Intra-S phase checkpoint kinase Chk1 dissociates replication proteins Treslin and TopBP1 through multiple mechanisms during replication stress.

Replication stress impedes DNA polymerase progression causing activation of the ataxia telangiectasia and Rad3-related signaling pathway, which promotes the intra-S phase checkpoint activity through phosphorylation of checkpoint kinase 1 (Chk1). Chk1 suppresses replication origin firing, in part, by disrupting the interaction between the preinitiation complex components Treslin and TopBP1, an interaction that is mediated by TopBP1 BRCT domain-binding to two cyclin-dependent kinase (CDK) phosphorylation sites, T968 and S1000, in Treslin. Two nonexclusive models for how Chk1 regulates the Treslin-TopBP1 interaction have been proposed in the literature: in one model, these proteins dissociate due to a Chk1-induced decrease in CDK activity that reduces phosphorylation of the Treslin sites that bind TopBP1 and in the second model, Chk1 directly phosphorylates Treslin, resulting in dissociation of TopBP1. However, these models have not been formally examined. We show here that Treslin T968 phosphorylation was decreased in a Chk1-dependent manner, while Treslin S1000 phosphorylation was unchanged, demonstrating that T968 and S1000 are differentially regulated. However, CDK2-mediated phosphorylation alone did not fully account for Chk1 regulation of the Treslin-TopBP1 interaction. We also identified additional Chk1 phosphorylation sites on Treslin that contributed to disruption of the Treslin-TopBP1 interaction, including S1114. Finally, we showed that both of the proposed mechanisms regulate origin firing in cancer cell line models undergoing replication stress, with the relative roles of each mechanism varying among cell lines. This study demonstrates that Chk1 regulates Treslin through multiple mechanisms to promote efficient dissociation of Treslin and TopBP1 and furthers our understanding of Treslin regulation during the intra-S phase checkpoint.

Repurposing Ceritinib Induces DNA Damage and Enhances PARP Inhibitor Responses in High-Grade Serous Ovarian Carcinoma.

PARP inhibitors (PARPi) have activity in homologous recombination (HR) repair-deficient, high-grade serous ovarian cancers (HGSOC). However, even responsive tumors develop PARPi resistance, highlighting the need to delay or prevent the appearance of PARPi resistance. Here, we showed that the ALK kinase inhibitor ceritinib synergizes with PARPis by inhibiting complex I of the mitochondrial electron transport chain, which increases production of reactive oxygen species (ROS) and subsequent induction of oxidative DNA damage that is repaired in a PARP-dependent manner. In addition, combined treatment with ceritinib and PARPi synergized in HGSOC cell lines irrespective of HR status, and a combination of ceritinib with the PARPi olaparib induced tumor regression more effectively than olaparib alone in HGSOC patient-derived xenograft (PDX) models. Notably, the ceritinib and olaparib combination was most effective in PDX models with preexisting PARPi sensitivity and was well tolerated. These findings unveil suppression of mitochondrial respiration, accumulation of ROS, and subsequent induction of DNA damage as novel effects of ceritinib. They also suggest that the ceritinib and PARPi combination warrants further investigation as a means to enhance PARPi activity in HGSOC, particularly in tumors with preexisting HR defects. SIGNIFICANCE: The kinase inhibitor ceritinib synergizes with PARPi to induce tumor regression in ovarian cancer models, suggesting that ceritinib combined with PARPi may be an effective strategy for treating ovarian cancer.

VLX600 Disrupts Homologous Recombination and Synergizes with PARP Inhibitors and Cisplatin by Inhibiting Histone Lysine Demethylases.

Tumors with defective homologous recombination (HR) DNA repair are more sensitive to chemotherapies that induce lesions repaired by HR as well as PARP inhibitors (PARPis). However, these therapies have limited activity in HR-proficient cells. Accordingly, agents that disrupt HR may be a means to augment the activities of these therapies in HR-proficient tumors. Here we show that VLX600, a small molecule that has been in a phase I clinical trial, disrupts HR and synergizes with PARPis and platinum compounds in ovarian cancer cells. We further found that VLX600 and other iron chelators disrupt HR, in part, by inhibiting iron-dependent histone lysine demethylases (KDM) family members, thus blocking recruitment of HR repair proteins, including RAD51, to double-strand DNA breaks. Collectively, these findings suggest that pharmacologically targeting KDM family members with VLX600 may be a potential novel strategy to therapeutically induce HR defects in ovarian cancers and correspondingly sensitize them to platinum agents and PARPis, two standard-of-care therapies for ovarian cancer.

Effect of CHK1 Inhibition on CPX-351 Cytotoxicity in vitro and ex vivo.

CPX-351 is a liposomally encapsulated 5:1 molar ratio of cytarabine and daunorubicin that recently received regulatory approval for the treatment of therapy-related acute myeloid leukemia (AML) or AML with myelodysplasia-related changes based on improved overall survival compared to standard cytarabine/daunorubicin therapy. Checkpoint kinase 1 (CHK1), which is activated by DNA damage and replication stress, diminishes sensitivity to cytarabine and anthracyclines as single agents, suggesting that CHK1 inhibitors might increase the effectiveness of CPX-351. The present studies show that CPX-351 activates CHK1 as well as the S and G2/M cell cycle checkpoints. Conversely, CHK1 inhibition diminishes the cell cycle effects of CPX-351. Moreover, CHK1 knockdown or addition of a CHK1 inhibitor such as MK-8776, rabusertib or prexasertib enhances CPX-351-induced apoptosis in multiple TP53-null and TP53-wildtype AML cell lines. Likewise, CHK1 inhibition increases the antiproliferative effect of CPX-351 on primary AML specimens ex vivo, offering the possibility that CPX-351 may be well suited to combine with CHK1-targeted agents.

NKG2D Ligand-Targeted Bispecific T-Cell Engagers Lead to Robust Antitumor Activity against Diverse Human Tumors.

Two new bispecific T-cell engaging (BiTE) molecules with specificity for NKG2D ligands were developed and functionally characterized. One, huNKG2D-OKT3, was derived from the extracellular portion of the human NKG2D receptor fused to a CD3ε binding single-chain variable fragment (scFv), known as OKT3. NKG2D has multiple ligands, including MICA, which are expressed by a variety of malignant cells. A second molecule, B2-OKT3, was created in the tandem scFv BiTE format that targets MICA on tumor cells and CD3ε on human T cells. Both BiTEs specifically activated T cells to kill human tumor cell lines. Cytotoxicity by B2-OKT3, but not huNKG2D-OKT3, is blocked by soluble rMICA. The huNKG2D-OKT3 induced greater T-cell cytokine production in comparison with B2-OKT3. No T-cell pretreatment was required for IFNγ production upon coculture of B2-OKT3 or huNKG2D-OKT3 with T cells and target cells. The effector memory T-cell compartment was the primary source of IFNγ, and culture of T cells and these BiTEs with plate-bound rMICA showed ligand density-dependent production of IFNγ from both CD4+ and CD8+ T cells. There was 2-fold more IFNγ produced per CD8+ T cell and 5-fold greater percentage of CD8+ T cells producing IFNγ compared with CD4+ T cells. In addition, both BiTEs elicited significant antitumor responses against human metastatic melanoma tumor samples using autologous or healthy donor T cells. These data demonstrate the robust antitumor activity of these NKG2D ligand-binding bispecific proteins and support their further development for clinical use. Mol Cancer Ther; 16(7); 1335-46. ©2017 AACR.

Genomically Incorporated 5-Fluorouracil that Escapes UNG-Initiated Base Excision Repair Blocks DNA Replication and Activates Homologous Recombination.

5-Fluorouracil (5-FU) and its metabolite 5-fluorodeoxyuridine (FdUrd, floxuridine) are chemotherapy agents that are converted to 5-fluorodeoxyuridine monophosphate (FdUMP) and 5-fluorodeoxyuridine triphosphate (FdUTP). FdUMP inhibits thymidylate synthase and causes the accumulation of uracil in the genome, whereas FdUTP is incorporated by DNA polymerases as 5-FU in the genome; however, it remains unclear how either genomically incorporated U or 5-FU contributes to killing. We show that depletion of the uracil DNA glycosylase (UNG) sensitizes tumor cells to FdUrd. Furthermore, we show that UNG depletion does not sensitize cells to the thymidylate synthase inhibitor (raltitrexed), which induces uracil but not 5-FU accumulation, thus indicating that genomically incorporated 5-FU plays a major role in the antineoplastic effects of FdUrd. We also show that 5-FU metabolites do not block the first round of DNA synthesis but instead arrest cells at the G1/S border when cells again attempt replication and activate homologous recombination (HR). This arrest is not due to 5-FU lesions blocking DNA polymerase δ but instead depends, in part, on the thymine DNA glycosylase. Consistent with the activation of HR repair, disruption of HR sensitized cells to FdUrd, especially when UNG was disabled. These results show that 5-FU lesions that escape UNG repair activate HR, which promotes cell survival.

Bispecific T-cell engagers for cancer immunotherapy.

Bispecific T-cell engagers (BiTEs) are a new class of immunotherapeutic molecules intended for the treatment of cancer. These molecules enhance the patient's immune response to tumors by retargeting T cells to tumor cells. BiTEs are constructed of two single-chain variable fragments (scFv) connected in tandem by a flexible linker. One scFv binds to a T-cell-specific molecule, usually CD3, whereas the second scFv binds to a tumor-associated antigen. This structure and specificity allows a BiTE to physically link a T cell to a tumor cell, ultimately stimulating T-cell activation, tumor killing and cytokine production. BiTEs have been developed, which target several tumor-associated antigens, for a variety of both hematological and solid tumors. Several BiTEs are currently in clinical trials for their therapeutic efficacy and safety. This review examines the salient structural and functional features of BiTEs, as well as the current state of their clinical and preclinical development.

ATR inhibition broadly sensitizes ovarian cancer cells to chemotherapy independent of BRCA status.

Replication stress and DNA damage activate the ATR-Chk1 checkpoint signaling pathway that licenses repair and cell survival processes. In this study, we examined the respective roles of the ATR and Chk1 kinases in ovarian cancer cells using genetic and pharmacologic inhibitors in combination with cisplatin, topotecan, gemcitabine, and the PARP inhibitor veliparib (ABT-888), four agents with clinical activity in ovarian cancer. RNA interference (RNAi)-mediated depletion or inhibition of ATR sensitized ovarian cancer cells to all four agents. In contrast, while cisplatin, topotecan, and gemcitabine each activated Chk1, RNAi-mediated depletion or inhibition of this kinase in cells sensitized them only to gemcitabine. Unexpectedly, we found that neither the ATR kinase inhibitor VE-821 nor the Chk1 inhibitor MK-8776 blocked ATR-mediated Chk1 phosphorylation or autophosphorylation, two commonly used readouts for inhibition of the ATR-Chk1 pathway. Instead, their ability to sensitize cells correlated with enhanced CDC25A levels. In addition, we also found that VE-821 could further sensitize BRCA1-depleted cells to cisplatin, topotecan, and veliparib beyond the potent sensitization already caused by their deficiency in homologous recombination. Taken together, our results established that ATR and Chk1 inhibitors differentially sensitize ovarian cancer cells to commonly used chemotherapy agents and that Chk1 phosphorylation status may not offer a reliable marker for inhibition of the ATR-Chk1 pathway. A key implication of our work is the clinical rationale it provides to evaluate ATR inhibitors in combination with PARP inhibitors in BRCA1/2-deficient cells.

Identification of DNA repair pathways that affect the survival of ovarian cancer cells treated with a poly(ADP-ribose) polymerase inhibitor in a novel drug combination.

Floxuridine (5-fluorodeoxyuridine, FdUrd), a U.S. Food and Drug Administration-approved drug and metabolite of 5-fluorouracil, causes DNA damage that is repaired by base excision repair (BER). Thus, poly(ADP-ribose) polymerase (PARP) inhibitors, which disrupt BER, markedly sensitize ovarian cancer cells to FdUrd, suggesting that this combination may have activity in this disease. It remains unclear, however, which DNA repair and checkpoint signaling pathways affect killing by these agents individually and in combination. Here we show that depleting ATR, BRCA1, BRCA2, or RAD51 sensitized to ABT-888 (veliparib) alone, FdUrd alone, and FdUrd + ABT-888 (F+A), suggesting that homologous recombination (HR) repair protects cells exposed to these agents. In contrast, disabling the mismatch, nucleotide excision, Fanconi anemia, nonhomologous end joining, or translesion synthesis repair pathways did not sensitize to these agents alone (including ABT-888) or in combination. Further studies demonstrated that in BRCA1-depleted cells, F+A was more effective than other chemotherapy+ABT-888 combinations. Taken together, these studies 1) identify DNA repair and checkpoint pathways that are important in ovarian cancer cells treated with FdUrd, ABT-888, and F+A, 2) show that disabling HR at the level of ATR, BRCA1, BRCA2, or RAD51, but not Chk1, ATM, PTEN, or FANCD2, sensitizes cells to ABT-888, and 3) demonstrate that even though ABT-888 sensitizes ovarian tumor cells with functional HR to FdUrd, the effects of this drug combination are more profound in tumors with HR defects, even compared with other chemotherapy + ABT-888 combinations, including cisplatin + ABT-888.

Checkpoint signaling, base excision repair, and PARP promote survival of colon cancer cells treated with 5-fluorodeoxyuridine but not 5-fluorouracil.

The fluoropyrimidines 5-fluorouracil (5-FU) and FdUrd (5-fluorodeoxyuridine; floxuridine) are the backbone of chemotherapy regimens for colon cancer and other tumors. Despite their widespread use, it remains unclear how these agents kill tumor cells. Here, we have analyzed the checkpoint and DNA repair pathways that affect colon tumor responses to 5-FU and FdUrd. These studies demonstrate that both FdUrd and 5-FU activate the ATR and ATM checkpoint signaling pathways, indicating that they cause genotoxic damage. Notably, however, depletion of ATM or ATR does not sensitize colon cancer cells to 5-FU, whereas these checkpoint pathways promote the survival of cells treated with FdUrd, suggesting that FdUrd exerts cytotoxicity by disrupting DNA replication and/or inducing DNA damage, whereas 5-FU does not. We also found that disabling the base excision (BER) repair pathway by depleting XRCC1 or APE1 sensitized colon cancer cells to FdUrd but not 5-FU. Consistent with a role for the BER pathway, we show that small molecule poly(ADP-ribose) polymerase 1/2 (PARP) inhibitors, AZD2281 and ABT-888, remarkably sensitized both mismatch repair (MMR)-proficient and -deficient colon cancer cell lines to FdUrd but not to 5-FU. Taken together, these studies demonstrate that the roles of genotoxin-induced checkpoint signaling and DNA repair differ significantly for these agents and also suggest a novel approach to colon cancer therapy in which FdUrd is combined with a small molecule PARP inhibitor.

Poly(ADP-Ribose) polymerase inhibition synergizes with 5-fluorodeoxyuridine but not 5-fluorouracil in ovarian cancer cells.

5-Fluorouracil (5-FU) and 5-fluorodeoxyuridine (FdUrd, floxuridine) have activity in multiple tumors, and both agents undergo intracellular processing to active metabolites that disrupt RNA and DNA metabolism. These agents cause imbalances in deoxynucleotide triphosphate levels and the accumulation of uracil and 5-FU in the genome, events that activate the ATR- and ATM-dependent checkpoint signaling pathways and the base excision repair (BER) pathway. Here, we assessed which DNA damage response and repair processes influence 5-FU and FdUrd toxicity in ovarian cancer cells. These studies revealed that disabling the ATM, ATR, or BER pathways using small inhibitory RNAs did not affect 5-FU cytotoxicity. In stark contrast, ATR and a functional BER pathway protected FdUrd-treated cells. Consistent with a role for the BER pathway, the poly(ADP-ribose) polymerase (PARP) inhibitors ABT-888 (veliparib) and AZD2281 (olaparib) markedly synergized with FdUrd but not with 5-FU in ovarian cancer cell lines. Furthermore, ABT-888 synergized with FdUrd far more effectively than other agents commonly used to treat ovarian cancer. These findings underscore differences in the cytotoxic mechanisms of 5-FU and FdUrd and suggest that combining FdUrd and PARP inhibitors may be an innovative therapeutic strategy for ovarian tumors.

The role of metal ion binding in generating 8-hydroxy-2'-deoxyguanosine from the nucleoside 2'-deoxyguanosine and the nucleotide 2'-deoxyguanosine-5'-monophosphate.

The metal ions Cu(II), Fe(II), and Cr(III) were allowed to react with H(2)O(2) in the presence of either the mononucleoside 2'-deoxyguanosine (dG) or the mononucleotide 2'-deoxyguanosine-5'-monophosphate (dGMP). The percentage of reacted dG or dGMP that formed the oxidative damage marker 8-hydroxy-2'-deoxyguanosine (8-OH-dG) was monitored. Oxidative damage from reactions involving Cu(II) appear dependent on an interaction between copper and N7 on the guanine base. Any interactions involving the phosphate group have little additional effect on overall oxidative damage or 8-OH-dG production. Reactions involving Fe(II) seem very dependent on an interaction that may involve both N7 on the guanine base and the phosphate group. This interaction may slow oxidation of Fe(II) to Fe(III) in solution, keeping iron in a readily available form to undergo the Fenton reaction. Chromium(III) appears to interact with the phosphate group of dGMP, resulting in significant overall oxidative damage. However, production of 8-OH-dG appears to be very dependent on the ability of Cr(III) to interact with N7 on the guanine base, an interaction that seems to be weak for both the mononucleoside and mononucleotide.