Oncogenic Stress Research Articles

The Power Behind the Throne: Senescence and the Hallmarks of Cancer

Cellular senescence is a state of stable proliferative arrest triggered by various stimuli, including oncogenic and other cellular stress. Senescent cells are highly metabolically active and have diverse and profound nonautonomous effects through the senescence-associated secretory phenotype (SASP). It has become increasingly evident that senescent cells can have tumour suppressive or pro-oncogenic effects on adjacent cancer cells and other players in the tumor microenvironment such as the stroma, vasculature, and immune system. Thus, the last decade or so has witnessed a huge leap forward in our understanding of the biology of senescence, promoting it from an autonomous tumor suppressor to a complex, dynamic, and interactive phenotype. It is perhaps not a coincidence that the concept of the “hallmarks of cancer” has also evolved during this period, with the latest iteration ( Hanahan & Weinberg 2011 ) focusing more on the microenvironment. Here, we suggest that cellular senescence could underpin the biology of many of the hallmarks of cancer, making it the true power behind the throne.

Abstract A16: Potent and selective ATR inhibitors for the treatment of homologous-recombination deficient and PARPi-resistant cancers

Abstract Ataxia Telangiectasia and Rad3-related (ATR) and Checkpoint kinase 1 (CHK1) stabilize stalled replication forks and prevent their collapse into DNA double strand breaks (DSBs). Inhibition of ATR in cells experiencing oncogenic stress or harboring other cancer-associated defects synergistically increases the formation of DSBs and causes synthetic lethality. Thus specific targeting of ATR represents an emerging strategy to treat a broad spectrum of cancers, most notably those that currently lack effective treatments. We and others have shown that inhibition of the ATR checkpoint kinase is synthetically lethal with multiple distinct cancer-associated mutations, including p53 loss, oncogenic stress (HRAS-G12V, KRAS-G12D, NRAS-G12D, MYC and CCNE1), deficiency in homologous recombination (BRCA1/2, PALB2, ATM loss), alternative lengthening of telomeres (ALT), chromatin modification (SETD2 loss), and others. The specificity of ATR inhibitors is vital to their successful clinical application, since off-targeting increases toxicity to normal cells and limits value in personalized treatments. We report the generation of a novel class of highly potent and specific ATR inhibitors (ATRN series) that exhibit low nanomolar activity in cultured cells (IC50 = 2-8 nM) and do not detectably inhibit ATM, DNA-PK or mTOR (IC50 > 10 µM). In side-by-side comparisons, both the potency and selectivity of the ATRN series is superior to previously reported ATR inhibitors (VE821, VE822, AZD20, AZD6738, ETP-46464). In addition, the ATRN series has sufficient bioavailability and stability for in vivo application. Our lead compound (ATRN-119) slows progression of human BRCA2-deficient PDAC (CAPAN1) and RAS oncogene-driven p53-null colon tumors in mice with minimal toxicity to tissues under normal proliferative control, including the bone marrow and intestine. Additionally, mice engrafted with BRCA2-mutant patient-derived xenograft (PDX) ovarian tumors show a significant reduction in tumor progression after 5 weeks treatment of ATRN-119, and display no toxicity or significant weight loss. In addition, cancers that maintain or reacquire HR function, and thus are resistant to treatment with PARPi or cisplatin, remain responsive to ATRN series inhibitors. Thus, ATRN-119 is highly efficacious in suppressing tumor growth in multiple murine models, including suppression of patient-derived BRCA2-mutant ovarian, and PARP resistant cancers, suggesting that the clinical application of the ATRN series will provide a new and effective treatment for human malignancies with fewer side effects than conventional chemotherapies. In summary, the ATRN series is a highly selective and potent class of ATR inhibitors with therapeutic potential for treating a broad range of cancers. The potentially identified biomarkers, in particular HR-deficiency, will inform patient selection (a critical component for delivering medical benefit) for treatment with our agent and could reduce future clinical risk and side-effects. Thus targeting patients with HR-deficient cancers is a particularly promising strategy in treating a broad range of cancers. Citation Format: Laura R. Butler, Ryan L. Ragland, Hank J. Breslin, Erin George, Tina Gill, Matthew Scheiwer, Nicolas Gordon, Karen Knudsen, Fiona Simpkins, Oren Gilad and Eric J. Brown. Potent and selective ATR inhibitors for the treatment of homologous-recombination deficient and PARPi-resistant cancers [abstract]. In: Proceedings of the AACR Special Conference on DNA Repair: Tumor Development and Therapeutic Response; 2016 Nov 2-5; Montreal, QC, Canada. Philadelphia (PA): AACR; Mol Cancer Res 2017;15(4_Suppl):Abstract nr A16.

Suppression of PKC causes oncogenic stress for triggering apoptosis in cancer cells.

Gain of functional mutations in ras occurs in more than 30% of human malignancies and in particular 90% of pancreatic cancer. Mutant ras, via activating multiple effector pathways, not only promote cell growth or survival, but also apoptosis, depending upon cell types or circumstances. In order to further study the mechanisms of apoptosis induced by oncogenic ras, we employed the ras loop mutant genes and demonstrated that Akt functioned downstream of Ras in human pancreatic cancer or HPNE cells ectopically expressing mutated K-ras for the induction of apoptosis after the concurrent suppression of PKC α and β. In this apoptotic process, the redox machinery was aberrantly switched on in the pancreatic cancer cells as well as prostate cancer DU145 cells. p73 was phosphorylated and translocated to the nucleus, accompanied with UPR activation and induction of apoptosis. The in vitro results were corroborated by the in vivo data. Thus, our study indicated that PKC α and β appeared coping with oncogenic Ras or mutated Akt to maintain the balance of the homeostasis in cancer cells. Once these PKC isoforms were suppressed, the redox state in the cancer cells was disrupted, which elicited persistent oncogenic stress and subsequent apoptotic crisis.

Abstract B33: Loss of transcriptional fidelity in cancers confers immunotherapy resistance

Abstract Most cancers are immunogenic, either due to neo-antigenic mutational load that makes them susceptible to rejection by the adaptive immune system, or due to oncogenic stress pathways that makes them vulnerable to innate anti-tumor responses. The therapeutic strategies to reactivate the host anti-tumor immunity have a great promise for cancer therapy. However, despite the unprecedented promise of a cure and durable disease management in select patients, the response rates are still low in advanced cases, most of which also relapse. These findings reflect the multitude of mechanisms of tumor cell evasion of anti-tumor immunity, and that mechanisms beyond inactivation of local lymphocyte infiltrates might also play a role. Through comprehensive computational and follow-up experimental validations, we have found that a subset of cancers (~15-20% of all cancers) is characterized by severe defects in almost the entire epigenetic and transcriptional apparatus, resulting in genome-wide deregulation of histone modifications, mRNA transcription elongation, splicing and processing (Loss of Transcriptional Fidelity: LTF). LTF impaired the transcription of long genes in the genome at both mRNA and protein levels; and the pathways that are primarily regulated by long genes, such as immune-related (FasL response, TNF/NF-kB signaling, interferon signaling), MAP kinase and tyrosine kinase signaling pathways, were significantly suppressed in LTF+ (i.e. those with LTF) tumors. We validated the LTF phenotype and associated epigenetic and transcriptional defects in some cancer cell lines, and showed that severe loss of transcriptional fidelity can confer resistance to pro-inflammatory anti-tumor mechanisms in vitro, and suppress immune-mediated tumor rejection in vivo. As such, we found that LTF predicted poor response to immunotherapeutic, but not to chemo- or targeted therapy, agents in the clinic, including immune checkpoint inhibitors, in 3 different cohorts. Therefore, LTF is a previously unknown global phenotype in cancer cells that confers cell-autonomous resistance to anti-tumor immune attacks. Tumor lymphocyte infiltration is one of the best markers of immunotherapy response in the clinic. However, LTF+ tumors were characterized by a higher rate of immune cell infiltration, but paradoxically, less immune-mediated local tumor cell lysis, further supporting the notion that LTF is a tumor cell-autonomous mechanism of immune resistance. As such, combining tumor lymphocyte infiltration with LTF had superior power in predicting the progression-free and overall survival of melanoma patients treated with the anti-CTLA4 antibody ipilimumab. This study uncovers a major phenotype in cancers that dictates global transcriptional and signaling state in the cell, and response to immunotherapeutic agents in the clinic. Further, this study warrants assessment of markers of LTF in immunotherapy-candidate patients, especially given that LTF+ kidney cancer patients had significantly better response to targeted therapy agents. Citation Format: Navneet Singh, Vishnu Modur, Vakul Mohanty, Kwangmin Choi, Edith M. Janssen, Lisa Privette-Vinnedge, Gang Huang, Kakajan Komurov. Loss of transcriptional fidelity in cancers confers immunotherapy resistance. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2016 Oct 20-23; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2017;5(3 Suppl):Abstract nr B33.

The caspase-3/p120 RasGAP stress-sensing module reduces liver cancer incidence but does not affect overall survival in gamma-irradiated and carcinogen-treated mice.

Activation of oncogenes is the initial step in cellular transformation. Oncogenes favor aberrant proliferation, which, at least initially, induces cellular stress. This oncogenic stress can act as a safeguard mechanism against further transformation by inducing senescence or apoptosis. Yet, the few premalignant cells that tolerate and escape these senescent or apoptotic responses are those that will ultimately generate tumors. The caspase-3/p120 RasGAP module is a stress-sensing device that promotes survival under mild stress conditions. A point mutation in RasGAP that prevents its cleavage by caspase-3 inactivates the pro-survival capacity of the device. When the mice homozygous for this mutation (D455A knock-in mice) are patho-physiologically challenged, they experience much stronger cellular damage than their wild-type counterparts and the affected organs rapidly lose their functionality. We reasoned that the caspase-3/p120 RasGAP module could help premalignant cells to cope with oncogenic stress and hence favor the development of tumors. Using gamma-irradiation and N-ethyl-N-nitrosourea (ENU) as tumor initiators, we assessed the survival advantage that the caspase-3/p120 RasGAP module could provide to premalignant cells. No difference in overall mortality between wild-type and D455A knock-in mice were observed. However, the number of ENU-induced liver tumors in the knock-in mice was higher than in control mice. These results indicate that the caspase-3/p120 RasGAP stress-sensing module impacts on carcinogen-induced liver cancer incidence but not sufficiently so as to affect overall survival. Hence, gamma irradiation and ENU-induced tumorigenesis processes do not critically rely on a survival mechanism that contributes to the maintenance of organ homeostasis in stressed healthy tissues.

Stat5 deficiency decreases transcriptional heterogeneity and supports emergence of hematopoietic sub-populations.

Aging is associated with significant changes in hematopoiesis, including clonal dominance, anemia, myeloid malignancies, and reduced activation of signal transducer and activator of transcription 5 (Stat5). In previous studies, Stat5 deletion surprisingly amplified FLT3/ITD+ myeloid expansion or Myc-driven lymphoid expansion. Here we show that Stat5 deficiency has a strong impact upon transcriptional heterogeneity in single sorted c-Kit+Lin−Sca-1+ (KLS) cells or CD150+CD48− KLS long-term repopulating hematopoietic stem cells (LT-HSC). Single cell polymerase chain reaction (PCR) was performed on selected regulators of multi-lineage hematopoiesis. At least two dominant sub-populations were identified by increased expression of cell cycle regulatory and leukemia-associated genes. Furthermore, in the top expressing quartile of cells, the majority of genes were proportionally overrepresented. In wild-type KLS cells, Stat5 mRNA levels were also strongly correlated with several genes. Since heterogeneity decreases with age or inflammatory or oncogenic stress, these results provide a potential mechanistic linkage to Stat5 expression.

STAT3 and ERK pathways are involved in cell growth stimulation of the ST2/IL1RL1 promoter.

The ST2 gene was originally identified as a primary responsive gene induced by stimulation with growth factors and by oncogenic stress. The ST2 gene harbors two distinct promoters – a distal promoter and a proximal promoter. In this study, we identified a novel type of serum‐responsive element in the ST2 proximal promoter using reporter gene analysis; this element includes a possible responsive element for STAT family proteins. Indeed, enforced expression of constitutively active STAT3 activated this promoter element and induced the expression of ST2 gene products. Furthermore, an oncogenic Ras (G12V) mutant also caused the expression of ST2 gene products by utilizing the proximal promoter. We also clarified that activation of the ST2 promoter by either growth stimulation or oncogenic Ras was suppressed by the inhibitors for STAT3 and ERK pathways. Our observations strongly suggest the importance of STAT family and ERK pathways for the induction of ST2 gene products by cell growth stimulation.

Autophagy Is Pro-Senescence When Seen in Close-Up, but Anti-Senescence in Long-Shot.

When mammalian cells and animals face a variety of internal or external stresses, they need to make homeostatic changes so as to cope with various stresses. To this end, mammalian cells are equipped with two critical stress responses, autophagy and cellular senescence. Autophagy and cellular senescence share a number of stimuli including telomere shortening, DNA damage, oncogenic stress and oxidative stress, suggesting their intimate relationship. Autophagy is originally thought to suppress cellular senescence by removing damaged macromolecules or organelles, yet recent studies also indicated that autophagy promotes cellular senescence by facilitating the synthesis of senescence-associated secretory proteins. These seemingly opposite roles of autophagy may reflect a complex picture of autophagic regulation on cellular senescence, including different types of autophagy or a unique spatiotemporal activation of autophagy. Thus, a better understanding of autophagy process will lead us to not only elucidate the conundrum how autophagy plays dual roles in the regulation of cellular senescence but also helps the development of new therapeutic strategies for many human diseases associated with cellular senescence. We address the pro-senescence and anti-senescence roles of autophagy while focusing on the potential mechanistic aspects of this complex relationship between autophagy and cellular senescence.

DNA Oncogenic Virus-Induced Oxidative Stress, Genomic Damage, and Aberrant Epigenetic Alterations.

Approximately 20% of human cancers is attributable to DNA oncogenic viruses such as human papillomavirus (HPV), hepatitis B virus (HBV), and Epstein-Barr virus (EBV). Unrepaired DNA damage is the most common and overlapping feature of these DNA oncogenic viruses and a source of genomic instability and tumour development. Sustained DNA damage results from unceasing production of reactive oxygen species and activation of inflammasome cascades that trigger genomic changes and increased propensity of epigenetic alterations. Accumulation of epigenetic alterations may interfere with genome-wide cellular signalling machineries and promote malignant transformation leading to cancer development. Untangling and understanding the underlying mechanisms that promote these detrimental effects remain the major objectives for ongoing research and hope for effective virus-induced cancer therapy. Here, we review current literature with an emphasis on how DNA damage influences HPV, HVB, and EBV replication and epigenetic alterations that are associated with carcinogenesis.

Abstract A38: MTH1 Inhibition as an effective tumor-suppressive strategy in RAS-driven cancer

Abstract Oncogenic RAS mutations confer multiple malignant traits to cancer cells, many of which are mediated by oncogene-induced reactive oxygen species (ROS). However, oncogenic ROS also create vulnerabilities in the cancer cells, sensitizing them to oxidative DNA damage and strand breaks which can trigger cellular senescence or cell death. Thus RAS-driven tumor cells require adaptive redox protective mechanisms to inhibit ROS-associated tumor suppression. We previously reported that MutT Homolog 1 (MTH1), the mammalian 8-oxodGTPase, comprises one such critical adaptation. We have shown MTH1 is important for facilitating the full gamut of RAS-driven malignancy by promoting evasion of the first barrier to transformation, viz. oncogene-induced senescence (OIS), enhancing RAS-mediated transformation and related pro-malignant traits, and maintaining proliferation and tumorigenicity in established RAS-driven tumor cells. Recently chemical inhibitors against MTH1 have been developed, although there is some controversy surrounding their efficacy and mode of action. Our work here discusses RAS regulation of MTH1 expression, and the molecular contexts under which MTH1 inhibition can synergize with oncogenic oxidative stress in RAS-driven tumor cells for optimal tumor suppressive response. Citation Format: Govindi Jayanika Samaranayake, Clara Issabella Troccoli, Tyler Andrew Cunningham, Priyamvada Rai. MTH1 Inhibition as an effective tumor-suppressive strategy in RAS-driven cancer. [abstract]. In: Proceedings of the AACR Precision Medicine Series: Targeting the Vulnerabilities of Cancer; May 16-19, 2016; Miami, FL. Philadelphia (PA): AACR; Clin Cancer Res 2017;23(1_Suppl):Abstract nr A38.

DNA repair factor RAD18 and DNA polymerase Polκ confer tolerance of oncogenic DNA replication stress

DNA repair factor RAD18 and DNA polymerase Polκ confer tolerance of oncogenic DNA replication stress

JMJD3 Plays Essential Roles in the Maintenance of Hematopoietic Stem Cells and Leukemic Stem Cells through the Regulation of p16INK4a

Hematopoiesis is a complex process that involves the interplay between lineage-specific transcription and epigenetic regulation, including histone modifications. Tri-methylation of histone H3 at Lys27 (H3K27me3) is an epigenetic mark for transcriptional repression. Jumonji domain-containing 3 (JMJD3) acts as a histone demethylase for H3K27 and contributes to various cellular processes including senescence and differentiation through transcriptional regulation. In the hematopoietic system, JMJD3 has been reported to be required for M2 macrophage development and terminal thymocyte differentiation. However, the roles of JMJD3 in normal hematopoiesis and leukemogenesis are still largely elusive.To address this issue, we generated pIpC-inducible Jmjd3 conditional KO (cKO) mice. Jmjd3-deficient (Jmjd3Δ/Δ) mice grew healthy and did not show obvious hematopoietic abnormalities, except a slight decrease of myeloid cells. To investigate the role of JMJD3in hematopoietic stem cell (HSC) function, a competitive repopulation assay was performed using control and Jmjd3Δ/Δ HSCs. The results showed that the chimerism of Jmjd3Δ/Δ cells was significantly decreased compared with that of control cells in all the hematopoietic lineages, indicating that JMJD3 is essential for long-term repopulating ability of HSCs. To further investigate the effect of Jmjd3 deletion in leukemogenesis, c-kit+ bone marrow (BM) cells from control and Jmjd3 cKO mice were transduced with MLL-AF9 fusion protein that rapidly induces acute leukemia. L-GMPs (the fraction containing leukemic stem cells (LSCs)) were sorted from MLL-AF9-transduced BM cells and subjected to colony replating and bone marrow transplantation (BMT) assays. In contrast control L-GMPs that continued to form colonies after multiple rounds of replating, Jmjd3Δ/Δ L-GMPs ceased to proliferate after third rounds of replating. In addition, recipients transplanted with Jmjd3Δ/Δ L-GMPs exhibited a significant delay in the onset of leukemia compared with those transplanted with controlL-GMPs. These results indicate that JMJD3 plays essential roles in maintaining stem cell properties not only in normal HSCs but also in LSCs.We next investigated underlying molecular mechanisms. Previous studies demonstrated the INK4a/ARF locus, a key executor of cellular senescence, is regulated by JMJD3. Thus, we examined whether JMJD3 regulates INK4a/ARF locus in hematopoietic cells under proliferative and oncogenic stresses. We found that enforced expression of Jmjd3 in in vitro-cultured and cytokine-stimulated hematopoietic stem-progenitor cells (HSPCs) significantly upregulated the expression of p16INK4a compared with control cells. In addition, transformation of HSPCs by MLL-AF9 induced expression of Jmjd3, but not other H3K27me3-related genes, such as Utx and EZH2, which was accompanied by the upregulation of p16INK4a. In contrast, no obvious expressional change was observed in p19ARF in both cases. In Jmjd3Δ/Δ HSPCs, no upregulation of p16INK4a was detected in HSPCs by cytokine-induced proliferation or MLL-AF9-induced transformation, where H3K27me3 was tightly associated with promoter region of p16INK4a locus. These results strongly suggest that proliferative and oncogenic stresses induces the expression of Jmjd3 in HSPCs, which subsequently upregulates p16INK4a through demethylating H3K27me3 on the p16INK4a promoter and consequently maintains stem cell potential by inhibiting excessive entry into cell cycle. Deficiency of Jmjd3 fails upregulation of p16INK4a, which induces continuous and excessive cell proliferation and finally causes exhaustion of stem cell pool. In conclusion, we propose the idea that JMJD3-p16INK4a axis plays essential roles in maintaining HSC and LSC pool size under proliferative and oncogenic stresses. DisclosuresNo relevant conflicts of interest to declare.

Evasion of Necroptosis and Inflammasome Activation Promotes Myeloid Leukemogenesis

Acute myeloid leukemia (AML) is a heterogeneous group of hematopoietic neoplasms driven partly by the loss of differentiation and theblockade of cell death. AML is sustained by leukemia-initiating cells (LICs) that arise from pre-leukemic hematopoietic stem and progenitor cells (HSPCs) that carry genetic alterations being selected for during leukemogenesis. The resistance of LICs to standard chemotherapies presents a major clinical challenge as they eventually cause disease relapse and death. Understanding the mechanisms of LIC resistance to undergoing cell death is therefore critical for a curative therapy of AML.While the regulatory factors that maintain HSPC proliferation and differentiation under normal conditions are well understood, significantly less is known about how LIC fate is regulated. As many hematopoietic disorders are characterized by the overproduction of pro-inflammatory cytokines, we hypothesized that necroptosis controlled cytokine secretion and inflammatory cell death might influence AML development. We therefore addressed the role of MLKL and XIAP in AML and tested whether deletion of Mlkl or Xiap would affect disease progression.Here we show that MLKL limits oncogene-mediated leukemogenesis by promoting the inflammatory cell death of common myeloid progenitors (CMPs) and short-term hematopoietic stem cells (HSCs) in experimental mice. Upon oncogenic stress MLKL-dependent necroptosis and subsequent inflammasome activation were triggered, promoting the production of IL-1β, a potent stimulator of HSPC differentiation and maturation, thus, suppressing the emergence of LICs and limiting leukemogenesis.In a murine bone marrow transplantation model of AML the absence of MLKL accelerated AML development significantly. The enhanced disease was due to the expansion of common myeloid progenitors (CMPs) and short-term hematopoietic stem cells (ST-HSCs), being the cellular compartments to contain LICs. The survival advantage of Mlkl-/- HSPCs became apparent in colony-forming assays and liquid cultures specifically within the CMP and ST-HSC compartments. Sorted ST-HSCs from Mlkl-/- produced more GEMM colonies than WT, the colony type harboring the multipotential myeloid progenitor cells, and both ST-HSCs and CMPs retained significantly more lineage-negative cells in liquid culture. In addition, Mlkl-/- colonies showed a reduction in propidium iodide (PI)-positive dead cells compared with WT colonies. Importantly, WT cells showed caspase activation and produced substantial amounts of the inflammatory cytokine IL-1β which was severely blunted by Mlkl deficiency. We also observed reduced expression of MLKL in leukemic cells on both mRNA and protein level, implying that suppression of cell death was beneficial for the survival of LICs.In contrast, deletion of Xiap did not alter survival or differentiation of leukemic cells when compared with WT cells. Furthermore, XIAP was not differentially expressed on mRNA or protein level compared with WT, indicating that XIAP does not play a critical role in leukemogenesis.In agreement with the murine data, gene expression analysis from primary leukemia cells from two large patient cohorts newly diagnosed with AML showed significantly lower expression of MLKL, but not XIAP, in a variety of AML subtypes compared to healthy controls.Overall, our data demonstrate a key role for MLKL-mediated cell death and activation of the inflammasome in AML and represents a novel tumor-suppressive mechanism. DisclosuresPeschel:MophoSys: Honoraria.

MDMX under stress: the MDMX-MDM2 complex as stress signals hub.

The tumor suppressor p53 plays a central role in safeguarding cellular homeostasis. Upon various types of stress signals such as DNA damage or oncogenic stress, p53 is promptly activated to prevent and repair damages that can threaten the genome stability. The two major negative regulators of p53 are MDM2 and MDMX, two homolog proteins that control p53 activity and turnover, hence keeping it in check during normal cell cycling. In the event of cellular stress, they have to be inhibited in order to relieve p53 from their suppression and allow its activation. As the essential upstream modulator of p53, the MDMX-MDM2 complex integrates multiple signaling pathways regulating p53 response to perturbations of cellular homeostasis. Given its predominantly cytoplasmic localization in normal conditions, we hypothesize that MDMX, rather than MDM2, is the first recipient of signaling cues directed towards the MDMX-MDM2 complex and aimed at modulating p53. In this review we give a synthetic overview of the phosphorylation sites of MDMX that are known to affect its degradation, ubiquitination, intracellular localization and interaction with MDM2 and p53, ultimately modulating the stability and activity of p53. The role of MDMX in response to the main types of cellular stress is also briefly discussed, along with the potential of the MDMX-MDM2 complex as therapeutic target to restore p53 activity.

Dissecting cellular senescence and SASP in Drosophila.

Cellular senescence can act as both tumor suppressor and tumor promoter depending on the cellular contexts. On one hand, premature senescence has been considered as an innate host defense mechanism against carcinogenesis in mammals. In response to various stresses including oxidative stress, DNA damage, and oncogenic stress, suffered cells undergo irreversible cell cycle arrest, leading to tumor suppression. On the other hand, recent studies in mammalian systems have revealed that senescent cells can drive oncogenesis by secreting diverse proteins such as inflammatory cytokines, matrix remodeling factors, and growth factors, the phenomenon called senescence-associated secretory phenotype (SASP). However, the mechanisms by which these contradictory effects regulate tumor growth and metastasis in vivo have been elusive. Here, we review the recent discovery of cellular senescence in Drosophila and the mechanisms underlying senescence-mediated tumor regulation dissected by Drosophila genetics.

Abstract B025: p53 responsive endogeneous retrovirus as early mediators of immunosurveillance

Abstract Triggering the immune system to attack the cancerous cells is the overarching goal of cancer immunotherapy. The key aspect of cancer immunosurveillance theory argues that our immune system acts as sentinels in recognizing and destroying the newly transformed cells that are continuously arising in vivo. Immediately after an oncogenic assault, the cell fires up various intrinsic cellular mechanisms in response to the genotoxic stress. This includes activation of DNA damage response proteins, senescence programs and tumor suppressor genes. These intrinsic responses have to be linked to the extrinsic mechanisms leading to activation of different components of the immune system. This intimate relationship between the intrinsic and the extrinsic mechanisms help to maintain tissue homeostasis and prevent further deleterious effect to the organ. However, very little is known about the critical molecular players that touch off the activation of immune system following the intrinsic response elicited by a genotoxic stress, for example, activation of tumor suppressor. These key molecular intermediaries should ideally possess the following features: 1) their expression should be upregulated in response to cellular stress 2) their upregulation should lead to a distinct immune response in vivo. We have recently identified a molecule that fits both of these criteria. We discovered a murine endogenous retrovirus (ERV) whose expression is dependent on the activity of p53. We found that components of this ERV are sufficient to induce changes in cell viability. It is characterized by a domain which is found in human T-cell leukemia virus type 1, a retrovirus found in blood cancer patients and are known to cause chronic inflammation. We found that this protein domain family is endogenized in the genomes of multiple vertebrates, including humans, potentially highlighting the evolutionary and functional significance of this element. Functional analysis of the murine ERV and homologous human ERV components suggest that these endogenous retroviral elements are early transducers of oncogenic stress, which brokers an immune response at the earliest stage of cellular transformation. We propose that these ERV components play a critical role in developing the immunogenicity against cancer, and may illuminate the mechanistic details of the first few events leading to activation of immune response following an oncogenic assault. Till recently, ERVs have been relegated as a part of the so-called ‘junk DNA’. Only recently have researchers begun revisiting this field to find the significance of ERVs in vertebrate biology. Our work sheds new light on the potential role of ERV remnants in response to neoplastic transformation. Citation Format: Kojiro Tashiro, Robyn Leary, Anindya Bagchi. p53 responsive endogeneous retrovirus as early mediators of immunosurveillance [abstract]. In: Proceedings of the Second CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; 2016 Sept 25-28; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(11 Suppl):Abstract nr B025.

An in silico algorithm for identifying stabilizing pockets in proteins: test case, the Y220C mutant of the p53 tumor suppressor protein.

The p53 tumor suppressor protein performs a critical role in stimulating apoptosis and cell cycle arrest in response to oncogenic stress. The function of p53 can be compromised by mutation, leading to increased risk of cancer; approximately 50% of cancers are associated with mutations in the p53 gene, the majority of which are in the core DNA-binding domain. The Y220C mutation of p53, for example, destabilizes the core domain by 4 kcal/mol, leading to rapid denaturation and aggregation. The associated loss of tumor suppressor functionality is associated with approximately 75 000 new cancer cases every year. Destabilized p53 mutants can be 'rescued' and their function restored; binding of a small molecule into a pocket on the surface of mutant p53 can stabilize its wild-type structure and restore its function. Here, we describe an in silico algorithm for identifying potential rescue pockets, including the algorithm's integration with the Dynameomics molecular dynamics data warehouse and the DIVE visual analytics engine. We discuss the results of the application of the method to the Y220C p53 mutant, entailing finding a putative rescue pocket through MD simulations followed by an in silico search for stabilizing ligands that dock into the putative rescue pocket. The top three compounds from this search were tested experimentally and one of them bound in the pocket, as shown by nuclear magnetic resonance, and weakly stabilized the mutant.

The Fanconi anemia pathway controls oncogenic response in hematopoietic stem and progenitor cells by regulating PRMT5-mediated p53 arginine methylation.

The Fanconi anemia (FA) pathway is involved in DNA damage and other cellular stress responses. We have investigated the role of the FA pathway in oncogenic stress response by employing an in vivo stress-response model expressing the Gadd45β-luciferase transgene. Using two inducible models of oncogenic activation (LSL-K-rasG12D and MycER), we show that hematopoietic stem and progenitor cells (HSPCs) from mice deficient for the FA core complex components Fanca or Fancc exhibit aberrant short-lived response to oncogenic insults. Mechanistic studies reveal that FA deficiency in HSPCs impairs oncogenic stress-induced G1 cell-cycle checkpoint, resulting from a compromised K-rasG12D-induced arginine methylation of p53 mediated by the protein arginine methyltransferase 5 (PRMT5). Furthermore, forced expression of PRMT5 in HSPCs from LSL-K-rasG12D/CreER-Fanca−/− mice prolongs oncogenic response and delays leukemia development in recipient mice. Our study defines an arginine methylation-dependent FA-p53 interplay that controls oncogenic stress response.

Abstract 1226: Highly specific ATR inhibitors as a therapeutic approach for a broad spectrum of cancers

Abstract Ataxia Telangiectasia and Rad3-related (ATR) and Checkpoint kinase 1 (CHK1) stabilize stalled replication forks and prevent their collapse into DNA double strand breaks (DSBs). Inhibition of ATR in cells experiencing oncogenic stress or harboring other cancer-associated defects synergistically increases the formation of DSBs and causes synthetic lethality. Thus specific targeting of ATR represents an emerging strategy to treat a broad spectrum of cancers, most notably those that currently lack effective treatments. Atrin Pharmaceuticals has synthesized a novel series of small molecules that inhibit ATR at low nanomolar concentrations in cultured cells. These compounds have the highest known potency for inhibiting ATR and maintain >800-fold lower cellular activity towards other kinases of the same family (ATM, DNA-PKcs and mTOR), which are substantially off-targeted by previously reported ATR inhibitors. Atrin's lead compound (ATRN-119), as a single agent, selectively kills cancer cells subjected to oncogenic stress, alternative lengthening of telomeres (ALT) or loss of double strand break (DSB) homologous recombination repair mechanism (BRCA1 or BRCA2 deficiency). Furthermore, ATRN-119 cytotoxicity is synergistically enhanced when combined with conventional chemotherapeutics, such as etoposide, cisplatin and olaparib. Therefore, this inhibitor can be used in combination with other therapeutics to potentiate its anti-tumor activity. In vivo studies of RAS oncogene driven HCT116 p53-null flank tumors in mice demonstrate the ability of our lead compound to slow tumor progression, with minimal toxicity to tissues under normal proliferative control, including the bone marrow and intestine. Additionally, mice engrafted with BRCA2-mutant patient-derived ovarian tumors show a significant reduction in progression after 5 weeks treatment (100 mg/kg BID) of ATRN-119, and display no toxicity or significant weight loss. Thus, ATRN-119 is highly efficacious in suppressing tumor growth in multiple murine models, including suppression of patient-derived BRCA2-mutant ovarian cancer, suggesting that the clinical application of the ATRN series will provide a new and effective treatment for human malignancies with fewer side effects than conventional chemotherapies. Citation Format: Laura R. Butler, Ryan L. Ragland, Hank J. Breslin, Tina Gill, Erin George, Fiona Simpkins, Eric J. Brown, Oren Gilad. Highly specific ATR inhibitors as a therapeutic approach for a broad spectrum of cancers. [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 1226.

Abstract 3683: The stress response mediator ATF3 stabilizes p53 by competing with it for MDM2-mediated ubiquitination

Abstract While the tumor suppressor p53 is crucial for the maintenance of genetic integrity upon DNA damage and oncogenic stresses, its level and activity are tightly controlled by a complex network largely mediated by the E3 ubiquitin ligase MDM2. MDM2 catalyzes the addition of ubiqutin chains to the C-terminus of p53 and is the major ubiquitin ligase to promote proteosomal degradation of p53. We previously demonstrated that ATF3, a common stress response mediator, can bind p53 and prevent it from MDM2-mediated ubiquitination. As a consequence, ATF3 can stabilize p53 upon DNA damage and prevent cells from oncogene-induced transformation by promoting p53-mediated senescence, in line with accumulating evidence supporting that ATF3 can act as a tumor suppressor. Interestingly, ATF3 is also a bona fide substrate of MDM2, and thus, the ATF3-MDM2 interaction can fine tune p53 activity in the DNA damage response. However, the mechanism by which ATF3 blocks p53 ubiquitination to activate the latter protein remains largely undefined. Efficient ubiquitination of p53 requires the binding of MDM2 to the N-terminus of p53, and the MDM2 binding to MDMX. Here we show that neither the p53-MDM2 interaction nor the MDM2-MDMX interaction is disrupted by the binding of ATF3 to p53 or MDM2. As both ATF3 and p53 are substrates of MDM2, we explored a possibility that ATF3 competes with p53 for MDM2-mediated ubiquitination. By in-vitro ubiquitination assays, we identified that the 5 lysine residues clustering in aa 100-115 of ATF3 are required for MDM2-mediated ubiquitination. Site-specific mutagenesis indicates that MDM2 mainly ubiquitinates ATF3 at lysine 107 (K107) and lysine 108 (K108) as MDM2 fails to add ubiquitin chains to ATF3 when these two residues were mutated to arginine. Importantly, while mutations at these two residues do not affect the ATF3 binding to MDM2 or p53, the ATF3 mutant devoid of ubiquitination fails to stabilize p53 and prevent p53 from MDM2-mediated ubiquitination. These results thus demonstrate that ATF3 can activate p53 by competing with the tumor suppressor for MDM2-mediated ubiquitination. Citation Format: Xingyao Li, Chunhong Yan. The stress response mediator ATF3 stabilizes p53 by competing with it for MDM2-mediated ubiquitination. [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 3683.