDT is well tolerated in vivo and elicits impressive complete response rates in various models of cancer when a short drug-light interval is applied. Herein, the mechanism of action of ADPM06-PDT in vitro and in vivo is outlined. Using a drug and light combination that reduces the clonogenicity of MDA-MB-231 cells by >90%, we detected a well-orchestrated apoptotic response accompanied by the activation of various caspases in vitro. The generation of reactive oxygen species (ROS) upon photosensitizer irradiation was found to be the key instigator in the observed apoptotic response, with the endoplasmic reticulum (ER) found to be the intracellular site of initial PDT damage, as determined by induction of a rapid ER stress response post-PDT. PDT-induced apoptosis was also found to be independent of p53 tumor suppressor status. A robust therapeutic response in vivo was demonstrated, with a substantial reduction in tumor proliferation observed, as well as a rapid induction of apoptosis and initiation of ER stress, mirroring numerous aspects of the mechanism of action of ADPM06 in vitro. Finally, using a combination of (18) F-labeled 3'-deoxy-3'-fluorothymidine ((18) F-FLT) nuclear and optical imaging, a considerable decrease in tumor proliferation over 24-hr in two models of human cancer was observed. Taken together, this data clearly establishes ADPM06 as an exciting novel PDT agent with significant potential for further translational development.

The hydroxylase inhibitor dimethyloxallyl glycine attenuates endotoxic shock via alternative activation of macrophages and IL-10 production by B1 cells.

Localized tissue hypoxia is a feature of infection and inflammation, resulting in the upregulation of the transcription factors hypoxia-inducible factor 1α and nuclear factor κB (NF-κB) via inhibition of oxygen sensing hydroxylase enzymes. Previous studies have demonstrated a beneficial role for the hydroxylase inhibitor dimethyloxallyl glycine (DMOG) in inflammatory conditions, including experimental colitis, by regulating the activity of hypoxia-inducible factor 1 and NF-κB. We have demonstrated in vivo that pretreatment with DMOG attenuates systemic LPS-induced activation of the NF-κB pathway. Furthermore, mice treated with DMOG had significantly increased survival in LPS-induced shock. Conversely, in models of polymicrobial sepsis, DMOG exacerbates disease severity. Dimethyloxallyl glycine treatment of mice promotes M2 polarization in macrophages within the peritoneal cavity, resulting in the downregulation of proinflammatory cytokines such as TNF-α. In addition, in vivo DMOG treatment upregulates IL-10 expression, specifically in the peritoneal B1 cell population. This study demonstrates cell type-specific roles for hydroxylase inhibition in vivo and provides insight into the mechanism underlying the protection conveyed by DMOG in models of endotoxic shock.

Synergistic interaction between trastuzumab and EGFR/HER-2 tyrosine kinase inhibitors in HER-2 positive breast cancer cells.

Overexpression of HER-2 in breast cancer is frequently associated with expression of EGFR, and EGFR expression influences response to HER-2 inhibition. The aim of this study was to examine the effects of combining dual inhibition of EGFR and HER-2, using trastuzumab, gefitinib and lapatinib, in HER-2 overexpressing breast cancer cells. Combination proliferation assays were performed in two HER-2 positive breast cancer cell lines, SKBR-3 and BT-474. Trastuzumab combined with lapatinib was also tested in BT-474 xenografts. In proliferation assays, dual targeting with trastuzumab and gefitinib or lapatinib showed synergy or additivity in both SKBR-3 and BT-474 cells. Trastuzumab (10 nM) or gefitinib (5 µM) alone did not induce significant apoptosis, whereas lapatinib (0.75 µM) induced significant apoptosis in SKBR-3 cells. Trastuzumab combined with lapatinib further enhanced apoptosis induction. Trastuzumab (10 nM) and gefitinib (5 µM) induced apoptosis comparable to lapatinib alone (0.75 µM), suggesting that inhibition of both EGFR and HER-2 may be required to induce apoptosis in these cells. Pre-treatment with trastuzumab and gefitinib or lapatinib enhanced response to chemotherapy in vitro. The combination of trastuzumab and lapatinib also effectively blocked tumour growth in vivo. Dual targeting of EGFR and HER-2, by combining trastuzumab with EGFR/HER-2 tyrosine kinase inhibitors, may improve response in HER-2 overexpressing breast cancer cells that also express EGFR.

Recent advances in molecular imaging biomarkers in cancer: application of bench to bedside technologies.

Molecular imaging is the visualization, characterization and measurement of biological processes at the molecular and cellular level. In oncology, molecular imaging approaches can be directly applied as translational biomarkers of disease progression. In this article, selected imaging modalities are discussed with respect to this role. Recent studies focusing on emerging imaging biomarkers and new developments in the field are highlighted. Importantly, because ex vivo or tissue-based imaging now represents an important tool in the discovery and validation of oncology biomarkers, special attention is given to this resurgent field.

Porphyrin and nonporphyrin photosensitizers in oncology: preclinical and clinical advances in photodynamic therapy.

Photodynamic therapy (PDT) is now a well-recognized modality for the treatment of cancer. While PDT has developed progressively over the last century, great advances have been observed in the field in recent years. The concept of dual selectivity of PDT agents is now widely accepted due to the relative specificity and selectivity of PDT along with the absence of harmful side effects often encountered with chemotherapy or radiotherapy. Traditionally, porphyrin-based photosensitizers have dominated the PDT field but these first generation photosensitizers have several disadvantages, with poor light absorption and cutaneous photosensitivity being the predominant side effects. As a result, the requirement for new photosensitizers, including second generation porphyrins and porphyrin derivatives as well as third generation photosensitizers has arisen, with the aim of alleviating the problems encountered with first generation porphyrins and improving the efficacy of PDT. The investigation of nonporphyrin photosensitizers for the development of novel PDT agents has been considerably less extensive than porphyrin-based compounds; however, structural modification of nonporphyrin photosensitizers has allowed for manipulation of the photochemotherapeutic properties. The aim of this review is to provide an insight into PDT photosensitizers clinically approved for application in oncology, as well as those which show significant potential in ongoing preclinical studies.