S-phase DNA Damage Research Articles

A modified nucleoside O6-methyl-2′-deoxyguanosine-5′-triphosphate exhibits anti-glioblastoma activity in a caspase-independent manner

Resistance to temozolomide (TMZ), the frontline chemotherapeutic agent for glioblastoma (GBM), has emerged as a formidable obstacle, underscoring the imperative to identify alternative therapeutic strategies to improve patient outcomes. In this study, we comprehensively evaluated a novel agent, O6-methyl-2′-deoxyguanosine-5′-triphosphate (O6-methyl-dGTP) for its anti-GBM activity both in vitro and in vivo. Notably, O6-methyl-dGTP exhibited pronounced cytotoxicity against GBM cells, including those resistant to TMZ and overexpressing O6-methylguanine-DNA methyltransferase (MGMT). Mechanistic investigations revealed that O6-methyl-dGTP could be incorporated into genomic DNA, disrupting nucleotide pools balance, and inducing replication stress, resulting in S-phase arrest and DNA damage. The compound exerted its anti-tumor properties through the activation of AIF-mediated apoptosis and the parthanatos pathway. In vivo studies using U251 and Ln229 cell xenografts supported the robust tumor-inhibitory capacity of O6-methyl-dGTP. In an orthotopic transplantation model with U87MG cells, O6-methyl-dGTP showcased marginally superior tumor-suppressive activity compared to TMZ. In summary, our research, for the first time, underscores the potential of O6-methyl-dGTP as an effective candidate against GBM, laying a robust scientific groundwork for its potential clinical adoption in GBM treatment regimens.

Downregulation of ASF1B inhibits tumor progression and enhances efficacy of cisplatin in pancreatic cancer.

Pancreatic cancer is an aggressive and lethal cancer with the highest mortality rate. Hence, the development of new targeting and innovative treatment strategies is needed. Recent studies reported that the histone chaperone anti-silencing function 1B (ASF1B) can be used as a diagnosis and prognosis cancer biomarker. However, functional studies of ASF1B in pancreatic cancer have not been performed. This study compared expression levels of ASF1B in pancreatic cancer specimens with those of normal tissues using publicly available online databases. We found that ASF1B was commonly overexpressed in pancreatic cancer specimens, which is associated with poor prognosis. ASF1B downregulation in pancreatic cancer cells reduced their colony formation, proliferation, migration, and invasion abilities, and inhibited MMP9 activity. Furthermore, ASF1B expression downregulation increased cell cycle S-phase arrest and DNA damage though activation of the checkpoint kinases Chk1 and Chk2 pathways. Additionally, increased caspase (caspases-3 and -9) activation and PARP cleavage led to enhanced caspase-dependent apoptosis and improved cisplatin sensitivity. Collectively, our results indicate that ASF1B may serve as a potential biomarker of pancreatic cancer and a novel therapeutic target.

PTEN loss drives resistance to the neddylation inhibitor MLN4924 in glioblastoma and can be overcome with TOP2A inhibitors.

Neddylation inhibition, affecting posttranslational protein function and turnover, is a promising therapeutic approach to cancer. We report vulnerability to MLN4924 or pevonedistat (a neddylation inhibitor) in a subset of glioblastoma (GBM) preclinical models and identify biomarkers, mechanisms, and signatures of differential response. GBM sequencing data were queried for genes associated with MLN4924 response status; candidates were validated by molecular techniques. Time-course transcriptomics and proteomics revealed processes implicated in MLN4924 response. Vulnerability to MLN4924 is associated with elevated S-phase populations, re-replication, and DNA damage. Transcriptomics and shotgun proteomics depict PTEN signaling, DNA replication, and chromatin instability pathways as significant differentiators between sensitive and resistant models. Loss of PTEN and its nuclear functions is associated with resistance to MLN4924. Time-course proteomics identified elevated TOP2A in resistant models through treatment. TOP2A inhibitors combined with MLN4924 prove synergistic. We show that PTEN status serves as both a novel biomarker for MLN4924 response in GBM and reveals a vulnerability to TOP2A inhibitors in combination with MLN4924.

YTHDF1 promotes breast cancer cell growth, DNA damage repair and chemoresistance

Chemoresistance represents a major obstacle to the treatment of human cancers. Increased DNA repair capacity is one of the important mechanisms underlying chemoresistance. In silico analysis indicated that YTHDF1, an m6A binding protein, is a putative tumor promoter in breast cancer. Loss of function studies further showed that YTHDF1 promotes breast cancer cell growth in vitro and in vivo. YTHDF1 facilitates S-phase entry, DNA replication and DNA damage repair, and accordingly YTHDF1 knockdown sensitizes breast cancer cells to Adriamycin and Cisplatin as well as Olaparib, a PARP inhibitor. E2F8 is a target molecule by YTHDF1 which modulates E2F8 mRNA stability and DNA damage repair in a METTL14-dependent manner. These data demonstrate that YTHDF1 has a tumor-promoting role in breast cancer, and is a novel target to overcome chemoresistance.

Abstract P5-13-14: Antitumor activity of patritumab deruxtecan (HER3-DXd), a HER3-directed antibody drug conjugate (ADC) across a diverse panel of breast cancer (BC) patient-derived xenografts (PDXs)

Abstract Background: HER3 is overexpressed in 30-50% of breast cancers and has been associated with poor prognosis. Patritumab deruxtecan (HER3-DXd; U3-1402) is a HER3-directed ADC with a potent topoisomerase I (TOP1) inhibitor payload. A phase 1/2 study of HER3-DXd (NCT02980341) demonstrated promising antitumor activity in hormone receptor positive (HR+) metastatic BC patients with clinical activity observed across baseline levels of HER3 protein or mRNA expression. A window of opportunity clinical trial is currently ongoing to evaluate the biological activity of HER3-DXd in patients with treatment naïve BC according to HER3 mRNA/protein expression levels (NCT04610528). Here, we aimed to describe the activity of HER3-DXd in PDX models to identify robust biomarkers of response. Methods: The antitumor activity of HER3-DXd was assessed in 21 BC PDX models (14 HR+ and 7 triple negative). HER3-DXd sensitivity was established as a complete response that lasted longer than 120 days, following 4 weekly doses of 10 mg/kg. HER3-DXd antitumor activity was compared to the antitumor activity of irinotecan (50 mg/kg dosed once weekly), which was evaluated according to modified RECIST criteria in a subset of 14 BC PDX models. PDX models of acquired resistance to HER3-DXd were generated by repeated treatment cycles. Pharmacodynamic (PD) experiments were conducted by collecting tumor samples from PDXs after a single dose of HER3-DXd or irinotecan. Baseline HER3 expression was assessed by immunohistochemistry (IHC) and Western blot. mRNA expression of 72 genes including ERBB3 and genes from the PAM50 signature were measured using the nCounter platform. Two-class unpaired significance analysis of microarrays (SAM), using a false-discovery rate<5%, identified differential gene expression across response groups. Genetic alterations harbored by PDX models were determined using the MSK-IMPACTTM targeted exome panel. Quantification of proliferation (% of Ki67-positive cells) and of DNA damage during the S-phase of the cell cycle (γH2AX nuclear foci in geminin-positive cells) was evaluated in untreated/treated PD samples by IHC or immunofluorescence (IF), respectively. Western blot was used to assess HER3-pathway downmodulation and induction of apoptosis. Results: Eight out of 21 (38%) PDXs were highly sensitive to HER3-DXd, and in 5/14 (36%) models HER3-DXd showed a superior antitumor activity when compared to irinotecan. We observed an enrichment of basal-like models amongst the non-relapsed PDXs, compared to the relapsed ones (6/8 (75%) vs. 3/13 (20%), p=0.0195). Baseline levels of HER3/ERBB3 were not associated with treatment response and a model of acquired-resistance did not exhibit a reduction in baseline HER3 expression. Interestingly, relapsed models showed increased expression of genes related with chemotherapy-resistance (MDM2, NAT1, MAPT, GRB7, BCL-2). Mechanistically, treatment with HER3-DXd did not reduce the level of Ki67-proliferating cells but resulted in a significantly higher induction of S-phase DNA damage measured as γH2AX nuclear foci in non-relapsed models, compared to relapsed ones. This was accompanied by a reduction of HER3 protein levels and downmodulation of pERK1/2 T202/Y204, along with activation of PARP cleavage. Conclusions: HER3-DXd exerts a potent antitumor response in BC PDXs, independently of baseline HER3/ERBB3 levels, in line with the clinical data of NCT02980341. Basal-like tumors were more sensitive to HER3-DXd than luminal B models. Mechanistically, our data suggests that treatment with HER3-DXd results in parallel HER3/ERK signaling downmodulation and induction of S-phase DNA damage, resulting in tumor cell death. Citation Format: Andreu Òdena, Laia Monserrat, Fara Brasó-Maristany, Marta Guzmán, Judit Grueso, Olga Rodríguez, Maurizio Scaltriti, Sarat Chandarlapaty, Yang Qiu, Kumiko Koyama, Mafalda Oliveira, Aleix Prat, Violeta Serra. Antitumor activity of patritumab deruxtecan (HER3-DXd), a HER3-directed antibody drug conjugate (ADC) across a diverse panel of breast cancer (BC) patient-derived xenografts (PDXs) [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P5-13-14.

Differential Effects of Combined ATR/WEE1 Inhibition in Cancer Cells.

Simple SummaryCancer cells often show elevated replication stress and loss of cell cycle checkpoints. The ataxia telangiectasia and Rad3-related (ATR) and WEE1 kinases play roles in protecting cancer cells from high replication stress and in regulating the remaining cell cycle checkpoints. Inhibitors of ATR or WEE1 therefore have the potential to selectively kill cancer cells and are currently being tested in clinical trials. However, more studies are needed to understand how these inhibitors work in various types of cancer and to find the most effective ways of using them. Here, we have explored whether simultaneous treatment with ATR and WEE1 inhibitors is a promising approach. Effects were investigated in cell lines from osteosarcoma and lung cancer. We expect our results to be of importance for future treatment strategies with these inhibitors.Inhibitors of WEE1 and ATR kinases are considered promising for cancer treatment, either as monotherapy or in combination with chemo- or radiotherapy. Here, we addressed whether simultaneous inhibition of WEE1 and ATR might be advantageous. Effects of the WEE1 inhibitor MK1775 and ATR inhibitor VE822 were investigated in U2OS osteosarcoma cells and in four lung cancer cell lines, H460, A549, H1975, and SW900, with different sensitivities to the WEE1 inhibitor. Despite the differences in cytotoxic effects, the WEE1 inhibitor reduced the inhibitory phosphorylation of CDK, leading to increased CDK activity accompanied by ATR activation in all cell lines. However, combining ATR inhibition with WEE1 inhibition could not fully compensate for cell resistance to the WEE1 inhibitor and reduced cell viability to a variable extent. The decreased cell viability upon the combined treatment correlated with a synergistic induction of DNA damage in S-phase in U2OS cells but not in the lung cancer cells. Moreover, less synergy was found between ATR and WEE1 inhibitors upon co-treatment with radiation, suggesting that single inhibitors may be preferable together with radiotherapy. Altogether, our results support that combining WEE1 and ATR inhibitors may be beneficial for cancer treatment in some cases, but also highlight that the effects vary between cancer cell lines.

DNA Damage Response during Replication Correlates with CIN70 Score and Determines Survival in HNSCC Patients.

Simple SummaryFunctional aneuploidy as determined by expression of the 70 genes of the CIN70 score, influences prognosis. The importance of DNA repair protein expression and activity in this context is unclear. We can show here that a high CIN70 score is associated with increased expression of 44 proteins of the known DNA repair complexes. Among these, an association with survival was only observed for 12 proteins of DNA replication and replication-associated DNA repair. This suggests that it is not the expression of individual DNA repair proteins of a DNA repair complex that causes resistance to therapy, but rather a balanced expression and coordinated activation of corresponding signaling cascades. Inhibitors to generally block the S-phase DNA damage response should therefore be used to develop therapeutic strategies in the future.Aneuploidy is a consequence of chromosomal instability (CIN) that affects prognosis. Gene expression levels associated with aneuploidy provide insight into the molecular mechanisms underlying CIN. Based on the gene signature whose expression was consistent with functional aneuploidy, the CIN70 score was established. We observed an association of CIN70 score and survival in 519 HNSCC patients in the TCGA dataset; the 15% patients with the lowest CIN70 score showed better survival (p = 0.11), but association was statistically non-significant. This correlated with the expression of 39 proteins of the major repair complexes. A positive association with survival was observed for MSH2, XRCC1, MRE11A, BRCA1, BRCA2, LIG1, DNA2, POLD1, MCM2, RAD54B, claspin, a negative for ERCC1, all related with replication. We hypothesized that expression of these factors leads to protection of replication through efficient repair and determines survival and resistance to therapy. Protein expression differences in HNSCC cell lines did not correlate with cellular sensitivity after treatment. Rather, it was observed that the stability of the DNA replication fork determined resistance, which was dependent on the ATR/CHK1-mediated S-phase signaling cascade. This suggests that it is not the expression of individual DNA repair proteins that causes therapy resistance, but rather a balanced expression and coordinated activation of corresponding signaling cascades.

Targeting OGG1 arrests cancer cell proliferation by inducing replication stress.

Altered oncogene expression in cancer cells causes loss of redox homeostasis resulting in oxidative DNA damage, e.g. 8-oxoguanine (8-oxoG), repaired by base excision repair (BER). PARP1 coordinates BER and relies on the upstream 8-oxoguanine-DNA glycosylase (OGG1) to recognise and excise 8-oxoG. Here we hypothesize that OGG1 may represent an attractive target to exploit reactive oxygen species (ROS) elevation in cancer. Although OGG1 depletion is well tolerated in non-transformed cells, we report here that OGG1 depletion obstructs A3 T-cell lymphoblastic acute leukemia growth in vitro and in vivo, validating OGG1 as a potential anti-cancer target. In line with this hypothesis, we show that OGG1 inhibitors (OGG1i) target a wide range of cancer cells, with a favourable therapeutic index compared to non-transformed cells. Mechanistically, OGG1i and shRNA depletion cause S-phase DNA damage, replication stress and proliferation arrest or cell death, representing a novel mechanistic approach to target cancer. This study adds OGG1 to the list of BER factors, e.g. PARP1, as potential targets for cancer treatment.

XPO1 Relieves MYC-Induced Replication Stress Limiting the Immunogenicity of DLBCL Cells

The soluble nuclear pore component XPO1 is a therapeutic target in DLBCL and its selective inhibitor selinexor received FDA approval in relapsed/refractory DLBCL. XPO1 inhibitors block the nuclear export of a subset of cargo proteins. Since the spectrum of XPO1 cargos varies among cell types, the mechanism underlying the anti-lymphoma effect of selinexor has not been fully elucidated. Lack of this knowledge hampers the clinical use of XPO1 inhibitors in terms of patient selection and development of rational therapeutic combinations. We reported that XPO1 is amplified and overexpressed in DLBCL. At the molecular level, we described that XPO1 overexpression functionally remodels the nuclear pore to sustain the expression of DNA damage repair proteins increasing lymphoma cells' tolerance to genotoxic stress. Here, we investigated whether this XPO1 role is relevant for the anti-lymphoma effect of selinexor. Aberrant MYC expression induces DNA damage resulting from replication stress; thus, we speculated that selinexor kills MYC expressing (MYC+) DLBCL cells by limiting their ability to tolerate replication stress. Using B-cells expressing MYC in an inducible manner, we demonstrated that exposure to selinexor (6 h) caused G2-M cell cycle arrest upon MYC expression (p<0.0001, vs. vehicle-treated cells). Cell cycle arrest was associated with increased levels of DNA damage in S-phase as measured by yH2AX (p<0.0001). Furthermore, although MYC increased the expression of key replication stress repair proteins (i.e. RAD51, WEE and BRCA1), this effect was blunted by selinexor. To determine the clinical relevance of this mechanism, we interrogated publicly available datasets of DLBCLs and found that XPO1 amplification and overexpression was associated with MYC translocation (p<0.005), double-hit lymphoma signature (p<0.005), and elevated levels of chromosomal instability (p<0.005). Overall, these observations suggest that MYC-driven lymphomas sustaining high levels of genomic instability may critically depend on XPO1. To therapeutically capitalized this mechanism, we evaluated the effect of selinexor on MYC+ DLBCLs. Clinically achievable doses of selinexor induced cell cycle arrest and apoptosis in 83% (5/6) of cell lines investigated in vitro. In a patient-derived xenograft (PDX) MYC+ DLBCL model, a human-equivalent dose of selinexor impaired tumor growth (p< 0.05, vs. vehicle) without significant toxicities. Reduced tumor growth was secondary to apoptosis as determined by TUNEL (p<0.05). To characterize the biological effects of XPO1 inhibition, we compared the transcriptome of PDX from the previous experiment. Pathway analysis of differentially expressed genes revealed that selinexor induced pathways regulating innate immune signaling. The upregulation of key genes from this pathway (i.e. DDX58, DDX60, IRF7 and STAT1) was validated by qRT-PCR. Since unrepaired DNA damage elicits an innate immune response, we hypothesized that these effects could be mechanistically linked. To test this notion, we exposed MYC+ DLBCL cells to sub-lethal doses of the DNA damaging agent etoposide, selinexor, or their combination. Expression of innate immune genes was significantly higher in cells exposed to the combination compared to either drug alone (p<0.001, for both comparisons), indicating that selinexor increases the immunogenicity of MYC+ DLBCL cells and this effect is enhanced by concomitant replication stress. Furthermore, by analyzing primary DLBCLs we found that poorly immunogenic lymphomas, characterized by an immune deserted microenvironment, expressed higher levels of XPO1 and MYC compared to DLBCL with an immune rich microenvironment (P<0.001, for both genes). To determine a potential effect of selinexor in the function of immune effector cells, we performed single cells secretomic analysis (Isolight) of circulating CD8+ T cells obtained from two DLBCL patients before and after selinexor treatment and found that selinexor increases the secretory activity of CD8+ T cells in both patients. Overall, our study reveals a key oncogenic role for XPO1 in enabling DLBCL cells to tolerate MYC-induced replication stress while limiting their immunogenicity. This suggest that patients with MYC+ DLBCL and elevated genomic instability may benefit from therapeutic approaches based on XPO1 inhibitors in combination with immunomodulatory drugs, an approach we will test prospectively. Disclosures Rutherford: AstraZeneca: Consultancy; Celgene: Consultancy; Dova: Consultancy; Genentech/Roche: Research Funding; Heron: Consultancy; Juno: Consultancy; Karyopharm: Consultancy, Research Funding; Kite: Consultancy; LAM Therapeutics: Research Funding; Regeneron: Research Funding; Seattle Genetics: Consultancy. Martin:Regeneron: Consultancy; Cellectar: Consultancy; Janssen: Consultancy; Karyopharm: Consultancy, Research Funding; Teneobio: Consultancy; Celgene: Consultancy; Bayer: Consultancy; Beigene: Consultancy; Sandoz: Consultancy; I-MAB: Consultancy; Morphosys: Consultancy; Kite: Consultancy; Incyte: Consultancy. Leonard:Miltenyi: Consultancy; Sutro: Consultancy; Roche/Genentech: Consultancy; BMS/Celgene: Consultancy; Regeneron: Consultancy; ADC Therapeutics: Consultancy; MEI Pharma: Consultancy; Bayer: Consultancy; Gilead/Kite: Consultancy; Karyopharm: Consultancy; GenMab: Consultancy; Epizyme: Consultancy; AstraZeneca: Consultancy. Cerchietti:Cellgene: Research Funding; Bristol Myers Squibb: Research Funding.

Abstract A32: Selective inhibition of AuroraA by LY3295668 erbumine in neuroblastoma models induces apoptosis through a combination of S-phase DNA damage and mitotic arrest

Abstract Aurora kinase A (AuroraA) is a serine/threonine protein kinase critical for centrosome maturation, mitotic spindle formation, and spindle checkpoint activation. Aberrant expression of AuroraA promotes tumor initiation and disease progression, making it a promising therapeutic target in several cancer types. However, recent clinical trials of purported selective AuroraA inhibitors failed due to lack of efficacy and/or hematologic toxicity, likely due to concurrent inhibition of Aurora kinase B (AuroraB). Selective AuroraA inhibition leads to mitotic arrest and apoptosis, while AuroraB inhibition causes persistent DNA endoreduplication and polyploidy leading to genomic instability. LY3295668 erbumine (LY3295668) is a selective AuroraA inhibitor with preclinical activity in multiple models of cancer including neuroblastoma (NBL). High-risk NBL, a devastating pediatric cancer with few effective treatment options, is mainly stratified by MYCN amplification status. In NBL, AuroraA stabilizes N-MYC post-transcriptionally; moreover, AuroraA also controls N-MYC-dependent transcription during S-phase. Here, we evaluated LY3295668 in multiple NBL cell lines and in vivo mouse models where treatment resulted in apoptosis and tumor regressions, respectively. We compared LY3295668 to alisertib (a dual AuroraA/B inhibitor) in multiple cell-based assays that differentiate AuroraA activity from that of AuroraB and showed that LY3295668 has a distinct pharmacologic profile from alisertib. In addition, we determined the mechanism of action for LY3295668 includes DNA damage prior to mitotic block. Our results further support that AuroraA controls MYC-dependent transcription in S-phase and selective inhibition of AuroraA with LY3295668 promotes transcription-replication conflicts leading to DNA damage. In addition to AuroraA-mediated mitotic arrest, this DNA damage may contribute to the hypersensitivity of MYC-driven tumors such as NBL. In summary, LY3295668 is a selective AuroraA inhibitor with a dual mechanism of action in NBL involving DNA damage in S-phase and a cell cycle block in mitosis. These combined activities contribute to sensitivity of preclinical NBL models. Citation Format: Michele Dowless, Jill Kremer, Caitlin Lowery, Xueqian Gong, Joseph Klahn, Jennifer Stephens, Yu-Hua Hui, Wayne Blosser, Ricardo Martinez, Jian Du, Louis Stancato. Selective inhibition of AuroraA by LY3295668 erbumine in neuroblastoma models induces apoptosis through a combination of S-phase DNA damage and mitotic arrest [abstract]. In: Proceedings of the AACR Special Conference on the Advances in Pediatric Cancer Research; 2019 Sep 17-20; Montreal, QC, Canada. Philadelphia (PA): AACR; Cancer Res 2020;80(14 Suppl):Abstract nr A32.

Systems approach to rational combination therapy: PARP inhibitors.

Poly (ADP-ribose) polymerase inhibitors (PARPi) have demonstrated activity across a broad spectrum of molecular backgrounds and tumor types, with the greatest activity observed in patients with aberrations in the homologous recombination DNA damage repair pathway. Despite remarkable responses in a subset of patients, the response is usually modest and transient due to the almost inevitable emergence of resistance. Tumors develop resistance through rapid adaptation to the effects of PARPi as well as by generation or selection of genomic aberration. Although adaptive responses results in drug resistance, it also induces therapeutic vulnerabilities that could be exploited with rational combination therapies. To fulfill this role, we established the combinatorial adaptive response therapy (CART) platform by performing reverse-phase protein arrays to characterize adaptive responses, and develop rational combination therapies. Our series of studies strongly support the efficacy of this strategy, wherein targeting the emerging adaptive responses to PARPi with MEK/ERK inhibitors, WEE1/ATR inhibition (inhibitors of S-phase and G2 DNA damage checkpoint), and PI3K/AKT/mTOR inhibition, and showed promising anti-tumor activity in various preclinical models. Importantly, this approach has been proven highly efficient, and several combinational therapies developed from the CART platform are being evaluated in ongoing clinical trials (NCT03801369, NCT03586661, NCT03162627, NCT03544125, NCT02659241, NCT02208375, NCT02316834, and NCT03637491).

Cordycepin inhibits pancreatic cancer cell growth in vitro and in vivo via targeting FGFR2 and blocking ERK signaling

Cordycepin (3'-deoxyadenosine) from Cordyceps militaris has been reported to have anti-tumor effects. However, the molecular target and mechanism underlying cordycepin impeding pancreatic cancer cell growth in vitro and in vivo remain vague. In this study, we reported functional target molecule of cordycepin which inhibited pancreatic cancer cells growth in vitro and in vivo. Cordycepin was confirmed to induce apoptosis by activating caspase-3, caspase-9 and cytochrome c. Further studies suggested that MAPK pathway was blocked by cordycepin via inhibiting the expression of Ras and the phosphorylation of Erk. Moreover, cordycepin caused S-phase arrest and DNA damage associated with activating Chk2 (checkpoint kinase 2) pathway and downregulating cyclin A2 and CDK2 phosphorylation. Very interestingly, we showed that cordycepin could bind to FGFR2 (KD = 7.77 × 10-9) very potently to inhibit pancreatic cancer cells growth by blocking Ras/ErK pathway. These results suggest that cordycepin could potentially be a leading compound which targeted FGFR2 to inhibit pancreatic cells growth by inducing cell apoptosis and causing cell cycle arrest via blocking FGFR/Ras/ERK signaling for anti-pancreatic cancer new drug development.

G-Quadruplex-Binding Small Molecule Induces Synthetic Lethality in Breast Cancer Cells by Inhibiting c-MYC and BCL2 Expression.

Herein, a prolinamide-derived peptidomimetic that preferentially binds to c-MYC and BCL2 G-quadruplexes present in the promoter regions of apoptosis-related genes (c-MYC and BCL2) over other DNA quadruplexes are described. Biological assays, such as real-time quantitative reverse transcription, western blot, dual luciferase, and small interfering RNA knockdown assays, indicate that the ligand triggers a synthetic lethal interaction by simultaneously inhibiting the expression of c-MYC and BCL2 genes through their promoter G-quadruplexes. The ligand shows antiproliferative activity in MCF-7 cells that overexpress both MYC and BCL2 genes, in comparison to cells that overexpress either of the two. Moreover, the ligand induces S-phase cell-cycle arrest, DNA damage, and apoptosis in MCF-7 cells.

DIBI, a novel 3-hydroxypyridin-4-one chelator iron-binding polymer, inhibits breast cancer cell growth and functions as a chemosensitizer by promoting S-phase DNA damage.

Breast cancer is a leading cause of cancer-related death in women; however, chemotherapy of breast cancer is often hindered by dose-limiting toxicities, demonstrating the need for less toxic approaches to treatment. Since the rapid growth and metabolism of breast cancer cells results in an increased requirement for iron, withdrawal of bioavailable iron using highly selective iron chelators has been suggested to represent a new approach to breast cancer treatment. Here we show that the recently developed iron-binding polymer DIBI inhibited the growth of five different breast cancer cell lines (SK-BR3, MDA-MB-468, MDA-MB-231, MCF-7, and T47D). In cultures of MDA-MB-468 breast cancer cells, which were most sensitive to DIBI-mediated growth inhibition, iron withdrawal was associated with increased expression of transferrin receptor 1 and ferritin H mRNA but decreased expression of ferroportin mRNA. MDA-MB-468 cells that were exposed to DIBI experienced double-strand DNA breaks during the S phase of the cell cycle. DNA damage was not mediated by reactive oxygen species (ROS) since DIBI-treated MDA-MB-468 cells exhibited a reduction in intracellular ROS. DIBI-treated MDA-MB-468 cells also showed increased sensitivity to growth inhibition by the chemotherapeutic drugs cisplatin, doxorubicin, and 4-hydroperoxy cyclophosphamide (active metabolite of cyclophosphamide). Combination treatment of MDA-MB-468 cells with DIBI and cisplatin caused greater DNA damage than either treatment alone, which was also associated with an increase in apoptotic cell death. Taken together, these findings suggest that DIBI-mediated iron withdrawal may enhance the effect of chemotherapeutic agents used in breast cancer treatment.

Abstract 3503: Biomarkers for inhibitors of the replication stress response proteins WEE1 and ATR in triple negative breast cancer

Abstract Replication stress (RS) is a hallmark of cancer and has the potential to be exploited by selective DNA Damage Response inhibitors. During replication stress, the DNA polymerase is uncoupled from the replisome helicase activity resulting in extended regions of single strand DNA that lead to the initiation of a replication stress response (RSR). Two key regulators of the RSR are the ATR and WEE1 kinases. Here, we aimed to identify patient selection biomarkers for WEE1 and ATR inhibitors (WEE1i, ATRi) from a cohort of 37 patient-derived tumor xenografts (PDX) from ovarian or triple negative breast cancer, 20 of which harbored BRCA1/BRCA2deleterious mutations. The antitumor response of the WEE1i AZD1775 and the ATRi AZD6837 was evaluated, and PDX tumors were characterized by exome sequencing, metabolomics, western blot and immunohistochemistry. AZD1775 treatment response in PDXs, measured as tumor regressions (≤-30% change in tumor volume), was observed in 28% of models tested. Exome sequencing identified an association between treatment response and concomitant alterations in RB1 and STK11 (LKB1), whose functional relevance was validated in an in vitro model. Immunoblots showed that WEE1i treatment resulted in downregulation of the ribonucleotide reductase subunit RRM2 and induction of an S-phase DNA damage response (DDR) in vivo. Metabolomics identified a profound change in the purine and pyrimidine synthesis pathways in WEE1i-sensitive PDXs and these changes appear functionally relevant since PDX ex vivo data demonstrate rescue of the WEE1i effects after addition of nucleosides. Another important association with WEE1i sensitivity was increased cyclin E expression. Together, these data suggest that early entry into S phase and an imbalance of origin firing to available dNTPs represent drivers of sensitivity to the WEE1i. Interestingly by contrast, ATRi-sensitive PDXs (representing 10% of the models) were characterized primarily by those tumors harboring ATM alterations, suggesting that synthetic lethality with this major DDR pathway is the primary mechanism of sensitivity to the ATR inhibitor in ovarian and triple negative breast cancer. This study therefore highlights differences between the drivers of sensitivity to inhibitors of these two important RSR proteins. Citation Format: Violeta Serra, Cristina Cruz, Zhongwu Lai, Marta Castroviejo-Bermejo, Marta Palafox, Urszula M. Polanska, Gemma N. Jones, Anderson Wang, Filippos Michopoulos, Rachel Brough, Brian Dougherty, Elaine Cadogan, Susan Critchlow, Alejandra Bruna, J. Carl Barrett, Cristina Saura, Christopher J. Lord, Carlos Caldas, Joaquin Arribas, Judith Balmaña, Mark J. O’Connor. Biomarkers for inhibitors of the replication stress response proteins WEE1 and ATR in triple negative breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3503.

The ATR Inhibitor AZD6738 Synergizes with Gemcitabine In Vitro and In Vivo to Induce Pancreatic Ductal Adenocarcinoma Regression.

Pancreatic ductal adenocarcinoma (PDAC) is among the deadliest cancers, and overall survival rates have barely improved over the past five decades. The antimetabolite gemcitabine remains part of the standard of care but shows very limited antitumor efficacy. Ataxia telangiectasia and Rad3-related protein (ATR), the apical kinase of the intra-S-phase DNA damage response, plays a central role in safeguarding cells from replication stress and can therefore limit the efficacy of antimetabolite drug therapies. We investigated the ability of the ATR inhibitor, AZD6738, to prevent the gemcitabine-induced intra-S-phase checkpoint activation and evaluated the antitumor potential of this combination in vitro and in vivo In PDAC cell lines, AZD6738 inhibited gemcitabine-induced Chk1 activation, prevented cell-cycle arrest, and restrained RRM2 accumulation, leading to the strong induction of replication stress markers only with the combination. Moreover, synergistic growth inhibition was identified in a panel of 5 mouse and 7 human PDAC cell lines using both Bliss Independence and Loewe models. In clonogenic assays, the combination abrogated survival at concentrations for which single agents had minor effects. In vivo, AZD6738 in combination with gemcitabine was well tolerated and induced tumor regression in a subcutaneous allograft model of a KrasG12D; Trp53R172H; Pdx-Cre (KPC) mouse cancer cell line, significantly extending survival. Remarkably, the combination also induced regression of a subgroup of KPC autochthonous tumors, which generally do not respond well to conventional chemotherapy. Altogether, our data suggest that AZD6738 in combination with gemcitabine merits evaluation in a clinical trial in patients with PDAC. Mol Cancer Ther; 17(8); 1670-82. ©2018 AACR.

Integrated genome analysis of uterine leiomyosarcoma to identify novel driver genes and targetable pathways.

Uterine leiomyosarcomas (uLMS) are rare, aggressive malignancies for which limited treatment options are available. To gain novel molecular insights into uLMS and identify potential novel therapeutic targets, we characterized 84 uLMS samples for genome-wide somatic copy number alterations, mutations, gene fusions and gene expression and performed a data integration analysis. We found that alterations affecting TP53, RB1, PTEN, MED12, YWHAE and VIPR2 were present in the majority of uLMS. Pathway analyses additionally revealed that the PI3K/AKT/mTOR, estrogen-mediated S-phase entry and DNA damage response signaling pathways, for which inhibitors have already been developed and approved, frequently harbored genetic changes. Furthermore, a significant proportion of uLMS was characterized by amplifications and overexpression of known oncogenes (CCNE1, TDO2), as well as deletions and reduced expression of tumor suppressor genes (PTEN, PRDM16). Overall, it emerged that the most frequently affected gene in our uLMS samples was VIPR2 (96%). Interestingly, VIPR2 deletion also correlated with unfavorable survival in uLMS patients (multivariate analysis; HR = 4.5, CI = 1.4-14.3, p = 1.2E-02), while VIPR2 protein expression was reduced in uLMS vs. normal myometrium. Moreover, stimulation of VIPR2 with its natural agonist VIP decreased SK-UT-1 uLMS cell proliferation in a dose-dependent manner. These data suggest that VIPR2, which is a negative regulator of smooth muscle cell proliferation, might be a novel tumor suppressor gene in uLMS. Our work further highlights the importance of integrative molecular analyses, through which we were able to uncover the genes and pathways most frequently affected by somatic alterations in uLMS.

Abstract 5110: A novel nucleoside analog therapeutically active against plasma cell malignancies and other ADK-expressing cancers including colon and pancreatic adenocarcinomas

Abstract A number of nucleoside analogues are used successfully for the treatment of several cancers, and in particular leukemias and lymphomas, but these have distinct efficacies for different tumor types, and many malignancies do not respond to currently available nucleoside analogues or other forms of chemotherapy. A high throughput screen conducted in our lab to search for inhibitors of primary effusion lymphoma (PEL) identified the nucleoside analog 6-ethylthioinosine (6-ETI) as a potent and selective inhibitor of PEL, a largely incurable malignancy of B cell origin with plasmacytic differentiation. 6-ETI induced necrosis and ATP-depletion accompanied by S-phase arrest, DNA damage and inhibition of DNA synthesis. To understand 6-ETI mechanism of selectivity, RNA-seq analysis of in vitro generated drug-resistant PEL clones revealed inactivating mutations and loss of expression of adenosine kinase (ADK) as the mechanism of resistance. In vitro assays showed that 6-ETI is a pro-drug that gets phosphorylated and activated by adenosine kinase (ADK) into its active form. We found high ADK expression in PEL cell lines and primary specimens of PEL, multiple myeloma (MM) and plasmablastic lymphoma (PBL) patient samples. 6-ETI was effective at killing multiple myeloma cell lines, primary MM specimens, and had a remarkable anti-tumor response in a disseminated multiple myeloma and PEL xenograft mouse models. Thus, ADK expression can serve as a predictive biomarker to help identify patients that are most likely to respond to 6-ETI treatment. To further assess the spectrum of activity and sensitivity of 6-ETI, we examined ADK expression in other cancer subtypes and found that colorectal and pancreatic adenocarcinomas also overexpress ADK and are highly sensitive to killing by 6-ETI at the low nanomolar concentration. We also found high ADK expression in primary colon and pancreatic adenocarcinoma patient specimens. We compared 6-ETI to other FDA-approved purine analogs and failed to find other compounds with similar potency or selectivity profile. Herein, we report the identification of a novel purine analog, 6-ethylthioinosine, as an effective therapeutic with exquisite sensitivity to plasma cell malignancies and other ADK-expressing cancers. We have successfully used RNASeq-based “resistome” analysis to identify its mechanism of specificity and discovered a new biomarker that can potentially impact patient care and the treatment of some of the most aggressive tumors. Citation Format: Jouliana Sadek, Utthara Nayar, Jonathan Reichel, Jennifer Totonchy, Shizuko Sei, Robert Shoemaker, David Warren, Olivier Elemento, Kenneth Kaye, Ethel Cesarman. A novel nucleoside analog therapeutically active against plasma cell malignancies and other ADK-expressing cancers including colon and pancreatic adenocarcinomas [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5110. doi:10.1158/1538-7445.AM2017-5110

Combined inhibition of Wee1 and Chk1 gives synergistic DNA damage in S-phase due to distinct regulation of CDK activity and CDC45 loading.

Recent studies have shown synergistic cytotoxic effects of simultaneous Chk1- and Wee1-inhibition. However, the mechanisms behind this synergy are not known. Here, we present a flow cytometry-based screen for compounds that cause increased DNA damage in S-phase when combined with the Wee1-inhibitor MK1775. Strikingly, the Chk1-inhibitors AZD7762 and LY2603618 were among the top candidate hits of 1664 tested compounds, suggesting that the synergistic cytotoxic effects are due to increased S-phase DNA damage. Combined Wee1- and Chk1-inhibition caused a strong synergy in induction of S-phase DNA damage and reduction of clonogenic survival. To address the underlying mechanisms, we developed a novel assay measuring CDK-dependent phosphorylations in single S-phase cells. Surprisingly, while Wee1-inhibition alone induced less DNA damage compared to Chk1-inhibition, Wee1-inhibition caused a bigger increase in S-phase CDK-activity. However, the loading of replication initiation factor CDC45 was more increased after Chk1- than Wee1-inhibition and further increased by the combined treatment, and thus correlated well with DNA damage. Therefore, when Wee1 alone is inhibited, Chk1 suppresses CDC45 loading and thereby limits the extent of unscheduled replication initiation and subsequent S-phase DNA damage, despite very high CDK-activity. These results can explain why combined treatment with Wee1- and Chk1-inhibitors gives synergistic anti-cancer effects.

Inhibition of Chk1 with the small molecule inhibitor V158411 induces DNA damage and cell death in an unperturbed S-phase.

Chk1 kinase is a critical component of the DNA damage response checkpoint and Chk1 inhibitors are currently under clinical investigation. Chk1 suppresses oncogene-induced replication stress with Chk1 inhibitors demonstrating activity as a monotherapy in numerous cancer types. Understanding the mechanism by which Chk1 inhibitors induce DNA damage and cancer cell death is essential for their future clinical development. Here we characterize the mechanism by which the novel Chk1 inhibitor (V158411) increased DNA damage and cell death in models of human cancer. V158411 induced a time- and concentration-dependent increase in γH2AX-positive nuclei that was restricted to cells actively undergoing DNA synthesis. γH2AX induction was an early event and correlated with activation of the ATR/ATM/DNA-PKcs DNA damage response pathways. The appearance of γH2AX positive nuclei preceded ssDNA appearance and RPA exhaustion. Complete and sustained inhibition of Chk1 kinase was necessary to activate a robust γH2AX induction and growth inhibition. Chk1 inhibitor cytotoxicity correlated with induction of DNA damage with cells undergoing apoptosis, mitotic slippage and DNA damage-induced permanent cell cycle arrest. We identified two distinct classes of Chk1 inhibitors: those that induced a strong increase in γH2AX, pChk1 (S317) and pRPA32 (S4/S8) (including V158411, LY2603618 and ARRY-1A) and those that did not (including MK-8776 and GNE-900). Tumor cell death, induced through increased DNA damage, coupled with abrogation of cell cycle checkpoints makes selective inhibitors of Chk1 a potentially useful therapeutic treatment for multiple human cancers.