S-phase Accumulation Research Articles

RSK Inhibition Suppresses AML Proliferation through Activation of DNA Damage Pathways and S Phase Arrest

The 90 kDa Ribosomal S6 Kinase (RSK), downstream of the ERK signaling pathway, has recently been implicated in a wide variety of cancers, ranging from lung cancer to medulloblastoma, as a driver of cancer cell proliferation and survival. However, its role in Acute Myeloid Leukemia (AML) remains unknown. Thus, the goal of this study was to characterize RSK-dependent signaling pathways in AML, with the overall hypothesis that disruption of this pathway represents a potential strategy for the treatment of AML. The RSK family consists of four gene isoforms, RSK1-4 (RPS6KA1 (RSK1), RPS6KA2 (RSK3), RPS6KA3 (RSK2), RPS6KA4 (RSK4). Knockdown (KD) of RSK1 by shRNA in HL-60 and KG-1 cell lines resulted in reduced AML cell growth in vitro. NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ (NSG) mice were injected with 2x106 HL-60 or KG-1 RSK1KD cells and vector control transduced cells in order to investigate the effects of RSK1 KD on AML cell growth and survival in vivo. Mice injected with RSK1 KD cells exhibited prolonged survival by 17 and 21 days respectively for HL-60 and KG-1 cell induced disease (p=0.0023 and 0.0018 respectively). These data indicate that RSK1 knockdown inhibits leukemia progression, and RSK1 is required for maximal proliferation of AML cells in vivo. Pharmacological inhibition of total RSK (RSK1-4) by the small molecule inhibitor BI-D1870 reduced AML cell growth and induced cell death in both AML cell lines and patient samples after treatment for 48 hours. The IC50 for growth inhibition was 1.8 uM for MOLM-13, 1.6 uM for MV-4-11, and 1.9 uM for HL-60 cells. In methylcellulose colony assays, normal hematopoietic stem and progenitor cell proliferation was not affected by RSK inhibition up to a concentration of 15 uM, establishing an approximately 10-fold therapeutic index. To elucidate the mechanism by which RSK inhibition suppresses AML proliferation, we performed cell cycle analysis with HL-60 cells. RSK inhibition by BI-D1870 resulted in delayed S-phase progression and accumulation of cells in late S-phase with increased pH2AX, cPARP, and CDK2/Cyclin A expression, as measured by flow cytometry. These data indicate that inhibition of RSK leads to activation of DNA damage pathways and arrest in S-phase, resulting in apoptosis. Inhibition of CDK activity rescued S-phase arrest, demonstrating that activation and dysregulation of CDK are crucial mediators of RSK inhibitor-induced S-phase arrest. In summary, this is the first study to demonstrate that RSK plays an important role in maintaining AML cell survival and proliferation and to position RSK as a promising target for treatment of AML. DisclosuresNo relevant conflicts of interest to declare.

Schedule-dependent increased efficiency of pemetrexed-ionizing radiation combination therapy elicits a differential DNA damage response in lung cancer cells.

BackgroundLung cancer causes the most cancer deaths worldwide, thus there is a urgent need to develop new treatment options. Concurrent chemoradiotherapy has become a common strategy for the treatment of non-resectable solid tumors including non-small cell lung cancer. Pemetrexed is a folic acid antagonist that inhibits the synthesis of precursor nucleotides, whereas ionizing radiation induces DNA damage, the repair of which is dependent on sufficiently high nucleotide levels. In the clinical setting, the pemetrexed-ionizing radiation combination therapy is administered concomitantly. We hypothesized that prolonged pretreatment with pemetrexed could be beneficial, as prior depletion of nucleotide pools could sensitize cancer cells to subsequent irradiation.MethodsNon-small cell lung cancer A549 cells were treated with 1 µM pemetrexed for 72 h. In addition, cells were exposed to five gray ionizing radiation either 1, 48 or 71 h after the initiation of the pemetrexed treatment. Cell growth, senescence induction, cell cycle distribution and DNA damage marker accumulation were analysed at different time points during the treatment and the recovery phase.ResultsStand-alone treatments of five gray ionizing radiation and 1 µM pemetrexed resulted in an intermediate cell growth inhibition of A549 cells and were therefore applied as the combination regimen. Prolonged pemetrexed pretreatment for 71 h resulted in a significant S-phase accumulation. Irradiation and prolonged pemetrexed pretreatment maximally delayed long term cell growth. Additionally, senescence was augmented and recovery from treatment-induced DNA damage was most prominently delayed by prolonged pemetrexed pretreatment.ConclusionsPretreatment with pemetrexed increases anticancer efficiency of pemetrexed-ionizing radiation combination therapy, which correlates with a persistence of treatment-induced DNA damage. Therefore, this study warrants further investigations to elucidate whether a similar adaptation to the standard treatment regimen could enhance the effectiveness of the non-small cell lung cancer clinical treatment regimen.Electronic supplementary materialThe online version of this article (doi:10.1186/s12935-016-0346-x) contains supplementary material, which is available to authorized users.

Abstract 2999: Cytotoxicity with hydroxamic acid HDACis in combination with antimetabolites is highly sequence dependent in solid tumor cells

Abstract Used alone or in combination with other chemotherapeutics, the antimetabolites 2’, 2’-difluoro-2’-deoxycytidine (gemcitabine, dFdCyd), and 5-fluorouracil (5-FU)/fluorodeoxyuridine (FdUrd) comprise the mainstay of treatment for gastrointestinal malignancies, such as pancreatic and colorectal cancer. Gemcitabine with cisplatin is also first-line therapy for non-small cell lung cancer (NSCLC). While these treatments can prolong survival, many patients still relapse and there is need for improvement to current therapies. Cytotoxicity elicited by dFdCyd and 5-FU/FdUrd is influenced by drug metabolism/activation, DNA repair proteins, cell cycle progression, and apoptosis. Histone deacetylase inhibitors (HDACi) target histone deacetylases, which regulate gene expression through deacetylation of lysine residues on histones and some non-histone proteins. HDACis have been shown to increase expression of p21 and increase apoptosis which could enhance cytotoxicity with antimetabolites. The success of HDACis, including vorinostat and panobinostat, in the treatment of hematologic malignancies has encouraged their use in solid tumors. However, results from clinical trials in patients treated with HDACis alone or with other drugs, including antimetabolites have been mixed and the mechanism(s) underlying successful therapy with HDACis is largely unknown. In the present studies, we used solid tumor cell lines that represent tumors commonly treated with antimetabolites; A549 (NSCLC) and PANC1 (pancreatic cancer) cells, and HT29 (colon cancer) cells to investigate the effect of an HDACi with either dFdCyd or FdUrd, respectively, and we measured clonogenic survival. The data demonstrate that cytotoxicity with an HDACi and an antimetabolite is highly sequence dependent; vorinostat added concurrently with dFdCyd or FdUrd or prior to either antimetabolite resulted in antagonistic cytotoxicity, whereas the addition of the antimetabolite followed by HDACi was additive/synergistic with dFdCyd. Cytotoxicity with panobinostat and antimetabolites was also sequence dependent. Cell cycle distribution studies revealed the accumulation of cells in G1 and G2/M following HDACi addition (≥90% of cells), thus rendering S-phase specific dFdCyd less cytotoxic upon its subsequent addition. Similarly, concurrent addition of HDACi and dFdCyd produced less S-phase accumulation than observed with dFdCyd alone. These data demonstrate that administration of the HDACis and antimetabolites must be sequenced appropriately so that cell cycle effects complement rather than antagonize drug mechanisms. Citation Format: Sheryl A. Flanagan, Jeffrey J. Ackroyd, Aaron Kramer, Jennifer Feigin, Donna S. Shewach. Cytotoxicity with hydroxamic acid HDACis in combination with antimetabolites is highly sequence dependent in solid tumor cells. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2999.

Design, synthesis and biological evaluation of 6-substituted pyrrolo[2,3-d]pyrimidines as dual inhibitors of TS and AICARFTase and as potential antitumor agents

A new series of 2-amino-4-oxo-6-substituted pyrrolo[2,3-d]pyrimidines, with an isosteric replacement of the side chain amide moiety to a sulfur atom, were designed and synthesized as multitargeted antifolates as well as potential antitumor agents. Starting from previously synthesized 2-amino-4-oxo-pyrrolo[2,3-d]pyrimidin-6-yl-acetic acid, a reduction by lithium triethylborohydride and successive mesylation afforded the key mesylate. Nucleophilic substitution by mercaptoacetic or mercaptopropionic acid methyl esters, followed by hydrolysis and condensation with pyridinyl-methylamines provided the nonclassical compounds 1–6, whereas condensation with glutamic acid diethyl ester hydrochloride and saponification afforded the classical analogs 7–8. All target compounds exhibited inhibitory activities toward KB, SW620 and A549 tumor cell lines. The most potent compounds of this series, 7 and 8, are better inhibitors against A549 cells than methotrexate (MTX) and pemetrexed (PMX). Nucleoside protection assays establish compound 8 a dual inhibitor of thymidylate synthase (TS) and 5-aminoimidazole-4-carboxamide ribonucleotide formyltransferase (AICARFTase) targeting both de novo thymidylate and purine nucleotide biosynthesis, which is further verified by the molecular modeling studies. Analogous to PMX, target compound 8 alternates the cell cycle of SW620 cells with S-phase accumulation and induces apoptosis, leading to cell death.

Palbociclib (PD-0332991), a Potent Selective CDK4/6 Inhibitor, Overcomes Therapy-Resistant and Sensitizes Diffuse Large B-Cell Lymphoma Pre-Clinical Models to Chemo-Reagents

Background: Results from the Collaborative trial in relapsed aggressive lymphoma (CORAL) study highlighted the poor clinical outcome of relapsed/refractory diffuse large B-cell lymphoma (DLBCL) patients previously treated with rituximab-based therapy. To better understand the molecular mechanisms underlining the acquired resistance to rituximab, we generated and characterized several rituximab-resistant DLBCL cell lines (RRCLs). We found that RRCL exhibited a unique cell cycle distribution (accumulation in S-phase) and an increase in cyclin D Kinase-4 (CDk4) mRNA/protein levels. In our current work, we evaluated Palbociclib, a novel orally bioavailable CDK4/6 inhibitor recently approved by the Food and Drug Administration for the treatment of breast cancer in rituximab-sensitive and -resistant lymphoma pre-clinical models.Methods: A panel of rituximab-sensitive cell lines (RSCL) or RRCL were exposed to escalating dosages of PD0332991 (0-80µM) for 24-72 hrs. Changes in cell viability and cell cycle distribution were evaluated using the Presto Blue assay and flow cytometry respectively. IC50 values were calculated using the GraphPad Prism6 software. Subsequently RSCL or RRCL were exposed to PD0332991 or vehicle control and doxorubicin (0.5, 1, 2µM), dexamethasone (1µM), ibrutinib (1µM), bortezomib (10-20nM) or carfilzomib (10nM) for 48 hours. Changes in cell viability were determined by the Presto Blue assay. Coefficient of synergy was calculated using the CalcuSyn software.Results: Palbociclib induced dose- and time-dependent decrease in cell viability in both RSCL and RRCL. Of interest, palbociclib was more potent in RRCL than RSCL. The IC50 values were lower in RRCL Raji-4RH and RL-4RH (12.12µM and 13.43µM) than in RSCL Raji and RL cells (14.7µM and 19.99µM). In vitro exposure of lymphoma cells to palbociclib resulted in G1 cell cycle arrested and it was more evident in RRCL than in RSCL. Synergistic activity was observed by combining palbociclib with doxorubicin, dexamethasone and proteasome inhibitors (bortezomib and carfilzomib) in vitro. Our data suggests that, palbociclib is active against in rituximab-sensitive or resistant pre-clinical models. In addition, CDK4/6 inhibition resulted in synergistic effects with several chemotherapeutic agents. A better understanding in the molecular events triggered by palbociclib is necessary to better develop optimal combination strategies using this novel agent in future clinical trials. (Research, in part, supported by a NIH grant R01 CA136907-01A1 awarded to Roswell Park Cancer Institute and The Eugene and Connie Corasanti Lymphoma Research Fund). DisclosuresCzuczman:Celgene: Employment; MorphoSys: Consultancy; Immunogen: Other: Advisory board; Boehringer-Ingelheim: Other: Advisory Board.

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.

IMGN779, a CD33-Targeted Antibody-Drug Conjugate (ADC) with a Novel DNA-Alkylating Effector Molecule, Induces DNA Damage, Cell Cycle Arrest, and Apoptosis in AML Cells

IMGN779 is a CD33-targeting ADC consisting of a humanized anti-CD33 antibody, Z4681A, conjugated to DGN462, a novel DNA-alkylating agent, through a cleavable disulfide linker, sulfo-SPDB. CD33 is broadly expressed on leukemic blasts of patients with AML, making it a promising target for AML therapy. DGN462 is a member of the novel IGN class of DNA-acting cytotoxic agents, that consists of an indolino-benzodiazepine dimer containing a mono-imine moiety.Potent killing of AML tumor cells by DGN462 and IMGN779 has previously been demonstrated in vitro (K. Whiteman et. al, ASH 2014 #2321). Here we describe studies elucidating the mechanism of action of this novel payload in the AML cell lines: MV4-11, HL60, and EOL-1. The ability of DGN462 to alkylate DNA was demonstrated in an AML cell line by isolation and analysis of the genomic DNA from cells. DGN462 was found to co-purify with DNA, demonstrating that it covalently reacts with cellular DNA. A comet assay was performed confirming that DGN462 does not cross-link DNA.H2AX is known to be phosphorylated on serine 139 in response to DNA damage. Using flow cytometry, we assessed the phosphorylation of H2AX in AML cell lines following exposure to DGN462 and IMGN779. Increases in phosphorylated H2AX levels were detected as early as 4 hours following exposure to DGN462-SMe and 12 hours following exposure to IMGN779. Additional flow cytometry analysis showed that exposure to DGN462-SMe and IMGN779 leads to S-phase accumulation, G2/M arrest, followed by induction of apoptotic markers (cleaved PARP and cleaved Caspase-3) at later time points. Ex vivo studies using AML patient samples also showed elevation of phosphorylated H2AX and an increase in apoptosis in myeloid blasts following exposure to DGN462-SMe and IMGN779.As a demonstration of proof-of-mechanism in AML disease models, our results suggest that cell killing by IMGN779 is mediated by DNA damage, as a consequence of DNA alkylation. The DNA damage response is accompanied by cell cycle arrest, which leads to apoptosis. This relationship will be further explored in additional AML preclinical models to support the use of phosphorylated H2AX as a pharmacodynamic biomarker for IMGN779 activity in future clinical studies. DisclosuresKrystal:ImmunoGen, Inc.: Employment, Equity Ownership. Walker:ImmunoGen, Inc.: Employment, Equity Ownership. Fishkin:ImmunoGen, Inc.: Employment, Equity Ownership. Audette:ImmunoGen, Inc.: Employment, Equity Ownership. Kovtun:ImmunoGen, Inc.: Employment, Equity Ownership. Romanelli:ImmunoGen, Inc.: Employment, Equity Ownership.

HHR23A is required to control the basal turnover of Chk1

Among other functions, the Chk1 protein plays an essential role in coordinating the cellular stress response by determining cell cycle arrest. The levels of Chk1 expression and activity are critical for its functions, especially in cell cycle progression, genomic integrity, cell viability and tissue development. Chk1 protein expression should therefore be tightly controlled both during normal growth and under stress situations. However, it is still unknown how Chk1 protein levels are regulated during normal cell cycle progression. In this study, we show that the effect of hHR23A on Chk1 protein turnover could impact the cell cycle progression observed in hHR23A-knockdown cells. Our results reveal that hHR23A associates with Chk1 through its UBA domains, and that knockdown of hHR23A increases and stabilizes the protein level of Chk1 and its phosphorylation at S347. Knockdown of hHR23A results in proliferation defects and S-phase accumulation. DNA damage reduces the interaction between Chk1 and hHR23A, releasing Chk1 from hHR23A and enhancing S-phase accumulation. Based on these novel findings, we propose that hHR23A acts as a carrier to promote Chk1 degradation through the Ubiquitin Proteasome System. These results strengthen the model in which DNA damage induces Chk1 phosphorylation on chromatin followed by releasing phospho-Chk1 from the chromatin into soluble nucleus and the cytoplasm where Chk1 activates the cell cycle checkpoints; and finally, Chk1 is degraded and checkpoint signaling is terminated.

Abstract 2628: Mithramycin analogs with reduced toxicity for EWS-FLI1 targeting

Abstract Introduction: EWS-FLI1 and related chromosomal translocations are prevalent in Ewing sarcoma and play a major role in modulating oncogenic transcription. Development of drugs that affect EWS-FLI1 oncoprotein function may lead to successful treatment for these patients. Mithramycin (MTM) was shown to inhibit transcriptional targets of EWS-FLI1, but it has a narrow therapeutic window attributed to its nonspecific toxicities. To overcome this, semisynthetic methods were developed to generate MTM analogs with unique pharmacologic properties. Mechanistic and pharmacologic studies are presented here. Methods: Studies were conducted using MTM and lead analogs (mithramycin-SK (MTM-SK), mithramycin-SA-tryptophan (MTM-SA-Trp), and mithramycin-SA-phenylalanine (MTM-SA-Phe)). EWS-FLI1 promoter occupancy was investigated using chromatin immunoprecipitation real-time PCR (ChIP-RTPCR). The effect of drug treatment on expression of genes controlled by EWS-FLI1 was evaluated by quantitative real-time PCR (qRT-PCR). The effect of treatment on cell cycle distribution was also compared among analogs. In vitro efficacy was evaluated by estimating GI50 parameters (72-hr). In addition, the maximum tolerated dose (MTD) and the effect of treatment on plasma total-calcium were used to assess relative toxicity in mice. Results: EWS-FLI1 promoter occupancy upstream from Nr0b1, Tgfbr2, and Rcor1 genes was evaluated in Ewing sarcoma cells (TC32) by ChIP-RTPCR. MTM and MTM-SA-Trp analog destabilized FLI1 binding to all three promoters and MTM-SA-Trp was shown to be the most destabilizing. Comparatively, MTM-SK appears to mostly stabilize FLI1. Additionally, qRTPCR showed that MTM and its analogs efficiently down-regulated mRNA expression in a dose-dependent manner (rank-order of efficiency: MTM-SA-Trp>MTM = MTM-SK). These data were in accord with the in vitro cytotoxicity data that show MTM-SA-Trp has relatively higher potency (lower GI50) among Ewing cell lines (n = 8) as compared to other analogs. Furthermore, the effect of drug treatment appears to lead to differences in cell-cycle progression. MTM and MTM-SK treated TC32 cells were primarily in G1/G2 phase, whereas MTM-SA-Trp treated cells showed increased S-phase accumulation. Compared to MTM, mice tolerated significantly higher single doses of the MTM analogs. Repeated dosing showed similar results except that MTM-SA-Trp was tolerated at lower doses. MTM treatment caused an acute drop in total-calcium, which also occurred with MTM-SA-Trp and MTM-SA-Phe analogs three days later within the two-week treatment. Comparatively, MTM-SK caused an increase in total-calcium. In all cases, total-calcium concentrations returned to baseline within two weeks following treatment. Conclusion: The work presented here demonstrates the ability to design more specific and less toxic analogs of MTM. Development of such analogs could lead to successful treatments of Ewing sarcoma. Citation Format: Joseph Eckenrode, Jamie Horn, Jhong-Min Chen, Jurgen Rohr, Markos Leggas. Mithramycin analogs with reduced toxicity for EWS-FLI1 targeting. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2628. doi:10.1158/1538-7445.AM2015-2628

Novel Pactamycin Analogs Induce p53 Dependent Cell-Cycle Arrest at S-Phase in Human Head and Neck Squamous Cell Carcinoma (HNSCC) Cells.

Pactamycin, although putatively touted as a potent antitumor agent, has never been used as an anticancer drug due to its high cytotoxicity. In this study, we characterized the effects of two novel biosynthetically engineered analogs of pactamycin, de-6MSA-7-demethyl-7-deoxypactamycin (TM-025) and 7-demethyl-7-deoxypactamycin (TM-026), in head and neck squamous cell carcinoma (HNSCC) cell lines SCC25 and SCC104. Both TM-025 and TM-026 exert growth inhibitory effects on HNSCC cells by inhibiting cell proliferation. Interestingly, unlike their parent compound pactamycin, the analogs do not inhibit synthesis of nascent protein in a cell-based assay. Furthermore, they do not induce apoptosis or autophagy in a dose- or a time-dependent manner, but induce mild senescence in the tested cell lines. Cell cycle analysis demonstrated that both analogs significantly induce cell cycle arrest of the HNSCC cells at S-phase resulting in reduced accumulation of G2/M-phase cells. The pactamycin analogs induce expression of cell cycle regulatory proteins including master regulator p53, its downstream target p21Cip1/WAF1, p27kip21, p19, cyclin E, total and phospho Cdc2 (Tyr15) and Cdc25C. Besides, the analogs mildly reduce cyclin D1 expression without affecting expression of cyclin B, Cdk2 and Cdk4. Specific inhibition of p53 by pifithrin-α reduces the percentage of cells accumulated in S-phase, suggesting contribution of p53 to S-phase increase. Altogether, our results demonstrate that Pactamycin analogs TM-025 and TM-026 induce senescence and inhibit proliferation of HNSCC cells via accumulation in S-phase through possible contribution of p53. The two PCT analogs can be widely used as research tools for cell cycle inhibition studies in proliferating cancer cells with specific mechanisms of action.

Benzo[a]pyrene-induced cell cycle progression occurs via ERK-induced Chk1 pathway activation in human lung cancer cells

Benzo[a]pyrene (B[a]P) is a potent lung carcinogen derived from tobacco smoking and environmental contamination. This study aimed to investigate the signal transduction pathway responsible for B[a]P-induced non-small cell lung cancer (NSCLC) development. We exposed the human NSCLC cell lines Calu-1, CL3, H1299, CH27, H23, and H1355 to B[a]P and assessed cell cycle progression using flow cytometry. Expression of cell cycle mediators was measured using Western blot analyses and electrophoretic mobility shift assays (EMSAs). B[a]P exposure dramatically induced S-phase accumulation in H1355 cells. Phospho-p53 (Ser15 and Ser20), phospho-ERK, phospho-p38, and Bax were significantly increased in H1355 cells whereas phospho-Rb was decreased in these cells. In addition, B[a]P induced phosphorylation of checkpoint kinase-1 (Chk1) but not Chk2. EMSA experiments revealed a slower migrating band after c-Myc bound the E-box in response to B[a]P treatment, which was abolished upon the addition of the ERK inhibitor PD98059 in H1355 cells. Phospho-ERK inhibition and dominant negative mutant Chk1 expression reversed B[a]P-induced S phase accumulation and downregulated phospho-Chk1 and phospho-ERK expression. Taken together, these results suggest that activation of ERK and its downstream mediator Chk1 may contribute to B[a]P-induced S phase accumulation in H1355 cells. The results could help in the development of lung cancer treatments that target the Chk1 pathway through ERK.

Drug metabolism and homologous recombination repair in radiosensitization with gemcitabine.

Gemcitabine (difluorodeoxycytidine; dFdCyd) is a potent radiosensitizer, noted for its ability to enhance cytotoxicity with radiation at noncytotoxic concentrations in vitro and subchemotherapeutic doses in patients. Radiosensitization in human tumor cells requires dFdCyd-mediated accumulation of cells in S phase with inhibition of ribonucleotide reductase, resulting in ≥80% deoxyadenosine triphosphate (dATP) depletion and errors of replication in DNA. Less is known of the role of specific DNA replication and repair pathways in the radiosensitization mechanism. Here the role of homologous recombination (HR) in relationship to the metabolic and cell cycle effects of dFdCyd was investigated using a matched pair of CHO cell lines that are either proficient (AA8 cells) or deficient (irs1SF cells) in HR based on expression of the HR protein XRCC3. The results demonstrated that the characteristics of radiosensitization in the rodent AA8 cells differed significantly from those in human tumor cells. In the AA8 cells, radiosensitization was achieved only under short (≤4 h) cytotoxic incubations, and S-phase accumulation did not appear to be required for radiosensitization. In contrast, human tumor cell lines were radiosensitized using noncytotoxic concentrations of dFdCyd and required early S-phase accumulation. Studies of the metabolic effects of dFdCyd demonstrated low dFdCyd concentrations did not deplete dATP by ≥80% in AA8 and irs1SF cells. However, at higher concentrations of dFdCyd, failure to radiosensitize the HR-deficient irs1SF cells could not be explained by a lack of dATP depletion or lack of S-phase accumulation. Thus, these parameters did not correspond to dFdCyd radiosensitization in the CHO cells. To evaluate directly the role of HR in radiosensitization, XRCC3 expression was suppressed in the AA8 cells with a lentiviral-delivered shRNA. Partial XRCC3 suppression significantly decreased radiosensitization [radiation enhancement ratio (RER) = 1.6 ± 0.15], compared to nontransduced (RER = 2.7 ± 0.27; P = 0.012), and a substantial decrease compared to nonspecific shRNA-transduced (RER = 2.5 ± 0.42; P = 0.056) AA8 cells. Although the results support a role for HR in radiosensitization with dFdCyd in CHO cells, the differences in the underlying metabolic and cell cycle characteristics suggest that dFdCyd radiosensitization in the nontumor-derived CHO cells is mechanistically distinct from that in human tumor cells.

Abstract 4091: Interfering with interferon: An axis of ATF2-mediated chemoresistance

Abstract Malignant melanoma is currently the most lethal skin cancer and comprises approximately 8% of total U.S. cancers cases. Despite the advent of even the most effective targeted monotherapies to date (e.g., mutant B-RAF inhibitors), clinical responses have largely been transient, as melanomas rapidly develop therapeutic resistance, highlighting the need to target other critical signaling pathways. In melanoma, Activating Transcription Factor 2 (ATF2) elicits an oncogenic function when nuclear but a tumor suppressor function when it is cytosolic/mitochondrial. In progressively malignant melanoma, increased PKCϵ-mediated phosphorylation of ATF2 on threonine 52 (T52) promotes its nuclear localization and transcriptional activity, and renders the cells more resistant to genotoxic stress. To determine how PKCϵ-phosphorylated ATF2 contributes to melanoma biology, we performed gene expression profiling of melanoma cells that were reconstituted with wild-type, T52 phosphomutant or phosphomimic ATF2, in the presence or absence of genotoxic stress (which attenuates the T52 phosphorylation of ATF2). We report that in melanoma cells, genotoxic stress induces the expression of a cluster of Interferon β1 (IFNβ1) and related genes, which is suppressed by the expression of ATF2T52E. ATF2 binds to the 5′-promoter region of IFNβ1, and we identified the ATF2/AP-1 binding site required for transcriptional repression. The stress-induced expression of IFNβ1 mediates S-phase accumulation during genotoxic stress with a concomitant induction of a senescence-like phenotype. In contrast, blockade of type-1 interferon signaling by shRNA-mediated depletion of the IFNAR receptor abrogates the S-phase accumulation, blocking cells in G1 phase and reducing the extent of stress-induced cell death. The expression of or treatment with exogenous IFNβ1 blunts the proliferation of melanoma cells in both 2- and 3-dimensional spheroid cultures. Notably, the treatment of melanoma cells with mutant BRAF inhibitor (Vemurafenib/PLX4720) also induces the expression of IFNβ1 and senescence markers, and the co-treatment of melanoma cells with IFNβ1 and etoposide or PLX4720 significantly enhances the cell death induced by either etoposide or PLX4720 alone. Lastly, we found that the stress-induced IFNβ1 expression by melanoma cells elicits paracrine effects, enhancing the melanoma cell clearance by lymphocytes in an in vitro co-culture system. Our data demonstrate a novel transcriptional regulatory role exhibited by ATF2 on IFNβ1, which influences cell cycle dynamics and stress/treatment resistance of melanoma cells. The therapeutic and prognostic implications of these findings will be discussed. Citation Format: Eric Lau, Giuseppina Claps, David S. Hoon, Ze'ev A. Ronai. Interfering with interferon: An axis of ATF2-mediated chemoresistance. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4091. doi:10.1158/1538-7445.AM2014-4091

Abstract 4556: A therapeutic strategy combining the Wee1 inhibitor MK1775 with HDAC inhibitors targets both p53 wild-type or mutant AML cells

Abstract Wee1 is an essential component of the G2-M cell cycle checkpoint machinery, providing a target for Wee1 inhibitors (e.g., MK1775) designed to potentiate the anti-tumor activity of genotoxic agents via mitotic lethality. In this setting, Wee1 inhibitor actions are primarily p53-dependent. However, recent studies have identified novel S-phase functions of Wee1 that operate independently of p53 status. Such findings present a rationale for combining Wee1 inhibitors with other targeted agents such as HDAC inhibitors (HDACIs) which interrupt DNA replication and repair. To test this hypothesis, interactions between MK1775 and the HDACIs SBHA or vorinostat were investigated in AML cells. Significantly, MK1775 interacted highly synergistically with either HDACI in both p53 wild type (wt) or mutant AML cells, manifested by pronounced apoptosis induction and attenuation of clonogenic survival. Consistent with these findings, shRNA knock-down of Wee1 significantly sensitized cells to HDACIs. Immunobloting revealed that HDACIs markedly enhanced Wee1 inhibition by MK1775, reflected by diminished Wee1 S642 phosphorylation and protein levels, as well as cdc2/Cdk1 Y15 phosphorylation, a direct downstream Wee1 kinase target. In p53-null (U937) or -mutant (MV-4-11) cells, MK1775 triggered S-phase arrest, whereas HDACI co-administration resulted in early S-phase accumulation accompanied by a marked increase in sub-G1 cells. In sharp contrast, in p53 wt MOLM-13 cells, HDACIs clearly induced G2/M arrest, an event abrogated by MK1775, arguing for context-specific cell cycle effects of this strategy. Interestingly, while MK1775/HDACI synergism persisted, shRNA p53 knock-down in p53 wt AML-3 cells partially attenuated lethal effects of combined treatment. Nevertheless, MK1775/HDACI co-treatment strikingly increased DNA damage, manifested by dramatically increases in γH2A.X generation, in both p53 wt and mutant cells. This event was associated with profound caspase activation and PARP cleavage. Notably, the MK1775/HDACI regimen was highly active against patient-derived AML cells harboring either wt or mutant p53, as well as various other mutations in cancer hotspot genes including FLT3, NRAS, KRAS, BRAF, PDGFRA, MET, STK11, SMARCB1, and APC. Furthermore, the CD34+/CD123+/CD38- population, enriched for leukemia-initiating progenitors, but not normal CD34+ hematopoietic cells, was highly susceptible to the Wee1/HDAC inhibition strategy. Finally, co-administration of MK1775 with vorinostat substantially suppressed tumor growth and significantly prolonged animal survival in a murine AML xenograft model. Together, these findings indicate that a strategy combining Wee1 inhibition (e.g., by MK1775) with HDACIs is highly active towards AML cells, including primitive progenitors, through context-specific p53-dependent and -independent mechanisms. Citation Format: Yu Zhang, Liang Zhou, Shuang Chen, Maciej Kmieciak, Hui Lin, Yun Dai, Steven Grant. A therapeutic strategy combining the Wee1 inhibitor MK1775 with HDAC inhibitors targets both p53 wild-type or mutant AML cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4556. doi:10.1158/1538-7445.AM2014-4556

Inhibition of STAT3 signaling targets both tumor-initiating and differentiated cell populations in prostate cancer.

Despite of tremendous research efforts to profile prostate cancer, the genetic alterations and biological processes that correlate with disease progression remain partially elusive. In this study we show that the STAT3 small molecule inhibitor Stattic caused S-phase accumulation at low-dose levels and led to massive apoptosis at a relatively high-dose level in prostate cancer cells. STAT3 knockdown led to the disruption of the microvascular niche which tumor-initiating cells (TICs) and non-tumor initiating cells (non-TICs)depend on. Primary human prostate cancer cells and prostate cancer cell line contained high aldehyde dehydrogenase activity (ALDH(high)) subpopulations with stem cell-like characteristics, which expressed higher levels of the active phosphorylated form of STAT3 (pSTAT3) than that of non-ALDH(high) subpopulations. Stattic could singnificantly decrease the population of ALDH(high) prostate cancer cells even at low-dose levels. IL-6 can convert non-ALDH(high) cells to ALDH(high) cells in prostate cancer cell line as well as from cells derived from human prostate tumors, the conversion mediated by IL-6 was abrogated in the presence of STAT3 inhibitor or upon STAT3 knockdown. STAT3 knockdown significantly impaired the ability of prostate cancer cells to initiate development of prostate adenocarcinoma. Moreover, blockade of STAT3 signaling was significantly effective in eradicating the tumor-initiating and bulk tumor cancer cell populations in both prostate cancer cell-line xenograft model and patient-derived tumor xenograft (PDTX) models. This data suggests that targeting both tumor initiating and differentiated cell populations by STAT3 inhibition is predicted to have greater efficacy for prostate cancer treatment.

NM23-H1 expression of head and neck squamous cell carcinoma in association with the response to cisplatin treatment.

We recently reported that low NM23-H1 expression of head and neck squamous cell carcinoma (HNSCC) correlated with poor patients' prognosis. Growing evidence has indicated that high tumor NM23-H1 expression contributes to a good response to chemotherapy. Therefore, we investigated the role of NM23-H1 in susceptibility of HNSCC cells to cisplatin and its clinical significance, as well as the in vitro study for validation was performed. Using immunohistochemistry, we analyzed NM23-H1 expression in surgical specimens from 46 HNSCC patients with cervical metastases receiving surgery and adjuvant chemoradiotherapy. Low tumor NM23-H1 expression correlated with locoregional recurrence of HNSCC following postoperative cisplatin-based therapy (p = 0.056) and poor patient prognosis (p = 0.001). To validate the clinical observation and the effect of NM23-H1 on cisplatin cytotoxicity, we established several stable clones derived from a human HNSCC cell line (SAS) by knockdown and overexpression. Knockdown of NM23-H1 attenuated the chemosensitivity of SAS cells to cisplatin, which was associated with reduced cisplatin-induced S-phase accumulation and downregulation of cyclin E1 and A. Overexpression of NM23-H1 reversed these results, indicating the essential role of NM23-H1 in treatment response to cisplatin. NM23-H1 may participate in HNSCC cell responses to cisplatin and be considered a potential therapeutic target.

FANCJ Localization by Mismatch Repair Is Vital to Maintain Genomic Integrity after UV Irradiation

Nucleotide excision repair (NER) is critical for the repair of DNA lesions induced by UV radiation, but its contribution in replicating cells is less clear. Here, we show that dual incision by NER endonucleases, including XPF and XPG, promotes the S-phase accumulation of the BRCA1 and Fanconi anemia-associated DNA helicase FANCJ to sites of UV-induced damage. FANCJ promotes replication protein A phosphorylation and the arrest of DNA synthesis following UV irradiation. Interaction defective mutants of FANCJ reveal that BRCA1 binding is not required for FANCJ localization, whereas interaction with the mismatch repair (MMR) protein MLH1 is essential. Correspondingly, we find that FANCJ, its direct interaction with MLH1, and the MMR protein MSH2 function in a common pathway in response to UV irradiation. FANCJ-deficient cells are not sensitive to killing by UV irradiation, yet we find that DNA mutations are significantly enhanced. Thus, we considered that FANCJ deficiency could be associated with skin cancer. Along these lines, in melanoma we found several somatic mutations in FANCJ, some of which were previously identified in hereditary breast cancer and Fanconi anemia. Given that, mutations in XPF can also lead to Fanconi anemia, we propose collaborations between Fanconi anemia, NER, and MMR are necessary to initiate checkpoint activation in replicating human cells to limit genomic instability.

Abstract P3-04-05: Amplification driven expression of KIAA0020, a PARP1 interacting gene, facilitates repair of replication associated DNA damage in triple-negative breast cancers

Abstract Approximately 25% of triple-negative breast cancers display increased copy number in the region of 9p24. Since several genes mapping to this genomic locus have been shown to be involved in the biology of triple-negative breast cancers, a copy number-dependent transcriptional influence of genes at 9p24 on the malignant phenotypes has been suggested. Given the limited options of targeted therapy for triple-negative breast cancer patients, further studies of genes located at this locus could therefore provide potential novel targets or companion biomarkers for this disease. By integrating microarray-based DNA copy number and gene expression of genes located on 9p24 in 111 triple-negative breast cancers, we identified among a wider set of genes three hypothetical genes, KIAA0020, KIAA1432 and KIAA2026 whose expression was highly correlated with their copy number status. External validation through analysis of several comprehensive breast cancer cohorts, confirmed KIAA0020 as being highly abundant in basal-like/ triple-negative breast cancers. Involvement of KIAA0020 in PARP1 activity and the DNA damage response has previously been suggested. To elucidate KIAA0020's functional involvement in cell growth, cell cycle progression, apoptosis and DNA damage response in this subtype of breast cancer, breast cancer cell lines with and without increased 9p24 copy number levels were used as in vitro models. In cell lines with increased DNA copy number at 9p24, depletion of KIAA0020 expression selectively impaired growth causing an accumulation in S-phase and a decrease in cell proliferation. Furthermore, KIAA0020-silencing in such cell lines resulted in decreased repair of hydroxyurea induced replication associated DNA damage, accumulation of DNA double strand breaks and decreased occurrence of RAD51 and PAR foci, all pointing to a decreased repair of inactivated replication forks. Taken together, our data supports the notion that genes of unknown function, residing in the 9p24 copy number aberrant region are involved in DNA repair and may thereby also contribute to the tumourigenesis of triple-negative breast cancers. A subset of such triple-negative tumours seemed to have developed a dependency on the expression of KIAA0020, a PARP1 interacting gene, for replication fork associated repair. Thus, greater understanding of KIAA0020's molecular function may provide additional information for patient selection with regards to DNA damaging chemotherapeutics or PARP inhibitors within triple-negative breast cancers. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P3-04-05.

Discovery of 5-Substituted Pyrrolo[2,3-d]pyrimidine Antifolates as Dual-Acting Inhibitors of Glycinamide Ribonucleotide Formyltransferase and 5-Aminoimidazole-4-carboxamide Ribonucleotide Formyltransferase in De Novo Purine Nucleotide Biosynthesis: Implications of Inhibiting 5-Aminoimidazole-4-carboxamide Ribonucleotide Formyltransferase to AMPK Activation and Antitumor Activity

We synthesized 5-substituted pyrrolo[2,3-d]pyrimidine antifolates (compounds 5-10) with one-to-six bridge carbons and a benozyl ring in the side chain as antitumor agents. Compound 8 with a 4-carbon bridge was the most active analogue and potently inhibited proliferation of folate receptor (FR) α-expressing Chinese hamster ovary and KB human tumor cells. Growth inhibition was reversed completely or in part by excess folic acid, indicating that FRα is involved in cellular uptake, and resulted in S-phase accumulation and apoptosis. Antiproliferative effects of compound 8 toward KB cells were protected by excess adenosine but not thymidine, establishing de novo purine nucleotide biosynthesis as the targeted pathway. However, 5-aminoimidazole-4-carboxamide (AICA) protection was incomplete, suggesting inhibition of both AICA ribonucleotide formyltransferase (AICARFTase) and glycinamide ribonucleotide formyltransferase (GARFTase). Inhibition of GARFTase and AICARFTase by compound 8 was confirmed by cellular metabolic assays and resulted in ATP pool depletion. To our knowledge, this is the first example of an antifolate that acts as a dual inhibitor of GARFTase and AICARFTase as its principal mechanism of action.

Flipping the phenotypic switch on a novel antimelanoma differentiation strategy

Flipping the phenotypic switch on a novel antimelanoma differentiation strategy