Search

This file contains supplementary figures S1-S10.

This excel file contains supplementary tables S1-S7.

This file contains supplementary methods and references.

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

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.

PDF file - 15K, Noxa, Puma and Bim are not cross-regulated

PDF file - 39K, Nutlin-3 sensitivity is correlated with Oct-4 and Noxa protein levels

PDF file - 86K, Patient characteristics and treatment regimens

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 G2-M arrest and by rapid induction of mitochondrial cell death after the first passage through G2. Imatinib abrogated ATM activation on cisplatin selectively in Bcr-Abl+ cells. As a consequence, phosphorylation of p53 on Ser15 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-xL was an additional requirement for cisplatin hypersensitivity, as p53-dependent cell death could be antagonized by exogenous Bcl-xL. 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-xL. [Cancer Res 2009;69(24):9337–45]

Supplementary Figure 3 from Imatinib Mesylate Induces Cisplatin Hypersensitivity in Bcr-Abl+ Cells by Differential Modulation of p53 Transcriptional and Proapoptotic Activity

More detailed information on materials and methods

Supplementary Figure 1 from Imatinib Mesylate Induces Cisplatin Hypersensitivity in Bcr-Abl+ Cells by Differential Modulation of p53 Transcriptional and Proapoptotic Activity

PDF file - 79K

PDF file - 56K, Reduction of proliferation and induction of cell death upon cisplatin treatment in category I tumors and tumors of categories II and III

PDF file - 31K, Oct-4 and Noxa determine the general sensitivity of TGCT cells to genotoxic and non-genotoxic agents

Supplementary Figures 1-3 from Imatinib Mesylate Induces Cisplatin Hypersensitivity in Bcr-Abl+ Cells by Differential Modulation of p53 Transcriptional and Proapoptotic Activity

Legends for Supplementary Figures 1-8

Supplemental Figures (Supplementary Fig. 1-8): Cell lines are HR proficient (Supplementary Fig. 1); Influence of hyperthermia on the quantity of intra-nuclear Pt-DNA adducts and intracellular doxorubicin concentration (Supplementary Fig. 2); Hyperthermia sensitizes tumor cells to drugs which induce PARylation without altering PARP of PARG protein levels (Supplementary Fig. 3); Hyperthermia leads to a delayed DNA strand break repair capacity in different cell lines from ovarian and colon cancer (Supplementary Fig. 4); HR competence in 14 of 15 samples from PC patients (Supplementary Fig. 5); Gamma-irradiation leads to induction of γH2AX foci in all cells independent of proliferation status (Supplementary Fig. 6); Hyperthermia has no acute effect on cisplatin or doxorubicin induced apoptosis (Supplementary Fig. 7); Combined treatment of cells with hyperthermia and PARPi slightly enhanced the potentiating effect of either treatment alone (Supplementary Fig. 8).

PDF file - 82K, TaqMan assays

PDF file - 61K, Constitutive levels of p53, p21 and TUNEL positivity in CAFs

PDF file - 56K, qPCR primer pairs

Supplementary Figure 2 from Imatinib Mesylate Induces Cisplatin Hypersensitivity in Bcr-Abl+ Cells by Differential Modulation of p53 Transcriptional and Proapoptotic Activity

Table of TaqMan Assays (Applied Biosystems) used in this study

PDF file - 20K, Constitutive Noxa mRNA levels are maintained via Oct-4-dependent transcriptional mechanisms

In intrinsic apoptosis, the interaction network of the Bcl-2 protein family controls the decision over life and death. Cells are sentenced to death when pro-apoptotic multidomain effector proteins BAX, BAK or BOK oligomerize and form pores in the mitochondrial outer membrane. This releases cytochrome c, which induces the activation of cell-wrecking proteases, the caspases. Interactions of Bcl-2 proteins with other family members essentially regulate cell death. The interaction via the BH3 region was intensively studied in the last decades. As a result, small-molecule drugs, BH3-mimetics, were developed which bind and inhibit anti-apoptotic Bcl-2 proteins. Since anti-apoptotic Bcl-2 proteins are often overexpressed in hematopoietic malignancies, BH3-mimetics e.g. Venetoclax are approved for anti-cancer therapy. Unlike the BH3 interaction site, the c-terminal α9-helix referred to as the transmembrane domain (TMD) is mostly neglected in Bcl-2 interaction studies. However, TMDs not only dictate subcellular localization, but also substantially influence protein-protein interactions. Interestingly, Bcl-2 TMDs can harbor several tumor-specific mutations. The functional basis for TMD interaction as well as the resulting functional relevance for apoptosis signaling, however, remains poorly understood. To unravel the Bcl-2 TMD interaction network, we developed a split luciferase assay system enabling us to detect Bcl-2 TMD interactions in living cells. Simultaneously encoding for the expression of a fluorophore and TMD fusion peptides this system was used to generate fluorescence-normalized luminescence-based interaction data. Here, we identified a homotypic interaction pattern among effector TMDs of BAX, BAK and BOK. Molecular modelling of effector TMD interaction in mimics of cellular membranes also supports these findings. TMD swap experiments show significant influence of TMD sequence on subcellular localization and cell death signaling as assessed via confocal laser scanning microscopy and flow cytometry-based cell death assays. Moreover, we tested previously described mutations of the BAX-TMD (S184A, S184D) as well as a tumor-specific mutation (V180G) in the novel interaction assay. S184 (de-)phosphorylation as mimicked with S184A/S184D affects subcellular localization. In accordance, we find that S184A enhanced and S184D abolished interaction with wildtype BAX-TMD. Intriguingly, V180G not only modulates BAX subcellular localization but also prevents interaction with wildtype BAX-TMD. These findings verify a crucial role of Bcl-2 TMDs in subcellular localization and furthermore strongly support a function in interaction and cell death regulation. Further efforts to explore the Bcl-2 TMD interaction network as well as functional analysis of tumor-specific TMD mutations could pave the way to establish TMDs as a target of cancer therapy. Citation Format: Tobias B. Beigl, Alexander Paul, Sandra Weller, Benjamin Schäfer, Walter E. Aulitzky, Hans-Georg Kopp, Thomas Fellmeth, Kristyna Pluhackova, Markus Rehm, Frank Essmann. Critical interactions and tumor-specific mutations of Bcl-2 transmembrane domains revealed by a novel split luciferase assay [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2524.

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.

Ovarian cancer is among the top ten leading causes of cancer death in women worldwide with a mortality rate of roughly 5% among all cancer deaths. So far, there are no recommended screening tests for the early detection of ovarian cancer. Therefore, patients are frequently diagnosed at late stages of ovarian cancer, which is associated with unfavorable 5-year survival. Patients suffering from ovarian cancer often show a good response to initial therapy with platinum-based compounds and/or taxanes subsequent to resection of the affected tissue. Unfortunately, ovarian cancer shows a high frequency of relapse and after chemotherapy persistent tumor cells re-expand and successively evolve into therapy-resistant tumors. We recently reported that cisplatin (cisPt) treatment of ovarian cancer cells enhances mitochondrial content and mitochondrial reactive oxygen species (Kleih et al. 2019). We therefore speculated that the enhanced mitochondrial content primes cisPt-treated cells for apoptosis induced by BH3 mimetic drugs, such as Venetoclax (ABT-199), that act on anti-apoptotic Bcl-2 proteins at the outer mitochondrial membrane. To explore this hypothesis, we investigated whether the combined administration of cisPt and ABT-199 leads to a synergistic cell death induction in ovarian cancer cells with different cisPt resistance and mitochondrial content. Detection of cell death by flow cytometric measurements of the mitochondrial membrane potential and exposure of phosphatidylserine demonstrated a synergistic cell death induction by cisPt and ABT-199 in OVCAR4 and OVCAR8 cells, irrespective of the cisPt resistance. Interestingly, Western blot analysis revealed that ABT-199 induced the accumulation of the BH3-only protein NOXA in both cancer cell lines. Accumulation of NOXA was dependent on ABT-199-mediated activation of the stress-responsive transcription factor ATF4. Knock-down experiments showed that apoptosis of cisPt-resistant OVCAR8 cells by the combined treatment with ABT-199 and cisPt was entirely dependent on NOXA. Moreover, also cisPt-sensitive OVCAR4 cells revealed an initially reduced apoptosis induction by the NOXA knock-down. Our data therefore elucidate the molecular mechanism of the synergistic efficacy of cisPt/ABT-199 combination therapies, which should be promising especially for the treatment of recurrent tumors developing reduced sensitivity to cisPt-based treatments. Citation Format: Benjamin Schaefer, Sandra Weller, Tobias B. Beigl, Klaus Schulze-Osthoff, Hans-Georg Kopp, Walter E. Aulitzky, Frank Essmann. Synergistic cell death induction in ovarian cancer by cisplatin and ABT-199 is mediated by expression of NOXA [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3710.

Mantle cell lymphomas (MCL) are rare B-cell neoplasms belonging to the non-Hodgkin’s lymphomas and often show an aggressive clinical course. MCL respond relatively briefly to conventional chemotherapy and are characterized by frequent relapses (Dreyling et al., Ann Oncol, 2017). Therapy for MCL remains a challenge also due to limited cultivation time of primary cells. In vitro suspension culture of primary human MCL cells is not easily feasible (Bryant et al., Lab Invest, 2000) and cells undergo spontaneous apoptosis ex vivo (Zhang et al., Blood, 2012; Medina et al., Heamatologica, 2012). Useful in vitro MCL models that allow long-term culture of primary MCL cells could be helpful in the biological and therapeutic study of MCL. For this purpose, we established a 3D hydrogel culture model for cultivation of primary MCL cells that were first tested with cell lines. After 3 days of culture, the cells cultured in the hydrogel can successfully be analysed for cell viability (live-dead confocal laser scanning microscope-based assay, flow cytometry), metabolism (pH, glucose, lactate), and biomarker expression by immunohistochemistry. The 3D hydrogel cultivation model allows a variety of analytical methods, also because it is reversible and the cells can easily be separated as single cell suspension from hydrogel for further analytical purposes, such as flow cytometric analyses. Next, we cultured primary MCL cells in the alginate hydrogel 3D culture system. MCL cells were isolated from blood of different patients and separated by Ficoll centrifugation. The isolated cell population was identified as MCL cells by double-staining of anti-CD20 and anti-CD5 followed by flow cytometric analysis. Cells were further cultured in medium as conventional suspension culture or in alginate hydrogel as 3D culture. Cell death was assessed with AnnexinV by staining phospholipids of apoptotic cells, allowing flow cytometric quantification. All samples had less than 6% AnnexinV positive (+) cells at day 0 before the experiment started. After 2 days cultivation, primary MCL cells showed a strong induction of cell death in suspension culture with 65.5% (±4.2%) AnnexinV+ cells versus 21.9% (±0.6%) AnnexinV+ cells in 3D hydrogel culture. Subsequently after 3 days cultivation, 74.2% (±12.2%) AnnexinV+ cells in suspension versus 33.5% (±1.3%) AnnexinV+ cells in the hydrogel 3D culture were determined. Therefore, we conclude that our novel 3D culture system might be a more suitable cultivation method for primary MCL cells than conventional suspension culture in cell culture medium. We have developed a 3D cultivation system with alginate hydrogel that allows nearly tripling the in vitro cultivation time of primary MCL. This could pave the way for new long-term treatments in research in the future and serve a better understanding of the cells. Citation Format: Kathrin Böpple, Annette M. Staiger, Heike Horn, German Ott, Walter E. Aulitzky, Meng Dong. A step closer to in-depth analyses: Cultivation of primary mantle cell lymphoma cells in 3D enables in vitro long-term experiments [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 199.

Soft-Tissue Sarcomas (STS) are mesenchymal malignancies with high heterogeneity and poor prognosis. As recently shown, ABT-199 and Bortezomib (BTZ) synergistically induce apoptosis in STS derived cells and cell lines (Muenchow et al. 2020). This is of great importance due to limited clinical therapeutic options and lower dependence on BCL-2 expression in solid cancers. To delineate the molecular basis for the synergism of ABT-199 and BTZ, we thought to elucidate the mechanism leading to enhanced expression of the BH3-only protein NOXA. We investigated whether proteasome inhibition augmented the expression of NOXA in a TP53-regulated manner. To clarify a possible role of TP53 in increased NOXA expression, we performed knock-down of TP53 and analyzed transcription of PMAIP1 by qRT-PCR and expression of NOXA protein by Western Blot in presence of ABT-199 alone and in combination with BTZ. Previous reports also show that ABT-199 impairs complex I and II activity of the electron transport chain (ETC), resulting in induction of metabolic reprogramming confirmed by enhanced reductive carboxylation (Roca-Portoles et al. 2020). The ratio of α-ketoglutarate : citrate, indicating disbalance of the ETC, was assessed by mass spectrometric analysis in SW982/WT cells after incubation with ABT-199 alone and in combination with BTZ. Moreover, we gained further insight into the molecular mechanism of action of enhanced NOXA expression by analyzing ABT-199 and BTZ induced cell death induction in time kinetics of cell death induction in knock-down experiments using CellTOX Green as indicator. We found that ABT-199 induced the expression of NOXA by a mainly TP53-independent mechanism at the transcriptional level. Indeed, our data suggest that ABT-199 induces metabolic reprogramming leading to enhanced reductive carboxylation, evidenced by an increased ratio of α-ketoglutarate : citrate. Subsequent activation of the integrated stress response (ISR) induces expression of the known ISR markers ATF3 and ATF4 at both the transcriptional and protein levels. Silencing of ATF3 and ATF4 significantly reduced NOXA expression and NOXA mediated cell death induction. Therefore, we propose that transactivation of NOXA by ABT-199 and subsequent sensitization to proteasome inhibitors effectively induces cell death in solid cancers. The double impact of ABT-199 by i) directly blocking anti-apoptotic BCL-2 and ii) inhibiting MCL-1 via transactivated NOXA is of high clinical relevance, because combined treatment with proteasome inhibitors might overcome intrinsic or acquired resistance to BH3-mimetics. Thus, the here described novel effect of ABT-199 to transactivate NOXA expression, should prompt future efforts in the clinical evaluation of combinatorial regimens in additional malignancies. We hypothesize that these effects represent generally active principles of a tumor-agnostic mechanism of action. Citation Format: Sandra Weller, Benjamin Schaefer, Tobias B. Beigl, Kathrin Böpple, Ute Hofmann, Walter E. Aulitzky, Hans-Georg Kopp, Frank Essmann. Double impact of ABT-199 by directly blocking anti-apoptotic BCL-2 and inhibiting MCL-1 via transactivation of NOXA [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3706.

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

Summary Background The German Hodgkin Study Group's HD18 trial established the safety and efficacy of PET-guided eBEACOPP (bleomycin, etoposide, doxorubicin, cyclophosphamide, vincristine, procarbazine, and prednisone in escalated doses) for the treatment of advanced-stage Hodgkin lymphoma. However, because of a protocol amendment during the enrolment period (June 1, 2011) that changed standard treatment from eight to six cycles, the results of the HD18 trial have been partially immature. We report a prespecified 5-year follow-up analysis of the completed HD18 trial. Methods HD18 was an international, open-label, randomised, phase 3 trial done in 301 hospitals and private practices in five European countries. Patients aged 18–60 years with newly diagnosed, advanced-stage Hodgkin lymphoma and an Eastern Cooperative Oncology Group performance status of 0–2 were recruited. After receiving an initial two cycles of eBEACOPP (1250 mg/m2 intravenous cyclophosphamide [day 1], 35 mg/m2 intravenous doxorubicin [day 1], 200 mg/m2 intravenous etoposide [day 1–3], 100 mg/m2 oral procarbazine [day 1–7], 40 mg/m2 oral prednisone [day 1–14], 1·4 mg/m2 intravenous vincristine [day 8], and 10 mg/m2 intravenous bleomycin [day 8]), patients underwent a contrast-enhanced CT and PET scan (PET-2). Patients with positive PET-2 were randomly assigned to receive standard therapy (an additional six cycles of eBEACOPP; ie, eight cycles in total) or experimental therapy (an additional six cycles of eBEACOPP plus 375 mg/m2 intravenous rituximab; ie, eight cycles in total) until June 1, 2011. After June 1, 2011, all patients with positive PET-2 were assigned to the updated standard therapy with an additional four cycles of eBEACOPP (ie, six cycles in total). Patients with negative PET-2 were randomly assigned (1:1) to receive standard therapy (an additional six cycles of eBEACOPP [ie, eight cycles in total] until June 1, 2011; an additional four cycles of eBEACOPP [ie, six cycles in total] after June 1, 2011) or experimental therapy (an additional two cycles of eBEACOPP; ie, four cycles in total). Randomisation was done centrally with the minimisation method, including a random component, stratified by centre, age, stage, international prognostic score, and sex. The primary endpoint was progression-free survival. HD18 aimed to improve 5-year progression-free survival by 15% in the PET-2-positive intention-to-treat cohort and to exclude inferiority of 6% or more in 5-year progression-free survival in the PET-2-negative per-protocol population. This study is registered with ClinicalTrials.gov , NCT00515554 , and is completed. Findings Between May 14, 2008, and July 18, 2014, 2101 patients were enrolled and 1945 were assigned to a treatment group according to their PET-2 result. In the PET-2-positive cohort, with a median follow-up of 73 months (IQR 59 to 94), 5-year progression-free survival was 89·9% (95% CI 85·7 to 94·1) in 217 patients assigned to eight cycles of eBEACOPP before the protocol amendment and 87·7% (83·1 to 92·4) in 217 patients assigned to eight cycles of rituximab plus eBEACOPP (p=0·40). Among 506 patients who received six cycles of eBEACOPP after the protocol amendment, 5-year progression-free survival was 90·1% (95% CI 87·2 to 92·9), with a median follow-up of 58 months (IQR 39 to 66). In the PET-2-negative cohort, with a median follow-up of 66 months (IQR 54 to 85) in the combined pre-amendment and post-amendment groups, 5-year progression-free survival was 91·2% (95% CI 88·4 to 93·9) in 446 patients who received eight or six cycles of eBEACOPP and 93·0% (90·6 to 95·4) in 474 patients who received four cycles of eBEACOPP (difference 1·9% [95% CI −1·8 to 5·5]). In the subgroup of PET-2-negative patients randomly assigned after protocol amendment, 5-year progression-free survival was 90·9% (95% CI 86·8 to 95·1) in 202 patients assigned to receive six cycles of eBEACOPP and 91·0% (86·6 to 95·5) in 200 patients assigned to receive four cycles of eBEACOPP (difference 0·1% [–5·9 to 6·2]). Interpretation Long-term follow-up confirms the efficacy and safety of PET-2-guided eBEACOPP in patients with advanced-stage Hodgkin lymphoma. The reduction from eight to four cycles of eBEACOPP represents a benchmark in the treatment of early-responding patients, who can now be potentially cured with a short and safe treatment approach. Funding Deutsche Krebshilfe, Swiss State Secretariat for Education, Research and Innovation SERI (Switzerland), and Roche Pharma. Translation For the German translation of the abstract see Supplementary Materials section.

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.

Early results of the randomized placebo-controlled SORAML trial showed that, in patients with newly diagnosed acute myeloid leukaemia (AML), sorafenib led to a significant improvement in event-free (EFS) and relapse-free survival (RFS). In order to describe second-line treatments and their implications on overall survival (OS), we performed a study after a median follow-up time of 78 months. Newly diagnosed fit AML patients aged ≤60 years received sorafenib (n = 134) or placebo (n = 133) in addition to standard chemotherapy and as maintenance treatment. The 5-year EFS was 41 versus 27% (HR 0.68; p = 0.011) and 5-year RFS was 53 versus 36% (HR 0.64; p = 0.035). Allogeneic stem cell transplantation (allo SCT) was performed in 88% of the relapsed patients. Four years after salvage allo SCT, the cumulative incidence of relapse was 54 versus 35%, and OS was 32 versus 50%. The 5-year OS from randomization in all study patients was 61 versus 53% (HR 0.82; p = 0.282). In conclusion, the addition of sorafenib to chemotherapy led to a significant prolongation of EFS and RFS. Although the OS benefit did not reach statistical significance, these results confirm the antileukaemic activity of sorafenib.

Within the tumor microenvironment, cancer cells coexist with noncancerous adjacent cells that constitute the tumor microenvironment and impact tumor growth through diverse mechanisms. In particular, cancer-associated fibroblasts (CAFs) promote tumor progression in multiple ways. Earlier studies have revealed that in normal fibroblasts (NFs), p53 plays a cell nonautonomous tumor-suppressive role to restrict tumor growth. We now wished to investigate the role of p53 in CAFs. Remarkably, we found that the transcriptional program supported by p53 is altered substantially in CAFs relative to NFs. In agreement, the p53-dependent secretome is also altered in CAFs. This transcriptional rewiring renders p53 a significant contributor to the distinct intrinsic features of CAFs, as well as promotes tumor cell migration and invasion in culture. Concordantly, the ability of CAFs to promote tumor growth in mice is greatly compromised by depletion of their endogenous p53. Furthermore, cocultivation of NFs with cancer cells renders their p53-dependent transcriptome partially more similar to that of CAFs. Our findings raise the intriguing possibility that tumor progression may entail a nonmutational conversion ("education") of stromal p53, from tumor suppressive to tumor supportive.

Solid tumors are often considered as abnormal organs composed of the cancerous cells and their surrounding tumor microenvironment (TME) containing fibroblasts, immune cells, blood and lymphatic vessels, and the extracellular matrix. The heterotypic interactions between this diversity of cell types within the TME are maintained through a wide variety of secreted proteins, resulting in a favorable milieu for the progression of the malignancy. The interactions between tumor cells and TME are complex and remain poorly understood. Here we investigated this by developing a unique nascent proteomic approach in tumor tissues.Precision cut cancer tissue slices (PCCTS) maintain tissue heterogeneity with different cell types and preserved TME. Cultivation of PCCTS provides an ex vivo model for tumor tissues. We developed an approach for PCCTS's nascent proteome analysis, using pulsed-SILAC (stable isotope labeling with amino acids in cell culture) labeling combined with click-chemistry to selectively isolate and quantify newly synthesized proteins in the TME upon applying a cellular perturbation. It is a powerful tool to selectively enrich secretory proteins from culture media even with presence of serum. Primary human ovarian tumors (phOVT) and patient derived xenografts (PDX) were used to produce the PCCTS with thickness of 150µm to 300µm. The different cell types and extracellular matrix of PCCTS make the depletion period of cells from the amino acids (methionine, lysine and arginine) prior the AHA-SILAC treatment difficult to define. The PCCTS need longer depletion periods than the 2D cell culture, the longer the depletion period the better depletion efficiency. Following, PCCTS were cultured in AHA-SILAC media and treated with cisplatin. PCCTS and culture media (containing secreted proteins) were harvested; newly synthesized proteins were enriched via click-chemistry and analyzed with mass spectrometry. The labeling time of 10 to 12h showed a good labeling efficiency of more than 60%, still further optimizations are needed. Different PCCTS showed various labeling efficiency indicating the patients heterogeneity. The nascent proteome analysis with cisplatin treatment demonstrated different protein regulations in patients suggesting different drug responses. STRING analysis can be applied to predict the protein-protein interactions. The immunohistochemical staining of the same PCCTS can be processed to further validate the results of the proteome analysis. In conclusion, we established a pulsed SILAC-AHA treatment approach for the PCCTS with the TME. This unique approach allows tracking the compositional and dynamic changes within the proteome and monitoring the direct proteome response at rapid time scale. It can be used to reveal a part of the proteome that has been poorly understood in the tumor tissues and contribute to studying cellular communication and finding new therapeutic targets. Citation Format: Meng Dong, Karim Aljakouch, Kathrin Böpple, Bernd Winkler, Julia Schüler, Frank Essmann, Hans-Georg Kopp, Jeroen Krijgsveld, Walter E. Aulitzky. Nascent proteome analysis of tumor cells and their microenvironment in cultured human tumor tissues [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 325.

50 Years after the generation of the most studied human breast cancer cell line, MCF-7, many research results fundamentally changed patient outcomes for the better. The mortality of breast cancer in women has decreased significantly, especially in the last 30 years. But the total number of deaths is rising again in the last ten years. Considering that breast cancer is the most common cancer in women and the second most common cancer overall, with incidences of 24,2% and 11,6%, respectively, total numbers of new cases are in the millions every year. First line drugs for the treatment of breast cancer often target microtubules (e.g. Vinblastine and Paclitaxel) or induce DNA damage (e.g. Carboplatin and Cisplatin (CisPt)). In addition to these classic therapeutic drugs new compounds that specifically target cancer-driving molecular alterations have been developed. Among these is the group of BH3-mimetics. The efficacy of several BH3-mimetics is investigated in numerous clinical trials while the BCL-2 specific inhibitor ABT-199 (Venetoclax) is already approved for clinical application in chronic lymphatic leukemia (CLL), acute myeloid leukemia (AML) and small lymphocytic lymphoma (SLL). BH3-mimetics target the apoptosis machinery by interacting with anti-apoptotic Bcl-2 proteins, mimicking the function of pro-apoptotic BH3-only proteins. In turn, the pro-apoptotic potential of pore-forming BCL-2 effectors BAX, BAK and BOK is released inducing mitochondrial outer membrane permeabilization and cell death. Recently we reported synergistic cell death induction by ABT-199 in combination with the proteasome inhibitor Bortezomib (BOZ) in soft tissue sarcoma (STS) cells. ABT-199 and BOZ induce accumulation of BOK and the BH3-only protein NOXA (Muenchow et al. 2020). Here, we combined ABT-199, CisPt and Nutlin-3 in breast cancer cell lines that differ in expression of the estrogen receptor (ER) and p53 activity. Similar to the results in STS cells synergistic induction of apoptotic cell death was observed by FACS analysis of the mitochondrial membrane potential (TMRM) and exposure of phosphatidyl serine (Annexin-V). Western blot analysis revealed that ABT-199 induced accumulation of NOXA and MCL-1, irrespective of the ER status or p53 mutation. Simultaneous treatment with ABT-199 and CisPt or Nutlin-3 further increased expression of NOXA and MCL-1. Interestingly, qRT-PCR revealed enhanced expression of NOXA and MCL-1 mRNA in ABT-199 treated cells indicating transcriptional regulation. However, transcriptional induction of NOXA was also detected independent of p53 activity. Thus, combined therapeutic approaches using CisPt and ABT-199 should be effective irrespective of ER and p53 expression. Nevertheless, in p53 proficient tumors Nutlins might increase therapeutic efficacy. Citation Format: Benjamin Schaefer, Sandra Weller, Tobias Beigl, Kathrin Boepple, Reiner Hoppe, Klaus Schulze-Osthoff, Hans-Georg Kopp, Walter E. Aulitzky, Frank Essmann. Synergistic cell death induction in breast cancer cell lines by combining ABT-199 and cisplatin irrespective of estrogen receptor and p53 expression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1946.

Metastatic Soft-Tissue Sarcomas (STS) are a rare and highly heterogeneous group of mesenchymal malignancies that carry a poor prognosis. Therapy is hampered by a limited number of effective treatments, and the almost non-existing long-term survival rate illustrates the need of effective targeted treatment. As a potential approach, we investigated combination of the clinically approved BH3-mimetic drug ABT-199 with different proteasome inhibitors (PIs): Bortezomib, Carfilzomib and Ixazomib, each with proven efficacy, e.g., in multiple myeloma. ABT-199 selectively inhibits the anti-apoptotic protein Bcl-2 while Bortezomib (BOZ) and Ixazomib (IXZ) bind reversible to the β1 and β5 subunits of the 20S proteasome and Carfilzomib (CFZ) irreversibly blocks the subunits β2 and β5. We investigated whether the combination of ABT-199 with CFZ or IXZ shows synergistic activity as recently published for BOZ. SW982 sarcoma cells were cultured with PIs alone or in combination with ABT-199. Cell death was detected by flow cytometric analysis of mitochondrial membrane potential (TMRM) and exposure of phosphatidyl serine (Annexin V). To elucidate potential differences due to specificity of PIs, we analyzed expression of BCL-2 family proteins by Western Blot and performed analogue experiments in knock-out (BAXKO, BAKKO, BOKKO) cell lines. In combination, ABT-199&BOZ or ABT-199&CFZ showed comparable synergistic cell death induction in SW982 while ABT-199&IXZ less efficiently induced cell death. Engineered SW982 knock-out cell lines suggests specific relevance of BOK in IXZ induced apoptosis, whereas all tested PIs crucially depend on BAX for apoptosis induction in combination with ABT-199. Also, as shown for ABT-199&BOZ, both CFZ and IXZ, alone and in combination with ABT-199, efficiently induced expression of NOXA. Strikingly, and in line with augmented cell death induction, ABT-199&CFZ resulted in highest expression of NOXA as compared to BOZ and IXZ. ABT-199&PIs synergistically induce apoptotic cell death in SW982 and corresponding knock-out cell lines with CFZ showing exacerbated expression of NOXA. ABT-199&CFZ also induced strongest stabilization of p53, a transcriptional key regulator of NOXA. We suggest that simultaneous inhibition of anti-apoptotic BCL-2 by ABT-199 and the enhanced expression of NOXA double-hit on the BCL-2 and the MCL-1 signaling axis force the cells over the threshold of apoptosis. Enhanced efficacy of CFZ is dually caused by reduced degradation of NOXA and enhanced stabilization of p53, which in turn transactivates expression of NOXA. In conclusion, PI specificity and efficacy modulate apoptosis induction by combination with ABT-199. Future efforts, e.g. knock-out of relevant BH3-only proteins, will elucidate the observed variable efficacy of these and other PIs for prospective translation in clinical applications. Citation Format: Sandra Weller, Benjamin Schaefer, Tobias Beigl, Kathrin Böpple, Walter E. Aulitzky, Hans-Georg Kopp, Frank Essmann. Impact of proteasome inhibitor specificity and efficacy on apoptosis induction by combination with ABT-199 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1398.

The intrinsic apoptosis signaling pathway comprises the intimate interaction network of the Bcl-2 protein family, in which both pro- and antiapoptotic members pushing cell fate to either survival or death. As a critical event, oligomerization of pore-forming effector proteins BAX, BAK and BOK drive mitochondrial outer membrane permeabilization (MOMP), which results in Cytochrome C release and ultimately apoptotic cell death. Thus far, investigations focus on protein-protein interactions mediated by the BH3-domain and hydrophobic groove. These interactions are targeted by BH3 mimetics that represent highly efficient anti-cancer drugs. In contrast, the C-terminal α-9 helices, known as transmembrane domains (TMD), tend to be neglected as a potential secondary interaction site with other Bcl-2 proteins. However, TMDs strongly affect subcellular localization and there is increasing evidence that TMDs crucially influence Bcl-2 protein interactions and therefore apoptosis signaling. Although TMDs are widely abundant in pro- and anti-apoptotic Bcl-2 proteins, their contribution to the complex Bcl-2 interaction network remains largely in the dark. Aiming to shed more light on the interactions of TMD among effector Bcl-2 proteins, we generated a bimolecular complementation assay in which effector TMD peptides are coupled to a bimolecular split-luciferase system. In addition, vectors encode for simultaneous expression of fluorophores to allow control of transgene expression. Human cell lines were co-transfected with combinations of TMD expression constructs yielding luminescence-based interaction data with a fluorescence-based normalization. Interestingly, we found an almost exclusively homotypic TMD interaction among the TMDs of BAX, BAK and BOK. Moreover, confocal microscopy with modified fluorophore-coupled TMD peptides revealed that TMD sequences were sufficient to locate BAX TMD and BAK TMD peptides effectively to mitochondria, while BOK TMD peptides were mainly localized to the endoplasmic reticulum. Strikingly, exchanging TMD sequences in full-length effector proteins profoundly modulated cell death induction. These results strongly suggest a central functional relevance of the α-9-helices in promoting homotypic interactions of effectors BAX, BAK and BOK during oligomerization and pore formation. In contrast, putative heterotypic interaction among effector proteins is not reflected by the newly generated TMD interaction assay. Thus, effector TMDs are a vital factor for specific protein localization and cell death induction displaying specific functionality in each effector protein. Our findings underline the importance of further investigating the TMD region as a potential oncogenic feature influencing interaction of Bcl-2 proteins and therefore as a potential target for therapeutic intervention. Citation Format: Tobias B. Beigl, Sandra Weller, Benjamin Schäfer, Kathrin Böpple, Lara-Sophie Rieder, Hans-Georg Kopp, Markus Rehm, Walter E. Aulitzky, Frank Essmann. Brought to light: the homotypic interaction pattern of Bcl-2 effector transmembrane domains in a bimolecular luciferase complementation assay [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1947.

Composite lymphoma (CL) is defined as the simultaneous presentation of two histologically distinct types of malignant lymphoma in the same patient, frequently in the same organ or tissue. With a fr...

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.

Summary Background CLEOPATRA was a phase 3 study comparing the efficacy and safety of pertuzumab, trastuzumab, and docetaxel with placebo, trastuzumab, and docetaxel in patients with HER2-positive metastatic breast cancer. In the primary analysis and subsequent reports, progression-free and overall survival were significantly improved in the pertuzumab group compared with the placebo group. Here, we report the end-of-study analysis of CLEOPATRA. Methods This was a double-blind, randomised, placebo-controlled, phase 3 trial that was done at 204 centres in 25 countries. Eligible patients were 18 years or older, had HER2-positive, metastatic breast cancer, had not received previous chemotherapy or biological treatment for their metastatic disease, and had an Eastern Cooperative Oncology Group performance status of 0 or 1. All study drugs were given intravenously, every 3 weeks. Patients were assigned to receive either pertuzumab or placebo at a loading dose of 840 mg, and 420 mg thereafter; plus trastuzumab at 8 mg/kg loading dose and 6 mg/kg thereafter; and docetaxel at 75 mg/m2, escalating to 100 mg/m2 if tolerated. Pertuzumab or placebo and trastuzumab were given until disease progression; docetaxel was given for six cycles, or longer at the investigators’ discretion. Randomisation (1:1) was done by use of an interactive voice-response system and was stratified by geographical region (Asia, Europe, North America, or South America) and previous treatment (previous adjuvant or neoadjuvant chemotherapy vs none). The primary endpoint was independent review facility-assessed progression-free survival, which has been reported previously. Since the confirmatory overall survival analysis had also occurred before this prespecified end-of-study analysis, analyses presented here are descriptive. Overall survival analyses were based on the intention-to-treat population with crossover patients analysed in the placebo group; analyses were not adjusted for crossover to the pertuzumab group and are likely to be conservative. Safety analyses were based on treatment received; crossover patients were counted in the placebo group up to the day before first pertuzumab dose. This trial is registered with ClinicalTrials.gov , number NCT00567190 . Findings Between Feb 12, 2008, and July 7, 2010, 1196 patients were assessed for eligibility, of whom 808 were enrolled and randomly assigned. 402 patients were assigned to receive docetaxel plus trastuzumab plus pertuzumab, and 406 patients were assigned to receive docetaxel plus trastuzumab plus placebo. Clinical cutoff for this analysis was Nov 23, 2018. Between July 2012 and clinical cutoff, 50 patients crossed from the placebo to the pertuzumab group. Median follow-up was 99·9 months in the pertuzumab group (IQR 92·9–106·4) and 98·7 months (90·9–105·7) in the placebo group. Median overall survival was 57·1 months (95% CI 50–72) in the pertuzumab group and 40·8 months (36–48) in the placebo group (hazard ratio 0·69, 95% CI 0·58–0·82); 8-year landmark overall survival rates were 37% (95% CI 31–42) in the pertuzumab group and 23% (19–28) in the placebo group. The most common grade 3–4 adverse event was neutropenia (200 [49%] of 408 patients in the pertuzumab group, 183 [46%] of 396 patients in the placebo group). Five (1%) of 408 patients in the pertuzumab group and six (2%) of 396 patients in the placebo group had treatment-related deaths. One new serious adverse event suggestive of congestive heart failure (pertuzumab group) and one new symptomatic left ventricular systolic dysfunction (post-crossover) occurred since the previous analysis. Interpretation Our analysis shows that the previously observed improvements in overall survival with pertuzumab, trastuzumab, and docetaxel versus placebo, trastuzumab, and docetaxel were maintained after a median of more than 8 years of follow-up. The long-term safety and cardiac safety profiles of pertuzumab, trastuzumab, and docetaxel were maintained in the overall safety population and within crossover patients. HER2-targeted therapy has changed the natural history of HER2-positive metastatic breast cancer, with the dual blockade of pertuzumab and trastuzumab, with docetaxel, demonstrating an 8-year landmark overall survival rate of 37%. Funding F Hoffmann-La Roche and Genentech.