Accumulation Of Cells In S-phase Research Articles

Abstract 1985: Ablation of adenylosuccinate lyase retarded the cell proliferation and induces mitochondrial dysfunction in endocrine therapy resistant breast cancer cells

Abstract Approximately 75% of breast tumors express estrogen receptors (ER+); recurrent ER+ breast cancer is generally incurable. Endocrine therapy (ET) is the mainstay of treatment for ER+ breast cancer and works by targeting either ER activity (Tamoxifen or Fulvestrant) or availability of its ligand estrogen (Letrozole, Exemestane). Unfortunately, endocrine therapies are limited by the emergence of resistance; resistance to endocrine therapies (ET) remains a significant clinical challenge. We have explored the role of adenylosuccinate lyase (ADSL), an enzyme of the de-novo purine biosynthesis pathway, in ET-resistant breast cancer cells. Protein expression of ADSL was significantly higher in two different ET-resistant cells (LCC9 and T47D-4HT) when compared with their respective parental ET-sensitive cells (MCF-7 and T47D). siRNA mediated ablation of ADSL in LCC9 and T47D-4HT cells blocked cell proliferation and both colony and spheroid formation. Depletion of ADSL in LCC9 cells obstructed cell cycle progression from G1 to S phase and elevated the protein expression of cyclin D1/D3, CDK2/4, and cyclin E. In T47D-4HT cells, ADSL depletion led to an accumulation of cells in S-phase with reduced protein expression of cyclin D1. Notably, ADSL ablation in LCC9 cells lead to DNA replication stress, resulting in activation (phosphorylation) of ataxia-telangiectasia-mutated-and-Rad3-related kinase (ATR) and its key downstream effector checkpoint kinase 1 (Chk1), which further limited the phosphatase activity of cdc25A leading to deactivation (phosphorylation) of Cyclin-Dependent Kinase 2 (CDK2). ADSL knockdown reduced the mitochondrial membrane potential and impaired the oxygen consumption (OCR) and extracellular acidification (ECAR) rates in both LCC9 and T47D-4HT cells. Of note, addition of AICAR to ADSL-depleted LCC9 cells significantly increased cell proliferation, colony formation, and restored mitochondrial membrane potential. RNA sequencing data revealed the effect of ADSL depletion on enrichment of different pathways in LCC9 and TH47D-4HT cells. Importantly, p53 signaling was significantly enriched in LCC9 cells but not in T47D-4HT cells after ADSL knockdown (T47D cells have a mutated p53 gene). ER+ breast cancer patients treated with ET showed high ADSL expression that is associated with poor recurrence-free survival. In summary, our findings indicate that ADSL-mediated de novo purine synthesis is crucial for cell proliferation. Therefore, targeting ADSL may be a novel therapeutic approach for ER, ET-resistant breast cancer. Citation Format: ANIL KUMAR Yadav, Lu Jin, Robert Clarke, Surojeet Sengupta. Ablation of adenylosuccinate lyase retarded the cell proliferation and induces mitochondrial dysfunction in endocrine therapy resistant breast cancer cells [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 1985.

Histone deacetylase inhibitor 2-hexyl-4-pentynoic acid enhances hydroxyurea therapeutic effect in triple-negative breast cancer cells

Triple-negative breast cancer (TNBC) treatment has only limited effect, and it causes a significant number of deaths. Histone deacetylase inhibitors (HDACis) are emerging as promising anti-tumor agents in many types of cancers. We thus hypothesized that 2-hexyl-4-pentynoic acid (HPTA), a novel HDACi, could sensitize TNBC to hydroxyurea (HU, a ribonucleotide reductase inhibitor). In the present study, we investigated the effect of HPTA, alone or in combination with HU on cell survival, DNA double-strand breaks (DSBs), key homologous recombination (HR) repair proteins and cell cycle progression in MDA-MB-468 and MDA-MB-231 human TNBC cell lines. HPTA and HU synergistically inhibited the survival of TNBC cell lines and resulted in the accumulation of DNA double-strand breaks (DSBs). HPTA can sensitize TNBC cells to HU by inhibiting replication protein A2 (RPA2) hyperphosphorylation-mediated HR repair, and lessen cell accumulation in S-phase by inhibiting ATR-CHK1 signaling pathway. Taken together, our data suggested that HPTA enhances HU therapeutic effect by blocking the HR repair and regulating cell cycle progression in TNBC.

ABL1 Kinase Plays an Important Role in Spontaneous and Melphalan-Induced Genomic Instability in Multiple Myeloma: Potential Therapeutic Application

Dysregulated homologous recombination (HR) contributes to the acquisition of genomic changes and development of drug resistance over time in multiple myeloma (MM). We have now further investigated the molecular intermediates that may play an important role in HR process. We observe that ABL kinase, which regulates RAD51 through its phosphorylation, contributes to increased HR activity and genomic instability in myeloma. Moreover, Cellular response after DSBs involves nuclear re-localization of ABL1 upon DNA damage. A cellular localization of ABL1 has been confirmed in MM. Consistently, we observe that ABL kinase inhibitor reduces HR activity and genomic instability (as assessed by micronucleus assay) in MM cells. Based on our observation that melphalan increases genomic instability (as assessed by micronuclei assay and by whole genome sequencing), we sought to investigate impact of melpahalan on HR and role of ABL1 kinase in this process. We show that treatment with melphalan leads to increase in RAD51 expression and HR activity in MM cells in a dose-dependent manner. Evaluation by RNA sequencing showed that treatment of MM1S cells with melphalan is associated with upregulation of p53 signaling (containing multiple genes involved in DNA damage, detection of damaged sites, recombination/repair and genomic instability) as the topmost pathway. These findings suggest that melphalan-induced DNA damage leads to a concerted overexpression of genes involved in DNA damage response, recombination, genomic instability and chemoresistance. With the role of ABL1-kinase following DSB, we investigated and report that treatment with melphalan induces micronuclei formation, whereas nilotinib significantly reduces both the basal and melphlalan-induced micronuclei in all MM cell lines tested. We have now confirmed these observations by evaluating copy number alterations using single nucleotide polymorphism (SNP) arrays. To evaluate the impact of nilotinib, melphalan and their combination on genomic instability, we cultured MM cells in the presence of nilotinib (2.5 µM), melphalan (1 µM) and combination of both drugs for three weeks and investigated the acquisition of new copy number events, relative to "day 0" cells (serving as baseline genome), using SNP arrays. Treatment with melphalan led to massive increase in the acquisition of amplification and deletion events, whereas nilotinib not only reduced the acquisition of copy number events under spontaneous condition but also almost completely reversed/prevented those induced by melphlalan. Importantly, the treatment with nilotinib could also significantly sensitized MM cell lines and bone marrow plasma cells from relapsed MM patients to melphalan treatment. These data confirm that ABL1 inhibition reduces spontaneous and melphalan-induced genomic instability in MM cells. Since components of cell cycle play critical role in the maintenance of genome stability and growth, we investigated the impact of ABL1-inhibitor nilotinib, alone and in combination with melphalan, on cell cycle. Treatment of MM1S cells with melphalan for 48hrs led to 2.5-fold increase in the accumulation of cells in S-phase, suggesting an increase in replication stress by melphalan. Nilotinib reduced both the spontaneous and melphalan-induced fraction of S-phase cells by 27% (±2%) and 30% (±1%), respectively. Combined treatment with nilotinib and melphalan also increased sub G1 fraction of cells by 2.3-fold compared to those treated with melphalan alone, suggesting increased apoptosis in these cells. These data are consistent with our observation that treatment of MM cells with melphalan increase the phosphorylation of RPA32, a marker of replication stress while nilotinib reduces both the endogenous and melphalan-induced phosphorylated RPA32 level, suggesting that nilotinib might also be helpful in combating replication stress-mediated genomic instability. Taken together, these data demonstrate the critical role of ABL1-kinase in both spontaneous and drug (melphalan)-induced genomic instability and its inhibition could reduce/delay genomic evolution while enhancing cytotoxicity in multiple myeloma. Disclosures Fulciniti: NIH: Research Funding. Munshi:Takeda: Consultancy; Karyopharm: Consultancy; AbbVie: Consultancy; Amgen: Consultancy; Legend: Consultancy; Adaptive: Consultancy; Janssen: Consultancy; C4: Current equity holder in private company; OncoPep: Consultancy, Current equity holder in private company, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties; BMS: Consultancy.

Anti-Proliferative and Pro-Apoptotic Effects of Short-Term Inhibition of Telomerase In Vivo and in Human Malignant B Cells Xenografted in Zebrafish.

Besides its canonical role in stabilizing telomeres, telomerase reverse transcriptase (TERT) may promote tumor growth/progression through extra-telomeric functions. Our previous in vitro studies demonstrated that short-term TERT inhibition by BIBR1532 (BIBR), an inhibitor of TERT catalytic activity, negatively impacts cell proliferation and viability via telomeres’ length-independent mechanism. Here we evaluate the anti-proliferative and pro-apoptotic effects of short-term telomerase inhibition in vivo in wild-type (wt) and tert mutant (terthu3430/hu3430; tert−/−) zebrafish embryos, and in malignant human B cells xenografted in casper zebrafish embryos. Short-term Tert inhibition by BIBR in wt embryos reduced cell proliferation, induced an accumulation of cells in S-phase and ultimately led to apoptosis associated with the activation of DNA damage response; all these effects were unrelated to telomere shortening/dysfunction. BIBR treatment showed no effects in tert−/− embryos. Xenografted untreated malignant B cells proliferated in zebrafish embryos, while BIBR pretreated cells constantly decreased and were significantly less than those in the controls from 24 to up to 72 h after xenotransplantation. Additionally, xenografted tumor cells, treated with BIBR prior- or post-transplantation, displayed a significant higher apoptotic rate compared to untreated control cells. In conclusion, our data demonstrate that short-term telomerase inhibition impairs proliferation and viability in vivo and in human malignant B cells xenografted in zebrafish, thus supporting therapeutic applications of TERT inhibitors in human malignancies.

Albendazole negatively regulates keratinocyte proliferation.

Increased keratinocyte proliferation occurs in the skin of psoriatic patients and is supposed to play a role in the pathogenesis of this disorder. Compounds interfering with keratinocyte proliferation could be useful in the management of psoriatic patients. To investigate whether albendazole, an anti-helmintic drug that regulates epithelial cell function in various systems, inhibits keratinocyte proliferation in models of psoriasis. Aldara-treated mice received daily topical application of albendazole. Keratinocyte proliferation and keratin (K) 6 and K16 expression were evaluated by immunohistochemistry and Western blotting and inflammatory cells/mediators were analysed by immunohistochemistry and real-time PCR. In human keratinocytes (HEKa and HaCaT) treated with albendazole, cell cycle and proliferation, keratins and cell cycle-associated factors were evaluated by flow cytometry, colorimetric assay and Western blotting respectively. Aldara-treated mice given albendazole exhibited reduced epidermal thickness, decreased number of proliferating keratinocytes and K6/K16 expression. Reduction of CD3- and Ly6G-positive cells in the skin of albendazole-treated mice associated with inhibition of IL-6, TNF-α, IL-1β, IL-17A, IL-36, CCL17, CXCL1, CXCL2 and CXCL5 expression. Treatment of keratinocytes with albendazole reduced K6/K16 expression and reversibly inhibited cell growth by promoting accumulation of cells in S-phase. This phenomenon was accompanied by down-regulation of CDC25A, a phosphatase regulating progression of cell cycle through S-phase, and PKR-dependent hyper-phosphorylation of eIF2α, an inhibitor of CDC25 translation. In Aldara-treated mice, albendazole activated PKR, enhanced eIF2α phosphorylation and reduced CDC25A expression. Data show that albendazole inhibits keratinocyte proliferation and exerts therapeutic effect in a murine model of psoriasis.

RelB plays an oncogenic role and conveys chemo-resistance to DLD-1 colon cancer cells

BackgroundNuclear transcription factor kappa B (NF-κB) subunits exhibit crucial roles in tumorigenesis and chemo-sensitivity. Recent studies suggest that RelB, the key subunit of the alternative NF-κB pathway, plays a critical role in the progression of diverse human malignancies. However, the significance of RelB in colorectal cancer (CRC) remains unclear. Here, we systematically explored the functions of the alternative NF-κB subunit RelB in colon cancer cells and its underlying mechanism.MethodsStably transfected RelB-shRNA DLD-1 cells were established using Lipofectamine 2000. NF-κB DNA-binding capability was quantified using an ELISA-based NF-κB activity assay. Cell growth was monitored by an x-Celligence system. Cell proliferation was analyzed by a CCK-8 and a Brdu proliferation assay. Response to 5-FU was assessed by an x-Celligence system. Cell apoptosis and cell cycle was detected using flow cytometry analyses. Cell migration and invasion abilities were detected by an x-Celligence system, Transwell inserts, and wound-healing assays. RelB expression and its clinical significance were analyzed using the CRC tissue microarray. The expression of NF-κB signaling subunits, AKT/mTOR signaling molecules, cell cycle related proteins, MMP2, MMP9, and Integrin β-1 were measured by Western blotting analyses.ResultsThe RelB-silencing inhibited cell growth of DLD-1 cells. The RelB-silencing exerted the anti-proliferative by downregulation of AKT/mTOR signaling. The RelB-silencing caused G0–G1 cell cycle arrested likely due to decreasing the expression of Cyclin D1 and CDK4, concomitant with increased expression of p27Kip1. The RelB-silencing enhanced cytotoxic effect of 5-FU and induced cell accumulation in S-phase. The RelB-silencing impaired the migration and invasion potential of DLD-1 cells, which was related to downregulation of MMP2, MMP9, and Integrin β-1. Importantly, the RelB expression was correlated with depth of tumor invasion, lymph node metastasis, metastasis stage, and pTNM stage. High-RelB expression was significantly correlated with poor overall survival in CRC patients.ConclusionOur studies here provided evidence that RelB plays an oncogenic role and conveys chemo-resistance to 5-FU. RelB can be considered as an independent indicator of prognosis in CRC.

Abstract 317: Role of KLLN in DNA damage-induced apoptosis is associated with the regulation of p53 phosphorylation and acetylation in breast cancer cells

Abstract KLLN was initially discovered as a target of p53 involved in S-phase checkpoint regulation. Germline hypermethylation in the promoter of KLLN is known to increase the predisposition for breast and clear cell renal cell carcinomas in PTEN-wildtype Cowden syndrome (CS) patients. KLLN is a known tumor suppressor protein and required for p53-mediated apoptosis. In this study, we explore the underlying mechanism by which KLLN regulates DNA damage response (DDR), specifically apoptosis. Our DDR studies were conducted on one breast normal and four breast cancer cell lines using doxorubicin as the DNA damaging agent. We initially showed that doxorubicin-induced DNA damage increases KLLN expression at 16h and 24h post-damage which could be abrogated by RNAi-mediated knockdown of KLLN expression. Response to DNA damage as determined by the presence of γH2AX foci formation and expression of γH2AX protein were both decreased after KLLN knockdown. Using TUNEL assay and cell viability studies, we demonstrated that a lack of KLLN expression decreases apoptosis and increases cell viability at 16h and 24h post-damage. Definitive markers of apoptosis such as cleavage of caspase 3 and PARP were also decreased. Since KLLN is a known transcription factor and regulates the expression of DDR genes such as TP53 and CHK1, we assessed the regulation of these genes in response to DNA damage and found that lack of KLLN expression did not affect the expression of either TP53 or CHK1. Cell cycle analysis results using flow cytometry also suggested that KLLN does not play a role in S-phase cell cycle regulation in response to DNA damage as was previously thought, since KLLN knockdown did not change accumulation of cells in S-phase after doxorubicin-induced DNA damage. DDR in cells is immediate and therefore we assessed the abrogation of DDR after KLLN knockdown at early timepoints (1h and 2h) post-damage. We found that lack of KLLN expression decreased Ser15 phosphorylation of p53 and Thr68 phosphorylation of CHK2 at 1h and 2h post-damage. KLLN regulation of p53-mediated apoptosis could also be associated with its interaction with deleted in breast cancer (DBC1). DBC1 is a known inhibitor of SIRT1, a deacetylase that is known for its inhibitory deacetylation of p53. Phosphorylated DBC1 binds to SIRT1 and inhibits its deacetylation of p53. We found that lack of KLLN expression decreased the phosphorylation of DBC1 on Thr454 in response to DNA damage. Therefore, KLLN displays a 2-pronged regulation of the activation of p53 and the downstream cascade of proteins involved in apoptosis. SIGNIFICANCE: Our study demonstrates a clear role for KLLN in DNA damage-induced apoptosis and elucidates the underlying mechanism through the regulation of p53 phosphorylation and deacetylation in response to DNA damage. Our observations add to the basis of KLLN as a tumor suppressor and as a susceptibility gene for inherited cancers. Citation Format: Madhav Sankunny, Charis Eng. Role of KLLN in DNA damage-induced apoptosis is associated with the regulation of p53 phosphorylation and acetylation in breast cancer cells [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 317.

Curcumin Analog Pentagamavunon-1 (PGV-1) Sensitizes Widr Cells to 5-Fluorouracil through Inhibition of NF-κB Activation

Cell cycle regulation and the NF-κB pathway in cancer cells are important in mediating resistance to 5-Fluorouracil (5-FU). Pentagamavunon-1 (PGV-1), a curcumin analog, is known to exhibit stronger growth inhibitory effects than curcumin itself in several cancer cells. In this study, we evaluated the potency of PGV-1 in combination with 5-FU in WiDr colon cancer cells. In MTT assays, PGV-1 did not only exhibit stronger growth inhibitory effects than both 5-FU and curcumin, but also enhanced the cytotoxicity of 5-FU. Flow cytometry demonstrated that single treatments with PGV-1 and 5-FU resulted in different effects on cell cycle profiles. PGV-1 induced G2/M arrest while 5-FU caused S-phase arrest at low concentration (1 μM) and G1-phase arrest at high concentration (100 μM). Interestingly, the combination of 5-FU and PGV-1 enhanced cell accumulation in S-phase. Although a single treatment with either 5-FU or PGV-1 increased cyclin D1 at the protein level, the combination treatment resulted in significant suppression. In addition, PGV-1 inhibited activation of NF-κB and suppressed the expression of cyclooxygenase-2, an NF-κB downstream protein. In conclusion, PGV-1 increased the cytotoxic effect of 5-FU on WiDr cells through inhibition of NF-κB activation.

DUTPase inhibition augments replication defects of 5-Fluorouracil.

The antimetabolite 5-Fluorouracil (5-FU) is used in the treatment of various forms of cancer and has a complex mode of action. Despite 6 decades in clinical application the contribution of 5-FdUTP and dUTP [(5-F)dUTP] and 5-FUTP misincorporation into DNA and RNA respectively, for 5-FU-induced toxicity is still under debate.This study investigates DNA replication defects induced by 5-FU treatment and how (5-F)dUTP accumulation contributes to this effect. We reveal that 5-FU treatment leads to extensive problems in DNA replication fork progression, causing accumulation of cells in S-phase, DNA damage and ultimately cell death. Interestingly, these effects can be reinforced by either depletion or inhibition of the deoxyuridine triphosphatase (dUTPase, also known as DUT), highlighting the importance of (5-F)dUTP accumulation for cytotoxicity.With this study, we not only extend the current understanding of the mechanism of action of 5-FU, but also contribute to the characterization of dUTPase inhibitors. We demonstrate that pharmacological inhibition of dUTPase is a promising approach that may improve the efficacy of 5-FU treatment in the clinic.

Checkpoint kinase inhibitor AZD7762 strongly sensitises urothelial carcinoma cells to gemcitabine

BackgroundMore effective chemotherapies are urgently needed for bladder cancer, a major cause of morbidity and mortality worldwide. We therefore explored the efficacy of the combination of gemcitabine and AZD7762, a checkpoint kinase 1/2 (CHK1/2) inhibitor, for bladder cancer.MethodsViability, clonogenicity, cell cycle distribution and apoptosis were assessed in urothelial cancer cell lines and various non-malignant urothelial cells treated with gemcitabine and AZD7762. DNA damage was assessed by γH2A.X and 53-BP1 staining and checkpoint activation was followed by Western blotting. Pharmacological inhibition of CHK1 and CHK2 was compared to downregulation of either CHK1 or CHK2 using siRNAs.ResultsCombined use of gemcitabine and AZD7762 synergistically reduced urothelial carcinoma cell viability and colony formation relative to either single treatment. Non-malignant urothelial cells were substantially less sensitive to this drug combination. Gemcitabine plus AZD7762 inhibited cell cycle progression causing cell accumulation in S-phase. Moreover, the combination induced pronounced levels of apoptosis as indicated by an increase in the fraction of sub-G1 cells, in the levels of cleaved PARP, and in caspase 3/7 activity. Mechanistic investigations showed that AZD7762 treatment inhibited the repair of gemcitabine-induced double strand breaks by interference with CHK1, since siRNA-mediated depletion of CHK1 but not of CHK2 mimicked the effects of AZD7762.ConclusionsAZD7762 enhanced sensitivity of urothelial carcinoma cells to gemcitabine by inhibiting DNA repair and disturbing checkpoints. Combining gemcitabine with CHK1 inhibition holds promise for urothelial cancer therapy.

Mitotic catastrophe and cell cycle arrest are alternative cell death pathways executed by bortezomib in rituximab resistant B-cell lymphoma cells.

The ubiqutin-proteasome system (UPS) plays a role in rituximab-chemotherapy resistance and bortezomib (BTZ) possesses caspase-dependent (i.e. Bak stabilization) and a less characterized caspase–independent mechanism-of-action(s). Here, we define BTZ-induced caspase-independent cell death pathways. A panel of rituximab-sensitive (RSCL), rituximab-resistant cell lines (RRCL) and primary tumor cells derived from lymphoma patients (N = 13) were exposed to BTZ. Changes in cell viability, cell-cycle, senescence, and mitotic index were quantified. In resting conditions, RRCL exhibits a low-proliferation rate, accumulation of cells in S-phase and senescence. Exposure of RRCL to BTZ reduces cell senescence, induced G2-M phase cell-cycle arrest, and is associated with mitotic catastrophe. BTZ stabilized p21, CDC2, and cyclin B in RRCL and in primary tumor cells. Transient p21 knockdown alleviates BTZ-induced senescence inhibition, G2-M cell cycle blockade, and mitotic catastrophe. Our data suggest that BTZ can induce apoptosis or mitotic catastrophe and that p21 has a pivotal role in BTZ activity against RRCL.

Abstract 3717: Defining optimal dose schedules for ATR inhibitors in combination with DNA damaging drugs: Informing clinical studies of VX-970, the first-in-class ATR inhibitor

Abstract Proficient repair of DNA damage is a cause of the poor response many patients experience when treated with commonly used DNA-damaging drugs such as cisplatin, carboplatin and gemcitabine. The protein kinase ataxia telangiectasia mutated and Rad3 related (ATR) is recruited to DNA damage lesions caused by such drugs during the S and G2 phase of cell cycle, where it coordinates a series of responses including checkpoint activation and DNA repair by homologous recombination. Inhibition of ATR potentiates the cytotoxic activity of DNA damaging drugs in many cancer cells. In stark contrast, non-cancer cells survive inhibition of ATR with just transient growth arrest. Cancer cells carrying common defects in a compensatory repair pathway mediated by the kinase ataxia telangiectasia mutated (ATM) and its principle substrate, p53, are especially sensitive to ATR inhibition. Two ATR inhibitors are in clinical development in combination with DNA damaging drugs, however a comprehensive assessment of dose schedule considerations has not been reported. In pre-clinical models, the efficacy of an ATR inhibitor in combination with multiple DNA damaging drugs was shown to be dependent on dose schedule. In vitro, transient exposure of cancer cells to an ATR inhibitor (2 hours) was highly effective when added after the DNA damaging drug. Maximum activity was observed when addition of the ATR inhibitor was timed to coincide with peak accumulation of cells in S-phase and concomitant activation of ATR (P-Chk1), following treatment with the DNA damaging drug. In mouse xenograft models, strong synergistic activity was achieved from just a single dose of the ATR inhibitor given once per cycle of the DNA damaging drug. Optimal efficacy was achieved by administering the ATR inhibitor 12-24 hours after the DNA damaging drug. Dosing the ATR inhibitor prior to, or greater than 48 hours after, the DNA damaging drug provided limited benefit. On this schedule, addition of the ATR inhibitor had minimal impact on the tolerability profile of the DNA damaging drug. VX-970, the first-in-class ATR inhibitor, is being assessed as monotherapy and in combination with gemcitabine, cisplatin and carboplatin in Ph1/2 clinical studies. Based on pre-clinical data, VX-970 is being dosed approximately 24 hours after the DNA damaging drug. Preliminary tumor biomarker data from three patients showed high P-Chk1 24 hours after treatment with carboplatin, which is inhibited by VX-970. These data suggest the importance of dose scheduling on the efficacy of ATR inhibitors and DNA damaging drug combinations and inform the design of ongoing clinical studies. Citation Format: John Pollard, Phil Reaper, Adele Peek, Stuart Hughes, Scott Gladwell, Julie Jones, Peter Chiu, Mark Wood, Crystal Tolman, Mac Johnson, Peter Littlewood, Marina Penney, Katherine McDermott, Brian Hare, Scott Z. Fields, Mohammed Asmal, Brent O’Carrigan, Timothy A. Yap. Defining optimal dose schedules for ATR inhibitors in combination with DNA damaging drugs: Informing clinical studies of VX-970, the first-in-class ATR inhibitor. [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 3717.

Role of STAT3 Phosphorylation in Ethanol-Mediated Proliferation of Breast Cancer Cells.

PurposeIn this study, we investigated the molecular mechanism involved in ethanol (EtOH)-mediated proliferation of breast cancer cells.MethodsEtOH concentration was optimized by studying its effect on cell proliferation in MCF-7 and MDA MB-231 cells. We used flow cytometry and immunoblot analysis to evaluate the increased proliferation caused by the optimized concentrations of EtOH. The mechanism of EtOH-mediated proliferation was determined using reactive oxygen species (ROS) release assay, reverse transcription polymerase chain reaction, and immunoblot studies. Gene silencing followed by quantitative real-time polymerase chain reaction studies and inhibitor studies indicated the involvement of signal transducer and activator of transcription 3 (STAT3) in EtOH-mediated breast cancer proliferation.ResultsExposure to EtOH caused an increase in cell proliferation and an accumulation of cells in S-phase in MCF-7 (347 µM EtOH) and MDA MB-231 (173 µM EtOH) cells. Additionally, increased release of ROS and the expression of pro-inflammatory cytokines, such as interleukin 6 and tumor necrosis factor α, confirmed that the proliferation was induced by the ROS-linked inflammatory response in breast cancer. The proinflammatory response was followed by phosphorylation of STAT3. The importance of STAT3 activation in EtOH-mediated proliferation was confirmed through the silencing of STAT3, followed by an investigation on the expression of cyclins and matrix metalloproteinases. Finally, studies using specific inhibitors indicated that the EtOH-mediated effect on STAT3 activation could be regulated by phosphoinositide-3-kinase and Janus kinase 2.ConclusionThe study demonstrates the involvement of STAT3 signaling in EtOH-mediated breast cancer proliferation.

Expression and regulation of COP1 in chronic lymphocytic leukemia cells for promotion of cell proliferation and tumorigenicity.

Chronic lymphocytic leukemia (CLL) is the most common leukemia in Western countries, and mainly originates from an accumulation of abnormal B cells caused by the dysregulation of cell proliferation and apoptosis. The aberration of proliferation-related gene in CLL cells induces cell arrest at G0/G1 phase, or a small section shows rapid cell growth, which further complicates the pathogenesis of CLL. The constitutively photomorphogenic 1 (COP1), as an E3 ubiquitin ligase, is involved in many biological processes in mammalian cells, but its role in chronic lymphocytic leukemia (CLL) progression remains unclear. In the present study, we analyzed the expression of COP1 in peripheral blood mononuclear cells (PBMCs) from 23 CLL patients and 3 healthy donors. The observed upregulated expression of COP1 in CLL patients was positively correlated with CLL clinical stage and ZAP-70 expression, but not del(13q14) and del(17q-). Overexpression of COP1 significantly promoted cell colony formation and proliferation, especially contributing to the accumulation of cells in S-phase by inhibition of FoxO1 and p21. Moreover, overexpression of COP1 accelerated tumorigenicity of HG3 cells and promoted xenograft growth. Therefore, the present study revealed that COP1 plays an important role in CLL cell proliferation and tumorigenicity, and may be a useful indicator of the chronic lymphocytic leukemia processes.

Novel Alkylating Agent, Ureidomustine Exhibit Pre-Clinical Efficacy in B-Cell Lymphoma with Minimal Dose-Limiting Myelotoxicity

Many patients with B-cell lymphomas, including mantle cell lymphoma (MCL) and diffuse large B-cell lymphoma (DLBCL), are not cured by conventional chemo-immunotherapy. One reason for this is because these drugs, while effective, are limited by their narrow therapeutic window and significant toxicities. B-cell lymphomas are highly dependent on DNA damage checkpoints, and hence are biologically responsive to drugs that trigger these checkpoints. Hence, In order to identify superior DNA damaging anti-lymphoma drugs we evaluated a series of novel, third generation, DNA-directed alkylating agents that have DNA specific binding domains chemically linked via urea, carbamate or hydrazinecarboxamide to N-mustard pharmacophores. The chemically favorable water-soluble ureidomustine (BO-1055) was evaluated for activity against 23 human lymphoma cell lines including MCL, DLBCL (GCB and ABC subtype) and a spontaneous murine B-cell lymphoma. Fifty % of these MCL and DLBCL cell lines exhibited BO-1055 IC50 values in the nanomolar range including even markedly chemo-resistant cell lines as OCI-Ly10 (IC50 0.117μM ±0.21). In marked contrast, against normal human tissues (lung fibroblast IMR90, kidney fibroblast CV1, mesenchymal stromal, bronchial epithelium, myofibroblast, bone marrow-derived endothelium) and hematopoietic cells (cord blood CD34+ cells, colony-forming assay for hematopoietic progenitors) and in cobblestone area-forming assay for hematopoietic stem cells, BO-1055 IC50s were > 10μM. Hence BO-1055 has a significant therapeutic window (50-100-fold) between its toxicity against B-cell lymphomas compared to normal human cells. We evaluated BO-1055 cardiotoxicity in the HL-1 cardiomyocyte line and observed a 227-fold less cytotoxicity compared to Doxorubicin. Drug combination studies with BO-1055 and an Hsp90 inhibitor (PU-H71), an Hsp70 inhibitor (TT46), doxorubicin and bortezomib (Velcade) demonstrated synergistic effects based on Compusyn analysis with very low combination indices in 50% of lymphoma cell lines. The synergistic effect was not observed in normal cells. Notably, BO-1055 caused downregulation of the critical lymphoma oncoproteins MYC and BCL6, but not Bcl2. BCL6 normally suppresses the ATR-driven S-phase checkpoint. Accordingly treatment with BO-1055 resulted in accumulation of cells in S-phase and up-regulation of proteins involved in DNA repair and intra-S-phase checkpoints [MRE11, p-P95/NBS1 (ser343), RAD50, p-ATR (ser428)]. Finally, xenograft experiments in NSG mice bearing MCL JEKO1 GFP/luciferase+ tumors treated with BO-1055 (30mg/kg) 3x/week showed complete tumor remission after 2 weeks of treatment as monitored by luminescent imaging. In summary, BO-1055 is emerging as a potent therapeutic agent for B-cell lymphomas, with little toxicity against normal tissues and hence potentially wider therapeutic window than current lymphoma drugs. DisclosuresCaldas Lopes:BOtique Biopharm: Employment.

The First-in-Class Alkylating Histone-Deacetylase Inhibitor (HDACi) Fusion Molecule Edo-S101 Exerts Potent Preclinical Activity Against Tumor Cells of Hodgkin Lymphoma (HL) Including Bendamustine-Resistant Clones

Background: The highly unfavorable outcome of patients with recurrent HL, who progress after stem cell transplantation or are ineligible for such procedure make the development of new active agents an impellent medical need in this clinical setting. EDO-S101 is fusion molecule combining the DNA damaging effects of bendamustine (BDM) with the pan-histone deacetylase (HDAC) inhibitor, vorinostat. Given that BDM and HDAC inhibitors are active agents in recurrent HL we investigated the preclinical activity of EDO-S101 in this malignancy.Methods: We assessed the patterns of EDO-S101 cytotoxicity (0.39 to 50 µmol/L) in a panel of HL-derived cell lines (L1236, L428, KMH2, HDLM2, L540) and its regulatory effects on genes involved in DNA-damage/repair response, apoptosis and cell cycle checkpoints. As a further model we exploited an L1236 cell clone (R100) selected for resistance (R) to BDM through continuous exposure to increasing concentrations of the agent. R100 cells display a growth pattern indistinguishable from parental L1236 cells when cultured in the presence of BDM (100 µmol/L). Clonal identity of R100 cells with parental L1236 was confirmed by sequencing of V3-21 (FR2/FR3) and JH3-JH4 Ig DNA regions.Results: EDO-S101 induced a significant time- and dose-dependent inhibition of growth and survival in all HL cell lines. L1236 cells displayed the highest sensitivity to the agent with an IC50, at 48 hrs, of 1.88 µmol/L, as opposed to KMH2, L428, L540 and HDLM2 cells with IC50 of 2.06, 2.53, 2.26 and 16.2 µmol/L, respectively. These values were about 10-fold lower than the IC50 of BDM in the same cell lines. While exposure of L1236 cells to EDO-S101 caused cell accumulation in S-phase, qRT-PCR disclosed that cell death was mainly dependent on triggering of apoptosis, as shown by the early (24 hrs) and sustained (48 hrs) upregulation of NOXA, p21 and p27 genes. Data were confirmed by the significant increase (>150%) of Annexin V-expressing L1236 cells. In contrast, expression levels of PLK1, AKA and cyclin B1 genes remained unchanged or were increased. This excluded induction of the mitotic catastrophe (MC) as a major determinant of cytotoxic activity for EDO-S101 in L1236 cells. Exposure to EDO-S101 induced a strong DNA stress/repair response as shown by the activation of pATR/pATM and increase of the downstream DNA damage checkpoint proteins pCHK1-/-2 and CCNB1, along with the upregulation of the EXO1 gene. Most intriguingly, BDM-resistant L1236 cells (R100) were highly sensitive to EDO-S101, with an IC50 of only 4.56 µmol/L, but less responsive to vorinostat (IC50: 6.17 µmol/L) than parental L1236 cells (IC50: 0.58 µmol/L). Differently from native cells, EDO-S101 induced a late downregulation of transcripts for PLK1 and AKA genes and of cyclin B1 gene and protein in R100 cells, along with the early induction of NOXA and p21, but not p27 genes. In both L1236 and R100 cells, expression of MC-genes was unaffected by exposure to vorinostat. This suggests a more complex mechanism for EDO-S101 in BDM-resistant HL cells involving activation of the both apoptotic and MC pathways. Notably, we documented that EDO-S101 corrected the constitutive ATM/ATR unbalance of R100 cells by triggering the early (24 hrs) upregulation of ATR and a late (48 hrs) downregulation of ATM transcripts and proteins, along with increased levels of EXO1 and MGMT at 24 hrs. Vorinostat induced a similar effect. Finally, while baseline expression levels of HDAC isoforms were comparable among HL cell lines, EDO-S101 caused a significant (>40%) late downregulation of transcripts for all HDAC isoforms (HDAC-1 to -8) in R100 cells but only of HDAC-6 in native L1236. This pattern diverged from results obtained in both L1236, i.e. increase of all HDAC isoform transcripts except HDAC-6, and R100 cells, i.e. upregulation of all isoforms and reduction of HDAC-6, with vorinostat and BDM as single agents.Conclusions: We have described for the first time that EDO-S101 is effective in preclinical models of HL including cells resistant to BDM. The combined functions of in one molecule of a bifunctional alkylator and panHDAC inhibitor confer this agent unique antitumor property different from both of its single drug components. Following a strong DNA damage response, triggering of apoptosis and/or MC may take place in HL cells according to their sensitivity status to BDM. A phase 1/2 study in recurrent HL, including patients pretreated with BDM, is next to be launched. DisclosuresMehrling:4Mundipharma-EDO GmbH, Basel, Switzerland: Employment. Pinto:Takeda, Celgene, Roche, TEVA: Honoraria; Takeda: Research Funding.

CHK1 Inhibition Synergizes with Gemcitabine Initially by Destabilizing the DNA Replication Apparatus.

Combining cell-cycle checkpoint kinase inhibitors with the DNA-damaging chemotherapeutic agent gemcitabine offers clinical appeal, with a mechanistic rationale based chiefly on abrogation of gemcitabine-induced G2-M checkpoint activation. However, evidence supporting this mechanistic rationale from chemosensitization studies has not been consistent. Here we report a systematic definition of how pancreatic cancer cells harboring mutant p53 respond to this combination therapy, by combining mathematical models with large-scale quantitative biologic analyses of single cells and cell populations. Notably, we uncovered a dynamic range of mechanistic effects at different ratios of gemcitabine and CHK1 inhibitors. Remarkably, effective synergy was attained even where cells exhibited an apparently functional G2-M surveillance mechanism, as exemplified by a lack of both overt premature CDK1 activation and S-phase mitotic entry. Consistent with these findings, S-G2 duration was extended in treated cells, leading to a definable set of lineage-dependent catastrophic fates. At synergistic drug concentrations, global replication stress was a distinct indicator of chemosensitization as characterized molecularly by an accumulation of S-phase cells with high levels of hyperphosphorylated RPA-loaded single-stranded DNA. In a fraction of these cells, persistent genomic damage was observed, including chromosomal fragmentation with a loss of centromeric regions that prevented proper kinetochore-microtubule attachment. Together, our results suggested a "foot-in-the-door" mechanism for drug synergy where cells were destroyed not by frank G2-M phase abrogation but rather by initiating a cumulative genotoxicity that deregulated DNA synthesis.

Pleiotropic effects of spongean alkaloids on mechanisms of cell death, cell cycle progression and DNA damage response (DDR) of acute myeloid leukemia (AML) cells

We investigated cytotoxic mechanisms evoked by the spongean alkaloids aaptamine (Aa) and aeroplysinin-1 (Ap), applied alone and in combination with daunorubicin, employing acute myeloid leukemia (AML) cells. Aa and Ap reduced the viability of AML cells in a dose dependent manner with IC50 of 10–20 µM. Ap triggered apoptotic cell death more efficiently than Aa. Both alkaloids increased the protein level of S139-phosphorylated H2AX (γH2AX), which however was independent of the induction of DNA damage. Expression of the senescence markers p21 and p16 was increased, while the phosphorylation level of p-Chk-2 was reduced following Aa treatment. As a function of dose, Aa and Ap protected or sensitized AML cells against daunorubicin. Protection by Aa was paralleled by reduced formation of ROS and lower level of DNA damage. Both Aa and Ap attenuated daunorubicin-stimulated activation of the DNA damage response (DDR) as reflected on the levels of γH2AX, p-Kap-1 and p-Chk-1. Specifically Ap restored the decrease in S10 phosphorylation of histone H3 resulting from daunorubicin treatment. The cytoprotective effects of Aa and Ap were independent of daunorubicin import/export. Both Aa and Ap abrogated daunorubicin-induced accumulation of cells in S-phase. Inhibition of DNA synthesis was specific for Ap. The data show that Aa and Ap have both congruent and agent-specific pleiotropic effects that are preferential for anticancer drugs. Since Ap showed a broader spectrum of anticancer activities, this compound is suggested as novel lead compound for forthcoming in vivo studies elucidating the usefulness of spongean alkaloids in AML therapy.

MYC is a critical target of FBXW7.

MYC deregulation is a driver of many human cancers. Altering the balance of MYC protein levels at the level of transcription, protein stability, or turnover is sufficient to transform cells to a tumorigenic phenotype. While direct targeting of MYC is difficult, specific genetic vulnerabilities of MYC-deregulated cells could be exploited to selectively inhibit their growth. Using a genome-wide shRNA screen, we identified 78 candidate genes, which are required for survival of human mammary epithelial cells with elevated MYC levels. Among the candidates, we validated and characterized FBXW7, a component of the SCF-like ubiquitin ligase complex that targets MYC for proteasomal degradation. Down-regulation of FBXW7 leads to synergistic accumulation of cellular and active chromatin-bound MYC, while protein levels of other FBXW7 targets appear unaffected. Over a four-week time course, continuous FBXW7 down-regulation and MYC activation together cause an accumulation of cells in S-phase and G2/M-phase of the cell cycle. Under these conditions, we also observe elevated chromatin-bound levels of CDC45, suggesting increased DNA replication stress. Consistent with these results, FBXW7 down-regulation alone decreases the survival of T47D breast cancer cells. These results establish that FBXW7 down-regulation is synthetic lethal with MYC, and that MYC is a critical target of FBXW7 in breast epithelial cells.

Pre-Clinical Evaluation of a Novel DNA Crosslinking Agent, Bo-1055 in B-Cell Lymphoma

Some B-cell lymphoma including mantle cell lymphoma (MCL) and diffuse large B-cell lymphoma (DLBCL) remain incurable using conventional chemotherapeutic approaches. Therefore it is important that new treatment strategies be developed. We have evaluated the efficacy of a number of novel, third generation, DNA-directed alkylating agents that have DNA specific binding domain by linking DNA-affinic molecules to N-mustard pharmacophore via a urea, carbamate or hydrazinecarboxamide linker (Kapuriya et al. Bioorganic & Medicinal Chem. 19:471–485; 2011). One of these, the water-soluble ureidomustine BO-1055, was screened for toxicity against a panel of human lymphoma cell lines including MCL (JEKO-1, Z-138, HBL-2), DLBCL (OCILy10, OCILy19) and a spontaneous murine B-cell lymphoma. We also screened BO1055 against a panel of normal human cells including mesenchymal stromal cells (IC50 >10uM), bone marrow-derived endothelium (IC50 >10uM) lung basal epithelium, bronchial epithelium and myofibroblasts (IC50s >10uM) and purified cord blood CD34+ cells in suspension culture or colony-forming assay (for hematopoietic progenitor cells) (IC50 >10uM) and in cobblestone area-forming assay (for hematopoietic stem cells) (IC50 9.1uM). The mean IC50±SD (uM) in MCL cell lines was JEKO-1 (0.266±0.27), Z-138 (0.182±0.15), HBL2 (0.161±0.34), In DLBCL lines OCILy10 (0.117±0.21), OCILy19 (0.287±0.17) and murine B-cell lymphoma (0.463±0.39). Our results indicated that BO-1055 has a significant therapeutic window (50-100-fold) between its toxicity against human B-cell lymphomas compared to various normal human cell types. We evaluated BO-1055 cardiotoxicity in the HL-1 cardiomyocyte line and observed a 227-fold less cytotoxicity compared to Doxorubicin. Treatment with BO-1055 resulted in accumulation of cells in S-phase and up-regulation of proteins involved in DNA repair [MRE11, p-P95/NBS1 (ser343), RAD50, p-ATR (ser428)] while Bcl-6, an important B-cell lymphoma biomarker, was down-regulated. Xenograft experiments in NSG mice bearing JEKO-1 GFP/luciferase+ tumors treated with BO-1055 (30mg/kg) 3x/week showed complete tumor remission after 2 weeks of treatment as monitored by luciferase image. Our results suggest that BO-1055 has potent activity against B-cell lymphoma and present a strong rationale for its further therapeutic development as an alkylating agent due to his low toxicity against normal tissue and high toxicity against hematopoietic tumors. DisclosuresNo relevant conflicts of interest to declare.