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Background: BI 2536, a novel Polo-like kinase 1 inhibitor, was assessed in patients with unresectable advanced exocrine adenocarcinoma of the pancreas.Methods: The study employed a two-stage design. Randomised first-line patients received BI 2536 200 mg on day 1 (n=43) or 60 mg on days 1-3 (n=43) every 21 days. Recruitment of second-line patients was planned for a second stage dependent on an interim analysis demonstrating ≥ 2 responses in the first 18 evaluable patients following 12 weeks of treatment and/or tumour control ≥ 12 weeks in 5 patients per schedule. Primary end point was objective response rate (ORR).Results: By independent review, ORR was 2.3% (all partial) and 24.4% had stable disease as confirmed best response. The second stage was not initiated. Median overall and progression-free survivals were 149 (95% confidence interval (CI), 91-307) and 46 days (95% CI, 44-56). Most common drug-related adverse events were neutropenia (37.2%), leukopenia (29.1%), fatigue (29.1%) and nausea (22.1%); most common grade 3/4-related events were neutropenia (36.0%), leukopenia (27.9%) and thrombocytopenia (8.1%).Conclusion: Given the low ORR and poor survival, further development of BI 2536 monotherapy is not warranted in this population. [ABSTRACT FROM AUTHOR]

Cancer peptide vaccination represents a promising therapeutic approach, but has been hampered by lack of suitable antigens and restricted applicability due to different HLA backgrounds of individual patients. We here introduce a novel warehouse-based concept for composition of personalized peptide vaccines and report on its successful application in a Phase II clinical trial in patients with chronic lymphocytic leukemia (CLL) after first-line therapy. 26 CLL patients in at least partial remission (PR) after 6 months of immuno-chemotherapy were vaccinated with a personalized vaccine compiled from a premanufactured peptide warehouse comprising immunopeptidome-defined CLL-associated peptides. Primary objective was evaluation of immunogenicity, secondary objectives were safety and minimal residual disease (MRD) response. Immunopeptidome-guided vaccine composition was throughout successful, proving the feasibility of warehouse-based vaccine design. Vaccination was well tolerated, with local injection site reactions being the most common adverse event. Only few patients showed vaccine-induced T cell responses, attributable to their inability to mount strong immune responses due to immune-chemotherapy and lack of potent adjuvant formulations. Both issues are addressed within a follow-up trial (NCT04688385), combining the immunopeptidome-guided warehouse-based vaccine design reported here with a potent novel adjuvant evaluating personalized multi- peptide vaccination in CLL patients under T cell supportive BTK inhibitor therapies., Clinical Trial Registration: www.clinicaltrialsregister.eu, identifier NCT02802943., Competing Interests: H-GR and JW are listed as inventors on a patent related to the CLL vaccine cocktail. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Heitmann, Jung, Wacker, Maringer, Nelde, Bauer, Denk, Hoenisch-Gravel, Richter, Oezbek, Dubbelaar, Bilich, Pumptow, Martus, Illerhaus, Denzlinger, Steinbach, Aulitzky, Müller, Dörfel, Rammensee, Salih and Walz.)

The Bcl-2 family controls apoptosis by direct interactions of pro- and anti-apoptotic proteins. The principle mechanism is binding of the BH3 domain of pro-apoptotic proteins to the hydrophobic groove of anti-apoptotic siblings, which is therapeutically exploited by approved BH3-mimetic anti-cancer drugs. Evidence suggests that also the transmembrane domain (TMD) of Bcl-2 proteins can mediate Bcl-2 interactions. We developed a highly-specific split luciferase assay enabling the analysis of TMD interactions of pore-forming apoptosis effectors BAX, BAK, and BOK with anti-apoptotic Bcl-2 proteins in living cells. We confirm homotypic interaction of the BAX-TMD, but also newly identify interaction of the TMD of anti-apoptotic BCL-2 with the TMD of BOK, a peculiar pro-apoptotic Bcl-2 protein. BOK-TMD and BCL-2-TMD interact at the endoplasmic reticulum. Molecular dynamics simulations confirm dynamic BOK-TMD and BCL-2-TMD dimers and stable heterotetramers. Mutation of BCL-2-TMD at predicted key residues abolishes interaction with BOK-TMD. Also, inhibition of BOK-induced apoptosis by BCL-2 depends specifically on their TMDs. Thus, TMDs of Bcl-2 proteins are a relevant interaction interface for apoptosis regulation and provide a novel potential drug target., (© 2024. The Author(s).)

High-grade serous ovarian cancer (HGSOC) represents the most common and lethal subtype of ovarian cancer. Despite initial response to platinum-based standard therapy, patients commonly suffer from relapse that likely originates from drug-tolerant persister (DTP) cells. We generated isogenic clones of treatment-naïve and cisplatin-tolerant persister HGSOC cells. In addition, single-cell RNA sequencing of barcoded cells was performed in a xenograft model with HGSOC cell lines after platinum-based therapy. Published single-cell RNA-sequencing data from neo-adjuvant and non-treated HGSOC patients and patient data from TCGA were analyzed. DTP-derived cells exhibited morphological alterations and upregulation of epithelial-mesenchymal transition (EMT) markers. An aggressive subpopulation of DTP-derived cells showed high expression of the stress marker ATF3. Knockdown of ATF3 enhanced the sensitivity of aggressive DTP-derived cells to cisplatin-induced cell death, implying a role for ATF3 stress response in promoting a drug tolerant persister cell state. Furthermore, single cell lineage tracing to detect transcriptional changes in a HGSOC cell line-derived xenograft relapse model showed that cells derived from relapsed solid tumors express increased levels of EMT and multiple endoplasmic reticulum (ER) stress markers, including ATF3. Single cell RNA sequencing of epithelial cells from four HGSOC patients also identified a small cell population resembling DTP cells in all samples. Moreover, analysis of TCGA data from 259 HGSOC patients revealed a significant progression-free survival advantage for patients with low expression of the ATF3-associated partial EMT genes. These findings suggest that increased ATF3 expression together with partial EMT promote the development of aggressive DTP, and thereby relapse in HGSOC patients., (© 2024. The Author(s).)

Precision-cut tumor slices (PCTS) maintain tissue heterogeneity concerning different cell types and preserve the tumor microenvironment (TME). Typically, PCTS are cultured statically on a filter support at an air-liquid interface, which gives rise to intra-slice gradients during culture. To overcome this problem, we developed a perfusion air culture (PAC) system that can provide a continuous and controlled oxygen medium, and drug supply. This makes it an adaptable ex vivo system for evaluating drug responses in a tissue-specific microenvironment. PCTS from mouse xenografts (MCF-7, H1437) and primary human ovarian tumors (primary OV) cultured in the PAC system maintained the morphology, proliferation, and TME for more than 7 days, and no intra-slice gradients were observed. Cultured PCTS were analyzed for DNA damage, apoptosis, and transcriptional biomarkers for the cellular stress response. For the primary OV slices, cisplatin treatment induced a diverse increase in the cleavage of caspase-3 and PD-L1 expression, indicating a heterogeneous response to drug treatment between patients. Immune cells were preserved throughout the culturing period, indicating that immune therapy can be analyzed. The novel PAC system is suitable for assessing individual drug responses and can thus be used as a preclinical model to predict in vivo therapy responses.

Breast cancer, the most frequent cancer in women, is generally classified into several distinct histological and molecular subtypes. However, single-cell technologies have revealed remarkable cellular and functional heterogeneity across subtypes and even within individual breast tumors. Much of this heterogeneity is attributable to dynamic alterations in the epigenetic landscape of the cancer cells, which promote phenotypic plasticity. Such plasticity, including transition from luminal to basal-like cell identity, can promote disease aggressiveness. We now report that the tumor suppressor LATS1, whose expression is often downregulated in human breast cancer, helps maintain luminal breast cancer cell identity by reducing the chromatin accessibility of genes that are characteristic of a "basal-like" state, preventing their spurious activation. This is achieved via interaction of LATS1 with the NCOR1 nuclear corepressor and recruitment of HDAC1, driving histone H3K27 deacetylation near NCOR1-repressed "basal-like" genes. Consequently, decreased expression of LATS1 elevates the expression of such genes and facilitates slippage towards a more basal-like phenotypic identity. We propose that by enforcing rigorous silencing of repressed genes, the LATS1-NCOR1 axis maintains luminal cell identity and restricts breast cancer progression., (© 2022. The Author(s).)

Background: Germline variant evaluation in precision oncology opens new paths toward the identification of patients with genetic tumor risk syndromes and the exploration of therapeutic relevance. Here, we present the results of germline variant analysis and their clinical implications in a precision oncology study for patients with predominantly rare cancers., Patients and Methods: Matched tumor and control genome/exome and RNA sequencing was carried out for 1485 patients with rare cancers (79%) and/or young adults (77% younger than 51 years) in the National Center for Tumor Diseases/German Cancer Consortium (NCT/DKTK) Molecularly Aided Stratification for Tumor Eradication Research (MASTER) trial, a German multicenter, prospective, observational precision oncology study. Clinical and therapeutic relevance of prospective pathogenic germline variant (PGV) evaluation was analyzed and compared to other precision oncology studies., Results: Ten percent of patients (n = 157) harbored PGVs in 35 genes associated with autosomal dominant cancer predisposition, whereof up to 75% were unknown before study participation. Another 5% of patients (n = 75) were heterozygous carriers for recessive genetic tumor risk syndromes. Particularly, high PGV yields were found in patients with gastrointestinal stromal tumors (GISTs) (28%, n = 11/40), and more specifically in wild-type GISTs (50%, n = 10/20), leiomyosarcomas (21%, n = 19/89), and hepatopancreaticobiliary cancers (16%, n = 16/97). Forty-five percent of PGVs (n = 100/221) supported treatment recommendations, and its implementation led to a clinical benefit in 40% of patients (n = 10/25). A comparison of different precision oncology studies revealed variable PGV yields and considerable differences in germline variant analysis workflows. We therefore propose a detailed workflow for germline variant evaluation., Conclusions: Genetic germline testing in patients with rare cancers can identify the very first patient in a hereditary cancer family and can lead to clinical benefit in a broad range of entities. Its routine implementation in precision oncology accompanied by the harmonization of germline variant evaluation workflows will increase clinical benefit and boost research., Competing Interests: Disclosure AJ: Honoraria: AstraZeneca. CH: Honoraria: Roche, Novartis; research funding: Boehringer Ingelheim; consulting or advisory board: Boehringer Ingelheim. CVG: Employed by Illumina since August 2021. LM reports non-financial support from Celgene outside the submitted work (travel expenses). MAu: Consulting or advisory board membership: Bristol-Myers Squibb, Ipsen, Merck KGaA, MSD Sharp & Dohme, Novartis, Pfizer, Roche, PharmaMar; research funding: AstraZeneca, Bristol-Myers Squibb, Eli Lilly, Exelixis, Ipsen, Merck KGaA, Novartis, Pfizer, PharmaMar, Roche; honoraria: Blueprint Medicines, Bristol-Myers Squibb, Ipsen, Janssen-Cilag, Merck KGaA, Pfizer, PharmaMar. PHoh: Advisory board membership: Roche, Deciphera, PharmaMar, Pfizer, BluMedicine, GSK; honoraria: Bristol-Meyers-Squibb, Astrazeneca; research funding: Novartis, Siemens. MK: Honoraria: Bayer, MSD, Eisai, Ipsen, Eli Lilly; research funding: Eli Lilly, Ipsen, Loxo Oncology. RFS: Consulting or advisory board membership: Astellas, Bristol-Myers Squibb, Celgene, Pfizer; research funding: AstraZeneca, Boehringer Ingelheim, Daiichi Sankyo, Pfizer, PharmaMar, Roche; honoraria: Daiichi Sankyo, Pfizer. GF: Honoraria: Amgen, Armo Biosciences, Bayer, Bristol-Myers Squibb, Eli Lilly, HRA Pharma, Merck KGaA, MSD Sharp & Dohme, Pierre Fabre, Roche, Sanofi, Servier, Shire; research funding: Merck KGaA. SB: Consulting or advisory board membership: ADC Therapeutics, Blueprint Medicines, Daichii-Sankyo, Deciphera, Eli Lilly, Exelixis, Janssen-Cilag, Mundipharma, Novartis, Plexxikon; honoraria: Bayer, Eli Lilly, GlaxoSmithKline, Novartis, Pfizer, PharmaMar; research funding: Blueprint Medicines, Incyte, Novartis. JTS: Consulting or advisory board membership: AstraZeneca, Bayer, Bristol Myers Squibb, Celgene, Immunocore, Novartis, Roche, Shire; honoraria: AstraZeneca, Aurikamed, Baxalta, Bristol Myers Squibb, Celgene, Falk Foundation, iomedico, Immunocore, Novartis, Roche, Shire; research funding: Bristol-Myers Squibb, Celgene, Roche; minor equity in iTheranostics and Pharma15; member of the Board of Directors for Pharma15, all outside the submitted work. ALI: Honoraria: Bristol-Myers Squibb, Janssen-Cilag, Takeda, Roche. NvB: Consulting or advisory board membership: Novartis; honoraria: Novartis, Takeda. PJJ: Consulting or advisory board membership, honoraria, research funding, travel or accommodation expenses: Abbvie, Bayer, Boehringer Ingelheim, Bristol-Myers Squibb, Celgene, Novartis, Pfizer, Roche, Servier. KHM: Consulting: Celgene/BMS, Novartis, Jazz Pharmaceuticals, Pfizer; honoraria: Celgene/BMS, Daiichi Sankyo, Astellas, AbbVie, Novartis; research funding: Celgene. AS: Consulting or advisory board membership, honoraria: AGCT, AstraZeneca, Bayer, Bristol-Myers Squibb, Chugai, Eli Lilly, Illumina, Janssen, MSD Sharp & Dohme, Novartis, Pfizer, Roche, Seattle Genetics, Takeda, Thermo Fisher; research funding: Bayer, Bristol-Myers Squibb, Chugai. WW: Consulting or advisory board membership, honoraria: Agilent, Amgen, Astellas, AstraZeneca, Bayer, Boehringer Ingelheim, Bristol-Myers Squibb, Eli Lilly, Illumina, Merck, MSD Sharp & Dohme, Pfizer, NewOncology, Novartis, Roche, Takeda; research funding: AstraZeneca, Bristol-Myers Squibb, MSD Sharp & Dohme, Roche. SF: Consulting or advisory board membership: Bayer, Illumina, Roche; honoraria: Amgen, Eli Lilly, PharmaMar, Roche; research funding: AstraZeneca, Pfizer, PharmaMar, Roche; travel or accommodation expenses: Amgen, Eli Lilly, Illumina, PharmaMar, Roche. ES: Honoraria: AstraZeneca, Illumina. All other authors have declared no conflicts of interest. Data sharing All evaluated germline variants can be found in Supplementary Table S5, available at https://doi.org/10.1016/j.annonc.2022.07.008. Sequencing data have been deposited in the European Genome-phenome Archive (https://www.ebi.ac.uk/ega/datasets) under accession EGAS00001005537., (Copyright © 2022 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Enhanced expression of anti-apoptotic B-cell lymphoma 2 (BCL-2) protein is frequent in cancer. Targeting of BCL-2 with the specific inhibitor ABT-199 (Venetoclax) has significant clinical activity in malignant diseases such as chronic lymphocytic leukemia and multiple myeloma. The small molecule drug ABT-199 mimics the pro-apoptotic BCL-2 homology domain 3 of BH3-only proteins and blocks the hydrophobic BC-groove in BCL-2. We have previously shown that ABT-199 synergizes with the proteasome inhibitor (PI) bortezomib in soft tissue sarcoma derived cells and cell lines to induce apoptosis. Synergistic apoptosis induction relies on the pore-forming effector BAX and expression of the pro-apoptotic BH3-only protein NOXA. Bortezomib augments expression of NOXA by blocking its proteasomal degradation. Interestingly, shown here for the first time, expression of NOXA is strongly enhanced by ABT-199 induced integrated stress response (ISR). ISR transcription factors ATF3 & ATF4 mediate transactivation of the BH3-only protein NOXA which specifically inhibits the anti-apoptotic MCL-1. Thus, NOXA potentiates the efficacy of the BCL-2 inhibitor ABT-199 by simultaneous inhibition of MCL-1. Hence, ABT-199 has a double impact by directly blocking anti-apoptotic BCL-2 and inhibiting MCL-1 via transactivated NOXA. By preventing degradation of NOXA PIs synergize with ABT-199. Synergism of ABT-199 and PIs therefore occurs on several, previously unexpected levels. This finding should prompt clinical evaluation of combinatorial regimens in further malignancies., (© 2022. The Author(s).)

The aim of this study was to characterize a large series of 154 patients with acute promyelocytic leukemia (median age, 53 years; range, 18-90 years) and evaluate real-life outcome after up-front treatment with arsenic trioxide and all-trans retinoic acid. All patients were included in the prospective NAPOLEON registry (NCT02192619) between 2013 and 2019. The acute promyelocytic leukemia was de novo in 91% (n=140) and therapy-related in 9% (n=14); 13% (n=20) of the patients were older than 70 years. At diagnosis bleeding/hemorrhage was present in 38% and thrombosis in 3%. Complete remission was achieved in 152 patients (99%), whereas two patients (1%) experienced induction death within 18 days after starting therapy. With a median follow-up of 1.99 years (95% confidence interval: 1.61-2.30 years) 1-year and 2-year overall survival rates were 97% (95% confidence interval: 94-100%) and 95% (95% confidence interval: 91-99%), respectively. Age above 70 years was associated with a significantly shorter overall survival (P<0.001) compared to that of younger patients. So far no relapses have been observed. Six patients (4%) died in complete remission at a median of 0.95 years after diagnosis (range, 0.18-2.38 years). Our data confirm the efficiency and durability of arsenic trioxide and all-trans retinoic acid therapy in the primary management of adults with low-/intermediate-risk acute promyelocytic leukemia in the real-life setting, irrespective of age.

The clinical relevance of comprehensive molecular analysis in rare cancers is not established. We analyzed the molecular profiles and clinical outcomes of 1,310 patients (rare cancers, 75.5%) enrolled in a prospective observational study by the German Cancer Consortium that applies whole-genome/exome and RNA sequencing to inform the care of adults with incurable cancers. On the basis of 472 single and six composite biomarkers, a cross-institutional molecular tumor board provided evidence-based management recommendations, including diagnostic reevaluation, genetic counseling, and experimental treatment, in 88% of cases. Recommended therapies were administered in 362 of 1,138 patients (31.8%) and resulted in significantly improved overall response and disease control rates (23.9% and 55.3%) compared with previous therapies, translating into a progression-free survival ratio >1.3 in 35.7% of patients. These data demonstrate the benefit of molecular stratification in rare cancers and represent a resource that may promote clinical trial access and drug approvals in this underserved patient population. SIGNIFICANCE: Rare cancers are difficult to treat; in particular, molecular pathogenesis-oriented medical therapies are often lacking. This study shows that whole-genome/exome and RNA sequencing enables molecularly informed treatments that lead to clinical benefit in a substantial proportion of patients with advanced rare cancers and paves the way for future clinical trials. See related commentary by Eggermont et al., p. 2677 . This article is highlighted in the In This Issue feature, p. 2659 ., (©2021 American Association for Cancer Research.)

Antigen-specific immunotherapies, in particular peptide vaccines, depend on the recognition of naturally presented antigens derived from mutated and unmutated gene products on human leukocyte antigens, and represent a promising low-side-effect concept for cancer treatment. So far, the broad application of peptide vaccines in cancer patients is hampered by challenges of time- and cost-intensive personalized vaccine design, and the lack of neoepitopes from tumor-specific mutations, especially in low-mutational burden malignancies. In this study, we developed an immunopeptidome-guided workflow for the design of tumor-associated off-the-shelf peptide warehouses for broadly applicable personalized therapeutics. Comparative mass spectrometry-based immunopeptidome analyses of primary chronic lymphocytic leukemia (CLL) samples, as representative example of low-mutational burden tumor entities, and a dataset of benign tissue samples enabled the identification of high-frequent non-mutated CLL-associated antigens. These antigens were further shown to be recognized by pre-existing and de novo induced T cells in CLL patients and healthy volunteers, and were evaluated as pre-manufactured warehouse for the construction of personalized multi-peptide vaccines in a first clinical trial for CLL (NCT04688385). This workflow for the design of peptide warehouses is easily transferable to other tumor entities and can provide the foundation for the development of broad personalized T cell-based immunotherapy approaches., Competing Interests: H-GR is shareholder of Immatics Biotechnologies GmbH, Synimmune GmbH, and Curevac AG, and holds a patent application on an adjuvant, XS15. AN, H-GR, and JW are listed as inventors on patents related to peptides described in this manuscript. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Nelde, Maringer, Bilich, Salih, Roerden, Heitmann, Marcu, Bauer, Neidert, Denzlinger, Illerhaus, Aulitzky, Rammensee and Walz.)

Soft tissue sarcomas (STS) are a heterogeneous group of malignancies predominantly affecting children and young adults. Despite improvements in multimodal therapies, 5-year survival rates are only 50% and new treatment options in STS are urgently needed. To develop a rational combination therapy for the treatment of STS we focused on ABT-199 (Venetoclax), a BCL-2 specific BH3-mimetic, in combination with the proteasome inhibitor bortezomib (BZB). Simultaneous inhibition of BCL-2 and the proteasome resulted in strongly synergistic apoptosis induction. Mechanistically, ABT-199 mainly affected the multidomain effector BAX by liberating it from BCL-2 inhibition. The combination with BZB additionally resulted in the accumulation of BOK, a BAX/BAK homologue, and of the BH3-only protein NOXA, which inhibits the anti-apoptotic protein MCL-1. Thus, the combination of ABT-199 and BZB sensitizes STS cells to apoptosis by simultaneously releasing several defined apoptotic restraints. This synergistic mechanism of action was verified by CRISPR/Cas9 knock-out, showing that both BAX and NOXA are crucial for ABT-199/BZB-induced apoptosis. Noteworthy, efficient induction of apoptosis by ABT-199/BZB was not affected by the p53 status and invariably detected in cell lines and patient-derived tumor cells of several sarcoma types, including rhabdomyo-, leiomyo-, lipo-, chondro-, osteo-, or synovial sarcomas. Hence, we propose the combination of ABT-199 and BZB as a promising strategy for the treatment of STS, which should warrant further clinical investigation.

Patients with high-grade serous ovarian cancer (HGSC) frequently receive platinum-based chemotherapeutics, such as cisplatin. Cisplatin binds to DNA and induces DNA-damage culminating in mitochondria-mediated apoptosis. Interestingly, mitochondrial DNA is critically affected by cisplatin but its relevance in cell death induction is scarcely investigated. We find that cisplatin sensitive HGSC cell lines contain higher mitochondrial content and higher levels of mitochondrial ROS (mtROS) than cells resistant to cisplatin induced cell death. In clonal sub-lines from OVCAR-3 mitochondrial content and basal oxygen consumption rate correlate with sensitivity to cisplatin induced apoptosis. Mitochondria are in two ways pivotal for cisplatin sensitivity because not only knock-down of BAX and BAK but also the ROS scavenger glutathione diminish cisplatin induced apoptosis. Mitochondrial ROS correlates with mitochondrial content and reduction of mitochondrial biogenesis by knock-down of transcription factors PGC1α or TFAM attenuates both mtROS induction and cisplatin induced apoptosis. Increasing mitochondrial ROS by inhibition or knock-down of the ROS-protective uncoupling protein UCP2 enhances cisplatin induced apoptosis. Similarly, enhancing ROS by high-dose ascorbic acid or H 2 O 2 augments cisplatin induced apoptosis. In summary, mitochondrial content and the resulting mitochondrial capacity to produce ROS critically determine HGSC cell sensitivity to cisplatin induced apoptosis. In line with this observation, data from the human protein atlas (www.proteinatlas.org) indicates that high expression of mitochondrial marker proteins (TFAM and TIMM23) is a favorable prognostic factor in ovarian cancer patients. Thus, we propose mitochondrial content as a biomarker for the response to platinum-based therapies. Functionally, this might be exploited by increasing mitochondrial content or mitochondrial ROS production to enhance sensitivity to cisplatin based anti-cancer therapies.

Background: NOTCH1 gene mutations in mantle cell lymphoma (MCL) have been described in about 5-10% of cases and are associated with significantly shorter survival rates. The present study aimed to investigate the biological impact of this mutation in MCL and its potential as a therapeutic target., Methods: Activation of Notch1 signaling upon ligand-stimulation and inhibitory effects of the monoclonal anti-Notch1 antibody OMP-52M51 in NOTCH1-mutated and -unmutated MCL cells were assessed by Western Blot and gene expression profiling. Effects of OMP-52M51 treatment on tumor cell migration and tumor angiogenesis were evaluated with chemotaxis and HUVEC tube formation assays. The expression of Delta-like ligand 4 (DLL4) in MCL lymph nodes was analyzed by immunofluorescence staining and confocal microscopy. A MCL mouse model was used to assess the activity of OMP-52M51 in vivo., Results: Notch1 expression can be effectively stimulated in NOTCH1-mutated Mino cells by DLL4, whereas in the NOTCH1-unmutated cell line JeKo-1, less effect was observed upon any ligand-stimulation. DLL4 was expressed by histiocytes in both, NOTCH1-mutated and -unmutated MCL lymph nodes. Treatment of NOTCH1-mutated MCL cells with the monoclonal anti-Notch1 antibody OMP-52M51 effectively prevented DLL4-dependent activation of Notch1 and suppressed the induction of numerous direct Notch target genes involved in lymphoid biology, lymphomagenesis and disease progression. Importantly, in lymph nodes from primary MCL cases with NOTCH1/2 mutations, we detected an upregulation of the same gene sets as observed in DLL4-stimulated Mino cells. Furthermore, DLL4 stimulation of NOTCH1-mutated Mino cells enhanced tumor cell migration and angiogenesis, which could be abolished by treatment with OMP-52M51. Importantly, the effects observed were specific for NOTCH1-mutated cells as they did not occur in the NOTCH1-wt cell line JeKo-1. Finally, we confirmed the potential activity of OMP-52M51 to inhibit DLL4-induced Notch1-Signaling in vivo in a xenograft mouse model of MCL., Conclusion: DLL4 effectively stimulates Notch1 signaling in NOTCH1-mutated MCL and is expressed by the microenvironment in MCL lymph nodes. Our results indicate that specific inhibition of the Notch1-ligand-receptor interaction might provide a therapeutic alternative for a subset of MCL patients.

Objectives: Domatinostat (4SC-202) is a selective class I histone deacetylase inhibitor (HDACi). This phase I study investigated safety, tolerability, pharmacokinetics (PK), pharmacodynamics, and antitumor activity in patients with advanced hematological malignancies., Methods: Domatinostat was administered orally once (QD) or twice daily (BID) on days 1-14 with 7 days off or continuously days 1-21 in a 3 + 3 design at 7 dose levels from 25 to 400 mg total daily dose (TDD). Twenty-four patients were treated with domatinostat., Results: No formal maximum tolerated dose (MTD) was determined. One dose-limiting toxicity (DLT, grade 4 hypercalcemia) occurred during 200 mg BID continuous treatment. Six patients were reported with ≥ grade 3 treatment-related adverse events (TRAE; grade 3 hematological in three patients, grade 3 and grade 4 liver enzyme increase in 2 patients, grade 4 pulmonary embolism, and grade 4 hypercalcemia in one patient each). Higher grade hepatic TRAE occurred in the 200 mg BID continuous treatment cohort. Out of 24 patients, 1 achieved a complete response, 1 achieved a partial response, and 18 had stable disease as best response., Conclusion: Administration of domatinostat was safe, well tolerated with signs of antitumor activity. Four hundred milligram TDD in a 200 mg BID schedule (14 + 7) is the recommended phase II dose for monotherapy., (© 2018 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Background: Mantle cell lymphoma (MCL) is an aggressive B-non-Hodgkin lymphoma with generally poor outcome. MCL is characterized by an aberrantly high cyclin D1-driven CDK4 activity. New molecular targeted therapies such as inhibitors of the ubiquitin-proteasome system (UPS) have shown promising results in preclinical studies and MCL patients. Our previous research revealed stabilization of the short-lived pro-apoptotic NOXA as a critical determinant for sensitivity to these inhibitors. It is currently unclear how cyclin D1 overexpression and aberrant CDK4 activity affect NOXA stabilization and treatment efficacy of UPS inhibitors in MCL., Methods: The effect of cyclin D1-driven CDK4 activity on response of MCL cell lines and primary cells to proteasome inhibitor treatment was investigated using survival assays (Flow cytometry, AnnexinV/PI) and Western blot analysis of NOXA protein. Half-life of NOXA protein was determined by cycloheximide treatment and subsequent Western blot analysis. The role of autophagy was analyzed by LC3-II protein expression and autophagolysosome detection. Furthermore, silencing of autophagy-related genes was performed using siRNA and MCL cells were treated with autophagy inhibitors in combination with proteasome and CDK4 inhibition., Results: In this study, we show that proteasome inhibitor-mediated cell death in MCL depends on cyclin D1-driven CDK4 activity. Inhibition of cyclin D1/CDK4 activity significantly reduced proteasome inhibitor-mediated stabilization of NOXA protein, mainly driven by an autophagy-mediated proteolysis. Bortezomib-induced cell death was significantly potentiated by compounds that interfere with autophagosomal function. Combined treatment with bortezomib and autophagy inhibitors enhanced NOXA stability leading to super-induction of NOXA protein. In addition to established autophagy modulators, we identified the fatty acid synthase inhibitor orlistat to be an efficient autophagy inhibitor when used in combination with bortezomib. Accordingly, this combination synergistically induced apoptosis both in MCL cell lines and in patient samples., Conclusion: Our data demonstrate that CDK4 activity in MCL is critical for NOXA stabilization upon treatment with UPS inhibitors allowing preferential induction of cell death in cyclin D transformed cells. Under UPS blocked conditions, autophagy appears as the critical regulator of NOXA induction. Therefore, inhibitors of autophagy are promising candidates to increase the activity of proteasome inhibitors in MCL.

Background: The value of allogeneic hematopoietic cell transplantation (alloHCT) as postremission treatment is not well defined for patients with intermediate-risk acute myeloid leukemia (AML) without FLT3-ITD, biallelic CEBPA-, or NPM1 mutations (here referred to as NPM1mut-neg/CEBPAdm-neg/FLT3-ITDneg AML) in first complete remission (CR1)., Patients and Methods: We addressed this question using data from two prospective randomized controlled trials on intensive induction- and risk-stratified postremission therapy. The NPM1mut-neg/CEBPAdm-neg/FLT3-ITDneg AML subgroup comprised 497 patients, aged 18-60 years., Results: In donor versus no-donor analyses, patients with a matched related donor had a longer relapse-free survival (HR 0.5; 95% CI 0.3-0.9, P = 0.02) and a trend toward better overall survival (HR 0.6, 95% CI 0.3-1.1, P = 0.08) compared with patients who received postremission chemotherapy. Notably, only 58% of patients in the donor group were transplanted in CR1. We therefore complemented the donor versus no-donor analysis with multivariable Cox regression analyses, where alloHCT was tested as a time-dependent covariate: overall survival (HR 0.58, 95% CI 0.37-0.9, P = 0.02) and relapse-free survival (HR 0.51, 95% CI 0.34-0.76; P = 0.001) for patients who received alloHCT compared with chemotherapy in CR1 were significantly longer., Conclusion: Outside clinical trials, alloHCT should be the preferred postremission treatment of patients with intermediate risk NPM1mut-neg/CEBPAdm-neg/FLT3-ITDneg AML in CR1., Cinicaltrials.gov Identifier: NCT00180115, NCT00180102., (© The Author 2017. Published by Oxford University Press on behalf of the European Society for Medical Oncology. [br]All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Treatment response of follicular lymphomas (FL) is highly variable. We, therefore, investigated the role of FL cancer-associated fibroblasts (CAFs) on tumor cell viability, in particular in response to treatment with cytotoxic drugs. Stromal cells outgrown from FL patients were characterized and pure CAF populations were co-cultivated with FL cells. To analyze fibroblast-mediated effects, cells in co-culture were treated with ABT-737 and Bortezomib. The adherent cell population was positive for all fibroblastic markers tested and showed increased mRNA-expression of the activation marker FAP. No effect on FL cell viability was noted when co-cultivating them with CAFs. However, stromal cells protected tumor cells from apoptosis in response to cytotoxic treatment. This might be explained by mRNA-induction of ABCC1 and ABCG2 and up-regulation of BCL2L1 in FL cells. Our finding of protective mechanisms mediated by CAFs is of pivotal impact for further studies of cytotoxic agents in FL.

Imbalances in the composition of BCL2 family proteins contribute to tumourigenesis and therapy resistance of mantle cell lymphoma (MCL), making these proteins attractive therapy targets. We studied the efficiency of dual targeting the NOXA/MCL1 axis by combining fatty acid synthase inhibitors (NOXA stabilization) with the CDK inhibitor Dinaciclib (MCL1 reduction). This combination synergistically induced apoptosis in cell lines and primary MCL cells and led to almost complete inhibition of tumour progression in a mouse model. Apoptosis was NOXA-dependent and correlated with the NOXA/MCL1 ratio, highlighting the importance of the NOXA/MCL1 balance for effective cell death induction in MCL., (© 2017 John Wiley & Sons Ltd.)

Although hyperthermia offers clinical appeal to sensitize cells to chemotherapy, this approach has been limited in terms of long-term outcome as well as economic and technical burden. Thus, a more detailed knowledge about how hyperthermia exerts its effects on chemotherapy may illuminate ways to improve the approach. Here, we asked whether hyperthermia alters the response to chemotherapy-induced DNA damage and whether this mechanism is involved in its sensitizing effect in BRCA-competent models of ovarian and colon cancer. Notably, we found that hyperthermia delayed the repair of DNA damage caused by cisplatin or doxorubicin, acting upstream of different repair pathways to block histone polyADP-ribosylation (PARylation), a known effect of chemotherapy. Furthermore, hyperthermia blocked this histone modification as efficiently as pharmacologic inhibitors of PARP (PARPi), producing comparable delay in DNA repair, induction of double-strand breaks (DSB), and cell cytotoxicity after chemotherapy. Mechanistic investigations indicated that inhibiting PARylation by either hyperthermia or PARPi induced lethal DSB upon chemotherapy treatment not only by reducing DNA repair but also by preventing replication fork slowing. Overall, our work reveals how PARP blockade, either by hyperthermia or small-molecule inhibition, can increase chemotherapy-induced damage in BRCA-competent cells. Cancer Res; 76(10); 2868-75. ©2016 AACR., (©2016 American Association for Cancer Research.)

Background: Hyperthermic intraperitoneal chemotherapy (HIPEC) following cytoreductive surgery is a radical but effective treatment option for patients with peritoneal carcinomatosis (PC). Unfortunately, a standardized HIPEC protocol is missing impeding systematic comparisons with regard to minimal effective temperatures., Objective: The purpose of the present study was to systematically analyse the precise minimal temperature needed for potentiation of chemotherapy effects in vitro and for patient survival., Methods: We established a cell line-based model to mimic HIPEC conditions used in clinical practice, and evaluated intracellular drug concentrations and long-term survival using different temperatures ranging from 38 to 42 °C combined with cisplatin or doxorubicin. In parallel, we evaluated the temperature reached in the clinical setting by measuring inflow and outflow, as well as in two locations in the peritoneal cavity in 34 patients. Finally, we determined the influence of different HIPEC temperatures on survival., Results: Long-term survival of cells treated with either cisplatin or doxorubicin was further improved only at temperatures above 40 °C. In patients, during HIPEC, constant temperatures were reached after 10 min in the peritoneal cavity. A temperature above 40 °C for at least 40 min was achieved in 68 % of patients over the 60 min duration of HIPEC. Importantly, we observed a significantly enhanced overall survival (OS) and progression-free survival (PFS) in those patients reaching temperatures above 40 °C., Conclusions: Hyperthermia significantly potentiated the chemotherapy effects only at temperatures above 40 °C in vitro. Importantly, this temperature threshold was also critical for OS and PFS of PC patients.

The optimal timing of allogeneic hematopoietic stem cell transplantation (HCT) in acute myeloid leukemia (AML) is controversial. We report on 1179 patients with a median age of 48 years who were randomized upfront. In the control arm, sibling HCT was scheduled in the first complete remission for intermediate-risk or high-risk AML and matched unrelated HCT in complex karyotype AML. In the experimental arm, matched unrelated HCT in first remission was offered also to patients with an FLT3-ITD (FMS-like tyrosine kinase 3-internal tandem duplication) allelic ratio >0.8, poor day +15 marrow blast clearance and adverse karyotypes. Further, allogeneic HCT was recommended in high-risk AML to be performed in aplasia after induction chemotherapy. In the intent-to-treat (ITT) analysis, superiority of the experimental transplant strategy could not be shown with respect to overall survival (OS) or event-free survival. As-treated analyses suggest a profound effect of allogeneic HCT on OS (HR 0.73; P=0.002) and event-free survival (HR 0.67; P<0.001). In high-risk patients, OS was significantly improved after allogeneic HCT in aplasia (HR 0.64; P=0.046) and after HCT in remission (HR 0.74; P=0.03). Although superiority of one study arm could not be demonstrated in the ITT analysis, secondary analyses suggest that early allogeneic HCT is a promising strategy for patients with high-risk AML.

Purpose: The presence of a mutated nucleophosmin-1 gene (NPM1(mut)) in acute myeloid leukemia (AML) is associated with a favorable prognosis. To assess the predictive value with regard to allogeneic stem-cell transplantation (SCT), we compared the clinical course of patients with NPM1(mut) AML eligible for allogeneic SCT in a donor versus no-donor analysis., Patients and Methods: Of 1,179 patients with AML (age 18 to 60 years) treated in the Study Alliance Leukemia AML 2003 trial, we identified all NPM1(mut) patients with an intermediate-risk karyotype. According to the trial protocol, patients were intended to receive an allogeneic SCT if an HLA-identical sibling donor was available. Patients with no available donor received consolidation or autologous SCT. We compared relapse-free survival (RFS) and overall survival (OS) depending on the availability of a suitable donor., Results: Of 304 eligible patients, 77 patients had a sibling donor and 227 had no available matched family donor. The 3-year RFS rates in the donor and no-donor groups were 71% and 47%, respectively (P = .005); OS rates were 70% and 60%, respectively (P = .114). In patients with normal karyotype and no FLT3 internal tandem duplication (n = 148), the 3-year RFS rates in the donor and no-donor groups were 83% and 53%, respectively (P = .004); and the 3-year OS rates were 81% and 75%, respectively (P = .300)., Conclusion: Allogeneic SCT led to a significantly prolonged RFS in patients with NPM1(mut) AML. The absence of a statistically significant difference in OS is most likely a result of the fact that NPM1(mut) patients who experienced relapse responded well to salvage treatment. Allogeneic SCT in first remission has potent antileukemic efficacy and is a valuable treatment option in patients with NPM1(mut) AML with a sibling donor., (© 2014 by American Society of Clinical Oncology.)

Background: Pancreatic ductal adenocarcinoma (PDAC) is one of the most common malignant tumours and is still associated with a poor prognosis in advanced disease. To improve the standard therapy with gemcitabine, we initiated a prospective randomised phase-II trial with gemcitabine (GEM) versus gemcitabine plus sunitinib (SUNGEM) based on data of in vitro trials and phase-I data for the combination treatment. The rational of adding sunitinib was its putative antiangiogenic mechanism of action., Methods: A total of 106 eligible patients with locally advanced, unresectable or metastatic PDAC without previous system therapy were randomised to receive GEM at a dosage of 1.000mg/m(2) d1, 8, 15 q28 versus a combination of SUNGEM at a dosage of GEM 1.000mg/m(2) d1+8 and sunitinib 50mg p.o. d1-14, q21d. The primary end-point was progression free survival (PFS), secondary end-points were overall survival (OS), toxicity and overall response rate (ORR)., Results: The confirmatory analysis of PFS was based on the intend-to-treat (ITT) population (N=106). The median PFS was 13.3 weeks (95% confidence interval (95%-CI): 10.4-18.1 weeks) for GEM and 11.6 weeks for SUNGEM (95%-CI: 7.0-18.0 weeks; p=0.78 one-sided log-rank). The ORR was 6.1% (95%-CI: 0.7-20.2%) for GEM and for 7.1% (95%-CI: 0.9-23.5%) for SUNGEM (p=0.87). The median time to progression (TTP) was 14.0 weeks (95%-CI: 12.4-22.3 weeks) for GEM and 18.0 weeks (95%-CI: 11.3-19.3 weeks) for SUNGEM (p=0.60; two-sided log-rank). The median OS was 36.7 weeks (95%-CI: 20.6-49.0 weeks) for the GEM arm and 30.4 weeks (95%-CI: 18.1-37.6 weeks) for the SUNGEM (p=0.78, one-sided log-rank). In regard to toxicities, suspected SAEs were reported in 53.7% in the GEM arm and 71.2% in the SUNGEM arm. Grade 3 and 4 neutropenia was statistically significantly higher in the SUNGEM arm with 48.1% versus 27.8% in the GEM arm (p=0.045, two sided log-rank)., Conclusions: The combination SUNGEM was not sufficient superior in locally advanced or metastatic PDAC compared to GEM alone in regard to efficacy but was associated with more toxicity., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Significant advances have been made in the development of small molecules blocking the p53/MDM2 interaction. The Mdm2 inhibitor Nutlin-3 is restricted to tumors carrying wtp53. In contrast, RITA, a compound that binds p53, has recently been shown also to restore transcriptional functions of mtp53. As more than 50% of solid tumors carry p53 mutations, RITA promises to be a more effective therapeutic strategy than Nutlin-3. We investigated effects of RITA on apoptosis, cell cycle and induction of 45 p53 target genes in a panel of 14 cell lines from different tumor entities with different p53 status as well as primary lymphocytes and fibroblasts. Nine cell strains expressed wtp53, four harbored mtp53, and three were characterized by the loss of p53 protein. A significant induction of cell death upon RITA was observed in 7 of 16 cell lines. The nonmalignant cells in our panel were substantially less sensitive. We found that in contrast to Nultin-3, RITA is capable to induce cell death not only in tumor cells harboring wtp53 and mtp53 but also in p53-null cells. Importantly, whereas p53 has a central role for RITA-mediated effects in wtp53 cells, neither p53 nor p63 or p73 were essential for the RITA response in mtp53 or p53-null cells in our panel demonstrating that besides the known p53-dependent action of RITA in wtp53 cells, RITA can induce cell death also independently of p53 in cells harboring defective p53. We identified an important role of both p38 and JNK/SAPK for sensitivity to RITA in these cells leading to a typical caspase- and BAX/BAK-dependent mitochondrial apoptosis. In conclusion, our data demonstrate that RITA can induce apoptosis through p38 and JNK/SAPK not only in tumor cells harboring wtp53 and mtp53 but also in p53-null cells, making RITA an interesting tumor-selective drug.

Mantle cell lymphoma (MCL) is an aggressive lymphoid neoplasm with transient response to conventional chemotherapy. We here investigated the role of the Bcl-2 homology domain 3-only protein NOXA for life-death decision in MCL. Surprisingly, NOXA (PMAIP1) mRNA and NOXA protein levels were extremely discrepant in MCL cells: NOXA mRNA was found to be highly expressed whereas NOXA protein levels were low. Chronic active B-cell receptor signaling and to a minor degree cyclin D1 overexpression contributed to high NOXA mRNA expression levels in MCL cells. The phoshatidyl-inositol-3 kinase/AKT/mammalian target of rapamycin pathway was identified as the major downstream signaling pathway involved in the maintenance of NOXA gene expression. Interestingly, MCL cells adapt to this constitutive pro-apoptotic signal by extensive ubiquitination and rapid proteasomal degradation of NOXA protein (T½∼15-30 min). In addition to the proteasome inhibitor Bortezomib, we identified the neddylation inhibitor MLN4924 and the fatty acid synthase inhibitor Orlistat as potent inducers of NOXA protein expression leading to apoptosis in MCL. All inhibitors targeted NOXA protein turnover. In contrast to Bortezomib, MLN4924 and Orlistat interfered with the ubiquitination process of NOXA protein thereby offering new strategies to kill Bortezomib-resistant MCL cells. Our data, therefore, highlight a critical role of NOXA in the balance between life and death in MCL. The discrepancy between NOXA transcript and protein levels is essential for sensitivity of MCL to ubiquitin-proteasome system inhibitors and could therefore provide a druggable Achilles' heel of MCL cells.

Testicular germ cell tumors (TGCT) are considered a paradigm of chemosensitive tumors. Embryonal carcinoma cells represent the pluripotent entity of TGCTs and are characterized by expression of Oct-4, a key regulator of pluripotency and a determinant of their inherent hypersensitivity to cisplatin. However, the mechanisms underlying this Oct-4-mediated sensitivity are poorly understood. We previously showed that p53 is a major player in cisplatin hypersensitivity and therefore investigated whether Oct-4 may directly affect p53 activity. Despite a significant decrease in sensitivity, depletion of Oct-4 neither did alter cisplatin-induced transactivation of p53 target genes nor its subcellular localization. These data indicate that, rather than directly modulating p53 activity, Oct-4 provides a cellular context that augments the proapoptotic activity of p53. As mitochondrial priming by the Bcl-2 family is a known determinant of chemosensitivity, we compared the constitutive levels of these proteins in Oct-4-positive and -depleted cells. We identified Noxa as the only Bcl-2 family protein to be highly correlated with Oct-4 status and cisplatin sensitivity. Compared with differentiated cells, constitutive Noxa levels were significantly higher in Oct-4-positive cell lines and cancer patient samples. Furthermore, RNA interference-mediated knockdown of Oct-4 resulted in reduced Noxa transcript, in an almost complete loss of constitutive Noxa protein and decreased cisplatin hypersensitivity to a similar extent as did Noxa depletion. In conclusion, our study indicates that Noxa is a central determinant of hypersensitivity to cisplatin. Oct-4-dependent high constitutive levels of this BH3-only protein prime embryonal carcinoma cells to undergo rapid and massive apoptosis in response to p53 activation., (©2012 AACR)

Current understanding of the p53 response is based mainly upon in vitro studies of homogeneous cell populations. However, there is little information on whether the same principles operate within heterogeneous tumor tissues that are comprised of cancer cells and other cell types, including cancer-associated fibroblasts (CAF). Using ex-vivo tissue cultures, we investigated p53 status and responses to cisplatin in tumor cells and CAFs from tissue specimens isolated from 32 lung cancer patients. By comparing cultivated tissue slices with the corresponding tumor tissues fixed immediately after surgery, we found that morphology, proliferation, and p53 staining pattern were preserved during cultivation. Unexpectedly, when CAFs were analyzed, p53 accumulation and induction of p21 was observed only in tumors with constitutively low p53 protein and accumulation upon cisplatin treatment. In contrast, in tumors with no p53 accumulation in cancer cells there was also no p53 accumulation or p21 induction in adjacent CAFs. Furthermore, induction of cisplatin-induced apoptosis in CAFs was selectively observed in tumors characterized by a parallel induction of cancer cell death. Our findings reveal an interdependence of the p53 response in cancer cells and adjacent CAFs within tumor tissues, arguing that cancer cells control the response of their microenvironment to DNA damage., (©2012 AACR.)

Consistent with the excellent clinical results in testicular germ cell tumors (TGCT), most cell lines derived from this cancer show an exquisite sensitivity to Cisplatin. It is well accepted that the high susceptibility of TGCT cells to apoptosis plays a central role in this hypersensitive phenotype. The role of the tumor suppressor p53 in this response, however, remains controversial. Here we show that siRNA-mediated silencing of p53 is sufficient to completely abrogate hypersensitivity not only to Cisplatin but also to non-genotoxic inducers of p53 such as the Mdm2 antagonist Nutlin-3 and the proteasome inhibitor Bortezomib. The close relationship between p53 protein levels and induction of apoptosis is lost upon short-term differentiation, indicating that this predominant pro-apoptotic function of p53 is unique in pluripotent embryonal carcinoma (EC) cells. RNA interference experiments as well as microarray analysis demonstrated a central role of the pro-apoptotic p53 target gene NOXA in the p53-dependent apoptotic response of these cells. In conclusion, our data indicate that the hypersensitivity of TGCT cells is a result of their unique sensitivity to p53 activation. Furthermore, in the very specific cellular context of germ cell-derived pluripotent EC cells, p53 function appears to be limited to induction of apoptosis.

Patients with secondary acute myeloid leukemia (sAML) are generally thought to have a poor prognosis. As there are no prognostic risk stratification models for patients with sAML available, the aim of this study was to obtain a scoring system. Prognostic factors influencing overall survival (OS) and event-free survival (EFS) were analyzed in 305 sAML patients treated in the prospective AML96 trial. The obtained prognostic scoring system was then validated in an independent patient cohort included in the AML2003 and AML60+ trials. In addition to the known risk factors for AML, age and karyotype, we identified the absolute platelet count and the Nucleophosmin 1 mutational status at diagnosis as prognostic factors of sAML patients. A pronounced distribution of sAML patients into three score groups was achieved showing a 2-year OS/EFS of 52/44% for patients in the low-risk group, 21/12% in the intermediate-risk group and 7/3% in the high-risk group (both P<0.001). Validation of this scoring system in a second independent set of sAML patients revealed similar significantly different survival results. In conclusion, for the first time, a prognostic scoring system is provided for sAML patients, allowing differential treatment strategies in the future.

In response to deregulated oncogene activation, mammalian cells activate disposal programs such as programmed cell death. To investigate the mechanisms behind this oncogenic stress response we used Bcr-Abl over-expressing cells cultivated in presence of imatinib. Imatinib deprivation led to rapid induction of Bcr-Abl activity and over-stimulation of PI3K/Akt-, Ras/MAPK-, and JAK/STAT pathways. This resulted in a delayed necrosis-like cell death starting not before 48 hours after imatinib withdrawal. Cell death was preceded by enhanced glycolysis, glutaminolysis, and amino acid metabolism leading to elevated ATP and protein levels. This enhanced metabolism could be linked to induction of cell death as inhibition of glycolysis or glutaminolysis was sufficient to sustain cell viability. Therefore, these data provide first evidence that metabolic changes induced by Bcr-Abl hyper-activation are important mediators of oncogenic stress-induced cell death.During the first 30 hours after imatinib deprivation, Bcr-Abl hyper-activation did not affect proliferation but resulted in cellular swelling, vacuolization, and induction of eIF2α phosphorylation, CHOP expression, as well as alternative splicing of XPB, indicating endoplasmic reticulum stress response. Cell death was dependent on p38 and RIP1 signaling, whereas classical death effectors of ER stress, namely CHOP-BIM were antagonized by concomitant up-regulation of Bcl-xL.Screening of 1,120 compounds for their potential effects on oncogenic stress-induced cell death uncovered that corticosteroids antagonize cell death upon Bcr-Abl hyper-activation by normalizing cellular metabolism. This protective effect is further demonstrated by the finding that corticosteroids rendered lymphocytes permissive to the transforming activity of Bcr-Abl. As corticosteroids are used together with imatinib for treatment of Bcr-Abl positive acute lymphoblastic leukemia these data could have important implications for the design of combination therapy protocols.In conclusion, excessive induction of Warburg type metabolic alterations can cause cell death. Our data indicate that these metabolic changes are major mediators of oncogenic stress induced by Bcr-Abl.

Cancer associated fibroblasts (CAFs) play a critical role for growth, invasion, and metastasis of cancer. Therefore, targeting CAFs with small molecule inhibitors may be an attractive anti-tumor strategy. The current study aims to identify small molecule kinase inhibitors affecting CAF's growth and to characterize the biological effects of active compounds on primary CAFs from lung cancer. We screened two individual CAF strains for their sensitivity to a panel of 160 kinase inhibitors. Five kinase inhibitors were identified inhibiting more than 50% of the growth of both cell lines. Three of them were inhibitors of PDGFR at nanomolar concentrations. Therefore, we further tested the FDA approved PDGFR inhibitors Dasatinib, Nilotinib, Sorafenib, and Imatinib. All 37 CAF strains investigated were highly sensitive to Dasatinib at clinically relevant concentrations. Imatinib was slightly less effective, whereas the inhibitory effects of Nilotinib and Sorafenib were significantly less pronounced.We investigated the effect of Dasatinib on the CAF transcriptome by microarray analysis of 9 individual CAF strains. 492 genes were identified whose expression was changed at least twofold. 104 of these encoded cell cycle related proteins with 97 of them being downregulated by Dasatinib. The majority of regulated genes, however, were of diverse biological functions not directly related to proliferation. We compared this Dasatinib expression signature to previously described differential signatures of normal tissue associated fibroblasts (NAFs) and CAFs and to a signature of fibroblast serum response. There was a significant overlap between genes regulated by Dasatinib and serum repression genes. More importantly, of the 313 genes downregulated by Dasatinib 64 were also reduced in NAFs compared to CAFs. Furthermore, 26 of 179 genes identified as upregulated by Dasatinib were also found to be elevated in NAFs compared to CAFs. These data demonstrate that Dasatinib partially reverses the phenotype of CAFs to a normal fibroblast like phenotype. This is further supported by the finding that incubation of tumor cells with conditioned medium from CAFs pre-incubated with Dasatinib significantly reduced tumor cell proliferation, suggesting that Dasatinib partially reverses the CAF mediated tumor promoting effect. Therefore, targeting CAFs with Dasatinib represents a promising therapeutic principle.

Imatinib is highly effective in inducing remission in chronic myelogenous leukemia (CML). However, complete eradication of the malignant clone by imatinib is rare. We investigated the efficacy of combining imatinib with cisplatin. Inhibition of Bcr-Abl by imatinib induced a hypersensitive phenotype both in Bcr-Abl(+) cell lines and in CD34(+) cells from CML patients. Importantly, cisplatin sensitivity of leukemic cells harboring an inactive Bcr-Abl greatly exceeded that of Bcr-Abl(-) parental cells. The cisplatin response of Bcr-Abl(+) cells treated with imatinib was characterized by an impaired G(2)-M arrest and by rapid induction of mitochondrial cell death after the first passage through G(2). Imatinib abrogated ATM activation on cisplatin selectively in Bcr-Abl(+) cells. As a consequence, phosphorylation of p53 on Ser(15) and its activity as a transcription factor was significantly diminished. Furthermore, p53 accumulated predominantly in the cytoplasm in Bcr-Abl(+) cells treated with imatinib and cisplatin. Silencing of p53 significantly reduced sensitivity to cisplatin in imatinib-treated Bcr-Abl(+) cells, indicating that p53 retains its proapoptotic activity. Simultaneous downregulation of Bcl-x(L) was an additional requirement for cisplatin hypersensitivity, as p53-dependent cell death could be antagonized by exogenous Bcl-x(L). We conclude that imatinib sensitizes Bcr-Abl(+) cells to cisplatin by simultaneous inhibition of p53 transactivation, induction of p53 accumulation predominantly in the cytoplasm, and reduction of Bcl-x(L).

Background: Carcinoma-associated fibroblasts (CAFs) can promote carcinogenesis and tumor progression. Only limited data on the response of CAFs to chemotherapy and their potential impact on therapy outcome are available. This study was undertaken to analyze the influence of chemotherapy on carcinoma-associated fibroblasts (CAFs) in vitro and in vivo., Methods: The in vivo response of stromal cells to chemotherapy was investigated in 22 neoadjuvant treated breast tumors on tissue sections before and after chemotherapy. Response to chemotherapy was analyzed in vitro in primary cultures of isolated CAFs from 28 human lung and 9 breast cancer tissues. The response was correlated to Mdm2, ERCC1 and TP53 polymorphisms and TP53 mutation status. Additionally, the cytotoxic effects were evaluated in an ex vivo experiment using cultured tissue slices from 16 lung and 17 breast cancer specimens., Results: Nine of 22 tumors showed a therapy-dependent reduction of stromal activity. Pathological response of tumor or stroma cells did not correlate with clinical response. Isolated CAFs showed little sensitivity to paclitaxel. In contrast, sensitivity of CAFs to cisplatinum was highly variable with a GI50 ranging from 2.8 to 29.0 microM which is comparable to the range observed in tumor cell lines. No somatic TP53 mutation was detected in any of the 28 CAFs from lung cancer tissue. In addition, response to cisplatinum was not significantly associated with the genotype of TP53 nor Mdm2 and ERCC1 polymorphisms. However, we observed a non-significant trend towards decreased sensitivity in the presence of TP53 variant genotype. In contrast to the results obtained in isolated cell culture, in tissue slice culture breast cancer CAFs responded to paclitaxel within their microenvironment in the majority of cases (9/14). The opposite was observed in lung cancer tissues: only few CAFs were sensitive to cisplatinum within their microenvironment (2/15) whereas a higher proportion responded to cisplatinum in isolated culture., Conclusion: Similar to cancer cells, CAF response to chemotherapy is highly variable. Beside significant individual/intrinsic differences the sensitivity of CAFs seems to depend also on the cancer type as well as the microenvironment.

Imatinib inhibits the kinase activity of Bcr-Abl and is currently the most effective drug for treatment of chronic myeloid leukemia (CML). Imatinib also blocks c-Abl, a physiological tyrosine kinase activated by a variety of stress signals including damaged DNA. We investigated the effect of pharmacological inhibition of c-Abl on the processing of irradiation-induced DNA damage in Bcr-Abl-negative cells. Cell lines and peripheral blood mononuclear cells (PBMCs) from healthy volunteers were treated with imatinib or dasatinib before gamma-irradiation. Inhibition of c-Abl caused an enhanced irradiation-induced mutation frequency and slowdown of DNA repair, whereas imatinib was ineffective in cells expressing a T315I variant of c-Abl. Mutation frequency and repair kinetics were also studied in c-Abl-/- murine embryonic fibroblasts (MEFs) retransfected with wild-type c-Abl (wt-Abl) or a kinase-defect variant of Abl (KD-Abl). Enhanced mutation frequency as well as delayed DNA repair was observed in cells expressing KD-Abl. These data indicate that pharmacological inhibition of c-Abl compromises DNA-damage response.

Treatment options for chronic myeloid leukemia (CML) have changed dramatically during the last decades. Interferon-alpha treatment and stem cell transplantation (SCT) clearly improved survival over conventional chemotherapy and offered the possibility of complete and durable responses. With the advent of the small molecule inhibitor imatinib mesylate (Glivec((R)), Gleevectrade mark) targeting the causative Bcr-Abl oncoprotein, the era of molecular cancer therapy began with remarkable success especially in chronic phase patients. Today, imatinib is the first-line treatment for CML. However, imatinib does not appear to be capable to eliminate all leukemia cells in the patients and pre-existing as well as acquired resistance to the drug has been increasingly recognized. To overcome these problems, several strategies involving dose escalation, combinations with other agents, and novel Bcr-Abl inhibitors have been developed.

Purpose: With a widening arsenal of cancer therapies available, it is important to develop therapy-specific predictive markers and methods to rapidly assess treatment efficacy. We here evaluated the use of cytokeratin-18 (CK18) as a serum biomarker for monitoring chemotherapy-induced cell death in breast cancer., Experimental Design: Different molecular forms of CK18 (caspase cleaved and total) were assessed by specific ELISA assays. Drug-induced release of CK18 was examined from breast carcinoma cells and tissue. CK18 protein composition was examined in serum. CK18 levels were determined in serum from 61 breast cancer patients during docetaxel or cyclophosphamide/epirubicin/5-fluorouracil (CEF) therapy., Results: Caspase-cleaved CK18 molecules were released from monolayer cultures and tumor organ cultures to the extracellular compartment. CK18 was present in complexes with other cytokeratins in serum. Such CK18 protein complexes are remarkably stable, leading to favorable performance of CK18 biomarker assays for clinical investigations. Docetaxel induced increased levels of caspase-cleaved CK18 in serum from breast cancer patients, indicating apoptosis. CEF therapy led to increases predominantly in uncleaved CK18, indicating induction of necrotic cell death in many tumors. The increase in total CK18 at 24 h of the first treatment cycle correlated to the clinical response to CEF therapy (P < 0.0001)., Conclusions: Induction of necrotic cell death may explain the clinical efficacy of anthracycline-based therapy for breast carcinomas with defective apoptosis pathways. We suggest that CK18 biomarkers are useful for early prediction of the response to CEF therapy in breast cancer and may be useful biomarkers for clinical trials.

The FIP1L1-PDGFRA fusion gene has been described in patients with eosinophilia-associated myeloproliferative disorders (Eos-MPD). Here, we report on seven FIP1L1-PDGFRA-positive patients who presented with acute myeloid leukemia (AML, n=5) or lymphoblastic T-cell non-Hodgkin-lymphoma (n=2) in conjunction with AML or Eos-MPD. All patients were male, the median age was 58 years (range, 40-66). AML patients were negative for common mutations of FLT3, NRAS, NPM1, KIT, MLL and JAK2; one patient revealed a splice mutation of RUNX1 exon 7. Patients were treated with imatinib (100 mg, n=5; 400 mg, n=2) either as monotherapy (n=2), as maintenance treatment after intensive chemotherapy (n=3) or in overt relapse 43 and 72 months, respectively, after primary diagnosis and treatment of FIP1L1-PDGFRA-positive disease (n=2). All patients are alive, disease-free and in complete hematologic and complete molecular remission after a median time of 20 months (range, 9-36) on imatinib. The median time to achievement of complete molecular remission was 6 months (range, 1-14). We conclude that all eosinophilia-associated hematological malignancies should be screened for the presence of the FIP1L1-PDGFRA fusion gene as they are excellent candidates for treatment with tyrosine kinase inhibitors even if they present with an aggressive phenotype such as AML.

Background: Safety, tolerability and efficacy of itraconazole and amphotericin B (AMB) were compared for empirical antifungal treatment of febrile neutropenic cancer patients., Patients and Methods: In an open, randomised study, 162 patients with at least 72 h of antimicrobial treatment received either intravenous followed by oral itraconazole suspension or intravenous AMB for a maximum of 28 days. Permanent discontinuation of study medication due to any adverse event was the primary safety parameter. Efficacy parameters included response and success rate for both treatment groups., Results: Significantly fewer itraconazole patients discontinued treatment due to any adverse event (22.2 vs. 56.8% AMB; p < 0.0001). The main reason for discontinuation was a rise in serum creatinine (1.2% itraconazole vs. 23.5% AMB). Renal toxicity was significantly higher and more drug-related adverse events occurred in the AMB group. Intention-to-treat (ITT) analysis showed favourable efficacy for itraconazole: response and success rate were both significantly higher than for AMB (61.7 vs. 42% and 70.4 vs. 49.3%, both p < 0.0001). Treatment failure was markedly reduced in itraconazole patients (25.9 vs. 43.2%), largely due to the better tolerability., Conclusions: Itraconazole was tolerated significantly better than conventional AMB and also showed advantages regarding efficacy. This study confirms the role of itraconazole as a useful and safe agent in empirical antifungal therapy of febrile neutropenic cancer patients.

Introduction: Sequential high dose (SHiDo) chemotherapy with stem cell support has been shown to prolong the event-free survival in patients with diffuse large B-cell lymphoma., Methods: To confirm this result in a multicenter trial, we randomized patients with aggressive NHL, to receive either eight cycles of CHOP or SHiDo. The primary endpoint was overall survival., Results: 129 evaluable patients were randomized to receive either CHOP or SHiDo: median age, 48 years; 62% male; stage III+IV: 73%; age adjusted International Prognostic Index 1/2/3: 21%/52%/27%. Toxicity grades 3+4 were more pronounced in the SHiDo-arm with 13% versus 3% of patients with fever; 34% versus 13% with infections; 13% versus 2% with esophagitis/dysphagia/gastric ulcer. The remission rates were similar in SHiDo and CHOP arms with 34%/37% complete remissions and 31%/31% partial remissions, respectively. After a median observation time of 48 months, there was no difference in overall survival at 3 years, with 46% for SHiDo and 53% for CHOP (P = 0.48)., Conclusion: In this multicenter trial, early intensification with SHiDo did not confer any survival benefit in previously untreated patients with aggressive NHL and was associated with a higher incidence of grades 3/4 toxicity.

Background: Sensitivity of breast tumors to anticancer drugs depends upon dynamic interactions between epithelial tumor cells and their microenvironment including stromal cells and extracellular matrix. To study drug-sensitivity within different compartments of an individual tumor ex vivo, culture models directly established from fresh tumor tissues are absolutely essential., Methods: We prepared 0.2 mm thick tissue slices from freshly excised tumor samples and cultivated them individually in the presence or absence of taxol for 4 days. To visualize viability, cell death, and expression of surface molecules in different compartments of non-fixed primary breast cancer tissues we established a method based on confocal imaging using mitochondria- and DNA-selective dyes and fluorescent-conjugated antibodies. Proliferation and apoptosis was assessed by immunohistochemistry in sections from paraffin-embedded slices. Overall viability was also analyzed in homogenized tissue slices by a combined ATP/DNA quantification assay., Results: We obtained a mean of 49 tissue slices from 22 breast cancer specimens allowing a wide range of experiments in each individual tumor. In our culture system, cells remained viable and proliferated for at least 4 days within their tissue environment. Viability of tissue slices decreased significantly in the presence of taxol in a dose-dependent manner. A three-color fluorescence viability assay enabled a rapid and authentic estimation of cell viability in the different tumor compartments within non-fixed tissue slices., Conclusion: We describe a tissue culture method combined with a novel read out system for both tissue cultivation and rapid assessment of drug efficacy together with the simultaneous identification of different cell types within non-fixed breast cancer tissues. This method has potential significance for studying tumor responses to anticancer drugs in the complex environment of a primary cancer tissue.

Imatinib targets Bcr-Abl, the causative event of chronic myelogenous leukemia (CML), and addresses leukemic cells to growth arrest and cell death. The exact mechanisms responsible for imatinib-induced cell death are still unclear. We investigated the role of poly(ADP-ribose) polymerase (PARP) activity in imatinib-induced cell death in Bcr-Abl-positive cells. Imatinib leads to a rapid increase of poly(ADP-ribosyl)ation (PAR) preceding loss of integrity of mitochondrial membrane and DNA fragmentation. The effect of imatinib on PAR can be mimicked by inhibition of phosphatidylinositol 3-kinase (PI3-K) implicating a central role of the PI3-K pathway in Bcr-Abl-mediated inhibition of PAR. Importantly, inhibition of PAR in imatinib-treated cells partially prevented cell death to an extent comparable to that observed after caspase inhibition. Simultaneous blockade of both caspases and PAR revealed additive cytoprotective effects indicating that both pathways function in parallel. In conclusion, our results suggest that in addition to the well-documented caspase-dependent pathway, imatinib also induces a PARP-mediated death process.

In Philadelphia-positive (Ph(+)) leukemia, point mutations within the Bcr-Abl kinase domain emerged as a major mechanism of resistance to imatinib mesylate. We established a cell-based screening strategy for detection of clinically relevant point mutations using Bcr-Abl-transformed Ba/F3 cells. We identified 32 different single-point mutations within the kinase domain of Bcr-Abl. The pattern and frequency of mutations in this cell culture-based screen resembled the pattern and frequency observed in resistant patients. We then applied this screen to an alternative Abl kinase inhibitor. Using PD166326, the frequency of resistant colonies emerging at 5 to 10 times the median growth inhibition (IC50) of PD166326 was significantly lower than with imatinib. In addition, PD166326 produced a distinct pattern of Bcr-Abl mutations. The majority of mutations that came up with both imatinib and PD166326 could effectively be suppressed by increasing the dose of PD166326 to 50 to 500 nM. In contrast, only a few mutations could be suppressed by increasing the imatinib dose to 5 to 10 microM. However, 3 mutations affecting F317 displayed complete resistance to PD166326, but could be effectively inhibited by standard concentrations of imatinib. Thus, this robust and simple screening system provides a rational basis for combinatorial and sequential treatment strategies in targeted cancer therapy.

Introduction: c-erbB2 (also known as HER-2/neu) and topoisomerase IIalpha are frequently overexpressed in breast cancer. The aim of the study was to analyze retrospectively whether the expression of c-erbB2 and topoisomerase IIalpha protein influences the long-term outcome of patients with primary breast cancer., Methods: In this study c-erbB2 and topoisomerase IIalpha protein were evaluated by immunohistochemistry in formalin-fixed paraffin-embedded tissue from 225 samples of primary breast cancer, obtained between 1986 and 1998. The prognostic value of these markers was analyzed., Results: Of 225 primary breast tumor samples, 78 (34.7%) showed overexpression of either c-erbB2 (9.8%) or topoisomerase IIalpha protein (24.9%), whereas in 21 tumors (9.3%) both proteins were found to be overexpressed. Patients lacking both c-erbB2 and topoisomerase IIalpha overexpression had the best long-term survival. Overexpression of either c-erbB2 or topoisomerase IIalpha was associated with shortened survival, whereas patients overexpressing both c-erbB2 and topoisomerase IIalpha showed the worst disease outcome (P < 0.0001). Treatment with anthracyclines was not capable of reversing the negative prognostic impact of topoisomerase IIalpha or c-erbB2 overexpression., Conclusion: The results of this exploratory study suggest that protein expression of c-erbB2 and topoisomerase IIalpha in primary breast cancer tissues are independent prognostic factors and are not exclusively predictive factors for anthracycline response in patients with primary breast cancer.

A number of highly specific small molecule inhibitors of oncogenic tyrosine kinases have been developed and may potentially improve the treatment of different malignant diseases. However, it became rapidly evident that multiple resistance mechanisms compromise the successful clinical application of these inhibitors, particularly in advanced solid tumors. To develop efficient therapeutic strategies with small molecule inhibitors, one must understand the causes for treatment failure. Three different types of resistance to small molecule inhibitors of oncogenic tyrosine kinases have been observed. The malignant phenotype may be independent of the activity of the target kinase (target-independent resistance). Alternatively, overexpression or mutation of the target kinase can counteract the inhibition of oncogenic tyrosine kinases (target-dependent resistance). Finally, alterations of drug transporters or drug-metabolizing pathways may block the bioavailability of the tyrosine kinase inhibitors (drug-dependent resistance). This article reviews the current knowledge of clinical resistance to small molecule inhibitors approved for treatment of cancer patients.