Michael A. Dengler

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.

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.

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

Abstract 2002: OCT-3/4 expression is associated with high levels of the pro-apoptotic BH3 only protein NOXA in testicular germ cell tumors (TGCTs)

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL Embryonal carcinoma (EC) cells are the pluripotent precursors of differentiated structures of testicular germ cell tumors (TGCT) characterized by expression of the embryonal transcription factor OCT-3/4. Loss of OCT-3/4 during differentiation abrogates the extraordinary high sensitivity to cisplatin in these cells. We previously observed a constitutively high expression level of NOXA and a major role of this pro-apoptotic BH3-only protein for cisplatin hypersensitivity in the EC cell lines NTERA-2D1 and 2102EP. We now investigated if these high NOXA protein levels may be directly linked to the OCT-3/4 status in TGCT cells. Therefore, we analyzed NOXA protein expression in an extended TGCT cell line panel of 5 OCT-3/4 negative and 5 OCT-3/4 positive cell lines. Importantly, NOXA protein was highly correlated with OCT-3/4 levels and also with cisplatin sensitivity. These data indicate that constitutively high NOXA levels might be responsible for the low threshold for cisplatin-induced apoptosis in OCT-3/4 positive pluripotent cells. A direct link between OCT-3/4 and NOXA could also be demonstrated by RNAi mediated silencing experiments performed in NTERA-2D1 and 2102EP cells. Silencing of OCT-3/4 resulted in a significant downregulation of NOXA transcript and an almost complete loss of NOXA protein accompanied by a loss of cisplatin sensitivity. This was observed in both differentiation competent NTERA-2D1 cells as well as in differentiation incompetent 2102EP cells pointing to a direct OCT-3/4 dependent NOXA regulation rather than a bystander effect of differentiation upon loss of OCT-3/4. We further validated our in vitro data by comparing OCT-3/4 and NOXA expression levels in patient samples derived from ECs with other non-seminomatous GCTs, seminomas, and tumor entities from lung, breast, and ovary. In agreement with our in vitro observations, OCT-3/4 and NOXA were found to be highly expressed selectively in seminomas and ECs. This could also be confirmed on protein level in primary tissue samples derived from 5 ECs and 8 seminomas by western blot analysis. In conclusion, our data for the first time demonstrate a close correlation between OCT-3/4 and NOXA protein levels and strengthen the previously hypothesized role of constitutively high NOXA levels for the exquisite sensitivity of TGCTs to cisplatin. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2002. doi:1538-7445.AM2012-2002

Abstract 4675: Fatty acid metabolism is a possible target for treatment of cyclin D1 over-expressing mantle cell lymphoma

Cyclin D1 over-expression has been linked to the development and progression of several types of cancer including mantle cell lymphoma (MCL), an aggressive type of B-cell lymphoma characterized by a t(11;14)(q13;q32) chromosomal translocation. Recent studies have shown that cyclin D1 is a multifunctional protein not only regulating the cell cycle but also affecting other cellular processes including DNA repair, apoptosis, as well as glucose, fatty acid, and lipid metabolism. In this study, we investigated the effects of the fatty acid synthase and lipase inhibitor Orlistat on cyclin D1 over-expressing MCL cell lines. In contrast to non-malignant peripheral blood lymphocytes and normal fibroblasts all MCL cell lines examined were sensitive to Orlistat. This enhanced sensitivity was dependent on cyclin D1 overexpression since siRNA mediated cyclin D1 knockdown almost completely rescued MCL cells from Orlistat induced apoptosis. Cell death upon Orlistat treatment was accompanied by loss of mitochondrial membrane potential and dependent on caspase activation since pre-incubation of the cells with the pan-caspase inhibitor zVAD-fmk completely blocked induction of apoptosis. We therefore investigated potential Orlistat-mediated changes in the expression levels of the pro- and anti-apoptotic Bcl-2 family proteins and found NOXA to be strongly induced whereas the expression of other Bcl-2 proteins did not change significantly. RNAi mediated knockdown of NOXA inhibited induction of cell death demonstrating the predominant role of this protein for the proapoptotic effect of Orlistat in MCL cells. Interestingly, silencing of cyclin D1 reduced the expression of NOXA upon Orlistat treatment further indicating a connection between cyclin D1, fatty acid metabolism, and the induction of NOXA. Since inhibition of fatty acid metabolism by Orlistat was found to disturb the balance between pro- and anti-apoptotic proteins in MCL cells we analyzed possible combinatory effects with Bcl-2 family modulators. Co-treatment of the cells with Orlistat and the BH3 mimetic ABT737 led to a rapid induction of cell death and an almost complete loss of cell viability in cyclin D1 over-expressing cells. A similar synergistic effect could be observed by combining Orlistat and 2-deoxy-D-glucose, a glycolysis inhibitor known to reduce MCL1 protein. These combinatory effects were selective for MCL cells as the same treatments had only minor or no effects on cell viability of primary PBMCs and fibroblasts from healthy donors. In summary, our results for the first time indicate that fatty acid metabolism may be an attractive target for therapy of cyclin D1 over-expressing MCL cells. Furthermore, these observations may contribute to the development of rational strategies combining fatty acid metabolism inhibitors with Bcl-2 family modulators for treatment of MCL. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4675. doi:1538-7445.AM2012-4675

Abstract 1266: Oncogenic stress induced by Bcr-Abl over-activation leads to cell death mediated by a massively enhanced aerobic glycolysis

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL It is well established that normal cells respond to over-activation of oncogenes by induction of genetically encoded programs such as apoptosis or senescence. Therefore, too much activity of a growth promoting oncogene seems to disturb the cellular homeostasis, a phenomenon which is known as oncogenic stress. To investigate the molecular mechanisms behind this phenomenon we used cell clones overexpressing the oncogene Bcr-Abl and cultivated them continuously in the presence of the Bcr-Abl inhibitor Imatinib. Removal of Imatinib led to a robust induction of Bcr-Abl autophosphorylation and concomitant overstimulation of the Bcr-Abl downstream pathways PI3K/AKT-, RAS/MAPK-, and JAK/STAT. Importantly, this acute over-activation of Bcr-Abl resulted in a delayed non-apoptotic cell death starting not before 48 hours after Imatinib withdrawal. This cell death was preceded by a massively enhanced aerobic glycolysis and glutaminolysis (Warburg effect) and amino acid metabolism leading to an elevated cellular ATP and protein content. During the first 30 hours after Imatinib deprivation the enhanced metabolism had no effect on proliferation but resulted in cellular swelling, cytoplasmic vacuolization, and massive induction of ER stress markers indicating that Bcr-Abl over-activation induces ER stress response. However, cell death upon Bcr-Abl over-activation was not dependent on this severe ER stress since siRNA mediated knockdown of CHOP and BIM had no effect on cell viability. In contrast, both inhibition of aerobic glycolysis by 2-deoxy-glucose as well as reduction of glutamine concentration in the medium were sufficient to block Imatinib deprivation induced cell death. These data demonstrate that the Bcr-Abl over-activation mediated enhanced metabolism is responsible for oncogenic stress induced cell death. Interestingly, screening of 1200 marketed small molecule inhibitors (Prestwick chemical library) for their potential effects on oncogenic stress response uncovered selectively steroids to effectively antagonize both ER stress and cell death upon Bcr-Abl over-activation. These agents were also able to normalize cellular metabolism upon Imatinib withdrawal, supporting the view that “overshooting” metabolism upon oncogenic over-activation leads to cell death. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1266. doi:10.1158/1538-7445.AM2011-1266

Abstract 4693: High expression levels of NOXA are important for p53-mediated hypersensitivity in testicular germ cell tumor (TGCT) cells

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL Consistent with the excellent clinical results in testicular germ cell tumors (TGCT), most cell lines derived from this cancer such as NTERA-2D1 show an exquisite sensitivity to Cisplatin. It is well accepted that the high susceptibility of TGCT cells to apoptosis is responsible for this hypersensitive phenotype. However, the role of the p53 pathway remains controversial. Using RNA interference-mediated gene silencing, we systematically investigated the impact of DNA damage kinases upstream of p53, p53 itself, as well as apoptosis-related p53 targets on this hypersensitivity. Unexpectedly, neither knockdown of ATM, ATR, or DNA-PK alone nor ATM/ATR double knockdown or ATM/ATR/DNA-PK triple knockdown had any significant protective effect on survival of NTERA-2D1 cells upon Cisplatin. However, siRNA-mediated silencing of p53 was sufficient to completely abrogate hypersensitivity demonstrating that in these cells p53 acts preferentially as a pro-apoptotic factor. This is also supported by the finding that NTERA-2D1 cells were not only hypersensitive to Cisplatin but also to non-genotoxic inducers of p53 such as the Mdm2 antagonist Nutlin-3 and the proteasome inhibitor Bortezomib. Screening of different p53 targets revealed that the pro-apoptotic function of p53 was dependent on NOXA, since silencing of this p53 target almost completely mimicked the effect of the p53 knockdown. Importantly, both constitutive and Cisplatin-induced levels of NOXA were significantly reduced in resistant NTERA-2D1 cells obtained by either short-term differentiation or cultivation with increasing Cisplatin concentrations. Furthermore, comparison of a panel of p53 wildtype tumor cell lines revealed exceptionally high NOXA levels in the most sensitive cell line NTERA-2D1. In conclusion, our data indicate that the hypersensitivity of TGCT cells is a result of their unique sensitivity to pro-apoptotic functions of p53 mediated by exceptionally high levels of NOXA. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4693. doi:10.1158/1538-7445.AM2011-4693