Abrogation Of Cell Cycle Checkpoints Research Articles

ATR, CHK1 and WEE1 inhibitors cause homologous recombination repair deficiency to induce synthetic lethality with PARP inhibitors.

PARP inhibitors (PARPi) are effective in homologous recombination repair (HRR) defective (HRD) cancers. To (re)sensitise HRR proficient (HRP) tumours to PARPi combinations with other drugs are being explored. Our aim was to determine the mechanism underpinning the sensitisation to PARPi by inhibitors of cell cycle checkpoint kinases ATR, CHK1 and WEE1. A panel of HRD and HRP cells (including matched BRCA1 or 2 mutant and corrected pairs) and ovarian cancer ascites cells were used. Rucaparib (PARPi) induced replication stress (RS) and HRR (immunofluorescence microscopy for γH2AX and RAD51 foci, respectively), cell cycle changes (flow cytometry), activation of ATR, CHK1 and WEE1 (Western Blot for pCHK1S345, pCHK1S296 and pCDK1Y15, respectively) and cytotoxicity (colony formation assay) was determined, followed by investigations of the impact on all of these parameters by inhibitors of ATR (VE-821, 1 µM), CHK1 (PF-477736, 50 nM) and WEE1 (MK-1775, 100 nM). Rucaparib induced RS (3 to10-fold), S-phase accumulation (2-fold) and ATR, CHK1 and WEE1 activation (up to 3-fold), and VE-821, PF-477736 and MK-1775 inhibited their targets and abrogated these rucaparib-induced cell cycle changes in HRP and HRD cells. Rucaparib activated HRR in HRP cells only and was (60-1,000x) more cytotoxic to HRD cells. VE-821, PF-477736 and MK-1775 blocked HRR and sensitised HRP but not HRD cells and primary ovarian ascites to rucaparib. Our data indicate that, rather than acting via abrogation of cell cycle checkpoints, ATR, CHK1 and WEE1 inhibitors cause an HRD phenotype and hence "induced synthetic lethality" with PARPi.

RTID-03. A PHASE 1/2 STUDY OF THE WEE1 INHIBITOR DEBIO 0123 IN COMBINATION WITH TEMOZOLOMIDE +- RADIOTHERAPY IN ADULTS WITH RECURRENT OR NEWLY DIAGNOSED GLIOBLASTOMA

Abstract BACKGROUND: Debio 0123 is an oral, brain-penetrant, highly selective WEE1 inhibitor. WEE1 inhibition leads to S phase and G2/M cell cycle checkpoint abrogation, permitting mitosis without DNA repair, leading to mitotic catastrophe and subsequent cell death. Preclinically, Debio 0123 has been shown to effectively penetrate the brain and demonstrated improved response to radiotherapy (RT) in vitro and temozolomide (TMZ) in mouse models of GBM, supporting the clinical investigation of Debio 0123 with TMZ +/- RT. Trial design: This Phase 1/2 multi-center study (NCT05765812) is evaluating Debio 0123 in combination with TMZ in patients (pts) with recurrent GBM isocitrate dehydrogenase (IDH)-wildtype (WT) or astrocytoma, IDH-mutant, Grade 3 (per WHO 2021 criteria) after first-line, TMZ-based chemoradiotherapy (CxRT) (Phase 1 Arm A) and Debio 0123 combined with TMZ-based CxRT in pts with newly diagnosed GBM IDH-WT (Phase 1 Arm B). In Arm A, pts will receive escalating doses of Debio 0123 with TMZ in 28-day cycles supported by a Bayesian logistic regression model (BLRM). Arm B is a Debio 0123 dose escalation with concomitant TMZ/RT for up to 6 weeks. The primary objective of both Phase 1 arms is to identify the recommended Phase 2 dose (RP2D). Secondary objectives include safety, pharmacokinetics, and preliminary antitumor activity. Key inclusion criteria are ≤2 prior treatment lines (only 1 systemic line allowed) and Karnofsky Performance Status (KPS) ≥60 (Phase 1 Arm A). In Phase 1 Arm B, pts with KPS ≥70 who are eligible for standard TMZ-based CxRT will be included. Phase 2 will be an externally controlled, open-label study comparing the efficacy (overall survival) of Debio 0123 combined with TMZ at the Phase 1 Arm A RP2D vs. standard of care in pts with recurrent GBM IDH-WT. The control arm will include patient-level data from recently completed clinical trials. Accrual is currently ongoing.

CSIG-15. DEFINING THE ROLE OF HISTONE H3 K27 METHYLATION AND H3.3 S31 PHOSPHORYLATION IN H3.3K27M MUTANT DIFFUSE MIDLINE GLIOMAS

Abstract Diffuse midline gliomas (DMG) are aggressive high-grade gliomas that characteristically occurs in children. Heterozygous K27M mutation occurs in histone H3.3 as early events in DMG and are sufficient to drive a global reduction of H3K27 methylation (H3K27me) on all histone H3 variants. Histone H3K27me is responsible for epigenetic gene repression, and loss of K27me is associated with epigenetic dysregulation. However, histone H3.3 has a unique Chk1 phosphorylation site, Serine 31, critical for mitotic checkpoint regulation. Masking of S31 phosphorylation results in the abrogation of cell cycle checkpoints that monitor the alignment and segregation of chromosomes. DMG cells have decreased levels of mitotic S31P and are chromosomally unstable and aneuploid. Genomic-editing of H3.3 to revert the K27M mutation restores mitotic S31P and K27me and significantly reduces the rate of chromosome instability (CIN). Expression of H3.3 K27M or a non-phosphorylatable S31A mutant in normal, diploid cells results in chromosome missegregation and cell cycle checkpoint defects; and cells fail to undergo G1 cycle arrest leading to aneuploidy. H3.3 K27M and S31A inhibit p53 accumulation. In a mouse model of DMG, H3.3K27M and H3.3S31A promoted the development of high-grade gliomas, whereas H3.3WT controls did not. H3.3S31A is WT for K27me, demonstrating that loss of S31P is oncogenic. It remains unclear if it is the H3.3 K27M mutations or the associated loss of H3K27me3 that drives the reduction of S31P. The PRC2 complex is responsible for H3K27me, catalyzed by methyltransferases subunits EZH1 and EZH2. We used genome editing to knockout EZH1 and EZH2 in DMG cells and untransformed human diploid cell lines. Deletion of EZH2 causes a loss in K27 tri-methylation, and EZH1/2 knockouts causes a global loss of K27me. Ongoing studies examine how the loss of K27me affects gene expression, proliferation, H3.3S31P, chromosome missegregation, and aneuploid.

P-47 Inhibition of phosphoprotein phosphatase 2A (PP2A) sensitizes pancreatic cancer to PARP inhibitors by modulation of homologous recombination repair

Pancreatic cancer remains one of the most difficult to treat cancers with a 5-years survival of less than 6%, representing one of the most lethal types of cancer, recent research has shown that PP2A inhibitors (LB-100) sensitize pancreatic cancer to chemotherapy and radiation. In addition, the finding from clinical trial study demonstrated that the effectiveness of single-agent Talazoparib for treatment of patients with and without germline BRCA1/2 mutation in ovarian, breast, small cell lung and Pancreatic cancers. The aim of this work was to Ι. investigate activity of LB-100 against pancreatic cancer cells as monotherapy and in combination with PARP inhibitions (Talazoparib). ΙΙ. Investigate the mechanism by which LB-100 and Talazoparib effect pancreatic cancer cells by studying effect of drugs on cell cycle and modulation of cell cycle regulatory proteins and assess the role of treatment combination in control DSB and HR repair through modulation of ATM phosphorylation. Human pancreatic cancer cell lines Panc-1, MIA-Pa-Ca-2 and BxPC-3 were obtained from European Collection of Authenticated Cell Culture (ECACC) and grown in either Dulbecco’s modified Eagle medium (DMEM) (MiaPaCa-2 and Panc-1) or RPMI medium (BxPC-3) with 10% FBS. Cell cultures were maintained in an atmosphere of 5% CO2/95% air at 37 C. PP2A activity was measured by PP2A Immunoprecipitation phosphatase assay Kit (Millipore), cell cytotoxicity were measured by MTT assay, cell cycle determined by flow cytometry. Western blot techniques were used to assess levels of proteins associated with regulation of cell cycle (Cdc2, p-Cdc2, and Cdc25c), apoptosis (caspase3) and DNA damage (γ-H2AX). Data are expressed as the mean ± (SEM) and analysed by an analysis of variance (ANOVA). The results show that LB-100 decreased PP2A activity in all pancreatic cancer cells in dose dependent manner. LB-100 significantly decreased cell viability of Panc-1, MIA-Pa-Ca-2 and BxPC-3 with IC50 (3.94 μM, 6.86 μM, and 10.87 μM) respectively. Interestingly adding 25 nm of Talazoparib further decreased IC50 in all cells (1.88 μM, 5.24 μM and 5.83 μM). Treatment combination attenuated pancreatic cancer cells growth through caspase activation and G2/M cell-cycle arrest. Combination therapy impaired cellular repair and induced DNA double-strand breaks by inducing Υ-H2AX. Our results suggest that treatment combination of LB100 and talazoparib in vitro inhibit cancer cells growth by modulation of the DNA damage response pathway and cell cycle checkpoint abrogation. The combination of PP2A inhibitor with PARP inhibitor has a synergistic effect in vitro. Further in vivo studies are needed to explore this combination as an effective option in the treatment of pancreatic cancer.

Dynamically Accumulating Homologous Recombination Deficiency Score Served as an Important Prognosis Factor in High-Grade Serous Ovarian Cancer.

Background: The homologous recombination (HR) pathway defects in cancers induced abrogation of cell cycle checkpoints, resulting in the accumulation of DNA damage, mitotic catastrophe, and cell death. Cancers with BRCA1/2 loss and other accumulation of similar genomic scars resulting in HRD displayed increased sensitivity to chemotherapy. Our study aimed to explore HRD score genetic mechanisms and subsequent clinical outcomes in human cancers, especially ovarian cancer. Methods: We analyzed TCGA data of HRD score in 33 cancer types and evaluated HRD score distribution and difference among tumor stages and between primary and recurrent tumor tissues. A weighted gene co-expression network analysis (WGCNA) was performed to identify highly correlated genes representing essential modules contributing to the HRD score and distinguish the hub genes and significant pathways. We verified HRD status predicting roles in patients’ overall survival (OS) with univariate and multivariate Cox regression analyses and built the predicting model for patient survival. Results: We found that the HRD score increased with the rise in tumor stage, except for stage IV. The HRD score tended to grow up higher in recurrent tumor tissue than in their primary counterparts (p = 0.083). We constructed 15 co-expression modules with WGCNA, identified co-expressed genes and pathways impacting the HRD score, and concluded that the HRD score was tightly associated with tumor cells replication and proliferation. A combined HRD score ≥42 was associated with shorter OS in 33 cancer types (HR = 1.010, 95% CI: 1.008–1.011, p < 0.001). However, in ovarian cancer, which ranked the highest HRD score among other cancers, HRD ≥42 cohort was significantly associated with longer OS (HR = 0.99, 95% CI: 0.98–0.99, p < 0.0001). We also built a predicting model for 3 and 5 years survival in HGSC patients. Conclusion: A quantitative HRD score representing the accumulated genomic scars was dynamically increasing in proliferating tumor cells since the HRD score was tightly correlated to tumor cell division and replication. We highlighted HRD score biomarker role in prognosis prediction of ovarian cancer.

Targeted inhibition of the WEE1 kinase as a novel therapeutic strategy in neuroendocrine neoplasms

Neuroendocrine neoplasms (NENs) represent a rare and heterogeneous group of malignancies, sharing features of both neural and endocrine cells. NENs G3 appear as a highly aggressive subset with a poor prognosis and limited therapeutic options. The small-molecule inhibitor of the WEE1 tyrosine kinase, adavosertib (AZD1775), has previously demonstrated potent anti-tumor effects on various types of cancer in preclinical and clinical studies. However, the role of adavosertib in NENs G3 had remained elusive. We evaluated the effects of adavosertib on pancreatic (BON-1, QGP-1) and bronchopulmonary (NCI-H720) neuroendocrine tumor cell lines applying 2D and 3D spheroid models. We newly demonstrated that adavosertib is sufficient to reduce cell viability and proliferation in neuroendocrine cell lines with features of high-grade NENs. As underlying mechanisms, we identified adavosertib-mediated DNA double-strand breaks and a G2/M cell cycle checkpoint abrogation leading into mitotic catastrophe and cancer cell apoptosis. Silencing of WEE1 via siRNA transfection resulted in a phenotype similar to adavosertib treatment. Together, inhibition of the WEE1 tyrosine kinase applying adavosertib on NENs G3 outlines a promising novel therapeutic strategy.

Targeting DNA damage response in head and neck cancers through abrogation of cell cycle checkpoints

Purpose Head and neck cancers (HNSCC) are routinely treated with radiotherapy; however, normal tissue toxicity remains a concern. Therefore, it is important to validate treatment modalities combining molecularly targeted agents with radiotherapy to improve the therapeutic ratio. The aim of this study was to assess the ability of the PARP inhibitor niraparib (MK-4827) alone, or in combination with cell cycle checkpoint abrogating drugs targeting Chk1 (MK-8776) or Wee1 (MK-1775), to radiosensitize HNSCCs in the context of HPV status. Materials and methods PARP1, PARP2, Chk1 or Wee1 shRNA constructs were analyzed from an in vivo shRNA screen of HNSCC xenografts comparing radiosensitization differences between HPV(+) and HPV(−) tumors. Radiosensitization by niraparib alone or in combination with MK-8776 or MK-1775 was assessed by clonogenic survival in HPV(−) and HPV(+) cells; and the role of p16 in determining response was explored. Relative expressions of DNA repair genes were compared by PCR array in HPV(+) and HPV(−) cells, and following siRNA-mediated knockdown of TRIP12 in HPV(−) cells. Results In vivo shRNA screening showed a modest preferential radiosensitization by Wee1 and PARP2 in HPV(−) and Chk1 in HPV(+) tumor models. Niraparib alone enhanced the radiosensitivity of all HNSCC cell lines tested. However, HPV(−) cells were sensitized to a greater degree, as suggested by the shRNA screen. When combined with MK-8776 or MK-1775, radiosensitization was further enhanced in an HPV dependent manner with HPV(+) cells enhanced by MK-8776 and HPV(−) cells enhanced by MK-1775. A PCR array for DNA repair genes showed PARP and HR proteins BRCA1 and RAD51 were much lower in HPV(+) cells than in HPV(−). Similarly, directly knocking down p16-dependent TRIP12 decreased expression of these same genes. Overexpressing p16 decreased TRIP12 expression and increased radiosensitivity in HPV(−) HN5. However, while PARP inhibition led to significant radiosensitization in the control, it led to no further significant radiosensitization in p16 overexpressing cells. Forced p16 expression in HPV(−) HN5 increased accumulation in G1 and subG1 and limited progression to S phase, thus reducing effectiveness of PARP inhibition. Conclusions Niraparib effectively radiosensitizes HNSCCs with a greater benefit seen in HPV(−). HPV status also plays a role in response to MK-8776 or MK-1775 when combined with niraparib due to differences in DNA repair mechanisms. This study suggests that using cell cycle abrogators in combination with PARP inhibitors may be a beneficial treatment option in HNSCC, but also emphasizes the importance of HPV status when considering effective treatment strategies.

Abstract 2355: Palbociclib enhances the antitumor activity of taxanes by abrogating cell cycle checkpoints and alleviating hypoxia in squamous cell lung cancer

Abstract Lung cancer remains one of the leading causes of cancer-related mortality. Squamous cell lung cancer (SqCLC) is the second most common subtype of non-small cell lung cancer (NSCLC) and is responsible for ~100,000 deaths in the US and EU. Most SqCLC patients receive chemotherapy as 1st line treatments and have a high un-met medical need for new therapies. Therapeutic approaches that enhance the efficacy of chemotherapy would therefore improve clinical outcomes for this patient population. CDK inhibitors comprise a class of drugs that target the dysregulated cell cycle in malignant cells. Treatment of tumor cells with the CDK4/6 inhibitor palbociclib inhibits tumor growth by decreasing retinoblastoma (RB) protein phosphorylation and inducing cell cycle arrest at the G1/S phase transition. Based on promising clinical trial results, palbociclib in combination with letrozole was granted accelerated approval by the US FDA for the treatment of postmenopausal women with ER-positive, HER2-negative advanced breast cancer. Like hormone receptor positive breast cancer patients, the vast majority of SqCLC patients harbor wild type RB and thus may also benefit from palbociclib treatment. Previously, we reported robust cytotoxicity and antitumor effects of palbociclib plus taxanes, including nanoparticle albumin-bound paclitaxel (Nab-PTX) or docetaxel (DTX), in several preclinical models of SqCLC. In the present study, we extended our efficacy studies of this combination to additional RB+ SqCLC models with diverse molecular genetic backgrounds. In search of mechanisms of action underlying the observed combinatorial effects, we identified several novel mechanisms, including cell cycle checkpoint abrogation as well as reduction of hypoxia-inducible factor 1 alpha (HIF-1α). Decrease in HIF-1α protein led to strong modulation of downstream genes involved in angiogenesis, resulting in reduced blood vessel size in tumor vasculature. Our results suggest that palbociclib enhances the antitumor activity of taxanes by abrogating cell cycle checkpoints and alleviating hypoxia in SqCLC. Citation Format: Joan Cao, Zhou Zhu, Hui Wang, Tim Nichols, Edward Rosfjord, Christine Hopf, Erik Upeslacis, Paul Rejto, Scott Weinrich, Todd Vanarsdale, James Hardwick, Ping Wei. Palbociclib enhances the antitumor activity of taxanes by abrogating cell cycle checkpoints and alleviating hypoxia in squamous cell lung cancer [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 2355. doi:10.1158/1538-7445.AM2017-2355

HSP90 inhibition sensitizes head and neck cancer to platin-based chemoradiotherapy by modulation of the DNA damage response resulting in chromosomal fragmentation

BackgroundConcurrent cisplatin radiotherapy (CCRT) is a current standard-of-care for locally advanced head and neck squamous cell carcinoma (HNSCC). However, CCRT is frequently ineffective in patients with advanced disease. It has previously been shown that HSP90 inhibitors act as radiosensitizers, but these studies have not focused on CCRT in HNSCC. Here, we evaluated the HSP90 inhibitor, AUY922, combined with CCRT.MethodsThe ability of AUY922 to sensitize to CCRT was assessed in p53 mutant head and neck cell lines by clonogenic assay. Modulation of the CCRT induced DNA damage response (DDR) by AUY922 was characterized by confocal image analysis of RAD51, BRCA1, 53BP1, ATM and mutant p53 signaling. The role of FANCA depletion by AUY922 was examined using shRNA. Cell cycle checkpoint abrogation and chromosomal fragmentation was assessed by western blot, FACS and confocal. The role of ATM was also assessed by shRNA. AUY922 in combination with CCRT was assessed in vivo.ResultsThe combination of AUY922 with cisplatin, radiation and CCRT was found to be synergistic in p53 mutant HNSCC. AUY922 leads to significant alterations to the DDR induced by CCRT. This comprises inhibition of homologous recombination through decreased RAD51 and pS1524 BRCA1 with a corresponding increase in 53BP1 foci, activation of ATM and signaling into mutant p53. A shift to more error prone repair combined with a loss of checkpoint function leads to fragmentation of chromosomal material. The degree of disruption to DDR signalling correlated to chromosomal fragmentation and loss of clonogenicity. ATM shRNA indicated a possible rationale for the combination of AUY922 and CCRT in cells lacking ATM function.ConclusionsThis study supports future clinical studies combining AUY922 and CCRT in p53 mutant HNSCC. Modulation of the DDR and chromosomal fragmentation are likely to be analytical points of interest in such trials.

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.

Prolongation of the remission period and reversal of new onset type 1 diabetes with the selective elimination of autoreactive T cells

Abstract Organ-specific autoimmune diseases such as type 1 diabetes (T1D) are principally mediated by autoreactive T lymphocytes targeting self-antigens. The rapid proliferation and concomitant genomic stress that these T cells undergo during their active immune response to self-antigens affords a unique opportunity for their elimination, since these autoreactive T cells have distinct apoptotic sensitivity as a result of intrinsic DNA damage response upon activation. This sensitivity can be harnessed with a combination of p53 potentiation and cell-cycle checkpoint abrogation (PPCA), selectively eliminating these pathogenic cells while leaving naïve and regulatory T cell populations largely untouched. Using this therapy at the onset of T1D, we are able to significantly reverse new onset T1D (≥200 mg/dL blood glucose) and prolong the remission period in the Nonobese Diabetes (NOD) mouse, representing a novel therapeutic approach with significant translational potential for not only type 1 diabetes, but also other T cell-mediated autoimmune diseases.

The protein phosphatase 2A inhibitor LB100 sensitizes ovarian carcinoma cells to cisplatin-mediated cytotoxicity.

Despite early positive response to platinum-based chemotherapy, the majority of ovarian carcinomas develop resistance and progress to fatal disease. Protein phosphatase 2A (PP2A) is a ubiquitous phosphatase involved in the regulation of DNA-damage response (DDR) and cell-cycle checkpoint pathways. Recent studies have shown that LB100, a small-molecule inhibitor of PP2A, sensitizes cancer cells to radiation-mediated DNA damage. We hypothesized that LB100 could sensitize ovarian cancer cells to cisplatin treatment. We performed in vitro studies in SKOV-3, OVCAR-8, and PEO1, -4, and -6 ovarian cancer lines to assess cytotoxicity potentiation, cell-death mechanism(s), cell-cycle regulation, and DDR signaling. In vivo studies were conducted in an intraperitoneal metastatic mouse model using SKOV-3/f-Luc cells. LB100 sensitized ovarian carcinoma lines to cisplatin-mediated cell death. Sensitization via LB100 was mediated by abrogation of cell-cycle arrest induced by cisplatin. Loss of the cisplatin-induced checkpoint correlated with decreased Wee1 expression, increased cdc2 activation, and increased mitotic entry (p-histone H3). LB100 also induced constitutive hyperphosphorylation of DDR proteins (BRCA1, Chk2, and γH2AX), altered the chronology and persistence of JNK activation, and modulated the expression of 14-3-3 binding sites. In vivo, cisplatin sensitization via LB100 significantly enhanced tumor growth inhibition and prevented disease progression after treatment cessation. Our results suggest that LB100 sensitizes ovarian cancer cells to cisplatin in vitro and in vivo by modulation of the DDR pathway and cell-cycle checkpoint abrogation.

Alisertib added to rituximab and vincristine is synthetic lethal and potentially curative in mice with aggressive DLBCL co-overexpressing MYC and BCL2.

Pearson correlation coefficient for expression analysis of the Lymphoma/Leukemia Molecular Profiling Project (LLMPP) demonstrated Aurora A and B are highly correlated with MYC in DLBCL and mantle cell lymphoma (MCL), while both Auroras correlate with BCL2 only in DLBCL. Auroras are up-regulated by MYC dysregulation with associated aneuploidy and resistance to microtubule targeted agents such as vincristine. Myc and Bcl2 are differentially expressed in U-2932, TMD-8, OCI-Ly10 and Granta-519, but only U-2932 cells over-express mutated p53. Alisertib [MLN8237 or M], a highly selective small molecule inhibitor of Aurora A kinase, was synergistic with vincristine [VCR] and rituximab [R] for inhibition of cell proliferation, abrogation of cell cycle checkpoints and enhanced apoptosis versus single agent or doublet therapy. A DLBCL (U-2932) mouse model showed tumor growth inhibition (TGI) of ∼10–20% (p = 0.001) for M, VCR and M-VCR respectively, while R alone showed ∼50% TGI (p = 0.001). M-R and VCR-R led to tumor regression [TR], but relapsed 10 days after discontinuing therapy. In contrast, M-VCR-R demonstrated TR with no relapse >40 days after stopping therapy with a Kaplan-Meier survival of 100%. Genes that are modulated by M-VCR-R (CENP-C, Auroras) play a role in centromere-kinetochore function in an attempt to maintain mitosis in the presence of synthetic lethality. Together, our data suggest that the interaction between alisertib plus VCR plus rituximab is synergistic and synthetic lethal in Myc and Bcl-2 co-expressing DLBCL. Alisertib plus vincristine plus rituximab [M-VCR-R] may represent a new strategy for DLBCL therapy.

Molecular Pathways: Targeting PARP in Cancer Treatment

Poly (ADP-ribose) polymerases (PARP) are a family of nuclear protein enzymes involved in the DNA damage response. The role of PARP-1 in base excisional repair has been extensively characterized. More recent in vitro studies additionally implicate a role for PARP-1 in facilitating homologous recombination and nonhomologous end-joining. The more faithful process of homologous recombination repair of double-stranded DNA breaks involves localization of BRCA-1 and BRCA-2 to sites of DNA damage, resection of the double-stranded break, and gap-filling DNA synthesis using the homologous sister chromatid as a template. Simultaneous dysfunction of both DNA repair pathways decreases the ability of cells to compensate, and forms the basis for the concept of synthetic lethality. Treatment strategies, thus far, have focused on two main principles: (i) exploitation of the concept of synthetic lethality in homologous recombination-deficient tumors, primarily in breast and ovarian cancer patients with BRCA mutation, and (ii) as radiosensitizers and chemosensitizers. BRCA deficiency accounts for only a fraction of dysfunction in homologous recombination repair. Epigenetic alterations of BRCA function and defects within the Fanconi anemia pathway also result in defective DNA repair. Rational therapeutic combinations exploiting alternate mechanisms of defective DNA repair, abrogation of cell-cycle checkpoints, and additional functions of PARP-1 present novel opportunities for further clinical development of PARP inhibitors. On the basis of the results of clinical studies of PARP inhibitors thus far, it is imperative that future development of PARP inhibitors take a more refined approach, identifying the unique subset of patients that would most benefit from these agents, determining the optimal time for use, and identifying the optimal combination partner in any particular setting.

Cyclin Dependent Kinase as Significant Target for Cancer Treatment

Cyclin Dependent Kinase (CDKs) regulates cell cycle commitment and DNA synthesis. Cell division in mammalian cells is driven by protein kinase that regulates progression through the various phases of cell cycle. The activity of cyclins and their associated CDKs are frequently deranged in human cancers. For this reason, Cyclin-CDK complexes have been considered as very promising therapeutic targets in human malignancies. An obvious concern whether, blocking cyclin-CDK function would preferentially affects cancer cells but not normal and non-transformed cells. The cell cycle represents a series of tightly integrated events that allow the cell to grow and proliferate. Critical part of the cell cycle machinery is the CDK, which, when activated, provide a means for the cell to move from one phase of the cell cycle to next. The cell cycle also serves to protect the cell from DNA damage. Thus, cell cycle arrest represents a survival mechanism that provides tumour cell, to repair its own damaged DNA Thus, abrogation of cell cycle checkpoints, before DNA repair is complete can activate the apoptotic cascade leading to cell death. Misregulation of CDK is one of the most frequent alterations in human cancer. CDK are critical regulators of cell cycle progression and RNA transcription. A series of targeted agents that directly inhibit the CDKs, inhibit unrestricted cell growth, and induce growth arrest. Recent attention has also focused on these drugs as inhibitors of transcription. In this review we are summarizing that why CDK is important target for cancer chemotherapy and why finding out the best and potent kinase inhibitor is essential. Keywords: Cancer, CAK, Cell cycle, CDK, Cyclin, Kinases, Phosporylation, Threonine

Akt-dependent Skp2 mRNA translation is required for exiting contact inhibition, oncogenesis, and adipogenesis

The requirement of Akt for cell proliferation and oncogenesis is mammalian target of rapamycin complex 1 (mTORC1) dependent. SV40 large T expression in Akt-deficient cells restores cell proliferation rate, but is insufficient for exiting contact inhibition and oncogene-induced anchorage-independent growth, because of a failure to promote Skp2 mRNA translation. Skp2 mRNA and protein are induced upon exiting contact inhibition, which enables entry into mitosis. While Skp2 mRNA is induced in Akt-deficient cells, it is not translated, preventing entry into mitosis. Restoring Skp2 expression in Akt-deficient cells is sufficient to restore exit from contact inhibition and oncogenesis. Skp2 mRNA translation is dependent on mTORC1 and the eukaryotic translation initiation factor 4E (eIF4E). Thus, the requirement of Akt for exiting contact inhibition is mediated by the induction of Skp2 mRNA translation in eIF4E-dependent mechanism. These results provide a new insight into the role of the Akt/mTORC1/eIF4E axis in tumourigenesis. Akt-dependent Skp2 mRNA translation is also required for mitotic clonal expansion (MCE)--the earliest event in adipogenesis. Skp2 re-expression in Akt-deficient preadipocytes, which are impaired in adipogenesis, is sufficient to restore adipogenesis. These results uncover the mechanism by which Akt mediates adipogenesis.

Abstract C204: Potentiation of the novel topoisomerase I inhibitor indenoisoquinoline LMP-400 by the Chk1/2 inhibitor, AZD7762.

Abstract Novel topoisomerase I (Top1) inhibitors are in clinical development to circumvent the drawbacks of camptothecins (1–3). Here we report molecular investigations of the clinical indenoisoquinoline Top1 inhibitor, LMP-400 by itself and in combination with the cell cycle checkpoint inhibitor, AZD7762 (4). Drug effects on DNA replication and killing of cancer cells were examined. LMP-400 shows synergistic antiproliferative activity when combined with AZD7762 in human colon carcinoma HT-29 and HCT-15 cells. Inhibition of S-phase progression and bromodeoxyuridine incorporation was similarly induced by LMP-400 and CPT and was abrogated by AZD7762. Replication studied by single DNA molecule analyses and immunofluorescence microscopy (molecular combing) (5) showed rapid inhibition of fork progression in response to LMP-400 treatment with subsequent recapitulation after AZD7762 addition. Consistent with cell cycle checkpoint abrogation, AZD7762 inhibited both the activation of Chk1 and Chk2 autophosphorylations in LMP-400-treated cells. In spite of the potent dual inhibition of Chk1 and Chk2 at nanomolar concentrations of AZD7762, cell cycle inhibition and synergism with LMP-400 were only observed at higher concentrations and independently of Chk2 both in Chk2-complemented HT-15 and Chk2 knockout HCT-116 cells. Our study demonstrates the rationale for combining checkpoint kinase inhibitors and the novel non-camptothecin indenoisoquinoline inhibitors.

Oncogenic YAP promotes radioresistance and genomic instability in medulloblastoma through IGF2-mediated Akt activation

Radiation therapy remains the standard of care for many cancers, including the malignant pediatric brain tumor medulloblastoma. Radiation leads to long-term side effects, while radio-resistance contributes to tumor recurrence. Radio-resistant medulloblastoma cells occupy the peri-vascular niche. They express Yes-associated protein (YAP), a Sonic hedgehog (Shh) target markedly elevated in Shh-driven medulloblastomas. Here we report that YAP accelerates tumor growth and confers radio-resistance, promoting ongoing proliferation after radiation. YAP activity enables cells to enter mitosis with un-repaired DNA through driving IGF2 expression and Akt activation, resulting in ATM/Chk2 inactivation and abrogation of cell cycle checkpoints. Our results establish a central role for YAP in counteracting radiation-based therapies and driving genomic instability, and indicate the YAP/IGF2/Akt axis as a therapeutic target in medulloblastoma.

Context-Dependent Cell Cycle Checkpoint Abrogation by a Novel Kinase Inhibitor

BackgroundCheckpoint kinase 1 and 2 (Chk1/Chk2), and the Aurora kinases play a critical role in the activation of the DNA damage response and mitotic spindle checkpoints. We have identified a novel inhibitor of these kinases and utilized this molecule to probe the functional interplay between these two checkpoints.Principal FindingsFragment screening, structure guided design, and kinase cross screening resulted in the identification of a novel, potent small molecule kinase inhibitor (VER-150548) of Chk1 and Chk2 kinases with IC50s of 35 and 34 nM as well as the Aurora A and Aurora B kinases with IC50s of 101 and 38 nM. The structural rationale for this kinase specificity could be clearly elucidated through the X-ray crystal structure. In human carcinoma cells, VER-150548 induced reduplication and the accumulation of cells with >4N DNA content, inhibited histone H3 phosphorylation and ultimately gave way to cell death after 120 hour exposure; a phenotype consistent with cellular Aurora inhibition. In the presence of DNA damage induced by cytotoxic chemotherapeutic drugs, VER-150548 abrogated DNA damage induced cell cycle checkpoints. Abrogation of these checkpoints correlated with increased DNA damage and rapid cell death in p53 defective HT29 cells. In the presence of DNA damage, reduplication could not be observed. These observations are consistent with the Chk1 and Chk2 inhibitory activity of this molecule.ConclusionsIn the presence of DNA damage, we suggest that VER-150548 abrogates the DNA damage induced checkpoints forcing cells to undergo a lethal mitosis. The timing of this premature cell death induced by Chk1 inhibition negates Aurora inhibition thereby preventing re-entry into the cell cycle and subsequent DNA reduplication. This novel kinase inhibitor therefore serves as a useful chemical probe to further understand the temporal relationship between cell cycle checkpoint pathways, chemotherapeutic agent induced DNA damage and cell death.

DNA breakage and cell cycle checkpoint abrogation induced by a therapeutic thiopurine and UVA radiation

The frequency of squamous cell skin carcinoma in organ transplant patients is around 100-fold higher than normal. This dramatic example of therapy-related cancer reflects exposure to sunlight and to immunosuppressive drugs. Here we demonstrate that the interaction between low doses of UVA, the major ultraviolet component of incident sunlight, and 6-TG, a UVA chromophore that is introduced into DNA by one of the most widely-prescribed immunosuppressive drugs, causes DNA single- and double strand breaks (DSB). S phase cells are particularly vulnerable to this DNA breakage and cells defective in rejoining of S-phase DSB are hypersensitive to the combination of low dose UVA and DNA 6-TG. 6-TG/UVA-induced DNA lesions provoke canonical DNA damage responses involving activation of the ATM/Chk2 and ATR/Chk1 pathways and appropriate cell cycle checkpoints. Higher levels of photochemical DNA damage induce a proteasome-mediated degradation of Chk1 and checkpoint abrogation that is consistent with persistent unrepaired DNA damage. These findings indicate that the interaction between UVA and an immunosuppressant drug causes photochemical DNA lesions, including DNA breaks, and can compromise cell cycle checkpoints. These two properties could contribute to the high risk of sunlight-related skin cancer in long-term immunosuppressed patients.