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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.

Suppl. fig. 1: Effect of CP-d/n-ATF5-S1 on ATF5 expression and stability Suppl. fig. 2: Effect of CP-d/n-ATF5-S1 on ASNS mRNA expression Suppl. fig. 3: Effect of CP-d/n-ATF5-S1 on cell viability in HL-60 cells Suppl. fig. 4: Pro-apoptotic activity of CP-d/n-ATF5-S1 in SF188 and GBM12 cells Suppl. fig. 5: Effect of CP-d/n-ATF5-S1 in HCT116 colorectal cancer Suppl. fig. 6: Effect of pan-caspase inhibition on CP-d/n-ATF5-S1 treatment Suppl. fig. 7: Effect of CP-d/n-ATF5-S1 on Bcl-2 family and Usp9X/Bag3 protein expression in A375 and PC3 Suppl. fig. 8: Effect of pan-caspase inhibition on CP-d/n-ATF5-S1-mediated down-regulation of Usp9X Suppl. fig. 9: Effect of Usp9X knock-down on apoptosis in LN229 Suppl. fig. 10: Isobologram for CP-d/n-ATF5-S1 and ABT263 Suppl. fig. 11: Effect of CP-d/n-ATF5-S1 and ABT263 on GBM12 cells Suppl. fig. 12: Knock-down of Mcl-1 sensitizes for ABT263-mediated apoptosis Suppl. fig. 13: Effects of CP-d/n-ATF5-S1 on U87MG xenograft model Suppl. fig. 14: Effects of CP-d/n-ATF5-S1 on PANC-1 and MDA-MB-231 xenograft model Suppl. fig. 15: Effect of CP-d/n-ATF5-S1 on organ toxicity

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Background: Spreading depolarization (SD) occurs nearly ubiquitously in malignant hemispheric stroke (MHS) and is strongly implicated in edema progression and lesion expansion. Due to this high burden of SD after infarct, it is of great interest whether SD in MHS patients can be mitigated by physiologic or pharmacologic means and whether this intervention improves clinical outcomes. Here we describe the association between physiological variables and risk of SD in MHS patients who had undergone decompressive craniectomy and present an initial case of using ketamine to target SD in MHS. Methods: We recorded SD using subdural electrodes and time-linked with continuous physiological recordings in five subjects. We assessed physiologic variables in time bins preceding SD compared to those with no SD. Results: Using multivariable logistic regression, we found that increased ETCO2 (OR 0.772, 95% CI 0.655–0.910) and DBP (OR 0.958, 95% CI 0.941–0.991) were protective against SD, while elevated temperature (OR 2.048, 95% CI 1.442–2.909) and WBC (OR 1.113, 95% CI 1.081–1.922) were associated with increased risk of SD. In a subject with recurrent SD, ketamine at a dose of 2 mg/kg/h was found to completely inhibit SD. Conclusion: Fluctuations in physiological variables can be associated with risk of SD after MHS. Ketamine was also found to completely inhibit SD in one subject. These data suggest that use of physiological optimization strategies and/or pharmacologic therapy could inhibit SD in MHS patients, and thereby limit edema and infarct progression. Clinical trials using individualized approaches to target this novel mechanism are warranted.

// Georg Karpel-Massler 1 , Chang Shu 1 , Lily Chau 1 , Matei Banu 2 , Marc-Eric Halatsch 3 , Mike-Andrew Westhoff 4 , Yulian Ramirez 5 , Alonzo H. Ross 5 , Jeffrey N. Bruce 2 , Peter Canoll 1 and Markus D. Siegelin 1 1 Department of Pathology and Cell Biology, Columbia University Medical Center, New York, USA 2 Department of Neurosurgery, Columbia University Medical Center, New York, USA 3 Department of Neurosurgery, Ulm University Medical Center, Ulm, Germany 4 Department of Pediatrics and Adolescent Medicine, Ulm University Medical Center, Ulm, Germany 5 Department of Biochemistry and Molecular Pharmacology, University of Massachusetts Medical School, Massachusetts, USA Correspondence to: Markus D. Siegelin, email: // Keywords : glioblastoma, BH3-mimetic, apoptotic resistance, ABT263, GX15-070 Received : March 12, 2015 Accepted : April 10, 2015 Published : May 04, 2015 Abstract Despite great efforts taken to advance therapeutic measures for patients with glioblastoma, the clinical prognosis remains grim. The antiapoptotic Bcl-2 family protein Mcl-1 is overexpressed in glioblastoma and represents an important resistance factor to the BH-3 mimetic ABT263. In this study, we show that combined treatment with ABT263 and GX15-070 overcomes apoptotic resistance in established glioblastoma cell lines, glioma stem-like cells and primary cultures. Moreover, this treatment regimen also proves to be advantageous in vivo . On the molecular level, GX15-070 enhanced apoptosis by posttranslational down-regulation of the deubiquitinase, Usp9X, and the chaperone Bag3, leading to a sustained depletion of Mcl-1 protein levels. Moreover, knock-down of Usp9X or Bag3 depleted endogenous Mcl-1 protein levels and in turn enhanced apoptosis induced through Bcl-2/Bcl-xL inhibition. In conclusion, combined treatment with ABT263 and GX15-070 results in a significantly enhanced anti-cancer activity in vitro as well as in vivo in the setting of glioblastoma. Both drugs, ABT263 and GX15-070 have been evaluated in clinical studies which facilitates the translational aspect of taking this combinatorial approach to the clinical setting. Furthermore we present a novel mechanism by which GX15-070 counteracts Mcl-1 expression which may lay a foundation for a novel target in cancer therapy.

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.

Deformable image registration of four head and neck cancer patients has been conducted using a biomechanical-based model. Patient-specific 3D finite element models have been developed using CT and cone-beam CT image data of the planning and a radiation treatment session. The model consists of seven vertebrae (C1 to C7), mandible, larynx, left and right parotid glands, tumor and body. Different combinations of boundary conditions are applied in the model in order to find the configuration with a minimum registration error. Each vertebra in the planning session is individually aligned with its correspondence in the treatment session. Rigid alignment is used for each individual vertebra and the mandible since no deformation is expected in the bones. In addition, the effect of morphological differences in the external body between the two image sessions is investigated. The accuracy of the registration is evaluated using the tumor and both parotid glands by comparing the calculated Dice similarity index of these structures following deformation in relation to their true surface defined in the image of the second session. The registration is improved when the vertebrae and mandible are aligned in the two sessions with the highest average Dice index of 0.86 ± 0.08, 0.84 ± 0.11 and 0.89 ± 0.04 for the tumor, left and right parotid glands, respectively. The accuracy of the center of mass location of tumor and parotid glands is also improved by deformable image registration where the errors in the tumor and parotid glands decrease from 4.0 ± 1.1, 3.4 ± 1.5 and 3.8 ± 0.9 mm using rigid registration to 2.3 ± 1.0, 2.5 ± 0.8 and 2.0 ± 0.9 mm in the deformable image registration when alignment of vertebrae and mandible is conducted in addition to the surface projection of the body.

Objectives An anti-osteoporotic herbal formula ELP containing Epimedii Herba (E), Ligustri Lucidi Fructus (L) and Psoraleae Fructus (P) was studied to investigate the herb–herb interaction (or the possible synergistic effect) among each component and to identify the principal herbs in different modes of action. Methods Rat osteoblast-like UMR-106 cells proliferation, rat MSCs-derived osteoblastogenesis and RANKL-induced RAW 264.7 osteoclastogenesis were adopted to investigate the bone-forming activity and bone-degrading activity of the herbal extracts. In the statistical aspect, a modified Tallarida's approach was employed to assess the synergistic effects in herbal combinations. Key findings Psoraleae Fructus is the active herb for stimulating osteoblast proliferation, and mild synergy was detected in the pairwise combinations EL, LP and formula ELP. In osteoblastogenesis assay, E and L are the principal herbs for promoting osteoblast differentiation and significant synergy was detected in the pairwise combination EL. For inhibiting osteoclast formation, L is the active herb and significant synergy was detected in the 3-way combination ELP. Conclusions The presence of E, L and P is essential for ELP formula as a whole to act against osteoporosis via enhancing bone formation and reducing bone reabsorption. An optimal dosage at 150 μg/ml was proposed for ELP based on our findings.

Background: Resistance to apoptosis is a paramount issue in the treatment of Glioblastoma (GBM). We show that targeting PARP by the small molecule inhibitors, Olaparib (AZD-2281) or PJ34, reduces proliferation and lowers the apoptotic threshold of GBM cells in vitro and in vivo. Methods: The sensitizing effects of PARP inhibition on TRAIL-mediated apoptosis and potential toxicity were analyzed using viability assays and flow cytometry in established GBM cell lines, low-passage neurospheres and astrocytes in vitro. Molecular analyses included western blots and gene silencing. In vivo, effects on tumor growth were examined in a murine subcutaneous xenograft model. Results: The combination treatment of PARP inhibitors and TRAIL led to an increased cell death with activation of caspases and inhibition of formation of neurospheres when compared to single-agent treatment. Mechanistically, pharmacological PARP inhibition elicited a nuclear stress response with upregulation of down-stream DNA-stress response proteins, e.g., CCAAT enhancer binding protein (C/EBP) homology protein (CHOP). Furthermore, Olaparib and PJ34 increased protein levels of DR5 in a concentration and time-dependent manner. In turn, siRNA-mediated suppression of DR5 mitigated the effects of TRAIL/PARP inhibitor-mediated apoptosis. In addition, suppression of PARP-1 levels enhanced TRAIL-mediated apoptosis in malignant glioma cells. Treatment of human astrocytes with the combination of TRAIL/PARP inhibitors did not cause toxicity. Finally, the combination treatment of TRAIL and PJ34 significantly reduced tumor growth in vivo when compared to treatment with each agent alone. Conclusions: PARP inhibition represents a promising avenue to overcome apoptotic resistance in GBM.

(Cell 146, 359–371; August 5, 2011)In this paper, we described the directed conversion of skin fibroblasts from unaffected individuals or familial Alzheimer’s disease patients into human induced neuronal cells. We also presented molecular analyses of Alzheimer’s-associated markers in these cells. Dr. Ryousuke Fujita, who was specifically and only responsible for the molecular analyses of Alzheimer’s-associated pathology, has acknowledged inappropriately manipulating image panels and data points, as well as misrepresenting the number of repeats performed, in the experiments presented in Figures 6 and 7 of the paper (and corresponding Figures S5 and S6). We are in the process of repeating these analyses. Given these issues, we believe that the most appropriate course of action is to retract the paper. We deeply regret this circumstance and apologize to the community.

Traditional Chinese Medicine (TCM) is a holistic approach to medicine which has been in use in China for thousands of years. The main treatment, Chinese Medicine Formulae is prescribed by combining sets of herbs to address the patient’s syndromes and symptoms based on clinical diagnosis. Although herbs are often combined based on various classical formulas, TCM practitioners personalize prescriptions by making adjustments to the formula. However, the underlying principles for the choice of herbs are not well understood. In this chapter, we introduce a framework to explore the complex relationships amongst herbs in TCM clinical prescriptions using Boolean logic. By logically analyzing variations of a large number of TCM herbal prescriptions, we have found that our framework was able to show some herbs may have different pathways to affect effectiveness and such herbs have often been overlooked but can play a weak yet non-trivial role in enhancing the overall effectiveness of the TCM treatment. To achieve this goal, two computational solutions are proposed. An efficient set-theoretic approach is first proposed to study the effectiveness of herbal formulations, and followed by complex network analysis to study the role each herb plays in affecting the outcome.

Traditional Chinese Medicine prescriptions depend not only on the individual herbs, but the interactions among herbs within an herbal formula. With examples, this chapter presents an innovative and comprehensive approach to studying the complex synergistic interactions among herbs in Chinese herbal formulae. In multi-targeted in vitro studies, we have integrated the use of the Combination Index and a statistical test of synergy to demonstrate not only the effectiveness of the combined use of herbs, but of the variation of their relationship on each mode of action. We report one of the first applications of statistical interpretations of combination effects of herbs in complex herbal formulae and the feasibility of applying this methodology in combinatory study of herbs.

Deformable image registration of four head and neck cancer patients was conducted using biomechanical based model. Patient specific 3D finite element models have been developed using CT and cone beam CT image data of the planning and a radiation treatment session. The model consists of seven vertebrae (C1 to C7), mandible, larynx, left and right parotid glands, tumor and body. Different combinations of boundary conditions are applied in the model in order to find the configuration with a minimum registration error. Each vertebra in the planning session is individually aligned with its correspondence in the treatment session. Rigid alignment is used for each individual vertebra and to the mandible since deformation is not expected in the bones. In addition, the effect of morphological differences in external body between the two image sessions is investigated. The accuracy of the registration is evaluated using the tumor, and left and right parotid glands by comparing the calculated Dice similarity index of these structures following deformation in relation to their true surface defined in the image of the second session. The registration improves when the vertebrae and mandible are aligned in the two sessions with the highest Dice index of 0.86±0.08, 0.84±0.11, and 0.89±0.04 for the tumor, left and right parotid glands, respectively. The accuracy of the center of mass location of tumor and parotid glands is also improved by deformable image registration where the error in the tumor and parotid glands decreases from 4.0±1.1, 3.4±1.5, and 3.8±0.9 mm using rigid registration to 2.3±1.0, 2.5±0.8 and 2.0±0.9 mm in the deformable image registration when alignment of vertebrae and mandible is conducted in addition to the surface projection of the body.

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

Directed conversion of mature human cells, as from fibroblasts to neurons, is of potential clinical utility for neurological disease modeling as well as cell therapeutics. Here, we describe the efficient generation of human-induced neuronal (hiN) cells from adult skin fibroblasts of unaffected individuals and Alzheimer's patients, using virally transduced transcription regulators and extrinsic support factors. hiN cells from unaffected individuals display morphological, electrophysiological, and gene expression profiles that typify glutamatergic forebrain neurons and are competent to integrate functionally into the rodent CNS. hiN cells from familial Alzheimer disease (FAD) patients with presenilin-1 or -2 mutations exhibit altered processing and localization of amyloid precursor protein (APP) and increased production of Aβ, relative to the source patient fibroblasts or hiN cells from unaffected individuals. Together, our findings demonstrate directed conversion of human fibroblasts to a neuronal phenotype and reveal cell type-selective pathology in hiN cells derived from FAD patients.