Matthias Gutekunst

p53 Hypersensitivity Is the Predominant Mechanism of the Unique Responsiveness of Testicular Germ Cell Tumor (TGCT) Cells to Cisplatin

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.

Capturing complex tumour biology in vitro: histological and molecular characterisation of precision cut slices

Precision-cut slices of in vivo tumours permit interrogation in vitro of heterogeneous cells from solid tumours together with their native microenvironment. They offer a low throughput but high content in vitro experimental platform. Using mouse models as surrogates for three common human solid tumours, we describe a standardised workflow for systematic comparison of tumour slice cultivation methods and a tissue microarray-based method to archive them. Cultivated slices were compared to their in vivo source tissue using immunohistochemical and transcriptional biomarkers, particularly of cellular stress. Mechanical slicing induced minimal stress. Cultivation of tumour slices required organotypic support materials and atmospheric oxygen for maintenance of integrity and was associated with significant temporal and loco-regional changes in protein expression, for example HIF-1α. We recommend adherence to the robust workflow described, with recognition of temporal-spatial changes in protein expression before interrogation of tumour slices by pharmacological or other means.

Cisplatin Hypersensitivity of Testicular Germ Cell Tumors Is Determined by High Constitutive Noxa Levels Mediated by Oct-4

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.

Oncogenic Stress Induced by Acute Hyper-Activation of Bcr-Abl Leads to Cell Death upon Induction of Excessive Aerobic Glycolysis

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.

Discrepant NOXA ( PMAIP1 ) transcript and NOXA protein levels: a potential Achilles’ heel in mantle cell lymphoma

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.

RITA can induce cell death in p53-defective cells independently of p53 function via activation of JNK/SAPK and p38.

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.

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

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).

Abstract 8: Differential roles of OCT3/4, SOX2 and NANOG for constitutive high NOXA expression levels in embryonal carcinoma (EC) cells

Proceedings: AACR 106th Annual Meeting 2015; April 18-22, 2015; Philadelphia, PA Recently we found that hypersensitivity of embryonal carcinoma (EC) to chemotherapy is mediated by high constitutive levels of NOXA protein. This pro-apoptotic BH3-only protein primes EC cells to undergo rapid and massive apoptosis in response to p53 activation. Both hypersensitivity as well as high NOXA protein levels were lost upon differentiation in these cells. We here investigated the role of three key regulators of pluripotency, namely OCT3/4, SOX2 and NANOG for NOXA protein and transcript (PMAIP1) expression in two EC cell lines, the pluripotent NTERA-2D1 and the nullipotent 2102EP. We found that siRNA-mediated silencing of POU5F1 (OCT3/4) led to a down-regulation of PMAIP1 mRNA by ∼80% and to an almost complete loss of NOXA protein (by >90%). On the other hand, silencing of SOX2 or NANOG only slightly reduced transcript levels (by ∼20% and ∼30%, respectively). At the same time, a distinct down-regulation of NOXA protein levels (by ∼75%) was observed in SOX2- and NANOG-deprived cells, respectively. These data indicate that the high constitutive levels of NOXA in EC cells depend on two independent mechanisms, (1) transcriptional regulation predominantly mediated by OCT3/4, and (2) post-transcriptional regulation mediated by either one of the stem cell factors. Indeed, we found that siRNA-mediated loss of one stem cell factor led to a ∼2fold reduction of NOXA protein stability in both cell lines reaching a NOXA half-life of approximately 2 hours, which was comparable to that observed in PHA stimulated lymphocytes from normal donors. These data demonstrate that the high constitutive levels of NOXA protein in EC cells are due to OCT3/4 dependent induction of the PMAIP1 transcript and a prolonged NOXA protein stability mediated by either one of the stem cell factors. Citation Format: Christine Bayha, Matthias Gutekunst, Walter E. Aulitzky, Heiko van der Kuip. Differential roles of OCT3/4, SOX2 and NANOG for constitutive high NOXA expression levels in embryonal carcinoma (EC) cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 8. doi:10.1158/1538-7445.AM2015-8

Abstract 1721: Testicular germ cell tumors are hypersensitive to p53 activation based on their Oct-4/Noxa-mediated cellular context rather than on differential p53 activity.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC TGCTs are highly apoptosis-prone cancers which can be cured by Cisplatin-based chemotherapy. We have previously shown that p53 plays a major role in this phenotype. Furthermore, we demonstrated that sensitivity of these tumors is dictated by p53 accumulation in general and is not restricted to Cisplatin. Hypersensitivity seems to be an intrinsic feature of pluripotent TGCTs, since both differentiation and loss of the pluripotency factor Oct-4 induce resistance. We sought to investigate the link between the Oct-4-mediated pluripotent context and the proapoptotic p53 response in these tumors. For this, we tested if loss of Oct-4 also reduces p53-mediated apoptosis upon non-genotoxic p53 activation. Indeed, RNAi-mediated silencing of Oct-4 significantly reduced sensitivity to the p53 activator Nutlin-3. Hence, we investigated a possible differential activation of p53 in the context of Oct-4-positive TGCT cells. For this, a qPCR system that allows simultaneous acquisition of 46 bona fide p53 target genes was used to detect p53’s transactivation capacity in NTERA-2D1 and 2102EP cells upon Cisplatin treatment in the presence and absence of Oct-4. In spite of p53’s central role in Cisplatin-induced apoptosis, no significant alterations in transactivation of p53 target genes were observed upon Cisplatin treatment in both cell lines. We also analyzed the cellular distribution of p53 upon Cisplatin treatment and found that p53 was not only accumulated in the nucleus but was increased to a similar extent in the cytoplasm. Importantly, RNAi-mediated silencing of Oct-4 did neither influence accumulation of p53 in the nucleus nor in the cytoplasm. These data indicate that Oct-4 does not directly modulate p53 activity but provides a cellular context that augments the proapoptotic activity of p53. We have previously proposed that Oct-4-dependent high constitutive Noxa protein levels account for the low apoptotic threshold in TGCTs. In contrast to Cisplatin or Nutlin-3, treatment with MG132 resulted in apoptosis in Oct-4-depleted NTERA-2D1 and 2102EP cells. This was due to simultaneous accumulation of p53 and Noxa, as RNAi-mediated silencing of either protein rescued the cells from death. It is of importance that p53 knockdown did not compromise Noxa accumulation upon MG132 treatment, indicating that both p53 activation and the Noxa-mediated apoptosis-prone context are required for high sensitivity. In conclusion, our data demonstrate that in spite of its impact on hypersensitivity, p53 activity is not altered in Oct-4-positive TGCT cells when compared to relatively resistant Oct-4-depleted cells. Although a robust p53 response is essential for hypersensitivity of TGCTs, its predominantly proapoptotic characteristic requires the apoptosis-prone cellular context of pluripotent Oct-4-positive TGCT cells, which includes high Noxa protein levels. Citation Format: Matthias Gutekunst, Thomas Mueller, Andrea Weilbacher, Michael A. Dengler, Moshe Oren, Walter E. Aulitzky, Heiko van der Kuip. Testicular germ cell tumors are hypersensitive to p53 activation based on their Oct-4/Noxa-mediated cellular context rather than on differential p53 activity. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1721. doi:10.1158/1538-7445.AM2013-1721

Abstract 1717: High NOXA (PMAIP1) transcript levels combined with a short-lived NOXA protein primes mantle cell lymphoma (MCL) cells for death by inhibition of the ubiquitin proteasome system.

MCL is an aggressive type of non-Hodgkin lymphoma with poor prognosis and short survival. The genetic hallmark of MCL is the t(11;14) translocation leading to an aberrant expression of the oncogene cyclin D1. Several other deregulated pathways also contribute to the pathogenesis of MCL, including the DNA damage response-, B-cell receptor- and PI3K/mTOR pathway. Deregulated oncogenic pathways not only induce proliferation but also frequently lead to a constitutive stress signal. A common response to cellular stress is the transcriptional up-regulation of pro-apoptotic genes. By analyzing mRNA expression of these genes in MCL cells we found high levels of the pro-apoptotic Bcl-2 family member NOXA (PMAIP1). NOXA transcript was significantly higher in MCL cell lines as well as in samples from MCL patients when compared to other cancer cell lines and PBMCs. To analyze if these high transcript levels depend on activation of oncogenic pathways, we treated the MCL cells with a panel of inhibitors of different signaling pathways and found that the inhibition of the PI3K/mTOR pathway led to a significant reduction of NOXA mRNA levels. These results indicate that this signaling axis is not only acting pro-survival but also mediates up-regulation of NOXA mRNA. In contrast to the high transcript levels, NOXA protein is low in MCL cells. We found that NOXA protein is unstable with half-life times of 15-30 min. Inhibitors of the ubiquitin proteasome system (UPS) such as bortezomib, MG-132 and the cullin-ubiquitin ligase inhibitor MLN4924 stabilized NOXA and led to a strong accumulation of the protein. This was accompanied by a rapid induction of cell death. Interestingly, similar effects could be observed using the fatty acid synthase inhibitor orlistat indicating that fatty acid metabolism is also involved in the UPS. Reducing the high NOXA mRNA levels by treating the cells with the PI3K/mTOR dual-inhibitor Bez235 or using RNA interference prior to treatment with UPS inhibitors significantly reduced cell death and NOXA protein accumulation. These results indicate that the high NOXA mRNA levels are essential for the response of MCL cells to proteasomal inhibitors. In summary, our results show that MCL have a constitutive signal mediated by the PI3K/mTOR pathway resulting in a high expression of NOXA mRNA. The cells survive by rapidly degrading the NOXA protein. Therefore, an effective strategy to kill MCL cells is to target the high NOXA protein turnover in these cells by inhibiting the proteasome ubiquitin system. This mechanism apparently underlies the in vitro activity of bortezomib which is already used in the clinic for treatment of patients with mantle cell lymphoma. Moreover, also other inhibitors such as MLN4924 or increasing the metabolic stress by orlistat might be promising to selectively kill MCL cells. Citation Format: Michael A. Dengler, Andrea Weilbacher, Matthias Gutekunst, Annette M. Staiger, Heike Horn, German Ott, Heiko van der Kuip, Walter E. Aulitzky. High NOXA (PMAIP1) transcript levels combined with a short-lived NOXA protein primes mantle cell lymphoma (MCL) cells for death by inhibition of the ubiquitin proteasome system. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1717. doi:10.1158/1538-7445.AM2013-1717