Prolonged Growth Arrest Research Articles

Abstract 2959: Signatures of therapy-induced senescence in glioblastoma-derived extracellular vesicles

Abstract The recalcitrant nature of glioblastoma (GBM) may be due, in part, to the promotion of therapy-induced senescence (TIS) by standard of care treatments. TIS is a prolonged growth arrest associated with epigenetic, metabolic, and biochemical alterations, as well as a robust secretory phenotype. Despite an initial period of tumor stasis due to the loss of cell proliferation, TIS is associated with the development of stemness, increased drug resistance, and immune suppression. Our preliminary data demonstrate the prominent induction of TIS in GBM tumor cells and astrocytes following standard of care therapies for GBM in vitro. However, studies of TIS in GBM patients have been limited due to i) the impracticality of receiving multiple GBM biopsies post-treatment but prior to recurrence and ii) the lack of robust detection methods for TIS even with appropriate biopsy. These challenges argue the need for novel methods to survey TIS in patients. Extracellular vesicles (EVs) are small membrane-bound particles that readily cross the blood brain barrier, and convey information about originating cell identity and cell state through their cargo content. We hypothesize that TIS in host and tumor cells drives the elaboration of senescence-associated EVs (senEVs) with quantifiable senescence protein cargo signatures, and consequently, that EVs can serve as a liquid biopsy for senescence in patients. In a preliminary experiment, two patient derived xenografts grown as short-term in vitro cultures were exposed to 6GY irradiation, with supernatant collected prior to radiation (D0), at peak senescence (D7), and at late senescence (D10). EVs were isolated and subjected to unbiased mass spectrometry. Following unsupervised clustering, EVs were grouped by treatment rather than by cell line. Approximately 6000 proteins were identified across the entire data set, with 105 proteins unique to D7 samples, 162 proteins unique to D10 samples, and 688 proteins exclusive to D7 and D10 samples. Further, proteins from two established senescence gene sets were identified as unique to the D7 and/or D10 conditions. Current work to more robustly identify enrichment of senescence gene sets in EV cargo post-irradiation is underway. We have also established shRNA knockdown cell lines with abrogated TIS to equip validation studies of the TIS-EV cargo relationship. We anticipate that these studies will aid in the development of EVs as a novel reporter of senescence in the clinic. Citation Format: Valerie DeLuca, Priya Digumarti, Krystine Garcia-Mansfield, Patrick Pirrotte, Michael E. Berens. Signatures of therapy-induced senescence in glioblastoma-derived extracellular vesicles [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2959.

DIPG-12. Induced mitotic abnormalities associated with BMI-1 modulation sensitize diffuse intrinsic pontine glioma cells to ionizing radiation

Abstract Diffuse intrinsic pontine glioma (DIPG) remains an incurable childhood brain cancer with a median overall survival of less than 12 months, affecting 200-300 children annually in the United States. Hence, there is an unmet need for the development of novel and effective targeted therapies. BMI-1 is a subunit of the multimeric protein complex Polycomb repressor complex 1 (PRC1) implicated in self-renewal of normal and cancer cells, and in DNA damage signaling. We have previously identified BMI-1 as a potential therapeutic target in DIPG and have shown that BMI-1 is highly expressed in DIPG tumors regardless of H3K27 mutational status. Treatment of DIPG cells with PTC596, a small molecule initially identified as a BMI-1 modulator, and ionizing radiation (IR) impairs the kinetics of DNA damage response. in vivo, treatment with PTC596 alone delayed tumor growth kinetics and induced in-tumor apoptosis. However, we observed tumor regrowth once PTC596 treatment is completed or discontinued. In the present study, we evaluated the use of PTC596 in combination with IR. Our in vivo results indicate that PTC596 sensitizes DIPG cells to IR inducing a prolonged cell growth arrest 14 days post-treatment compared to IR or PTC596 alone. The effectiveness of this combination is currently evaluated in murine orthotopic DIPG models and the results will be presented. PTC596 is being tested in newly diagnosed children with DIPG and high-grade gliomas (NCT03605550). Data collected from this study will support the development of a novel therapy including PTC596 in combination with radiotherapy to treat children with DIPG.

Trade-off between sex and growth in diatoms: Molecular mechanisms and demographic implications.

Diatoms are fast-growing and winning competitors in aquatic environments, possibly due to optimized growth performance. However, their life cycles are complex, heteromorphic, and not fully understood. Here, we report on the fine control of cell growth and physiology during the sexual phase of the marine diatom Pseudo-nitzschia multistriata. We found that mating, under nutrient replete conditions, induces a prolonged growth arrest in parental cells. Transcriptomic analyses revealed down-regulation of genes related to major metabolic functions from the early phases of mating. Single-cell photophysiology also pinpointed an inhibition of photosynthesis and storage lipids accumulated in the arrested population, especially in gametes and zygotes. Numerical simulations revealed that growth arrest affects the balance between parental cells and their siblings, possibly favoring the new generation. Thus, in addition to resources availability, life cycle traits contribute to shaping the species ecological niches and must be considered to describe and understand the structure of plankton communities.

DUSP6 regulates radiosensitivity in glioblastoma by modulating the recruitment of phosphorylated DNAPKcs at DNA double-strand breaks.

Glioblastoma (GBM) has poor median survival due to its resistance to chemoradiotherapy, which results in tumor recurrence. Recurrent GBMs currently lack effective treatments. DUSP6 is known to be pro-tumorigenic and is upregulated in GBM. We show that DUSP6 expression is significantly higher in recurrent GBM patient biopsies compared to expression levels in primary GBM biopsies. Importantly, although it has been reported to be a cytoplasmic protein, we found nuclear localization of DUSP6 in primary and recurrent patient samples and in parent and relapse populations of GBM cell lines generated from an in vitro radiation survival model. DUSP6 inhibition using BCI resulted in decreased proliferation and clonogenic survival of parent and relapse cells. Pharmacological or genetic inhibition of DUSP6 catalytic activity radiosensitized primary and, importantly, relapse GBM cells by inhibiting the recruitment of phosphorylated DNAPKcs (also known as PRKDC), subsequently downregulating the recruitment of phosphorylated histone H2AX (γH2AX) and 53BP1 (also known as TP53BP1). This resulted in decreased cell survival and prolonged growth arrest upon irradiation in vitro and significantly increased the progression-free survival in orthotopic mouse models of GBM. Our study highlights a non-canonical function of DUSP6, emphasizing the potential application of DUSP6 inhibitors in the treatment of recurrent GBM.

CGAS guards against chromosome end-to-end fusions during mitosis and facilitates replicative senescence

As a sensor of cytosolic DNA, the role of cyclic GMP-AMP synthase (cGAS) in innate immune response is well established, yet how its functions in different biological conditions remain to be elucidated. Here, we identify cGAS as an essential regulator in inhibiting mitotic DNA double-strand break (DSB) repair and protecting short telomeres from end-to-end fusion independent of the canonical cGAS-STING pathway. cGAS associates with telomeric/subtelomeric DNA during mitosis when TRF1/TRF2/POT1 are deficient on telomeres. Depletion of cGAS leads to mitotic chromosome end-to-end fusions predominantly occurring between short telomeres. Mechanistically, cGAS interacts with CDK1 and positions them to chromosome ends. Thus, CDK1 inhibits mitotic non-homologous end joining (NHEJ) by blocking the recruitment of RNF8. cGAS-deficient human primary cells are defective in entering replicative senescence and display chromosome end-to-end fusions, genome instability and prolonged growth arrest. Altogether, cGAS safeguards genome stability by controlling mitotic DSB repair to inhibit mitotic chromosome end-to-end fusions, thus facilitating replicative senescence.

Simultaneous Targeting Tumor Cells and Cancer-Associated Fibroblasts with a Paclitaxel-Hyaluronan Bioconjugate: In Vitro Evaluation in Non-Melanoma Skin Cancer.

Background: Cancer-associated fibroblasts (CAFs) facilitate many aspects of cancer development by providing a structural framework rich in bioactive compounds. There are emerging studies proposing a combination of conventional anti-cancer therapies directed against neoplastic cells to molecules targeting tumor microenvironments. Methods: The study evaluated the pharmacological properties of the anti-tumor agent paclitaxel conjugated to hyaluronic acid (HA) regarding non-melanoma skin cancer (NMSC) and the surrounding fibroblasts. This molecule, named Oncofid-P20 (Onco-P20), preferentially targets cells expressing high levels of CD44, the natural ligand of HA. Results: Consistent with paclitaxel’s mechanism of action involving interference with the breakdown of microtubules during cell division, highly sensitive carcinoma cells rapidly underwent apoptotic cell death. Interestingly, less sensitive cells, such as dermal fibroblasts, resisted the Onco-P20 treatment and experienced a prolonged growth arrest characterized by morphological change and significant modification of the gene expression profile. Onco-P20-treated fibroblasts exhibited reduced growth factor production, downmodulation of the Wnt signaling pathway, and the acquisition of a marked pro-inflammatory profile. Independently of direct exposure to taxol, in the presence of Onco-P20-treated fibroblasts or in their conditioned medium, carcinoma cells had a reduced proliferation rate. Similar to NHF, fibroblasts isolated from skin cancer lesions or from adjacent tissue acquired anti-neoplastic activity under Onco-P20 treatment. Conclusion: Collectively, our data demonstrate that Onco-P20, exerting both a direct and an NHF-mediated indirect effect on carcinoma cells, is a candidate for an innovative therapy alternative to surgery for the treatment of NMSC.

Rucaparib Treatment Alters p53 Oscillations in Single Cells to Enhance DNA-Double-Strand-Break-Induced Cell Cycle Arrest

DNA double strand breaks induce oscillatory expression of the transcription factor p53 that is dependent on ataxia telangiectasia mutated (ATM) activity and the rate of double strand break resolution. Although p53 dynamics are known to play a role in the regulation of cell fate determination, the consequences of the variability in dynamics associated with differences in repair rates and utilized repair pathways are unknown. Using single-cell time-lapse microscopy, we found that disruption of specific repair pathways has distinct impacts on p53 dynamics. The small-molecule rucaparib, an inhibitor of the alternative end-joining-associated protein poly (ADP-ribose) polymerase (PARP), increased p53 pulse duration, altering the temporal expression of multiple p53 target genes. As a result, combination treatments of the radiomimetic drug neocarzinostatin with rucaparib drove prolonged growth arrest beyond that of DNA damage alone. This study highlights how pharmacological manipulation of DNA repair pathways may be used to alter p53 dynamics to enhance therapeutic regimens.

37P - Adaption of pancreatic cancer cells to AKT1 inhibition induces the acquisition of cancer stem-cell like phenotype through upregulation of mitochondrial functions

BackgroundAKT/PKB is a protein kinase that plays a key role in pancreatic adenocarcinoma. Three isoforms with a similar structure but non-overlapping functions have been described: AKT1/PKBα, AKT2/PKBβ and AKT3/PKBγ. Our project studies the molecular routes of adaption to single AKT isoform silencing. MethodsWe have individually silenced AKT isoforms using short hairpin RNAs (shRNAs) delivered by lentivirus. A shRNA against an irrelevant gene was used as control. When cells adapted to the modification, high-throughput quantitative proteomics analyses were performed to evaluate the differentially altered molecular routes. Mitochondrial protein expression was determined by Western Blot. Mitochondrial function was evaluated using Agilent SeaHorse XF. Cancer stem-cell like phenotype was determined by CD44 and EpCAM expression. A subsequent silencing of the escape routes discovered was performed. ResultsOnly cells adapted to AKT1 silencing, but not AKT2 or AKT3, exhibited a cancer stem-cell like phenotype with a sharp increase in CD44/EpCAM expression compared to the other cell lines or control (p<0.0001). shAKT1 expressing cells presented a potentiation of mitochondrial functions determined both by quantitative proteomics, and by basal and maximal oxygen consumption rate (p<0.0001). Double silencing of AKT1 and the mitochondrial protein TFB2M caused a prolonged cell growth arrest. ConclusionsWhen exposed to stable AKT1 inhibition, pancreatic adenocarcinoma cells adapt by switching their metabolism from glycolysis to mitochondrial respiratorion, which suggests a reversion of Warburg effect, and adopting cancer stem-cell like phenotype. Cancer stem cells have been proposed as one of the main factors favouring therapy resistance. Targeting mitochondrial metabolism might improve the efficacy of conventional treatments. Legal entity responsible for the studyGrupo de Inmunomodulación. FundingGrupo de Inmunomodulación. DisclosureAll authors have declared no conflicts of interest.

Abstract 899: Selective clearance of cells undergoing therapy-induced senescence in a model of triple negative breast cancer

Abstract Despite the ability of chemotherapy (and/or radiation) to eliminate the majority of tumor cells, some residual surviving cells may be able to escape cell death by entering into a prolonged senescence-based growth arrest. In addition to apoptosis, the majority of anti-cancer therapies induce senescence that has long been considered to be a favorable outcome of treatment due to its perceived irreversibility. However, accumulating studies in the literature demonstrate that therapy induced senescence may reflect one form of tumor dormancy and disease recurrence, when senescent cells are able to regain proliferative capacity. Tumor cells that escape dormancy often demonstrate an aggressive phenotype and are refractory to further treatment. Senescent cells are characterized by a specific (though often heterogeneous) secretory profile (Senescence Associated Secretory Phenotype or SASP), some components of which have been shown to promote tumorigenesis and may assist in neoplastic escape after therapy. In the current work, we evaluated the capacity of the BCL protein family inhibitor, ABT 263, to selectively clear breast tumor cells induced into senescence by doxorubicin. MDA-MB231-231 human breast cancer cells (a model of triple negative breast cancer) were induced into senescence upon exposure to a clinical concentration of the chemotherapeutic agent, doxorubicin (Dox). While a fraction of the MDA-MB-231 cell population underwent cell death, a large subpopulation entered into a state of prolonged growth arrest that lasted for several days, after which time the cells recovered proliferative capacity and formed colonies. At the time points when cells were arrested, prominent Senescence Associated β-Galactosidase staining marker indicative of senescence. In addition, tumor tissue collected from mice that received injections of Dox also were positive for β-Galactosidase staining. Cells induced into senescence and treated with ABT263 for 18 hours showed a significant increase in apoptosis and did not regain colony forming capabilities. We therefore postulate that senescence and escape from senescence may represent a form of tumor dormancy and disease recurrence, respectively. Consequently, if Therapy Induced Senescence is a deleterious outcome of treatment, it is of importance to identify strategies that might prevent senescent cells from neoplastic escape and induce cell death in order to completely eliminate residual tumors and thereby interfere with disease recurrence. Citation Format: Liliya Tyutyunyk, Tareq Saleh, David A. Gewirtz. Selective clearance of cells undergoing therapy-induced senescence in a model of triple negative breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 899.

Abstract 460: Reversibility of chemotherapy-induced senescence in breast tumor cells in culture and in vivo

Abstract Despite the ability of chemotherapy (and/or radiation) to eliminate the majority of tumor cells, some residual surviving cells may be able to escape cell death by entering into a prolonged growth arrest from which they can ultimately re-emerge and proliferative . In this study, we evaluated two mechanisms, autophagy and senescence, that in theory could allow tumor cells to escape apoptotic cell death, and which therefore might be responsible for cancer recurrence. 4T1 murine breast cancer cells exposed to the chemotherapeutic agent, Adriamycin (doxorubicin), underwent both autophagy and senescence. Senescence induced by anticancer drugs or radiation is a state of prolonged growth arrest that is thought to be irreversible and is considered an alternative (but nevertheless desirable) response to apoptosis. Our studies, however, suggest that Therapy Induced Senescence (TIS) is reversible and serves as a mechanism of evading cell death. When subjected to acute treatment with Adriamycin, some of the 4T1 cells underwent apoptosis; however, a large population entered into a state of prolonged growth arrest that lasted for several days, after which time the cells recovered proliferative capacity and formed colonies. At the time points when cells were arrested, prominent Senescence Associated β-Galactosidase staining and polyploidy were detected, markers indicative of senescence. Inhibition of autophagy did not prevent the tumor cells from undergoing senescence and later escaping growth arrest. To further establish the reversibility of senescence, senescent cells that were isolated by Flow Cytometry (based on their enlarged morphology and detection of a fluorescent β-Gal substrate (C12FDG)), plated and monitored over time; these cells were also shown to recover, as was the case from with the non-sorted senescent cells. Sorted and isolated senescent cells implanted into either NSG (immunodeficient) or syngeneic (immunocompetent) mice also recovered and developed into tumors; recovery was accelerated in the NSG mice, suggesting that the immune system may (at least transiently) recognize and eliminate the tumor cells either at senescence or during recovery from senescence. Senescent cells are characterized by a specific (though often heterogeneous) secretory profile (Senescence Associated Secretory Phenotype or SASP), some components of which have been shown to promote tumorigenesis and may assist in neoplastic escape after therapy. Furthermore, senescence and escape from senescence may represent a form of tumor dormancy and disease recurrence, respectively. Therefore, it is important to treat Therapy Induced Senescence as a deleterious outcome of treatment and identify strategies that might prevent senescent cells from neoplastic escape and induce cell death to completely eliminate residual tumors. Citation Format: Liliya Tyutyunyk, Joseph Landry, Tareq Saleh, David Gewirtz. Reversibility of chemotherapy-induced senescence in breast tumor cells in culture and in vivo [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 460.

MEK inhibitors overcome resistance to BET inhibition across a number of solid and hematologic cancers

BET inhibitors exhibit broad activity in cancer models, making predictive biomarkers challenging to define. Here we investigate the biomarkers of activity of the clinical BET inhibitor GSK525762 (I-BET; I-BET762) across cancer cell lines and demonstrate that KRAS mutations are novel resistance biomarkers. This finding led us to combine BET with RAS pathway inhibition using MEK inhibitors to overcome resistance, which resulted in synergistic effects on growth and survival in RAS pathway mutant models as well as a subset of cell lines lacking RAS pathway mutations. GSK525762 treatment up-regulated p-ERK1/2 levels in both RAS pathway wild-type and mutant cell lines, suggesting that MEK/ERK pathway activation may also be a mechanism of adaptive BET inhibitor resistance. Importantly, gene expression studies demonstrated that the BET/MEK combination uniquely sustains down-regulation of genes associated with mitosis, leading to prolonged growth arrest that is not observed with either single agent therapy. These studies highlight a potential to enhance the clinical benefit of BET and MEK inhibitors and provide a strong rationale for clinical evaluation of BET/MEK combination therapies in cancer.

DNA Damage Response Regulates Initiation of Liver Regeneration Following Acetaminophen Overdose.

Acetaminophen (APAP) overdose is the leading cause of acute liver failure (ALF) with limited treatment options. It is known that liver regeneration following APAP-induced ALF is a deciding factor in the final outcome. Previous studies from our laboratory using an incremental dose model involving a regenerating (300 mg/kg, APAP300) and a nonregenerating (600 mg/kg, APAP600) dose of APAP in mice have revealed several proregenerative pathways that regulate regeneration after APAP overdose. Here we report that DNA damage and repair mechanisms regulate initiation of liver regeneration following APAP overdose. Mice treated with nonregenerating APAP600 dose showed prolonged expression of pH2AX, a marker of the DNA double-strand break (DSB), compared with APAP300. In regenerating APAP300 dose-treated mice, H2AX was rapidly dephosphorylated at Tyr142, indicating timely DNA repair. Expression of several DNA repair proteins was substantially lower with APAP600. Poly(ADP) ribose polymerase (PARP) activation, involved in DNA repair, was significantly higher in the APAP300 group compared to the APAP600 group. Activation of p53, the major cell cycle checkpoint protein, was significantly higher with APAP600 as demonstrated by substantially higher expression of its target genes. Taken together, these data show that massive DNA DSB occurs in high-dose APAP toxicity, and lack of prompt DSB repair after APAP overdose leads to prolonged growth arrest and proliferative senescence, resulting in inhibited liver regeneration.

Abstract 5459: Autophagy and senescence as possible mechanisms leading to proliferative recovery and escape from treatment-induced tumor dormancy

Abstract Despite the ability of chemotherapy to eliminate the majority of tumor cells, some are able to escape cell death and proliferate. In this study we evaluated mechanisms of autophagy and senescence, that in theory allow cells to escape apoptosis and might be responsible for cancer recurrence. Autophagy and senescence, either alone or in concert, may result in temporary growth arrest followed by proliferative recovery. With regard to autophagy, its function may be either cytoprotective, where autophagy inhibition results in sensitization to therapy or nonprotective, where autophagy inhibition does not alter sensitivity to chemotherapy and/or ionizing radiation. To determine the mode of autophagy in murine metastatic carcinoma 4T1 cell lines, autophagy was inhibited pharmacologically using chloroquine, and by genetic silencing of the Atg5 autophagy associated protein. Cells were treated with 1 μM Adriamycin, stained with Acridine Orange to assess autophagy by fluorescent microscopy (autophagosomes appear orange-red) and autophagy quantified by Flow Cytometry. Induction and blockade of autophagy by chloroquine was confirmed by Western blotting for the appearance of LC3B and degradation of p62. Senescence was monitored by β-galactosidase staining and quantified by Flow Cytometry based on C12FDG staining; senescence was confirmed based on the induction of p21 and p16. Adriamycin (doxorubicin) exposure resulted in breast tumor cell death as well as prolonged growth arrest; some of the arrested cells eventually recovered and formed colonies. After exposure to doxorubicin, 4T-1 cells were sorted based on intensity of β-galactosidase staining and increased size (senescent cells experience changes in size and morphology). β-galactosidase positive cells were plated and monitored over time during which some of the cells recovered the capacity to proliferate. In separate studies with mouse mammary carcinoma cells, radiation also promoted cell death and prolonged growth arrest from which some cells were able to recover proliferative capacity. Our data suggests that although chemotherapy and radiation induce prolonged growth arrest and senescence, these features are not permanent and cells are able to escape and re-emerge from the senescent state to generate proliferating daughter cells. As pharmacologic inhibition of autophagy did not result in increased sensitivity to doxorubicin in 4T1 cells (or to radiation in the mouse mammary tumor cells) or interfere with proliferative recovery, we postulate that inhibition of senescence associated pathways may block proliferative recovery and/or promote tumor cell killing in response to chemotherapy and/or radiation. Studies in progress are focused on modulation of c-myc and miR34 levels and the IL1/6/8 signaling axis as potential strategies for interference with senescence and suppression of residual (dormant) tumor growth and cancer recurrence. Citation Format: Liliya Tyutyunyk, Theresa Thekkudan, Tareq Saleh, David A. Gewirtz. Autophagy and senescence as possible mechanisms leading to proliferative recovery and escape from treatment-induced tumor dormancy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5459. doi:10.1158/1538-7445.AM2017-5459

Significance of Wild-Type p53 Signaling in Suppressing Apoptosis in Response to Chemical Genotoxic Agents: Impact on Chemotherapy Outcome.

Our genomes are subject to potentially deleterious alterations resulting from endogenous sources (e.g., cellular metabolism, routine errors in DNA replication and recombination), exogenous sources (e.g., radiation, chemical agents), and medical diagnostic and treatment applications. Genome integrity and cellular homeostasis are maintained through an intricate network of pathways that serve to recognize the DNA damage, activate cell cycle checkpoints and facilitate DNA repair, or eliminate highly injured cells from the proliferating population. The wild-type p53 tumor suppressor and its downstream effector p21WAF1 (p21) are key regulators of these responses. Although extensively studied for its ability to control cell cycle progression, p21 has emerged as a multifunctional protein capable of downregulating p53, suppressing apoptosis, and orchestrating prolonged growth arrest through stress-induced premature senescence. Studies with solid tumors and solid tumor-derived cell lines have revealed that such growth-arrested cancer cells remain viable, secrete growth-promoting factors, and can give rise to progeny with stem-cell-like properties. This article provides an overview of the mechanisms by which p53 signaling suppresses apoptosis following genotoxic stress, facilitating repair of genomic injury under physiological conditions but having the potential to promote tumor regrowth in response to cancer chemotherapy.

Tumor Cell Recovery from Senescence Induced by Radiation with PARP Inhibition.

Inhibitors of poly(ADP-ribose) polymerase (PARP) are clinically used as single-agent therapy for tumors with BRCA1 or BRCA2 mutations. One approach to expanding the use of PARP inhibitors to a wider range of tumors is to combine them with cytotoxic chemotherapy or radiotherapy. Preclinical studies in experimental animals and tumor cells in culture indicate that PARP inhibition modestly sensitizes most tumor cells to ionizing radiation. Studies of cell behavior after these combined treatments show that radiosensitization is manifested predominantly in an increase in prolonged growth arrest and senescence, with little or no contribution from apoptosis. The secretory phenotype associated with senescence can target these tumor cells for immune surveillance, and therefore increased senescence can effectively contribute to tumor control. However, the possible recovery of senescent cells and re-entry into cell cycle after prolonged arrest also needs to be considered. Such recovery could lead to tumor recurrence, yet may not be reflected in short-term assays commonly used to assess radiosensitization.

The time to progression ratio: a new individualized volumetric parameter for the early detection of clinical benefit of targeted therapies

Early signs of efficacy are critical in drug development. Response Evaluation Criteria in Solid Tumors (RECIST) are commonly used to determine the efficacy of anti-cancer therapy in clinical trials. RECIST, however, emphasizes the value of tumor shrinkage, while many targeted agents induce prolonged tumor growth arrest. This limits its use for the detection of treatment efficacy for these more cytostatic regimens. Therefore, we designed an individualized variant of a time to progression (TTP) end point based on prospective volumetric measurements and an intra-patient control, the TTP ratio. Patients with any metastatic malignancy, without regular treatment options, were treated with the mTOR inhibitor everolimus. Treatment response was determined using both RECIST and the TTP ratio. The TTP ratio was defined as the volumetric pretreatment TTP divided by the volumetric on-treatment TTP. A patient was classified as a responder if the TTP ratio was <0.7. Consistency and reproducibility of volumetric measurements were determined. Seventy-three patients were included of whom 59 started treatment. A TTP ratio could be established in 73% (n = 43) of the treated patients. The inter-observer agreement for volumetric progression was 0.78 (95% confidence interval 0.70-0.87) (Krippendorff's α-coefficient). According to RECIST, 35 patients (59%) had stable disease (SD) and 1 patient demonstrated a partial response (PR), whereas only 21 patients (36%) met the prespecified criteria for treatment efficacy according to the TTP ratio. Treatment response according to both the TTP ratio and RECIST (SD + PR) correlated with overall survival (OS) [P(log-rank) < 0.001]. The TTP ratio, however, was also able to differentiate which patients had a better OS within the RECIST SD group [P(log-rank) = 0.0496]. The TTP ratio had a high inter-observer agreement, correlated with OS and identified which patients within the RECIST SD group had a longer OS. NCT01566279.

Abstract 3528: Induction of cytoprotective autophagy and senescence in response to chemotherapy and ionizing-radiation as possible mechanisms of tumor dormancy

Abstract Proliferative recovery in tumor cells subsequent to chemotherapy and/or radiation-induced prolonged growth arrest may prove to represent a state of autophagy and/or senescence. This may also be useful as an in vitro model of tumor dormancy. To explore this hypothesis, studies were performed in primary mouse mammary carcinoma cells (MMC) treated with Adriamycin (ADR) (0.25uM - 1uM) for 3 days (2h each) and ionizing radiation (IR) (6Gy). Promotion of autophagy was confirmed based on detection of autophagosomes based on acridine orange staining, the appearance of the autophagy marker, LC3.B, associated with autophagosome, degradation of p62/SQSTM1, a hallmark of autophagic flux (i.e. the completion of autophagy and degradation of autophagosome content). Autophagosome formation was observed 24h post ADR treatment and 72h post IR treatment. Furthermore, to understand the function of autophagy (cytoprotective, nonprotective or cytotoxic) autophagy was blocked either pharmacologically using chloroquine (CQ) or genetically by silencing of the key autophagy gene, ATG5. LC3.B appearance and p62/SQSTM1 degradation were more pronounced in wt (autophagy proficient) cells compared to ATG5 silenced cells, confirming that autophagy was inhibited by these pharmacological and genetic strategies. Furthermore, interference with autophagy sensitized the MMC cells to ADR and radiation. These observations indicated that ADR and IR induced autophagy was cytoprotective in the MMC cells. Unexpectedly, apoptosis (as measured by PI/ Annexin V assay) was not increased in response to either ADR alone or the combination of ADR and CQ, suggesting that sensitization was likely occurring through alternative pathways. An evaluation of select markers of senescence indicated enhanced expression of p21 as well as enhanced secretion of the autophagy/senescence associated marker, HMGB1, in response to Adriamycin and IR. Overall, these data suggest that both autophagy and senescence are induced in breast cancer cells by conventional therapies and could contribute to tumor dormancy. Inhibition of autophagy and/or senescence associated signaling pathways could lead to therapeutic strategies for interfering with proliferative recovery of tumor cells after standard modes of therapy. Studies are currently in progress to determine whether the senescence associated secretory phenotype (SASP) accompanies the induction of senescence. SASP has been associated with promotion of tumor growth and alternatively, with activation of an immune response to therapy. Citation Format: Theresa Thekkudan, Supriya Joshi, David Gewirtz. Induction of cytoprotective autophagy and senescence in response to chemotherapy and ionizing-radiation as possible mechanisms of tumor dormancy. [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 3528.

DNA Damage Response Plays Critical Role In Liver Regeneration After Acetaminophen Overdose

Acetaminophen (APAP) overdose is a leading cause of Acute Liver Failure (ALF). Previous studies have shown that liver regeneration followed by APAP overdose plays a critical role in survival. Here we show that significant DNA double strand break leading to inhibited DNA repair results in prolonged activation of cell cycle checkpoint delaying liver regeneration. We used a novel incremental dose model developed in our laboratory using a regenerating (300 mg/kg, APAP300) and a non‐regenerating (600 mg/kg, APAP600) dose of APAP to identify pathways involved in regeneration after APAP overdose. Marker of liver injury, regeneration and microarray‐based global gene expression changes were studied in male C57BL6 mice over time course of 0 to 72 hr following treatment with either APAP300 or APAP600. The ingenuity pathway analysis of microarray data revealed significant differences in DNA damage, replication and checkpoint related pathways between both doses of APAP. Western blot and immunofluorescence staining of pH2AX Ser139, a hallmark of DNA double strand break, showed prolonged phosphorylation of histone H2AX in mice treated with APAP600 than APAP300. DNA repair proteins involved in Non Homologous End Joining (NHEJ) pathway showed a rapid induction in APAP300 but were significantly suppressed in APAP600, indicating delay or lack of DNA repair. Further, extensive stabilization and activation of p53, the DNA damage Response (DDR) effector protein, was observed in APAP600 as compared to APAP300. p53 stabilization was accompanied by significantly higher and prolonged activation of several p53 target genes such as GADD45α,PAI1, BAX, p21 in APAP600 treated mice as compared to APAP300 treated mice. Furthermore, studies in p53KO mice showed significantly higher injury than WT mice after APAP300 treatment at initial time points. Interestingly, despite the higher injury, p53KO mice recovered similarly as the WT mice due to faster cell cycle resulting in much higher liver regeneration. These data illustrate that lack of prompt DSB repair response after APAP overdose leads to p53‐mediated prolonged growth arrest and replicative senescence. These results suggest that improving DNA repair process to rapidly decrease cell cycle checkpoint alert resulting in faster initiation of liver regeneration following APAP overdose may have therapeutic benefit.Support or Funding InformationThese studies were supported by NIH‐COBRE (P20 RR021940‐03), NIEHS Toxicology Training Grant (T32ES007079‐34) and NIDDK R0198414

Is Senescence Reversible?

Senescence was originally identified by the finite lifespan of normal cells that is a consequence of telomere shortening with each cycle of DNA replication. Cells undergoing replicative senescence display pronounced morphological and biochemical changes such as flattening and/or enlargement, increases in p21(WAF1) and/or p16(INK4A), a senescence-associated secretory phenotype, and often senescence-associated heterochromatic foci. Senescence also occurs in tumor cells in response to various forms of chemotherapy or radiation (therapy-induced senescence), which could be the basis for prolonged or (ideally) permanent growth arrest. Alternatively, therapy-induced senescence could represent a means whereby tumor cells evade the potential toxicity of chemotherapy and radiation, allowing for the eventual re-emergence or escape from senescence that could lead to disease recurrence. This review discusses the experimental data in the literature that support the premise that senescence is potentially reversible through the inactivation of p53, p16(INK4A) and/or Rb, over-expression of Cdc2/cdk1 and survivin, the development of polyploidy, the survival of cancer stem cells and/or restoration of the nuclear landscape. If senescence is truly reversible, then the re-emergence of tumor cells from senescent arrest induced by chemotherapy or radiation could represent a barrier to the development of effective and curative cancer therapies.

Modulation of medium pH by Caulobacter crescentus facilitates recovery from uranium-induced growth arrest.

The oxidized form of uranium [U(VI)] predominates in oxic environments and poses a major threat to ecosystems. Due to its ability to mineralize U(VI), the oligotroph Caulobacter crescentus is an attractive candidate for U(VI) bioremediation. However, the physiological basis for U(VI) tolerance is unclear. Here we demonstrated that U(VI) caused a temporary growth arrest in C. crescentus and three other bacterial species, although the duration of growth arrest was significantly shorter for C. crescentus. During the majority of the growth arrest period, cell morphology was unaltered and DNA replication initiation was inhibited. However, during the transition from growth arrest to exponential phase, cells with shorter stalks were observed, suggesting a decoupling between stalk development and the cell cycle. Upon recovery from growth arrest, C. crescentus proliferated with a growth rate comparable to that of a control without U(VI), although a fraction of these cells appeared filamentous with multiple replication start sites. Normal cell morphology was restored by the end of exponential phase. Cells did not accumulate U(VI) resistance mutations during the prolonged growth arrest, but rather, a reduction in U(VI) toxicity occurred concomitantly with an increase in medium pH. Together, these data suggest that C. crescentus recovers from U(VI)-induced growth arrest by reducing U(VI) toxicity through pH modulation. Our finding represents a unique U(VI) detoxification strategy and provides insight into how microbes cope with U(VI) under nongrowing conditions, a metabolic state that is prevalent in natural environments.