Julio C. Morales

DNA mismatch repair (MMR)-dependent 5-fluorouracil cytotoxicity and the potential for new therapeutic targets

The metabolism and efficacy of 5‐fluorouracil (FUra) and other fluorinated pyrimidine (FP) derivatives have been intensively investigated for over fifty years. FUra and its antimetabolites can be incorporated at RNA‐ and DNA‐levels, with RNA level incorporation provoking toxic responses in human normal tissue, and DNA‐level antimetabolite formation and incorporation believed primarily responsible for tumour‐selective responses. Attempts to direct FUra into DNA‐level antimetabolites, based on mechanism‐of‐action studies, have led to gradual improvements in tumour therapy. These include the use of leukovorin to stabilize the inhibitory thymidylate synthase‐5‐fluoro‐2′‐deoxyuridine 5′ monophoshate (FdUMP)‐5,10‐methylene tetrahydrofolate (5,10‐CH2FH4) trimeric complex. FUra incorporated into DNA also contributes to antitumour activity in preclinical and clinical studies. This review examines our current state of knowledge regarding the mechanistic aspects of FUra:Gua lesion detection by DNA mismatch repair (MMR) machinery that ultimately results in lethality. MMR‐dependent direct cell death signalling or futile cycle responses will be discussed. As 10–30% of sporadic colon and endometrial tumours display MMR defects as a result of human MutL homologue‐1 (hMLH1) promoter hypermethylation, we discuss the use and manipulation of the hypomethylating agent, 5‐fluorodeoxycytidine (FdCyd), and our ability to manipulate its metabolism using the cytidine or deoxycytidylate (dCMP) deaminase inhibitors, tetrahydrouridine or deoxytetrahydrouridine, respectively, as a method for re‐expression of hMLH1 and re‐sensitization of tumours to FP therapy.

DNA Mismatch Repair-dependent Activation of c-Abl/p73α/GADD45α-mediated Apoptosis

Cells with functional DNA mismatch repair (MMR) stimulate G2 cell cycle checkpoint arrest and apoptosis in response to N-methyl-N′-nitro-N-nitrosoguanidine (MNNG). MMR-deficient cells fail to detect MNNG-induced DNA damage, resulting in the survival of “mutator” cells. The retrograde (nucleus-to-cytoplasm) signaling that initiates MMR-dependent G2 arrest and cell death remains undefined. Since MMR-dependent phosphorylation and stabilization of p53 were noted, we investigated its role(s) in G2 arrest and apoptosis. Loss of p53 function by E6 expression, dominant-negative p53, or stable p53 knockdown failed to prevent MMR-dependent G2 arrest, apoptosis, or lethality. MMR-dependent c-Abl-mediated p73α and GADD45α protein up-regulation after MNNG exposure prompted us to examine c-Abl/p73α/GADD45α signaling in cell death responses. STI571 (Gleevec™, a c-Abl tyrosine kinase inhibitor) and stable c-Abl, p73α, and GADD45α knockdown prevented MMR-dependent apoptosis. Interestingly, stable p73α knockdown blocked MMR-dependent apoptosis, but not G2 arrest, thereby uncoupling G2 arrest from lethality. Thus, MMR-dependent intrinsic apoptosis is p53-independent, but stimulated by hMLH1/c-Abl/p73α/GADD45α retrograde signaling.

Role of c-Abl Kinase in DNA Mismatch Repair-dependent G2 Cell Cycle Checkpoint Arrest Responses

Current published data suggest that DNA mismatch repair (MMR) triggers prolonged G2 cell cycle checkpoint arrest after alkylation damage from N-methyl-N′-nitro-N-nitrosoguanidine (MNNG) by activating ATR (ataxia telangiectasia-Rad3-related kinase). However, analyses of isogenic MMR-proficient and MMR-deficient human RKO colon cancer cells revealed that although ATR/Chk1 signaling controlled G2 arrest in MMR-deficient cells, ATR/Chk1 activation was not involved in MMR-dependent G2 arrest. Instead, we discovered that disrupting c-Abl activity using STI571 (Gleevec™, a c-Abl inhibitor) or stable c-Abl knockdown abolished MMR-dependent p73α stabilization, induction of GADD45α protein expression, and G2 arrest. In addition, inhibition of c-Abl also increased the survival of MNNG-exposed MMR-proficient cells to a level comparable with MMR-deficient cells. Furthermore, knocking down GADD45α (but not p73α) protein levels affected MMR-dependent G2 arrest responses. Thus, MMR-dependent G2 arrest responses triggered by MNNG are dependent on a human MLH1/c-Abl/GADD45α signaling pathway and activity. Furthermore, our data suggest that caution should be taken with therapies targeting c-Abl kinase because increased survival of mutator phenotypes may be an unwanted consequence.

ATM-dependent IGF-1 induction regulates secretory clusterin expression after DNA damage and in genetic instability

Secretory clusterin (sCLU) is a stress-induced, pro-survival glycoprotein elevated in early-stage cancers, in particular in APC/Min-defective colon cancers. sCLU is upregulated after exposure to various cytotoxic agents, including ionizing radiation (IR), leading to a survival advantage. We found that stimulation of insulin-like growth factor-1 (IGF-1) and IGF-1R protein kinase signaling was required for sCLU induction after IR exposure. Here, we show that activation of Ataxia telangiectasia-mutated kinase (ATM) by endogenous or exogenous forms of DNA damage was required to relieve basal repression of IGF-1 transcription by the p53/NF-YA complex, leading to sCLU expression. Although p53 levels were stabilized and elevated after DNA damage, dissociation of NF-YA, and thereby p53, from the IGF-1 promoter resulted in IGF-1 induction, indicating that NF-YA was rate limiting. Cells with elevated endogenous DNA damage (deficient in H2AX, MDC1, NBS1, mTR or hMLH1) or cells exposed to DNA-damaging agents had elevated IGF-1 expression, resulting in activation of IGF-1R signaling and sCLU induction. In contrast, ATM-deficient cells were unable to induce sCLU after DNA damage. Our results integrate DNA damage resulting from genetic instability, IR, or chemotherapeutic agents, to ATM activation and abrogation of p53/NF-YA-mediated IGF-1 transcriptional repression, that induces IGF-1–sCLU expression. Elucidation of this pathway should uncover new mechanisms for cancer progression and reveal new targets for drug development to overcome resistance to therapy.

Kub5-Hera, the human Rtt103 homolog, plays dual functional roles in transcription termination and DNA repair

Functions of Kub5-Hera (In Greek Mythology Hera controlled Artemis) (K-H), the human homolog of the yeast transcription termination factor Rtt103, remain undefined. Here, we show that K-H has functions in both transcription termination and DNA double-strand break (DSB) repair. K-H forms distinct protein complexes with factors that repair DSBs (e.g. Ku70, Ku86, Artemis) and terminate transcription (e.g. RNA polymerase II). K-H loss resulted in increased basal R-loop levels, DSBs, activated DNA-damage responses and enhanced genomic instability. Significantly lowered Artemis protein levels were detected in K-H knockdown cells, which were restored with specific K-H cDNA re-expression. K-H deficient cells were hypersensitive to cytotoxic agents that induce DSBs, unable to reseal complex DSB ends, and showed significantly delayed γ-H2AX and 53BP1 repair-related foci regression. Artemis re-expression in K-H-deficient cells restored DNA-repair function and resistance to DSB-inducing agents. However, R loops persisted consistent with dual roles of K-H in transcription termination and DSB repair.

Tumor-Selective, Futile Redox Cycle-Induced Bystander Effects Elicited by NQO1 Bioactivatable Radiosensitizing Drugs in Triple-Negative Breast Cancers

AIMS β-Lapachone (β-lap), a novel radiosensitizer with potent antitumor efficacy alone, selectively kills solid cancers that over-express NAD(P)H quinone oxidoreductase 1 (NQO1). Since breast or other solid cancers have heterogeneous NQO1 expression, therapies that reduce the resistance (e.g., NQO1(low)) of tumor cells will have significant clinical advantages. We tested whether NQO1-proficient (NQO1(+)) cells generated sufficient hydrogen peroxide (H2O2) after β-lap treatment to elicit bystander effects, DNA damage, and cell death in neighboring NQO1(low) cells. RESULTS β-Lap showed NQO1-dependent efficacy against two triple-negative breast cancer (TNBC) xenografts. NQO1 expression variations in human breast cancer patient samples were noted, where ~60% cancers over-expressed NQO1, with little or no expression in associated normal tissue. Differential DNA damage and lethality were noted in NQO1(+) versus NQO1-deficient (NQO1(-)) TNBC cells and xenografts after β-lap treatment. β-Lap-treated NQO1(+) cells died by programmed necrosis, whereas co-cultured NQO1(-) TNBC cells exhibited DNA damage and caspase-dependent apoptosis. NQO1 inhibition (dicoumarol) or H2O2 scavenging (catalase [CAT]) blocked all responses. Only NQO1(-) cells neighboring NQO1(+) TNBC cells responded to β-lap in vitro, and bystander effects correlated well with H2O2 diffusion. Bystander effects in NQO1(-) cells in vivo within mixed 50:50 co-cultured xenografts were dramatic and depended on NQO1(+) cells. However, normal human cells in vitro or in vivo did not show bystander effects, due to elevated endogenous CAT levels. Innovation and Conclusions: NQO1-dependent bystander effects elicited by NQO1 bioactivatable drugs (β-lap or deoxynyboquinone [DNQ]) likely contribute to their efficacies, killing NQO1(+) solid cancer cells and eliminating surrounding heterogeneous NQO1(low) cancer cells. Normal cells/tissue are protected by low NQO1:CAT ratios.

XRN2 Links Transcription Termination to DNA Damage and Replication Stress.

XRN2 is a 5’-3’ exoribonuclease implicated in transcription termination. Here we demonstrate an unexpected role for XRN2 in the DNA damage response involving resolution of R-loop structures and prevention of DNA double-strand breaks (DSBs). We show that XRN2 undergoes DNA damage-inducible nuclear re-localization, co-localizing with 53BP1 and R loops, in a transcription and R-loop-dependent process. XRN2 loss leads to increased R loops, genomic instability, replication stress, DSBs and hypersensitivity of cells to various DNA damaging agents. We demonstrate that the DSBs that arise with XRN2 loss occur at transcriptional pause sites. XRN2-deficient cells also exhibited an R-loop- and transcription-dependent delay in DSB repair after ionizing radiation, suggesting a novel role for XRN2 in R-loop resolution, suppression of replication stress, and maintenance of genomic stability. Our study highlights the importance of regulating transcription-related activities as a critical component in maintaining genetic stability.

The Kub5-Hera/RPRD1B interactome: a novel role in preserving genetic stability by regulating DNA mismatch repair

Ku70-binding protein 5 (Kub5)-Hera (K-H)/RPRD1B maintains genetic integrity by concomitantly minimizing persistent R-loops and promoting repair of DNA double strand breaks (DSBs). We used tandem affinity purification-mass spectrometry, co-immunoprecipitation and gel-filtration chromatography to define higher-order protein complexes containing K-H scaffolding protein to gain insight into its cellular functions. We confirmed known protein partners (Ku70, RNA Pol II, p15RS) and discovered several novel associated proteins that function in RNA metabolism (Topoisomerase 1 and RNA helicases), DNA repair/replication processes (PARP1, MSH2, Ku, DNA-PKcs, MCM proteins, PCNA and DNA Pol δ) and in protein metabolic processes, including translation. Notably, this approach directed us to investigate an unpredicted involvement of K-H in DNA mismatch repair (MMR) where K-H depletion led to concomitant MMR deficiency and compromised global microsatellite stability. Mechanistically, MMR deficiency in K-H-depleted cells was a consequence of reduced stability of the core MMR proteins (MLH1 and PMS2) caused by elevated basal caspase-dependent proteolysis. Pan-caspase inhibitor treatment restored MMR protein loss. These findings represent a novel mechanism to acquire MMR deficiency/microsatellite alterations. A significant proportion of colon, endometrial and ovarian cancers exhibit k-h expression/copy number loss and may have severe mutator phenotypes with enhanced malignancies that are currently overlooked based on sporadic MSI+ screening.

Focal adhesion kinase regulates the DNA damage response and its inhibition radiosensitizes mutant KRAS lung cancer

Purpose: Non–small cell lung cancer (NSCLC) is the leading cause of cancer-related deaths worldwide due to the limited availability of effective therapeutic options. For instance, there are no effective strategies for NSCLCs that harbor mutant KRAS, the most commonly mutated oncogene in NSCLC. Thus, our purpose was to make progress toward the generation of a novel therapeutic strategy for NSCLC. Experimental Design: We characterized the effects of suppressing focal adhesion kinase (FAK) by RNA interference (RNAi), CRISPR/CAS9 gene editing or pharmacologic approaches in NSCLC cells and in tumor xenografts. In addition, we tested the effects of suppressing FAK in association with ionizing radiation (IR), a standard-of-care treatment modality. Results: FAK is a critical requirement of mutant KRAS NSCLC cells. With functional experiments, we also found that, in mutant KRAS NSCLC cells, FAK inhibition resulted in persistent DNA damage and susceptibility to exposure to IR. Accordingly, administration of IR to FAK-null tumor xenografts causes a profound antitumor effect in vivo. Conclusions: FAK is a novel regulator of DNA damage repair in mutant KRAS NSCLC and its pharmacologic inhibition leads to radiosensitizing effects that could be beneficial in cancer therapy. Our results provide a framework for the rationale clinical testing of FAK inhibitors in NSCLC patients. Clin Cancer Res; 22(23); 5851–63. ©2016 AACR.

Cellular Stress Responses to DNA Damage: An Intracellular Balance between Life, Senescence, and Death

Recent cellular and molecular biological examination of DNA repair, cell cycle checkpoints, apoptosis, differentiation, and stress-induced premature senescence (SIPS) in response to genotoxic stress is largely responsible for the current understanding of the interplay between cell stress responses and human diseases, such as cancer. Human pathologies associated with older individuals show homeostatic alterations of several cell stress factors, including Bax, p53, Hdm2, and secretory clusterin (sCLU) that can influence survival, senescence, and ultimately the risk of cancer. Understanding cell stress responses in the context of genetic predispositions is vital to the understanding of cancer risk from environmental stress, such as exposure to low doses of ionizing radiation (IR). Increased expression of sCLU appears to be a sensitive marker of exposure. Its interplay and regulation with stress-induced alterations in Bax, Ku70, Hdm2, p53, and their roles in regulation of survival and carcinogenesis are discussed. Understanding the mechanisms of induction and repression of the cellular responses to genotoxic agents is essential for understanding downstream cancer initiation and progression.