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Developing novel therapeutics often follows three steps: target identification, design of strategies to suppress target activity and drug development to implement the strategies. In this review, we recount the evidence identifying the basic leucine zipper transcription factors ATF5, CEBPB, and CEBPD as targets for brain and other malignancies. We describe strategies that exploit the structures of the three factors to create inhibitory dominant-negative (DN) mutant forms that selectively suppress growth and survival of cancer cells. We then discuss and compare four peptides (CP-DN-ATF5, Dpep, Bpep and ST101) in which DN sequences are joined with cell-penetrating domains to create drugs that pass through tissue barriers and into cells. The peptide drugs show both efficacy and safety in suppressing growth and in the survival of brain and other cancers in vivo, and ST101 is currently in clinical trials for solid tumors, including GBM. We further consider known mechanisms by which the peptides act and how these have been exploited in rationally designed combination therapies. We additionally discuss lacunae in our knowledge about the peptides that merit further research. Finally, we suggest both short- and long-term directions for creating new generations of drugs targeting ATF5, CEBPB, CEBPD, and other transcription factors for treating brain and other malignancies.

Aberrant expression of protein arginine methyltransferases (PRMTs) has been implicated in a number of cancers, making PRMTs potential therapeutic targets. But it remains not well understood how PRMTs impact specific oncogenic pathways. We previously identified PRMTs as important regulators of cell growth in neuroblastoma, a deadly childhood tumor of the sympathetic nervous system. Here, we demonstrate a critical role for PRMT1 in neuroblastoma cell survival. PRMT1 depletion decreased the ability of murine neuroblastoma sphere cells to grow and form spheres, and suppressed proliferation and induced apoptosis of human neuroblastoma cells. Mechanistic studies reveal the prosurvival factor, activating transcription factor 5 (ATF5) as a downstream effector of PRMT1-mediated survival signaling. Furthermore, a diamidine class of PRMT1 inhibitors exhibited anti-neuroblastoma efficacy both in vitro and in vivo. Importantly, overexpression of ATF5 rescued cell apoptosis triggered by PRMT1 inhibition genetically or pharmacologically. Taken together, our findings shed new insights into PRMT1 signaling pathway, and provide evidence for PRMT1 as an actionable therapeutic target in neuroblastoma.

AMP-activated protein kinase (AMPK) is a eukaryotic energy sensor and protector from mitochondrial/energetic stress that is also a therapeutic target for cancer and metabolic disease. Metformin is an AMPK inducer that has been used in cancer therapeutic trials. Through screening we isolated cetylpyridinium chloride (CPC), a drug known to dose-dependently inhibit mitochondrial complex 1, as a potent and dose-dependent AMPK stimulator. Mitochondrial biogenesis and bioenergetics changes have also been implicated in glioblastoma, which is the most aggressive form of brain tumors. Cetylpyridinium chloride has been administered in humans as a safe drug-disinfectant for several decades, and we report here that under in vitro conditions, cetylpyridinium chloride kills glioblastoma cells in a dose dependent manner at a higher efficacy compared to current standard of care drug, temozolomide.

Simple Summary The gene-regulatory factors ATF5, CEBPB and CEBPD promote survival, growth, metastasis and treatment resistance of a range of cancer cell types. Presently, no drugs target all three at once. Here, with the aim of treating cancers, we designed novel cell-penetrating peptides that interact with and inactivate all three. The peptides Bpep and Dpep kill a range of cancer cell types in culture and in animals. In animals with tumors, they also significantly increase survival time. In contrast, they do not affect survival of non-cancer cells and have no apparent side effects in animals. The peptides work in combination with other anti-cancer treatments. Mechanism studies of how the peptides kill cancer cells indicate a decrease in survival proteins and increase in death proteins. These studies support the potential of Bpep and Dpep as novel, safe agents for the treatment of a variety of cancer types, both as mono- and combination therapies. Abstract Transcription factors are key players underlying cancer formation, growth, survival, metastasis and treatment resistance, yet few drugs exist to directly target them. Here, we characterized the in vitro and in vivo anti-cancer efficacy of novel synthetic cell-penetrating peptides (Bpep and Dpep) designed to interfere with the formation of active leucine-zipper-based dimers by CEBPB and CEBPD, transcription factors implicated in multiple malignancies. Both peptides similarly promoted apoptosis of multiple tumor lines of varying origins, without such effects on non-transformed cells. Combined with other treatments (radiation, Taxol, chloroquine, doxorubicin), the peptides acted additively to synergistically and were fully active on Taxol-resistant cells. The peptides suppressed expression of known direct CEBPB/CEBPD targets IL6, IL8 and asparagine synthetase (ASNS), supporting their inhibition of transcriptional activation. Mechanisms by which the peptides trigger apoptosis included depletion of pro-survival survivin and a required elevation of pro-apoptotic BMF. Bpep and Dpep significantly slowed tumor growth in mouse models without evident side effects. Dpep significantly prolonged survival in xenograft models. These findings indicate the efficacy and potential of Bpep and Dpep as novel agents to treat a variety of cancers as mono- or combination therapies.

Glioblastoma (GBM) is an aggressive tumor of the brain, with an average post-diagnosis survival of 15 months. GBM stem cells (GBMSC) resist the standard-of-care therapy, temozolomide, and are considered a major contributor to tumor resistance. Mammalian target of rapamycin Complex 1 (mTORC1) regulates cell proliferation and has been shown by others to have reduced activity in GBMSC. We recently identified a novel chemical series of human-safe piperazine-based brain-penetrant mTORC1-specific inhibitors. We assayed the piperazine-mTOR binding strength by two biophysical measurements, biolayer interferometry and field-effect biosensing, and these confirmed each other and demonstrated a structure&ndash, activity relationship. As mTORC1 is altered in human GBMSC, and as mTORC1 inhibitors have been tested in previous GBM clinical trials, we tested the killing potency of the tightest-binding piperazines and observed that these were potent GBMSC killers. GBMSCs are resistant to the standard-of-care temozolomide therapy, but temozolomide supplemented with tight-binding piperazine meclizine and flunarizine greatly enhanced GBMSC death over temozolomide alone. Lastly, we investigated IDH1-mutated GBMSC mutations that are known to affect mitochondrial and mTORC1 metabolism, and the tight-binding meclizine provoked &lsquo, synthetic lethality&rsquo, in IDH1-mutant GBMSCs. In other words, IDH1-mutated GBMSC showed greater sensitivity to the coadministration of temozolomide and meclizine. These data tend to support a novel clinical strategy for GBM, i.e., the co-administration of meclizine or flunarizine as adjuvant therapy in the treatment of GBM and IDH1-mutant GBM.

Increasing efforts are ongoing to deliver effective cancer care through integrated networks of services. Measuring patients' experience of care is essential to identify problematic areas that require organisational adjustments. The aim of the present study was to examine the validity of OPTION questionnaire, designed to measure patient's perceived continuity of care across different phases of their care pathway. The study was carried at the Institute for Cancer Treatment and Research, Meldola and the oncology departments of the Local Health Authority of Romagna, Italy. Principal component analysis (PCA) was performed to identify factors underlying patients' perception of continuity of care. Factor scores were compared between patients with or without a care coordinator using Mann-Whitney test. The study sample consisted of 214 patients with breast or colorectal cancer, with a mean age of 62.3 years. Most patients identified the oncologist as their care coordinator. Five factors were extracted using PCA: (1) "trustful relationship with health care staff," (2) "information on care pathway," (3) "information on changes related to the illness," (4) "feelings of abandonment" and (5) "collaboration among health care professionals." The scores of factors 2 and 3 were significantly higher among those with a care coordinator. The OPTION questionnaire is a reliable instrument that can help clinicians and administrative stakeholder target efforts and resources in the pursuit of quality of care.

Survivin (BIRC5, product of the BIRC5 gene) is highly expressed in many tumor types and has been widely identified as a potential target for cancer therapy. However, effective anti-survivin drugs remain to be developed. Here we report that both vector-delivered and cell-penetrating dominant-negative (dn) forms of the transcription factor ATF5 that promote selective death of cancer cells in vitro and in vivo cause survivin depletion in tumor cell lines of varying origins. dn-ATF5 decreases levels of both survivin mRNA and protein. The depletion of survivin protein appears to be driven at least in part by enhanced proteasomal turnover and depletion of the deubiquitinase USP9X. Survivin loss is rapid and precedes the onset of cell death triggered by dn-ATF5. Although survivin downregulation is sufficient to drive tumor cell death, survivin over-expression does not rescue cancer cells from dn-ATF5-promoted apoptosis. This indicates that dn-ATF5 kills malignant cells by multiple mechanisms that include, but are not limited to, survivin depletion. Cell-penetrating forms of dn-ATF5 are currently being developed for potential therapeutic use and the present findings suggest that they may pose an advantage over treatments that target only survivin.

The basic leucine zipper transcription factor ATF5 is overexpressed in many tumor types and interference with its expression or function inhibits cancer cell survival. As a potential therapeutic approach to exploit these findings, we created dominant-negative (DN) ATF5 forms lacking DNA-binding ability that retain the ATF5 leucine zipper, and thus associate with and sequester ATF5's requisite leucine zipper–binding partners. Preclinical studies with DN-ATF5, including a cell-penetrating form, show in vitro and in vivo efficacy in compromising cancer cell survival. However, DN-ATF5's targets, and particularly those required for tumor cell survival, have been unknown. We report that cells lacking ATF5 succumb to DN-ATF5, indicating that ATF5 itself is not DN-ATF5's obligate target. Unbiased pull-down assays coupled with mass spectrometry and immunoblotting revealed that DN-ATF5 associates in cells with the basic leucine zipper proteins CEBPB and CEBPD and coiled-coil protein CCDC6. Consistent with DN-ATF5 affecting tumor cell survival by suppressing CEBPB and CEBPD function, DN-ATF5 interferes with CEBPB and CEBPD transcriptional activity, while CEBPB or CEBPD knockdown promotes apoptotic death of multiple cancer cells lines, but not of normal astrocytes. We propose a two-pronged mechanism by which DN-ATF5 kills tumor cells. One is by inhibiting heterodimer formation between ATF5 and CEBPB and CDBPD, thus suppressing ATF5-dependent transcription. The other is by blocking the formation of transcriptionally active CEBPB and CEBPD homodimers as well as heterodimers with partners in addition to ATF5. Implications: This study indicates that the potential cancer therapeutic DN-ATF5 acts by associating with and blocking the transcriptional activities of CEBPB and CEBPD.

Glioblastoma Multiforme (GBM) tumors contain a small population of glioma stem-like cells (GSCs) among the various differentiated GBM cells (d-GCs). GSCs drive tumor recurrence, and resistance to Temozolomide (TMZ), the standard of care (SoC) for GBM chemotherapy. In order to investigate a potential link between GSC specific mitochondria function and SoC resistance, two patient-derived GSC lines were evaluated for differences in their mitochondrial metabolism. In both the lines, GSCs had significantly lower mitochondrial -content, and -function compared to d-GCs. In vitro, the standard mitochondrial-specific inhibitors oligomycin A, antimycin A, and rotenone selectively inhibited GSC proliferation to a greater extent than d-GCs and human primary astrocytes. These findings indicate that mitochondrial inhibition can be a potential GSC-targeted therapeutic strategy in GBM with minimal off-target toxicity. Mechanistically the standard mitochondrial inhibitors elicit their GSC-selective cytotoxic effects through the induction of apoptosis or autophagy pathways. We tested for GSC proliferation in the presence of 3 safe FDA-approved drugs--trifluoperazine, mitoxantrone, and pyrvinium pamoate, all of which are also known mitochondrial-targeting agents. The SoC GBM therapeutic TMZ did not trigger cytotoxicity in glioma stem cells, even at 100 μM concentration. By contrast, trifluoperazine, mitoxantrone, and pyrvinium pamoate exerted antiproliferative effects in GSCs about 30-50 fold more effectively than temozolomide. Thus, we hereby demonstrate that FDA-approved mitochondrial inhibitors induce GSC-selective cytotoxicity, and targeting mitochondrial function could present a potential therapeutic option for GBM treatment.

Activating transcription factor 5 (ATF5) is a cellular prosurvival transcription factor within the basic leucine zipper (bZip) family that is involved in cellular differentiation and promotes cellular adaptation to stress. Recent studies have characterized the oncogenic role of ATF5 in the development of several different types of cancer, notably glioblastoma. Preclinical assessment of a systemically deliverable dominant-negative ATF5 (dnATF5) biologic has found that targeting ATF5 results in tumor regression and tumor growth inhibition of glioblastoma xenografts in mouse models. In this review, we comprehensively and critically detail the current scientific literature on ATF5 in the context of cellular differentiation, survival, and response to stressors in normal tissues. Furthermore, we will discuss how the prosurvival role of ATF5 aides in cancer development, followed by current advances in targeting ATF5 using dominant-negative biologics, and perspectives on future research.

The lethality of the aggressive brain tumor glioblastoma multiforme (GBM) results in part from its strong propensity to invade surrounding normal brain tissue. Although oncogenic drivers such as epidermal growth factor receptor activation and Phosphatase and Tensin homolog inactivation are thought to promote the motility and invasiveness of GBM cells via phosphatidylinostitol 3-kinase activation, other unexplored mechanisms may also contribute to malignancy. Here we demonstrate that several components of the planar cell polarity (PCP) arm of non-canonical Wnt signaling including VANGL1, VANGL2 and FZD7 are transcriptionally upregulated in glioma and correlate with poorer patient outcome. Knockdown of the core PCP pathway component VANGL1 suppresses the motility of GBM cell lines, pointing to an important mechanistic role for this pathway in glioblastoma malignancy. We further observe that restoration of Nrdp1, a RING finger type E3 ubiquitin ligase whose suppression in GBM also correlates with poor prognosis, reduces GBM cell migration and invasiveness by suppressing PCP signaling. Our observations indicate that Nrdp1 physically interacts with the Vangl1 and Vangl2 proteins to mediate the K63-linked polyubiquitination of the Dishevelled, Egl-10 and Pleckstrin (DEP) domain of the Wnt pathway protein Dishevelled (Dvl). Ubiquitination hinders Dvl binding to phosphatidic acid, an interaction necessary for efficient Dvl recruitment to the plasma membrane upon Wnt stimulation of Fzd receptor and for the propagation of downstream signals. We conclude that the PCP pathway contributes significantly to the motility and hence the invasiveness of GBM cells, and that Nrdp1 acts as a negative regulator of PCP signaling by inhibiting Dvl through a novel polyubiquitination mechanism. We propose that the upregulation of core PCP components, together with the loss of the key negative regulator Nrdp1, act coordinately to promote GBM invasiveness and malignancy.

The expression of activating transcription factor 5 (ATF5) correlates negatively with patient survival in different types of cancer. ATF5 is important for the survival and proliferation of cancer cells, and can be targeted to selectively trigger cancer cell apoptosis while sparing normal cells. Cell-penetrating peptides combined with a dominant negative ATF5 cargo have recently shown efficacy against brain, breast, melanoma, and prostate cancers.

Purpose: Despite significant progress in cancer research, many tumor entities still have an unfavorable prognosis. Activating transcription factor 5 (ATF5) is upregulated in various malignancies and promotes apoptotic resistance. We evaluated the efficacy and mechanisms of the first described synthetic cell-penetrating inhibitor of ATF5 function, CP-d/n-ATF5-S1. Experimental Design: Preclinical drug testing was performed in various treatment-resistant cancer cells and in vivo xenograft models. Results: CP-d/n-ATF5-S1 reduced the transcript levels of several known direct ATF5 targets. It depleted endogenous ATF5 and induced apoptosis across a broad panel of treatment-refractory cancer cell lines, sparing non-neoplastic cells. CP-d/n-ATF5-S1 promoted tumor cell apoptotic susceptibility in part by reducing expression of the deubiquitinase Usp9X and led to diminished levels of antiapoptotic Bcl-2 family members Mcl-1 and Bcl-2. In line with this, CP-d/n-ATF5-S1 synergistically enhanced tumor cell apoptosis induced by the BH3-mimetic ABT263 and the death ligand TRAIL. In vivo, CP-d/n-ATF5-S1 attenuated tumor growth as a single compound in glioblastoma, melanoma, prostate cancer, and triple receptor–negative breast cancer xenograft models. Finally, the combination treatment of CP-d/n-ATF5-S1 and ABT263 significantly reduced tumor growth in vivo more efficiently than each reagent on its own. Conclusions: Our data support the idea that CP-d/n-ATF5-S1, administered as a single reagent or in combination with other drugs, holds promise as an innovative, safe, and efficient antineoplastic agent against treatment-resistant cancers. Clin Cancer Res; 22(18); 4698–711. ©2016 AACR.

Background The Italian Ministry of Health declared oncology a priority and stressed the importance of ensuring continuity and integration in cancer care pathways. In order to monitor the quality of cancer care pathways, we need to explore patients’ experience of the continuity of care, identifying the dimensions that define continuity Methods We found 886 relevant articles in the Pubmed database from 1987 to 5 November 2013. The search strategy for the electronic database was defined using the Population, Intervention, Comparison and Outcome(s) framework (PICO) to identify keywords. Two researchers independently reviewed records identified through the search strategy, analyzing continuity dimensions, specificity and/or transversal domains Results We selected 20 articles that measure the patients’ experience of continuity of care: 7 articles including 5 questionnaires [Questionnaire by King et al. 2008; Cancer care coordination Questionnaire (Cccq); Patient Continuity of Care Questionnaire (Pccq); Medical Care Questionnaire (Mcq); Continuity and Coordination of Care Questionnaire (CCCQ)]; 6 articles evaluating the relationship between patient and his/her physician (the same across the care pathway) in terms of frequency and/or dispersion; 6 articles considering one subscale of larger scales designed to evaluate the generic cancer care service patient experience; 1 revealing four organizational indicators of care pathway continuity / discontinuity Conclusion We traced 3 transversal dimensions across the individual analyses: informational, organizational, relational continuity. It follows that in order to cater to the needs of cancer patients, we need to simultaneously focus on these three dimensions along the cancer care pathway. In line with these results, we promoted the “R.In.Cu.ORAM.i” study (Networks for Integrated Treatment of colorectal and breast cancer), in Area Vasta Romagna Area (Italy), and developed a continuity of care patient-experience continuity tool

ATF5, a transcription factor important in differentiation, proliferation and survival, has been found to be highly expressed in neural progenitor cells and in certain tumors including glioblastomas (GBMs), but its expression in other normal and neoplastic tissues has not been extensively investigated. A tissue microarray immunostained for ATF5 showed diffuse nuclear expression (as defined by the presence in greater than 25% of cells) in 63% (117/186) of neoplastic samples, when compared to only 32% (20/62) in nonneoplastic tissues. When analyzed by histologic subtype, a significantly greater proportion of adenocarcinomas, transitional cell carcinomas, squamous cell carcinomas and metastatic carcinomas of various tissue origins had nuclear staining when compared to nonneoplastic tissues. There was no significant difference in ATF5 expression in renal cell carcinomas, lymphomas and seminomas, when compared to nonneoplastic tissues. An expanded series of nonarray breast resection specimens revealed a significantly greater proportion of ATF5 positivity in ductal and lobular carcinomas, when compared to normal breast tissue. Past work found that loss of ATF5 function triggers death of GBM cells, but not of normal activated astrocytes. Here, we observed that loss of ATF5 function caused significant apoptotic death of neoplastic breast cell lines, but not of nonneoplastic breast cell lines. Our data demonstrate elevated ATF5 expression in a wide variety of neoplasms and that interference with ATF5 function selectively triggers death of breast carcinoma cells. Such findings may have potential therapeutic application.

Background The purpose of this study was to validate the anti-neoplastic activity of a novel ATF5-targeted therapeutic approach against various recalcitrant human malignancies. Activating transcription factor 5 (ATF5) is a member of the cAMP response element binding (CREB)/ATF subfamily of basic leucine zipper transcription factors and has been shown to be overexpressed in many human cancers. CP-d/n-ATF5 is a novel peptide specifically designed to contain a cell-penetrating domain and a domain inhibiting the function of ATF5 in a unique and novel targeted therapeutic approach. This therapeutic strategy was tested among a large panel of different therapy refractory human cancers including glioblastoma, melanoma, breast, prostate-, lung- and pancreatic cancer. Methods Cellular viability was assessed by MTT-assays. The induction of apoptosis was examined by staining for annexin V/propidium iodide or JC-1 (loss of mitochondrial membrane potential) prior to flow cytometric analysis. Western blotting was performed for further molecular analysis detecting caspase cleavage and expression of Bcl-2 family members. Subcutaneous xenograft models were used to examine the anti-neoplastic effects of CP-d/n-ATF5 in vivo. Results Our data show that CP-d/n-ATF5 yields a significant increase in apoptosis across a panel of 10 different cancer cell lines when compared to treatment with a mock-mutated control peptide or penetratin. On the molecular level, treatment with CP-d/n-ATF5 resulted in a marked down-regulation of the anti-apoptotic Bcl-2 family members Mcl-1 and Bcl-2. This effect was amplified in a synergistic manner by adding either ABT-263 or TRAIL to a combined targeted therapeutic approach. In vivo, treatment with CP-d/n-ATF5 resulted in a significant reduction of tumor growth in a heterotopic glioblastoma- and an orthotopic melanoma model. Conclusions The novel ATF5-targeting compound CP-d/n-ATF5 provides a broad and pronounced anti-neoplastic activity against human cancers in vitro and in vivo. On the molecular level, this effect is at least in part due to a shift of the cellular homeostasis towards a pro-apoptotic cellular phenotype by inhibiting anti-apoptotic Bcl-2 family proteins. Overall, CP-d/n-ATF5 may represent a promising novel anti-cancer agent. Citation Format: Georg Karpel-Massler, Chang Shu, Lily Chau, James M. Angelastro, Lloyd A. Greene, Markus D. Siegelin. A novel cell-penetrating peptide targeting ATF5 (CP-d/n-ATF5) exerts anti-cancer activity in vitro and in vivo against a broad spectrum of human cancers. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2923. doi:10.1158/1538-7445.AM2015-2923