John J. Pink

Development of β-Lapachone Prodrugs for Therapy Against Human Cancer Cells with Elevated NAD(P)H:Quinone Oxidoreductase 1 Levels

β-Lapachone, an o-naphthoquinone, induces a novel caspase- and p53-independent apoptotic pathway dependent on NAD(P)H:quinone oxidoreductase 1 (NQO1). NQO1 reduces β-lapachone to an unstable hydroquinone that rapidly undergoes a two-step oxidation back to the parent compound, perpetuating a futile redox cycle. A deficiency or inhibition of NQO1 rendered cells resistant to β-lapachone. Thus, β-lapachone has great potential for the treatment of specific cancers with elevated NQO1 levels (e.g., breast, non–small cell lung, pancreatic, colon, and prostate cancers). We report the development of mono(arylimino) derivatives of β-lapachone as potential prodrugs. These derivatives are relatively nontoxic and not substrates for NQO1 when initially diluted in water. In solution, however, they undergo hydrolytic conversion to β-lapachone at rates dependent on the electron-withdrawing strength of their substituent groups and pH of the diluent. NQO1 enzyme assays, UV-visible spectrophotometry, high-performance liquid chromatography-electrospray ionization-mass spectrometry, and nuclear magnetic resonance analyses confirmed and monitored conversion of each derivative to β-lapachone. Once converted, β-lapachone derivatives caused NQO1-dependent, μ-calpain-mediated cell death in human cancer cells identical to that caused by β-lapachone. Interestingly, coadministration of N-acetyl-l-cysteine, prevented derivative-induced cytotoxicity but did not affect β-lapachone lethality. Nuclear magnetic resonance analyses indicated that prevention of β-lapachone derivative cytotoxicity was the result of direct modification of these derivatives by N-acetyl-l-cysteine, preventing their conversion to β-lapachone. The use of β-lapachone mono(arylimino) prodrug derivatives, or more specifically a derivative converted in a tumor-specific manner (i.e., in the acidic local environment of the tumor tissue), should reduce normal tissue toxicity while eliciting tumor-selective cell killing by NQO1 bioactivation.

μ-calpain activation in β-lapachone-mediated apoptosis

β-Lapachone (β-Lap) triggers apoptosis in a number of human breast and prostate cancer cell lines through a unique apoptotic pathway that is dependent upon NQO1, a two-electron reductase. Recently, our laboratory showed that β-lap-exposed MCF-7 cells exhibited an early increase in intracellular cytosolic Ca2+ from endoplasmic reticulum stores, and that BAPTA-AM (an intracellular Ca2+ chelator) blocked these early increases and partially inhibited all aspects of b-lap-induced apoptosis. We now show that exposure of NQO1-expressing breast cancer cells to β-lap stimulates a unique proteolytic apoptotic pathway involving μ-calpain activation. No apparent activation of μ-calpain was noted. Upon activation, m-calpain translocated to the nucleus concomitant with specific nuclear proteolytic events. Apoptotic responses in β-lap-exposed NQO1-expressing cells were significantly delayed and survival enhanced by exogenous over-expression of calpastatin, a natural inhibitor of m- and μ-calpains. Furthermore, purified μ-calpain cleaved PARP to a unique fragment (~60 kDa), not previously reported for calpains. We provide evidence that β-lap-induced, μ-calpain-stimulated apoptosis does not involve any known apoptotic caspases; the activated fragments of caspases were not observed after β-lap exposures, nor were there any changes in the pro-enzyme forms as measured by Western blot analyses. The ability of β-lap to trigger an apparently novel, p53-independent, calpain-mediated apoptotic cell death further support the development of this drug for improved breast cancer therapy. Keywords: β-Lapachone, apoptosis, Ca2+, calpain, breast cancer

Phase I Trial of Pelvic Radiation, Weekly Cisplatin, and 3-Aminopyridine-2-Carboxaldehyde Thiosemicarbazone (3-AP, NSC #663249) for Locally Advanced Cervical Cancer

Purpose: This study assessed the safety/tolerability, pharmacokinetics, and clinical activity of three times weekly i.v. 3-aminopyridine-2-carboxaldehyde thiosemicarbazone (3-AP, NSC #663249) in combination with once-weekly i.v. cisplatin and daily pelvic radiation in patients with gynecologic malignancies. 3-AP is a novel small-molecule inhibitor of ribonucleotide reductase (RNR) and is being tested as a potential radiosensitizer and chemosensitizer. Experimental Design: Patients with stage IB2 to IVB cervical cancer (n = 10) or recurrent uterine sarcoma (n = 1) were assigned to dose-finding cohorts of 2-hour 3-AP infusions during 5 weeks of cisplatin chemoradiation. Pharmacokinetic and methemoglobin samples and tumor biopsy for RNR activity were obtained on day 1 and day 10. Clinical response was assessed. Results: The maximum tolerated 3-AP dose was 25 mg/m2 given three times weekly during cisplatin and pelvic radiation. Two patients experienced manageable 3-AP–related grade 3 or 4 electrolyte abnormalities. 3-AP pharmacokinetics showed a 2-hour half-life, with median peak plasma concentrations of 277 ng/mL (25 mg/m2) and 467 ng/mL (50 mg/m2). Median methemoglobin levels peaked at 1% (25 mg/m2) and 6% (50 mg/m2) at 4 hours after initiating 3-AP infusions. No change in RNR activity was found on day 1 versus day 10 in six early complete responders, whereas elevated RNR activity was seen on day 10 as compared with day 1 in four late complete responders (P = 0.02). Ten (100%) patients with stage IB2 to IVB cervical cancer achieved complete clinical response and remained without disease relapse with a median 18 months of follow-up (6-32 months). Conclusions: 3-AP was well tolerated at a three times weekly i.v. 25 mg/m2 dose during cisplatin and pelvic radiation. Clin Cancer Res; 16(4); 1298–306

Irreversible loss of the oestrogen receptor in T47D breast cancer cells following prolonged oestrogen deprivation.

The development of antioestrogen resistance is a major clinical obstacle encountered in the treatment of breast cancer. By long-term growth in oestrogen-free medium, we have derived an oestrogen-independent, anti-oestrogen resistant cell line from the oestrogen receptor (ER)-positive, oestrogen-dependent T47D human breast cancer cell line. This cell line grows maximally in oestrogen-free medium and is resistant to all tested antioestrogens. This cell line does not express any measurable amounts of ER mRNA or protein and, in short-term studies, these cells show no response to either oestrogens or antioestrogens. However, return of these cells to oestrogen-containing medium for more than 8 weeks resulted in the re-expression of ER mRNA and protein. Subsequent limiting dilution subcloning of the T47D:C4 line revealed two phenotypically distinct clones, one which did not express measurable ER after long-term growth in oestrogen-containing medium and one which expressed ER mRNA and protein after a number of weeks in oestrogen-containing medium. In the absence of oestrogen, both types of cells are ER-negative as determined by Northern and Western blotting and lack of any oestrogen-dependent responses. The clone which re-expresses the ER (T47D:C4:5W) now responds to E2 with a 50% increase in growth and a 30-fold induction of an ER-responsive luciferase reporter construct. Long-term growth of the stably ER-negative clone (T47D:C4:2W) causes no measurable oestrogen-mediated responses, as assessed by ER expression, growth stimulation or luciferase induction. Interestingly, ER mRNA can be detected in both cell types by using reverse transcriptase-polymerase chain reaction (RT-PCR). This suggests that the ER mRNA present in the T47D:C4:2W clone is either inefficiently translated or is present at such a low level as to be functionally irrelevant. These novel clonal cell lines will prove to be invaluable in the study of the regulation of ER expression and regulatory pathways leading to oestrogen-independent growth.

Catalase Abrogates β-Lapachone–Induced PARP1 Hyperactivation–Directed Programmed Necrosis in NQO1-Positive Breast Cancers

Improving patient outcome by personalized therapy involves a thorough understanding of an agents mechanism of action. β-Lapachone (clinical forms, Arq501/Arq761) has been developed to exploit dramatic cancer-specific elevations in the phase II detoxifying enzyme NAD(P)H:quinone oxidoreductase (NQO1). NQO1 is dramatically elevated in solid cancers, including primary and metastatic [e.g., triple-negative (ER−, PR−, Her2/Neu−)] breast cancers. To define cellular factors that influence the efficacy of β-lapachone using knowledge of its mechanism of action, we confirmed that NQO1 was required for lethality and mediated a futile redox cycle where ∼120 moles of superoxide were formed per mole of β-lapachone in 2 minutes. β-Lapachone induced reactive oxygen species (ROS), stimulated DNA single-strand break-dependent poly(ADP-ribose) polymerase-1 (PARP1) hyperactivation, caused dramatic loss of essential nucleotides (NAD+/ATP), and elicited programmed necrosis in breast cancer cells. Although PARP1 hyperactivation and NQO1 expression were major determinants of β-lapachone–induced lethality, alterations in catalase expression, including treatment with exogenous enzyme, caused marked cytoprotection. Thus, catalase is an important resistance factor and highlights H2O2 as an obligate ROS for cell death from this agent. Exogenous superoxide dismutase enhanced catalase-induced cytoprotection. β-Lapachone–induced cell death included apoptosis-inducing factor (AIF) translocation from mitochondria to nuclei, TUNEL+ staining, atypical PARP1 cleavage, and glyceraldehyde 3-phosphate dehydrogenase S-nitrosylation, which were abrogated by catalase. We predict that the ratio of NQO1:catalase activities in breast cancer versus associated normal tissue are likely to be the major determinants affecting the therapeutic window of β-lapachone and other NQO1 bioactivatable drugs. Mol Cancer Ther; 12(10); 2110–20. ©2013 AACR.

Ribonucleotide Reductase Inhibition Enhances Chemoradiosensitivity of Human Cervical Cancers

Abstract For repair of damaged DNA, cells increase de novo synthesis of deoxyribonucleotide triphosphates through the rate-limiting, p53-regulated ribonucleotide reductase (RNR) enzyme. In this study we investigated whether pharmacological inhibition of RNR by 3-aminopyridine-2-carboxaldehyde thiosemicarbazone (3-AP, NSC #663249) enhanced chemoradiation sensitivity through a mechanism involving sustained DNA damage. RNR inactivation by 3-AP and resulting chemoradiosensitization were evaluated in human cervical (CaSki, C33-a) cancer cells through study of DNA damage (&ggr;-H2AX signal) by flow cytometry, RNR subunit p53R2 and p21 protein steady-state levels by Western blot analysis and laser scanning imaging cytometry, and cell survival by colony formation assays. 3-AP treatment led to sustained radiation- and cisplatin-induced DNA damage (i.e. increased &ggr;-H2AX signal) in both cell lines through a mechanism of inhibited RNR activity. Radiation, cisplatin and 3-AP exposure resulted in significantly elevated numbers and persistence of &ggr;-H2AX foci that were associated with reduced clonogenic survival. DNA damage was associated with a rise in p53R2 but not p21 protein levels 6 h after treatment with radiation and/or cisplatin plus 3-AP. We conclude that blockage of RNR activity by 3-AP impairs DNA damage responses that rely on deoxyribonucleotide production and thereby may substantially increase chemoradiosensitivity of human cervical cancers.

Modulating Radiation Resistance by Inhibiting Ribonucleotide Reductase in Cancers with Virally or Mutationally Silenced p53 Protein

Abstract Kunos, C. A., Chiu, S., Pink, J. and Kinsella, T. J. Modulating Radiation Resistance by Inhibiting Ribonucleotide Reductase in Cancers with Virally or Mutationally Silenced p53 Protein. Therapeutic ionizing radiation damages DNA, increasing p53-regulated ribonucleotide reductase (RNR) activity required for de novo synthesis of the deoxyribonucleotide triphosphates used during DNA repair. This study investigated the pharmacological inhibition of RNR in cells of virally or mutationally silenced p53 cancer cell lines using 3-aminopyridine-2-carboxaldehyde thiosemicarbazone (3-AP, Triapine®, NSC #663249), a chemotherapeutic radiosensitizer that equally inhibits RNR M2 and p53R2 small subunits. The effects of 3-AP on RNR inhibition and resulting radiosensitization were evaluated in cervical (CaSki, HeLa and C33-a) and colon (RKO, RKO-E6) cancer cells. 3-AP treatment significantly enhanced radiation-related cytotoxicity in cervical and colon cancer cells. 3-AP treatment significantly decreased RNR activity, caused prolonged radiation-induced DNA damage, and resulted in an extended G1/S-phase cell cycle arrest in all cell lines. Similar effects were observed in both RKO and RKO-E6 cells, suggesting a p53-independent mechanism of radiosensitization. We conclude that inhibition of ribonucleotide reductase by 3-AP enhances radiation-mediated cytotoxicity independent of p53 regulation by impairing repair processes that rely on deoxyribonucleotide production, thereby substantially increasing the radiation sensitivity of human cancers.

A synthetic peptide targeting the BH4 domain of Bcl-2 induces apoptosis in multiple myeloma and follicular lymphoma cells alone or in combination with agents targeting the BH3-binding pocket of Bcl-2

Bcl-2 inhibits apoptosis by two distinct mechanisms but only one is targeted to treat Bcl-2-positive malignancies. In this mechanism, the BH1-3 domains of Bcl-2 form a hydrophobic pocket, binding and inhibiting pro-apoptotic proteins, including Bim. In the other mechanism, the BH4 domain mediates interaction of Bcl-2 with inositol 1,4, 5-trisphosphate receptors (IP3Rs), inhibiting pro-apoptotic Ca2+ signals. The current anti-Bcl-2 agents, ABT-263 (Navitoclax) and ABT-199 (Venetoclax), induce apoptosis by displacing pro-apoptotic proteins from the hydrophobic pocket, but do not inhibit Bcl-2-IP3R interaction. Therefore, to target this interaction we developed BIRD-2 (Bcl-2 IP3 Receptor Disruptor-2), a decoy peptide that binds to the BH4 domain, blocking Bcl-2-IP3R interaction and thus inducing Ca2+-mediated apoptosis in chronic lymphocytic leukemia, multiple myeloma, and follicular lymphoma cells, including cells resistant to ABT-263, ABT-199, or the Bruton’s tyrosine kinase inhibitor Ibrutinib. Moreover, combining BIRD-2 with ABT-263 or ABT-199 enhances apoptosis induction compared to single agent treatment. Overall, these findings provide strong rationale for developing novel therapeutic agents that mimic the action of BIRD-2 in targeting the BH4 domain of Bcl-2 and disrupting Bcl-2-IP3R interaction.

Cellular and molecular responses to topoisomerase I poisons. Exploiting synergy for improved radiotherapy.

Abstract: The efficacy of topoisomerase (Topo) I‐active drugs may be improved by better understanding the molecular and cellular responses of tumor compared to normal cells after genotoxic insults. Ionizing radiation (IR) + Topo I‐active drugs (e.g., Topotecan) caused synergistic cell killing in various human cancer cells, even in cells from highly radioresistant tumors. Topo I poisons had to be added either during or immediately after IR. Synergy was caused by DNA lesion modification mechanisms as well as by concomitant stimulation of two pathways of cell death: necrosis (IR) + apoptosis (Topo I poisons). Cumulative data favor a mechanism of synergistic cell killing caused by altered DNA lesion modification and enhanced apoptosis. However, alterations in cell cycle regulation may also play a role in the synergy between these two agents in certain human cancers. We recently showed that NF‐κB, a known anti‐apoptotic factor, was activated in various cancer cells after poisoning Topo I using clinically active drugs. NF‐κB activation was dependent on initial nuclear DNA damage followed by cytoplasmic signaling events. Cytoplasmic signaling leading to NF‐κB activation after Topo I poisons was diminished in cytoplasts (lacking nuclei) and in CEM/C2 cells that expressed a mutant Topo I protein that did not interact with Topo I‐active drugs. NF‐κB activation was intensified in S‐phase and blocked by aphidicolin, suggesting that activation was a result of double‐strand break formation due to Topo I poisoning and DNA replication. Dominant‐negative IkB expression augmented Topo I poison‐ mediated apoptosis. Elucidation of molecular signal transduction pathways after Topo I drug‐IR combinations may lead to improved radiotherapy by blocking anti‐apoptotic NF‐κB responses. Recent data also indicate that synergy caused by IR + Topo I poisons is different from radiosensitization by β‐lapachone (β‐lap), a “reported” Topo I and II‐α poison in vitro. In fact, β‐lap does not kill cells by poisoning either Topo I or II‐αin vivo. Instead, the compound is “activated” by an IR (damage)‐inducible enzyme, NAD(P)H:quinone oxidoreductase (NQO1), a gene cloned as x‐ray‐inducible transcript #3, xip3. Unlike the lesion modification pathway induced by IR + Topo I drugs, β‐lap kills cells via NQO1 futile cycle metabolism. Downstream apoptosis caused by β‐lap appears to be noncaspase‐mediated, involving calpain or a calpain‐like protease. Thus, although Topo I poisons or β‐lap in combination with IR both synergistically kill cancer cells, the mechanisms are very different.

Quinacrine Overcomes Resistance to Erlotinib by Inhibiting FACT, NF-κB, and Cell-Cycle Progression in Non–Small Cell Lung Cancer

Erlotinib is a tyrosine kinase inhibitor approved for the treatment of patients with advanced non–small cell lung cancer (NSCLC). In these patients, erlotinib prolongs survival but its benefit remains modest because many tumors express wild-type (wt) EGFR or develop a second-site EGFR mutation. To test drug combinations that could improve the efficacy of erlotinib, we combined erlotinib with quinacrine, which inhibits the FACT (facilitates chromatin transcription) complex that is required for NF-κB transcriptional activity. In A549 (wtEGFR), H1975 (EGFR-L858R/T790M), and H1993 (MET amplification) NSCLC cells, this drug combination was highly synergistic, as quantified by Chou–Talalay combination indices, and slowed xenograft tumor growth. At a sub-IC50 but more clinically attainable concentration of erlotinib, quinacrine, alone or in combination with erlotinib, significantly inhibited colony formation and induced cell-cycle arrest and apoptosis. Quinacrine decreased the level of active FACT subunit SSRP1 and suppressed NF-κB–dependent luciferase activity. Knockdown of SSRP1 decreased cell growth and sensitized cells to erlotinib. Moreover, transcriptomic profiling showed that quinacrine or combination treatment significantly affected cell-cycle–related genes that contain binding sites for transcription factors that regulate SSRP1 target genes. As potential biomarkers of drug combination efficacy, we identified genes that were more strongly suppressed by the combination than by single treatment, and whose increased expression predicted poorer survival in patients with lung adenocarcinoma. This preclinical study shows that quinacrine overcomes erlotinib resistance by inhibiting FACT and cell-cycle progression, and supports a clinical trial testing erlotinib alone versus this combination in advanced NSCLC. Mol Cancer Ther; 13(9); 2203–14. ©2014 AACR.