PD Dr. Ina Oehme
Group leader "Functional Studies"
German Cancer Research Center
CCU Pediatric Oncology / G340
Im Neuenheimer Feld 280
Thanks to the enormous progress in the molecular genetic characterization and treatment of tumors, many pediatric cancers can be successfully treated today. However, a substantial number of patients suffer from tumors that either lack actionable therapeutic targets or become refractory to treatment. To help these patients in the future, we are currently pursuing two closely connected research approaches:
The aim of the Translational Drug Screening Unit (TDSU) is to complement molecular genetic analyses with functional analyses in the form of high-throughput drug screens. To that end, we use cells from tumor biopsies of patients from the INFORM study. We then culture these cells as three-dimensional multicellular spheroids (miniature tumors) and, within a few days of tumor resection, test them for sensitivity to a large number of clinically relevant anti-cancer therapeutics, thereby generating a drug sensitivity profile of each tumor sample. Additionally, whenever possible, we aim to establish molecularly defined long-term cell cultures from these samples to increase the number of available clinically relevant patient-derived models.
In parallel, we investigate resistance mechanisms and actionable cellular alterations in various pediatric nervous system tumors (neuroblastoma, medulloblastoma, glioma and ependymoma), with a special focus on highly treatment-resistant tumor subtypes. With the identification of mechanistic vulnerabilities, we aim to create novel treatment options for these tumors. Promising therapy approaches are rapidly transferred to in vivo testing in our zebrafish early larvae xenograft model.
The main focus of the Translational Drug Screening Unit (TDSU) is translational research from bench to bedside and back with the aim to establish personalized targeted treatment concepts.
Within the INFORM precision medicine registry study we generated a molecular diagnostics platform within the German Society for Pediatric Oncology and Hematology (GPOH) which enables us to identify therapeutic targets, biological tumor classifications and hereditary predisposition syndromes. However, therapeutic targets with a high evidence for a therapeutic response (genetic driver mutations) are only detected in a minority of the tumors. Thus, there is urgent need to complement the molecular analyses by functional information derived from vital tumor cells.
We have started a pilot drug sensitivity- and resistance-profiling phase on fresh tumor specimen from children which are registered in INFORM. This approach aims at identifying more therapeutic options for these pediatric cancer patients by integrating functional multi-parametric single and combination drug response data with our well established INFORM molecular profiling analysis.
Current preclinical, two-dimensional (2D) models in tumor biology are limited in their ability to recapitulate relevant (patho-) physiological processes. Therefore, the cells are cultured and tested as three-dimensional (3D) heterogeneous multicellular spheroids under serum-free conditions to preserve cellular interactions and cell-derived extracellular matrix composition. This approach helps more closely reflecting the physiologically relevant tumor architecture and the heterogeneity and individuality of each tumor. Drug screening is performed rapidly, within few days after tumor resection/biopsy from the patient, using a large number of clinically relevant anti-cancer therapeutics to generate a drug sensitivity and resistance profile (DSRP) of every individual tumor.
Whenever possible we additionally establish long-term cell cultures and, in collaboration with expert teams of the Hopp Children´s Tumor Center Heidelberg (KiTZ), patient–derived xenografts from the INFORM fresh tumor specimens with the aim to expand the panel of clinically relevant, molecularly defined cellular models for translational research. These models will allow testing the (in vivo) applicability of therapy options identified by our DSRP.
In close collaboration with other KiTZ groups, we additionally employ our platform to generate DSRP data on pediatric tumor cell lines and PDX tumor cell re-isolates. For these experiments, we use single and combination drug plate layouts tailored to the specific scientific questions. Further analyses such as genetic characterization of the tumor samples (identification of biomarkers, CRISPR/CAS screenings, single cell sequencing) are performed in parallel settings.
Altogether, our TDSU activities aim to complement diagnostics in the future, and to improve clinical success rates for pediatric cancer patients with yet un-met clinical need through the identification of novel (individual) vulnerabilities.
We use pediatric cancer early larvae zebrafish (danio rerio) models to investigate and monitor tumor growth and progression on a single cell level.
We validate ex vivo results using zebrafish xenograft model as an in vivo medium- to high-throughput approach. We use the zebrafish models in our lab for drug screening to identify potential therapeutic strategies for the treatment of children with relapsed cancers. We use established cell lines and patient derived xenograft models (zPDX) to model disease progression and to valuate/predict therapeutic response.
We have established a zebrafish human pediatric tumor cell xenograft model to investigate tumor growth and tumor progression (e.g. migration). This system is based on microinjection of pediatric tumor cells into the yolk sac and perivitelline space (PVS). Moreover, we are additionally establishing an orthotopic pediatric brain tumor zebrafish model through microinjection of cells into zebrafish blastulas.
Even though survival chances for children with malignant cancers have increased substantially over the last few decades, too many children still cannot be cured and succumb to disease. In many instances, this is either due to lack of suitable treatment options in certain tumor entities or due to treatment resistance either existing before treatment or developing during therapy. The aim of the below described projects is the identification of actionable cellular alterations in pediatric nervous system tumors (neuroblastoma, medulloblastoma, glioma, ependymoma) via high-throughput drug screening. In these drug screens, we use both primary patient material from the INFORM study, as well as various tumor models such as short-term cultures, PDX models and established cell lines.
In our currently running projects, we investigate for example how pediatric histone H3 mutant gliomas, brain tumors with a very poor prognosis, can be treated specifically using epigenetically active anti-cancer drugs. In case of treatment resistant neuroblastomas, we are currently investigating how inhibition of cell surface growth receptors as well as drug efflux pumps can re-sensitize resistant cells to chemotherapy.
An additional focus of our studies is the lysosome, a cellular organelle responsible for the degradation of cellular macromolecules. A number of studies suggest that lysosomes contribute to the chemoresistance of cancer cells by various mechanisms. In case of treatment resistant neuroblastoma, we could for instance show that a member of the histone deacetylase (HDAC) family, HDAC10, promotes autophagy as well as the secretion of drugs via lysosomal exocytosis (Oehme et al. 2013, Ridinger et al. 2018). We could further demonstrate that broad-spectrum HDAC inhibitors such as panobinostat and vorinostat modulate autophagy in neuroblastoma and that combination of these HDACi with autophagy inhibitors such as chloroquine effectively kills neuroblastoma cells (Korholz et al. 2021). By systematically correlating high-throughput drug screens with high-content microscopy data, we are currently investigating how frequently lysosomal resistance mechanisms occur in pediatric nervous system tumors and whether they can be exploited as a target for combination therapies.
In previous works, we have identified the HDAC member HDAC8 as a therapeutic target in neuroblastoma. Inhibition of HDAC8 promotes differentiation of aggressive neuroblastoma cells in vitro and in vivo (Oehme et al. 2009, Rettig et al. 2015). Additionally, inhibitors of the receptor tyrosine kinase ALK, which we identified as a potentially synthetic lethal target in a kinome-wide RNAi screen, proved to be synergistic combination partners with HDAC8 inhibitors. The simultaneous inhibition of HDAC8 and ALK signaling blocks tumor-relevant signaling pathways such as ERK signaling which effectively induces cell death in neuroblastoma cells (Shen et al. 2018). However, our studies also show that a subset of cells escape HDAC8 inhibitor treatment. We are currently investigating how these treatment-surviving cells differ in their gene expression profile from HDAC8 inhibitor sensitive cells in order to systematically screen for actionable targets in HDAC8i resistant cells (unpublished data). One potential way of killing such resistant cells is the simultaneous inhibition of HDAC8 and HDAC10 via highly selective HDAC6/8/10 inhibitors such as TH34 (Kolbinger et al. 2018).
Despite the successful identification of mechanistic vulnerabilities, many tumors respond poorly to single agent treatment. One major focus of our work thus lies in the systematical identification of potential combination therapies with clinically approved anti-cancer drugs (e.g. ATRA) in highly drug resistant tumor models. Promising therapy approaches are then readily transferred to in vivo testing in our zebrafish larvae xenograft model.
We have identified HDAC family member 8 (HDAC8) as a novel target in childhood neuroblastoma. The development of neuroblastoma, the most common extracranial solid tumor in children, is hypothesized to be related to maturation defects of neural crest derived precursor cells of the peripheral sympathetic nervous system. The long-term overall survival probability of high-risk neuroblastoma patients is less than 50%.
Inhibition of HDAC8 enzymatic activity with selective inhibitors exhibits anti-neuroblastoma activity. Selective HDAC8 inhibition leads to cell cycle arrest and differentiation in vitro and in vivo. Upon combination with retinoic acid, differentiation is enhanced. Thus, selective HDAC targeting can be effective in tumors exhibiting HDAC isotype dependent tumor growth and can be combined with differentiation-inducing agents. Future investigations will focus on the use of selective HDAC8 inhibitors as anti-neuroblastoma drugs on the one hand; on the other hand, we will use the selective inhibitors as a tool to further understand the mechanistic background of HDAC8 mediated malignancy of neuroblastoma. In addition, we will investigate cooperating signaling pathways using synthetic lethal screening approaches.
Korholz K, Ridinger J, Krunic D, Najafi S, Gerloff XF, Frese K, Meder B, Peterziel H, Vega-Rubin-de-Celis S, Witt O, Oehme I (2021). "Broad-Spectrum HDAC Inhibitors Promote Autophagy through FOXO Transcription Factors in Neuroblastoma." Cells 10(5).
Wrobel JK, Najafi S, Ayhan S, Gatzweiler, C, Krunic D, Ridinger J, Milde T, Westermann F, Peterziel H, Meder B, Distel M, Witt O, Oehme I (2020). "Rapid In Vivo Validation of HDAC Inhibitor-Based Treatments in Neuroblastoma Zebrafish Xenografts." Pharmaceuticals (Basel) 13(11).
Oehme, I., Deubzer, H. E., Wegener, D., Pickert, D., Linke, J. P., Hero, B., Kopp-Schneider, A., Westermann, F., Ulrich, S. M., von Deimling, A., et al. (2009). Histone deacetylase 8 in neuroblastoma tumorigenesis. Clin Cancer Res 15, 91-99.
Oehme I, Linke JP, Böck BC, Milde T, Lodrini M, Hartenstein B, Wiegand I, Eckert C, Roth W, Kool M, Kaden S, Gröne HJ, Schulte JH, Lindner S, Hamacher-Brady A, Brady NR, Deubzer HE, Witt O. (2013) Histone deacetylase 10 promotes autophagy-mediated cell survival. Proc Natl Acad Sci U S A 110(28): E2592-2601.
Oehme I, Lodrini M, Brady NR, Witt O (2013) Histone deacetylase 10-promoted autophagy as a druggable point of interference to improve the treatment response of advanced neuroblastomas. Autophagy 9(12):2163-2165.
Rettig I, Koeneke E, Trippel F, Mueller WC, Burhenne J, Kopp-Schneider A, Fabian J, Schober A, Fernekorn U, von Deimling A, Deubzer HE, Milde T, Witt O, Oehme I (2015) Selective inhibition of HDAC8 decreases neuroblastoma growth in vitro and in vivo and enhances retinoic acid-mediated differentiation. Cell Death Dis 6: e1657.
Bingel C, Koeneke E, Ridinger J, Bittmann A, Sill M, Peterziel H, Wrobel JK, Rettig I, Milde T, Fernekorn U, Weise F, Schober A, Witt O, Oehme I (2017) Three-dimensional tumor cell growth stimulates autophagic flux and recapitulates chemotherapy resistance. Cell Death Dis 8: e3013.
Kolbinger FR, Koeneke E, Ridinger J, Heimburg T, Müller M, Bayer T, Sippl W, Jung M, Gunkel N, Miller AK, Westermann F, Witt O, Oehme I (2018) The HDAC6/8/10 inhibitor TH34 induces DNA damage mediated cell death in human high-grade neuroblastoma cell lines. Arch Toxicol. 2018 Aug;92(8):2649-2664.
Shen J, Najafi S, Stäble S, Fabian J, Koeneke E, Kolbinger FR, Wrobel J, Meder B, Distel M, Heimburg T, Sippl W, Jung M, Peterziel H, Kranz D, Boutros M, Westermann F, Witt O, Oehme I (2018) A kinome-wide RNAi screen identifies ALK as a target to sensitize neuroblastoma cells for HDAC8-inhibitor treatment. Cell Death & Differentiation. Dec; 25(12): 2053–2070.
Ridinger J, Koeneke E, Kolbinger FR, Koerholz K, Mahboobi S, Hellweg L, Gunkel N, Miller AK, Peterziel H, Schmezer P, Hamacher-Brady A, Witt O, and Oehme I (2018) Dual role of HDAC10 in lysosomal exocytosis and DNA repair promotes neuroblastoma chemoresistance. Sci Rep. 2018 Jul 3;8(1):10039.