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Ferroptosis is a recently defined form of regulated cell death, which is biochemically and morphologically distinct from traditional forms of programmed cell death such as apoptosis or necrosis. It is driven by iron, reactive oxygen species, and phospholipids that are oxidatively damaged, ultimately resulting in mitochondrial damage and breakdown of membrane integrity. Numerous cellular signaling pathways and molecules are involved in the regulation of ferroptosis, including enzymes that control the cellular redox status. Alterations in the ferroptosis-regulating network can contribute to the development of various diseases, including cancer. Evidence suggests that ferroptosis is commonly suppressed in cancer cells, allowing them to survive and progress. However, cancer cells which are resistant to common chemotherapeutic drugs seem to be highly susceptible to ferroptosis inducers, highlighting the great potential of pharmacologic modulation of ferroptosis for cancer treatment. Non-coding RNAs (ncRNAs) are considered master regulators of various cellular processes, particularly in cancer where they have been implicated in all hallmarks of cancer. Recent work also demonstrated their involvement in the molecular control of ferroptosis. Hence, ncRNA-based therapeutics represent an exciting alternative to modulate ferroptosis for cancer therapy. This review summarizes the ncRNAs implicated in the regulation of ferroptosis in cancer and highlights their underlying molecular mechanisms in the light of potential therapeutic applications.

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 - 79K

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

PDF file - 82K, TaqMan assays

PDF file - 56K, qPCR primer pairs

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

The BCL2 inhibitor venetoclax has established therapeutic roles in chronic lymphocytic leukemia (CLL) and acute myeloid leukemia (AML). As BCL2 is an important determinant of survival of both myeloid progenitor and B cells, we investigated whether clinical and molecular abnormalities arise in the myeloid compartment during long-term continuous venetoclax treatment of CLL in 89 patients (87 with relapsed/refractory CLL). Over a median follow-up of 75 (range 21-98) months, persistent cytopenias (≥1 of neutropenia, thrombocytopenia, anemia) lasting ≥4 months and unrelated to CLL occurred in 25 patients (28%). Of these patients, 20 (80%) displayed clonal hematopoiesis, including 10 with therapy-related myeloid neoplasms (t-MNs). t-MNs occurred exclusively in patients previously exposed to fludarabine-alkylator combination therapy with a cumulative 5-year incidence of 10.4% after venetoclax initiation, consistent with rates reported for patients exposed to fludarabine-alkylator combination therapy without venetoclax. To determine whether the altered myelopoiesis reflected the acquisition of mutations, we analyzed samples from patients with no or minimal bone marrow CLL burden (n = 41). Mutations in the apoptosis effector BAX were identified in 32% (13/41). In cellular assays, C-terminal BAX mutants abrogated outer mitochondrial membrane localization of BAX and engendered resistance to venetoclax killing. BAX-mutated clonal hematopoiesis occurred independently of prior fludarabine-alkylator combination therapy exposure and was not associated with t-MNs. Single-cell sequencing revealed clonal co-occurrence of mutations in BAX with DNMT3A or ASXL1. We also observed simultaneous BCL2 mutations within CLL cells and BAX mutations in the myeloid compartment of the same patients, indicating lineage-specific adaptation to venetoclax therapy.

Randomized trials in acute myeloid leukemia (AML) have demonstrated improved survival by the BCL-2 inhibitor venetoclax combined with azacitidine in older patients, and clinical trials are actively exploring the role of venetoclax in combination with intensive chemotherapy in fitter patients with AML. As most patients still develop recurrent disease, improved understanding of relapse mechanisms is needed. We find that 17% of patients relapsing after venetoclax-based therapy for AML have acquired inactivating missense or frameshift/nonsense mutations in the apoptosis effector gene BAX. In contrast, such variants were rare after genotoxic chemotherapy. BAX variants arose within either leukemic or preleukemic compartments, with multiple mutations observed in some patients. In vitro, AML cells with mutated BAX were competitively selected during prolonged exposure to BCL-2 antagonists. In model systems, AML cells rendered deficient for BAX, but not its close relative BAK, displayed resistance to BCL-2 targeting, whereas sensitivity to conventional chemotherapy was variable. Acquired mutations in BAX during venetoclax-based therapy represent a novel mechanism of resistance to BH3-mimetics and a potential barrier to the long-term efficacy of drugs targeting BCL-2 in AML.

The conformational changes converting BAX from an inert cytosolic monomer into the homo-oligomers that permeabilize the mitochondrial outer membrane (MOM) are crucial steps toward apoptosis. Here, we have explored the potential role of the BAX α1-α2 loop in this process by three mutagenic approaches: replacing loop segments with cognate loop regions from closely related proteins, alanine scanning and analysis of BAX α1-α2 loop missense mutations observed in tumours. Responsiveness to a death signal, such as tBID, was reduced by mutations in the N-terminal but not C-terminal half of the loop. N-terminal loop variants, which were enriched in tumours, impaired MOM integration by allosterically reducing exposure of the BAX α9 transmembrane anchor. Most C-terminal loop variants reduced BAX stability, leading to increased BAX apoptotic function in some variants. Thus, our systematic mutagenesis suggests that the two halves of the α1-α2 loop have distinct functions. We show that the N-terminal half of the loop (its first nine residues) comprises an important allosteric regulator of BAX activation by setting the proportion of MOM-integrated BAX following a death signal. The enrichment of N-terminal loop mutations in tumours indicates that they may promote tumour cell survival and underscore the loop as a target for therapeutic manipulation of BAX function.

Selective targeting of BCL-2 with the BH3-mimetic venetoclax has been a transformative treatment for patients with various leukemias. TP-53 controls apoptosis upstream of where BCL-2 and its prosurvival relatives, such as MCL-1, act. Therefore, targeting these prosurvival proteins could trigger apoptosis across diverse blood cancers, irrespective of TP53 mutation status. Indeed, targeting BCL-2 has produced clinically relevant responses in blood cancers with aberrant TP-53. However, in our study, TP53-mutated or -deficient myeloid and lymphoid leukemias outcompeted isogenic controls with intact TP-53, unless sufficient concentrations of BH3-mimetics targeting BCL-2 or MCL-1 were applied. Strikingly, tumor cells with TP-53 dysfunction escaped and thrived over time if inhibition of BCL-2 or MCL-1 was sublethal, in part because of an increased threshold for BAX/BAK activation in these cells. Our study revealed the key role of TP-53 in shaping long-term responses to BH3-mimetic drugs and reconciled the disparate pattern of initial clinical response to venetoclax, followed by subsequent treatment failure among patients with TP53-mutant chronic lymphocytic leukemia or acute myeloid leukemia. In contrast to BH3-mimetics targeting just BCL-2 or MCL-1 at doses that are individually sublethal, a combined BH3-mimetic approach targeting both prosurvival proteins enhanced lethality and durably suppressed the leukemia burden, regardless of TP53 mutation status. Our findings highlight the importance of using sufficiently lethal treatment strategies to maximize outcomes of patients with TP53-mutant disease. In addition, our findings caution against use of sublethal BH3-mimetic drug regimens that may enhance the risk of disease progression driven by emergent TP53-mutant clones.

Ferroptosis is a recently defined form of regulated cell death, which is biochemically and morphologically distinct from traditional forms of programmed cell death such as apoptosis or necrosis. It is driven by iron, reactive oxygen species, and phospholipids that are oxidatively damaged, ultimately resulting in mitochondrial damage and breakdown of membrane integrity. Numerous cellular signaling pathways and molecules are involved in the regulation of ferroptosis, including enzymes that control the cellular redox status. Alterations in the ferroptosis-regulating network can contribute to the development of various diseases, including cancer. Evidence suggests that ferroptosis is commonly suppressed in cancer cells, allowing them to survive and progress. However, cancer cells which are resistant to common chemotherapeutic drugs seem to be highly susceptible to ferroptosis inducers, highlighting the great potential of pharmacologic modulation of ferroptosis for cancer treatment. Non-coding RNAs (ncRNAs) are considered master regulators of various cellular processes, particularly in cancer where they have been implicated in all hallmarks of cancer. Recent work also demonstrated their involvement in the molecular control of ferroptosis. Hence, ncRNA-based therapeutics represent an exciting alternative to modulate ferroptosis for cancer therapy. This review summarizes the ncRNAs implicated in the regulation of ferroptosis in cancer and highlights their underlying molecular mechanisms in the light of potential therapeutic applications.

Significance Neutralizing antibodies are important for immunity against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and as therapeutics for the prevention and treatment of COVID-19. We identified high-affinity nanobodies against SARS-CoV-2 receptor-binding domain and found that nanobody cocktails consisting of two noncompeting nanobodies were able to block ACE2 engagement with RBD variants present in human populations and potently neutralize both wild-type SARS-CoV-2 and the N501Y D614G variant at low concentrations. Prophylactic administration of nanobody cocktails reduced viral loads in mice infected with the N501Y D614G SARS-CoV-2 virus, showing that nanobody cocktails are useful as prophylactic agents against SARS-CoV-2., Neutralizing antibodies are important for immunity against SARS-CoV-2 and as therapeutics for the prevention and treatment of COVID-19. Here, we identified high-affinity nanobodies from alpacas immunized with coronavirus spike and receptor-binding domains (RBD) that disrupted RBD engagement with the human receptor angiotensin-converting enzyme 2 (ACE2) and potently neutralized SARS-CoV-2. Epitope mapping, X-ray crystallography, and cryo-electron microscopy revealed two distinct antigenic sites and showed two neutralizing nanobodies from different epitope classes bound simultaneously to the spike trimer. Nanobody-Fc fusions of the four most potent nanobodies blocked ACE2 engagement with RBD variants present in human populations and potently neutralized both wild-type SARS-CoV-2 and the N501Y D614G variant at concentrations as low as 0.1 nM. Prophylactic administration of either single nanobody-Fc or as mixtures reduced viral loads by up to 104-fold in mice infected with the N501Y D614G SARS-CoV-2 virus. These results suggest a role for nanobody-Fc fusions as prophylactic agents against SARS-CoV-2.

Summary Programmed cell death contributes to host defense against pathogens. To investigate the relative importance of pyroptosis, necroptosis, and apoptosis during Salmonella infection, we infected mice and macrophages deficient for diverse combinations of caspases-1, -11, -12, and -8 and receptor interacting serine/threonine kinase 3 (RIPK3). Loss of pyroptosis, caspase-8-driven apoptosis, or necroptosis had minor impact on Salmonella control. However, combined deficiency of these cell death pathways caused loss of bacterial control in mice and their macrophages, demonstrating that host defense can employ varying components of several cell death pathways to limit intracellular infections. This flexible use of distinct cell death pathways involved extensive cross-talk between initiators and effectors of pyroptosis and apoptosis, where initiator caspases-1 and -8 also functioned as executioners when all known effectors of cell death were absent. These findings uncover a highly coordinated and flexible cell death system with in-built fail-safe processes that protect the host from intracellular infections., Graphical Abstract, Highlights • Mice lacking pyroptosis and apoptosis cannot control Salmonella infection • Macrophages lacking pyroptosis and apoptosis resist Salmonella-induced killing • Caspase-1 kills Salmonella-infected cells by activating GSDMD, BID, or other caspases • Caspase-1 and -8 act as cell death executioners when all cell death effectors are lost, The clearance of intracellular pathogens requires the killing of infected cells, but it remains unclear why host cells have so many different means of inducing programmed cell death. Doerflinger et al. demonstrate that interconnectivity between pyroptosis and apoptosis involving flexible deployment of caspases ensures control of Salmonella infection in mice.

BAK and BAX are the essential effectors of apoptosis because without them a cell is resistant to most apoptotic stimuli. BAK and BAX undergo conformation changes to homooligomerize then permeabilize the mitochondrial outer membrane during apoptosis. How BCL-2 homology 3 (BH3)-only proteins bind to activate BAK and BAX is unclear. We report that BH3-only proteins bind inactive full-length BAK at mitochondria and then dissociate following exposure of the BAK BH3 and BH4 domains before BAK homodimerization. Using a functional obstructive labeling approach, we show that activation of BAK involves important interactions of BH3-only proteins with both the canonical hydrophobic binding groove (α2-5) and α6 at the rear of BAK, with interaction at α6 promoting an open groove to receive a BH3-only protein. Once activated, how BAK homodimers multimerize to form the putative apoptotic pore is unknown. Obstructive labeling of BAK beyond the BH3 domain and hydrophobic groove did not inhibit multimerization and mitochondrial damage, indicating that critical protein-protein interfaces in BAK self-association are limited to the α2-5 homodimerization domain.

Apoptosis-regulating BCL-2 family members, which can promote malignant transformation and resistance to therapy, have become prime therapeutic targets, as illustrated by the striking efficacy in certain lymphoid malignancies of the BCL-2-specific inhibitor venetoclax. In other lymphoid malignancies, however, such as the aggressive mantle cell lymphoma (MCL), cell survival might rely instead or also on BCL-2 relative MCL-1. We have explored MCL-1 as a target for killing MCL cells by both genetic and pharmacologic approaches. In several MCL cell lines, MCL-1 knockout with an inducible CRISPR/Cas9 system triggered spontaneous apoptosis. Accordingly, most MCL cell lines proved sensitive to the specific MCL-1 inhibitor S63845, and MCL-1 inhibition also proved efficacious in an MCL xenograft model. Furthermore, its killing efficacy rose on combination with venetoclax, the BCL-XL-specific inhibitor A-1331852, or Bruton's tyrosine kinase (BTK) inhibitor ibrutinib, which reduced pro-survival signals. We also tested the MCL-1 inhibitor in primary samples from 13 MCL patients, using CD40L-expressing feeder cells to model their microenvironmental support. Notably, all unstimulated primary MCL samples were very sensitive to S63845, but the CD40L stimulation attenuated their sensitivity. Mass cytometric analysis revealed that the stimulation likely conveyed protection by elevating BCL-XL and MCL-1. Accordingly, sensitivity of the CD40L-stimulated cells to S63845 was substantially restored by co-treatment with venetoclax, the BCL-XL-specific inhibitor or ibrutinib. Overall, our findings indicate that MCL-1 is very important for survival of MCL cells and that the MCL-1 inhibitor, both alone and together with ibrutinib, venetoclax or a BCL-XL inhibitor, offers promise for novel improved MCL therapies.

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

Summary: Apoptotic cell death removes unwanted cells and is regulated by interactions between pro-survival and pro-apoptotic members of the BCL-2 protein family. The regulation of apoptosis is thought to be crucial for normal embryonic development. Accordingly, complete loss of pro-survival MCL-1 or BCL-XL (BCL2L1) causes embryonic lethality. However, it is not known whether minor reductions in pro-survival proteins could cause developmental abnormalities. We explored the rate-limiting roles of MCL-1 and BCL-XL in development and show that combined loss of single alleles of Mcl-1 and Bcl-x causes neonatal lethality. Mcl-1+/–;Bcl-x+/– mice display craniofacial anomalies, but additional loss of a single allele of pro-apoptotic Bim (Bcl2l11) restores normal development. These findings demonstrate that the control of cell survival during embryogenesis is finely balanced and suggest that some human craniofacial defects, for which causes are currently unknown, may be due to subtle imbalances between pro-survival and pro-apoptotic BCL-2 family members. : Grabow et al. find that combined loss of single alleles of pro-survival Mcl-1 and Bcl-x causes craniofacial defects, including holoprosencephaly, a severe birth defect. Normal development is restored by concomitant loss of a single allele of pro-apoptotic Bim, revealing that cell survival and cell death during embryogenesis are finely balanced. Keywords: MCL-1, MCL1, BCL-XL, BCL2L1, BIM, BCL2L11, apoptosis, embryonic development, holoprosencephaly, cyclopia

Aims The purpose of this clinical trial was to investigate whether cardiovascular magnetic resonance imaging (CMR) using ferumoxytol (Feraheme™, FH), an ultrasmall superparamagnetic iron oxide nanoparticle (USPIO), allows more detailed characterization of infarct pathology compared with conventional gadolinium-based necrosis/fibrosis imaging in patients with acute myocardial infarction. Methods and results Fourteen patients who had experienced an acute ST-elevation myocardial infarction were included in this study. Following coronary angiography, a first baseline study (pre-FH) was performed followed by subsequent CMR studies (post-FH) 48 h after intravenous ferumoxytol administration. The CMR studies comprised cine-CMR, T 2-weighted short tau inversion recovery spin echo imaging, T 2*-mapping, and T 1-weighted late gadolinium enhancement (LGE) imaging. The median extent of short-axis in-plane LGE was 30% [inter-quartile range (IQR) 26–40%]. The median in-plane extent of T 2-weighted ‘hypoenhancement’ in the region of myocardial infarction, which was not present prior to ferumoxytol administration in any patient, was 19% (IQR 14–22%; P < 0.001 compared with the extent of LGE). The median in-plane extent of areas showing signal void in T 2*-mapping images post-FH in the region of myocardial infarction was 16% (IQR 12–18%; P < 0.001 compared with the extent of LGE; P = 0.34 compared with the extent of T 2-weighted hypoenhancement). A substantial drop in absolute T 2*-values was observed not only in the infarct core and peri-infarct zone, but also in the remote ‘healthy’ myocardium, although there was only a minor change in the skeletal muscle. Substantial ferumoxytol uptake was detected only in cultured macrophages, but not in peripheral blood monocytes from study patients. Conclusion We could demonstrate in humans that USPIO-based contrast agents enable a more detailed characterization of myocardial infarct pathology mainly by detecting infiltrating macrophages. Considering the multi-functionality of USPIO-based particles and their superior safety profile compared with gadolinium-based compounds, these observations open up new vistas for the clinical application of USPIO.

An insoluble graft copolymer composed of the covalently bound polyoxyethylene to cross-linked polystyrene-divinylbenzene was employed as the solid support for peptide synthesis. The physical properties of this new polymeric support were similar to those of the cross-linked polystyrene. Thus, identical manipulations such as shaking and filtration could be performed on this resin. The graft copolymer was used for the synthesis of the C-terminal decapeptide of bovine insulin B-chain in the usual solid-phase manner. The protected peptide was cleaved from the polymeric support by photolysis and was purified by chromatography on silica gel and Sephadex LH-20. Samples of the decapeptide were treated with liquid HF and the unprotected peptide was purified by ion-exchange chromatography. The free peptide was shown to be homogeneous by HPLC, electrophoresis and t.l.c. The identity of this peptide was also confirmed by FAB-MS and amino acid analysis. The synthesized peptides were shown to be free of racemization. The free peptide possesses a disordered conformation as revealed by the CD measurement.

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. Cancer Res; 73(5); 1460–9. ©2012 AACR.

Mantle cell lymphoma (MCL) is clinically characterized by an aggressive course and only transient response to chemotherapy. Therefore, new treatment strategies for this malignancy are warranted. The genetic hallmark of MCL is the t(11;14)(q13;q32) translocation leading to deregulated expression of cyclin D1. It has been reported recently that cyclin D1 overexpression in MCL is associated with constitutively high levels of GSTP1 and PMAIP1 transcripts, coding for the glutathion S-transferase pi (GSTpi) and for the proapoptotic protein NOXA, respectively. Whereas GSTpi protein is stably expressed, NOXA half-life turned out to be extremely short in this B cell lymphoma leading to constitutively low levels of this BH3-only protein in these cells. We asked if this MCL typical phenotype can be utilized for a selective treatment by stabilizing NOXA protein and concurrent inhibition of GSTpi activity. By screening different compounds known to stabilize NOXA protein and GSTpi inhibitors we found that combination of the fatty acid synthase (FASN) inhibitor Cerulenin together with Ezatiostat or Ethacrynic Acid is not only effective but also selective for MCL cells. Cell death induced by lethal doses of Cerulenin was dependent on Cyclin D1 since siRNA-mediated knockdown of Cyclin D1 partially rescued cells from apoptosis. Combinatory treatment of cells with sub-lethal doses of Cerulenin together with Ezatiostat/Ethacrynic Acid had no effect on viability of fibroblasts from normal donors but effectively killed both MCL cell lines as well as primary cells from MCL patients. Importantly, Cerulenin-mediated NOXA accumulation was significantly further elevated upon combination with GSTpi inhibitors and associated with a concomitant induction of reactive oxygen species (ROS) levels. Cell death was dependent on both NOXA and ROS since either inhibition of ROS by GSH or Catalase or siRNA-mediated knockdown of NOXA was sufficient to almost completely rescue cells from Cerulenin/GSTpi inhibitor induced apoptosis. In conclusion we demonstrate that combined pharmacological inhibition of GSTpi activity and stabilization of NOXA protein via inhibition of fatty acid synthase might be an effective strategy to selectively kill MCL cells and offer novel treatment options. Citation Format: Markus Kleih, Simon Heine, Michael Dengler, Lea Schaaf, Elisabeth Hoering, Heike Horn, German Ott, Walter E. Aulitzky, Heiko van der Kuip. Combined targeting of NOXA and GSTpi effectively kills mantle cell lymphoma cells. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3732.

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 ofNOXA gene expression. Interestingly, MCL cells adapt to this constitutive pro-apoptotic signal by extensive ubiquitination and rapid proteasomal degradation of NOXA protein (T½B15–30 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. Cell Death and Disease (2014) 5, e1013; doi:10.1038/cddis.2013.552; published online 23 January 2014 Subject Category: Cancer

Mantle cell lymphoma (MCL) is an aggressive type of non-Hodgkin lymphoma with comparatively short response to chemotherapy and frequent relapses. New treatment strategies for this malignancy are urgently needed. The genetic hallmark of MCL is the t(11;14)(q13;q32) translocation. This alteration leads to deregulated expression of the oncogene Cyclin D1 and is considered the primary event in the pathogenesis of MCL. Additionally, deregulations of different oncogenic signaling and cell death pathways have been described in MCL. In this study, we investigated the role of the BH3-only protein NOXA for life/death decision in MCL. We found a stunning discrepancy between constitutive Noxa (PMAIP1) mRNA and NOXA protein levels in MCL cell lines and primary cells. Noxa mRNA was found to be highly expressed whereas NOXA protein levels were low. Remarkably, constitutive high gene expression of this pro-apoptotic Bcl2 family member was dependent on Cyclin D1 overexpression and chronic active B cell receptor (BCR) signaling, two of the major oncogenic alterations in MCL. We identified the PI3K/AKT/mTOR pathway to be crucial in the maintenance of Noxa transcript levels downstream of BCR. Cyclin D1 overexpression contributed to the high Noxa mRNA levels by exerting a positive feedback loop on PI3K/AKT/mTOR signaling. Intriguingly, the high constitutive Noxa transcript levels do not impair cell viability. MCL cells adapt to this constitutive pro-apoptotic signal by keeping NOXA protein low due to extensive ubiquitination and rapid proteasomal degradation of NOXA protein (T½ ∼ 15-30 min). As expected, treatment of the cells with the proteasome inhibitor Bortezomib accumulated NOXA protein and efficiently induced cell death. Additionally, we identified the neddylation inhibitor MNL4924 and the fatty acid synthase (FASN) inhibitor Orlistat as potent inducers of NOXA protein, and thereby apoptosis, in MCL. Cell death upon Bortezomib as well as MLN4924 and Orlistat treatment was dependent on NOXA since RNAi mediated silencing of the pro-apoptotic protein significantly reduced induction of apoptosis. We found that all three inhibitors targeted the rapid NOXA protein turnover by stabilizing the preexisting pool of NOXA. In contrast to Bortezomib, however, MLN4924 and Orlistat inhibited the ubiqutination process of NOXA protein and stabilized the pro-apoptotic protein by a proteasome independent manner. These findings could be of great clinical relevance as Bortezomib resistance is a frequently observed phenomenon. Indeed, both inhibitors were still able to induce NOXA and cell death in Bortezomib resistant clones through targeting ubiquitine-proteasome system-mediated NOXA turnover upstream of the proteasome. Interestingly, active PI3K/AKT/mTOR signaling was needed for effective accumulation of NOXA protein and induction of cell death by all these compounds indicating that the high constitutive Noxa mRNA levels are essential for sensitivity of MCL cells. Together, our data highlight that NOXA regulation may represent an important Achilles heel of MCL cells. Stabilization of NOXA protein by inhibition of the ubiquitin-proteasome system on different levels might be an effective strategy to kill MCL cells and offer novel treatment options. Disclosures: No relevant conflicts of interest to declare.

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

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

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

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 1656 Mantle cell lymphoma (MCL) is an aggressive type of B-cell lymphoma characterized by a t(11;14)(q13;q32) chromosomal translocation resulting in a constitutive over-expression of cyclin D1. Cyclin D1 is a multifunctional protein not only regulating cell cycle progression but also affecting other cellular functions including glucose and lipid metabolism. In this study, we investigated the effects of the fatty acid synthase (FASN) inhibitors orlistat and C75 on viability of different MCL cell lines including JEKO-1, MINO, Granta-519, JVM-2, and Rec-1. In all cell lines inhibition of FASN alone induced apoptosis. In contrast, normal peripheral blood lymphocytes were resistant to these compounds. This proapoptotic effect was dependent on cyclin D1 as silencing of cyclin D1 partially rescued cells from induction of cell death following FASN inhibition. Inhibition of FASN led to a strong induction of the proapoptotic protein NOXA (PMAIP) in all MCL cell lines investigated independent of the p53 status. Pre-treatment of cells with NOXA siRNA significantly reduced induction of cell death demonstrating the predominant role of this proapoptotic protein for the observed effects. We then analyzed the combined effects of inhibition of FASN with a panel of compounds with potential clinical relevance. Combination of FASN inhibitors with the BH3 mimetic ABT737, the proteasome inhibitor Bortezomib, and the glycolysis inhibitor 2-deoxy-D-glucose (2-DG) led to an almost complete loss of viability. These combinatory effects were selective for MCL cells as the same treatments had almost no effects on cell viability of primary PBMCs or fibroblasts from healthy donors. Bortezomib, 2-DG, and ABT737 enhanced the proapoptotic effect of FASN inhibitors by further disturbing the balance of pro- and antiapoptotic Bcl-2 family proteins: FASN inhibitor mediated induction of NOXA was enhanced by Bortezomib whereas 2-DG significantly reduced the NOXA antagonist Mcl-1. In summary, our results suggest that FASN inhibitors exert significant antitumoral activity especially in combination with Bcl-2 family modulators in different MCL cell lines and may therefore represent an attractive model for the design of new therapeutic approaches for this entity. Disclosures: No relevant conflicts of interest to declare.

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. © 2011 Gutekunst et al.

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

Abstract 3150 Imatinib resistance is a major problem in treatment of Bcr-Abl positive leukemias, particularly in patients with ALL and advanced CML. One mechanism of this resistance is overexpression of Bcr-Abl. We investigated the effects of Imatinib deprivation in Bcr-Abl overexpressing Imatinib-resistant ALL cell lines. Removal of Imatinib from culture medium led to induction of a non-apoptotic cell death starting approximately 40 hours after Imatinib withdrawal. This cell death was preceded by a rapid and robust induction of Bcr-Abl autophosphorylation and concomitant overstimulation of PI3K-, MAPK-, and JAK/STAT signalling. Over-activation of Bcr-Abl downstream signalling was accompanied by a significant enhanced glucose and amino acid metabolism and an elevated intracellular protein and nucleic acid content. As a result of this enhanced metabolisms we observed a massive increase in cell size, multinucleation, cytoplasmic vacuolization, and reduced intracellular ATP level. The phase-lucent vacuoles did not incorporate the endosome/lysosome tracker, Lyso Tracker Red indicating that the vacuoles were formed by dilation of ER cisternae. To determine if Imatinib deprivation induces an ER stress response concurrent with vacuolization we investigated typical ER stress markers. Upon removal of Imatinib a massive induction of CHOP expression and eIF2 alpha phosphorylation, as well as alternative splicing of XPB could be detected. It has been demonstrated that severe ER stress promotes cell death either by induction of a BIM-dependent apoptitic process or by a TRAF2-RIP1 dependent pathway. Despite of a robust induction of BIM and posttranslational modification of RIP1, siRNA-mediated suppression of BIM and RIP1 had no effect on Imatinib deprivation-induced cell death whereas downmodulation of CHOP partially rescued those cells. Using different small molecule inhibitor libraries, we also identified inhibitors of glycogen synthase kinase-3 (GSK3) and p38-MAPK, as well as glucocorticoids as potent compounds which not only completely prevented metabolic stress and induction of cell death upon removal of Imatinib but also partially repressed induction of CHOP upon Imatinib withdrawal. Importantly, in the presence of glucocorticoids, we found a significant enhanced number of cell clones with Bcr-Abl overexpression in lympoid cell lines upon de novo transfection with Bcr-Abl. In conclusion, acute over-activation of the oncogene Bcr-Abl led to metabolic stress and ER stress followed by a delayed induction of a necrosis-like cell death. These cellular responses to Bcr-Abl-mediated oncogenic stress could be completely blocked by glucocorticoids. Therefore, treatment of glucocorticoid resistant ALL may generate selective pressure towards Bcr-Abl overexpession. Disclosures: No relevant conflicts of interest to declare.

Imatinib resistance is a major problem in treatment of Bcr-Abl positive leukemias, particularly in patients with ALL and advanced CML. One of the major mechanisms of this resistance is overexpression of Bcr-Abl. We investigated the effects of Imatinib deprivation in Bcr-Abl overexpressing Imatinib-resistant ALL cell lines. Removal of Imatinib from culture medium led to a huge induction of Bcr-Abl autophosphorylation and concomitant overstimulation of PI3K-, MAPK-, and JAK/STAT signalling leading to an elevated cell size and cellular protein content. Massive induction of cell death was then observed at later time points (48 hours after Imatinib deprivation) accompanied by loss of outer and inner mitochondrial membrane integrity. Using a protein kinase inhibitor library, we identified inhibitors of glycogen synthase kinase-3 (GSK3) and p38-MAPK as the most potent compounds which completely prevented induction of cell death upon removal of Imatinib whereas the same inhibitors had synergistic effects with Imatinib in Imatinib-sensitive ALL cells. In contrast to GSK3- and p38, inhibition of PI3K had no effect on oncogenic stress upon Imatinib withdrawal in the resistant clones. Importantly, oncogenic stress-induced cell death could not be blocked by the pan-caspase inhibitor z-VAD-fmk whereas the RIP1 inhibitor Necrostatin partially rescued the cells upon Imatinib deprivation. We also detected a posttranslational modification of RIP1 upon Imatinib withdrawal that was completely absent in cells pre-treated with GSK3- or p38 inhibitors indicating a central role of RIP1 for oncogenic stress-induced cell death. In conclusion, we found a massive induction of a necroptosis-like cell death in Bcr-Abl overexpressing ALL cells as a consequence of oncogenic stress upon Imatinib deprivation. GSK3 and p38 turned out to play the most prominent role for oncogenic stress induced cell death. These data implicate that a discontinuous treatment with Imatinib (periods of Imatinib treatment followed by short periods without Imatinib) may prevent the appearance of cell clones with Bcr-Abl overexpression. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4522.