Targeting Cytotoxic Agents through EGFR-Mediated Covalent Binding and Release.

A major drawback of cytotoxic chemotherapy is the lack of selectivity toward noncancerous cells. The targeted delivery of cytotoxic drugs to tumor cells is a longstanding goal in cancer research. We proposed that covalent inhibitors could be adapted to deliver cytotoxic agents, conjugated to the ß-position of the Michael acceptor, via an addition-elimination mechanism promoted by covalent binding. Studies on model systems showed that conjugated 5-fluorouracil (5FU) could be released upon thiol addition in relevant time scales. A series of covalent epidermal growth factor receptor (EGFR) inhibitors were synthesized as their 5FU derivatives. Achieving the desired release of 5FU was demonstrated to depend on the electronics and geometry of the compounds. Mass spectrometry and NMR studies demonstrated an anilinoquinazoline acrylate ester conjugate bound to EGFR with the release of 5FU. This work establishes that acrylates can be used to release conjugated molecules upon covalent binding to proteins and could be used to develop targeted therapeutics.

An Alkynylpyrimidine-Based Covalent Inhibitor That Targets a Unique Cysteine in NF-κB-Inducing Kinase.

NF-κB-inducing kinase (NIK) is a key enzyme in the noncanonical NF-κB pathway, of interest in the treatment of a variety of diseases including cancer. Validation of NIK as a drug target requires potent and selective inhibitors. The protein contains a cysteine residue at position 444 in the back pocket of the active site, unique within the kinome. Analysis of existing inhibitor scaffolds and early structure-activity relationships (SARs) led to the design of C444-targeting covalent inhibitors based on alkynyl heterocycle warheads. Mass spectrometry provided proof of the covalent mechanism, and the SAR was rationalized by computational modeling. Profiling of more potent analogues in tumor cell lines with constitutively activated NIK signaling induced a weak antiproliferative effect, suggesting that kinase inhibition may have limited impact on cancer cell growth. This study shows that alkynyl heterocycles are potential cysteine traps, which may be employed where common Michael acceptors, such as acrylamides, are not tolerated.

IKBKE activity enhances AR levels in advanced prostate cancer via modulation of the Hippo pathway.

Resistance to androgen receptor (AR) targeting therapeutics in prostate cancer (PC) is a significant clinical problem. Mechanisms by which this is accomplished include AR amplification and expression of AR splice variants, demonstrating that AR remains a key therapeutic target in advanced disease. For the first time we show that IKBKE drives AR signalling in advanced PC. Significant inhibition of AR regulated gene expression was observed upon siRNA-mediated IKBKE depletion or pharmacological inhibition due to inhibited AR gene expression in multiple cell line models including a LNCaP derivative cell line resistant to the anti-androgen, enzalutamide (LNCaP-EnzR). Phenotypically, this resulted in significant inhibition of proliferation, migration and colony forming ability suggesting that targeting IKBKE could circumvent resistance to AR targeting therapies. Indeed, pharmacological inhibition in the CWR22Rv1 xenograft mouse model reduced tumour size and enhanced survival. Critically, this was validated in patient-derived explants where enzymatic inactivation of IKBKE reduced cell proliferation and AR expression. Mechanistically, we provide evidence that IKBKE regulates AR levels via Hippo pathway inhibition to reduce c-MYC levels at cis-regulatory elements within the AR gene. Thus, IKBKE is a therapeutic target in advanced PC suggesting repurposing of clinically tested IKBKE inhibitors could be beneficial to castrate resistant PC patients.

Selective DNA-PKcs inhibition extends the therapeutic index of localized radiotherapy and chemotherapy.

Potentiating radiotherapy and chemotherapy by inhibiting DNA damage repair is proposed as a therapeutic strategy to improve outcomes for patients with solid tumors. However, this approach risks enhancing normal tissue toxicity as much as tumor toxicity, thereby limiting its translational impact. Using NU5455, a newly identified highly selective oral inhibitor of DNA-dependent protein kinase catalytic subunit (DNA-PKcs) activity, we found that it was indeed possible to preferentially augment the effect of targeted radiotherapy on human orthotopic lung tumors without influencing acute DNA damage or a late radiation-induced toxicity (fibrosis) to normal mouse lung. Furthermore, while NU5455 administration increased both the efficacy and the toxicity of a parenterally administered topoisomerase inhibitor, it enhanced the activity of doxorubicin released locally in liver tumor xenografts without inducing any adverse effect. This strategy is particularly relevant to hepatocellular cancer, which is treated clinically with localized drug-eluting beads and for which DNA-PKcs activity is reported to confer resistance to treatment. We conclude that transient pharmacological inhibition of DNA-PKcs activity is effective and tolerable when combined with localized DNA-damaging therapies and thus has promising clinical potential.

Identification of a novel orally bioavailable ERK5 inhibitor with selectivity over p38α and BRD4.

Extracellular regulated kinase 5 (ERK5) signalling has been implicated in driving a number of cellular phenotypes including endothelial cell angiogenesis and tumour cell motility. Novel ERK5 inhibitors were identified using high throughput screening, with a series of pyrrole-2-carboxamides substituted at the 4-position with an aroyl group being found to exhibit IC50 values in the micromolar range, but having no selectivity against p38α MAP kinase. Truncation of the N-substituent marginally enhanced potency (∼3-fold) against ERK5, but importantly attenuated inhibition of p38α. Systematic variation of the substituents on the aroyl group led to the selective inhibitor 4-(2-bromo-6-fluorobenzoyl)-N-(pyridin-3-yl)-1H-pyrrole-2-carboxamide (IC50 0.82 µM for ERK5; IC50 > 120 µM for p38α). The crystal structure (PDB 5O7I) of this compound in complex with ERK5 has been solved. This compound was orally bioavailable and inhibited bFGF-driven Matrigel plug angiogenesis and tumour xenograft growth. The selective ERK5 inhibitor described herein provides a lead for further development into a tool compound for more extensive studies seeking to examine the role of ERK5 signalling in cancer and other diseases.

Preclinical evaluation of the first intravenous small molecule MDM2 antagonist alone and in combination with temozolomide in neuroblastoma.

High-risk neuroblastoma, a predominantly TP53 wild-type (wt) tumour, is incurable in >50% patients supporting the use of MDM2 antagonists as novel therapeutics. Idasanutlin (RG7388) shows in vitro synergy with chemotherapies used to treat neuroblastoma. This is the first study to evaluate the in vivo efficacy of the intravenous idasanutlin prodrug, RO6839921 (RG7775), both alone and in combination with temozolomide in TP53 wt orthotopic neuroblastoma models. Detection of active idasanutlin using liquid chromatography-mass spectrometry and p53 pathway activation by ELISA assays and Western analysis showed peak plasma levels 1 h post-treatment with maximal p53 pathway activation 3-6 h post-treatment. RO6839921 and temozolomide, alone or in combination in mice implanted with TP53 wt SHSY5Y-Luc and NB1691-Luc cells showed that combined RO6839921 and temozolomide led to greater tumour growth inhibition and increase in survival compared to vehicle control. Overall, RO6839921 had a favourable pharmacokinetic profile consistent with intermittent dosing and was well tolerated alone and in combination. These preclinical studies support the further development of idasanutlin in combination with temozolomide in neuroblastoma in early phase clinical trials.

PARP inhibitor rucaparib induces changes in NAD levels in cells and liver tissues as assessed by MRS.

Poly(adenosine diphosphate ribose) polymerases (PARPs) are multifunctional proteins which play a role in many cellular processes. Namely, PARP1 and PARP2 have been shown to be involved in DNA repair, and therefore are valid targets in cancer treatment with PARP inhibitors, such as rucaparib, currently in clinical trials. Proton magnetic resonance spectroscopy (1 H-MRS) was used to study the impact of rucaparib in vitro and ex vivo in liver tissue from mice, via quantitative analysis of nicotinamide adenosine diphosphate (NAD+ ) spectra, to assess the potential of MRS as a biomarker of the PARP inhibitor response. SW620 (colorectal) and A2780 (ovarian) cancer cell lines, and PARP1 wild-type (WT) and PARP1 knock-out (KO) mice, were treated with rucaparib, temozolomide (methylating agent) or a combination of both drugs. 1 H-MRS spectra were obtained from perchloric acid extracts of tumour cells and mouse liver. Both cell lines showed an increase in NAD+ levels following PARP inhibitor treatment in comparison with temozolomide treatment. Liver extracts from PARP1 WT mice showed a significant increase in NAD+ levels after rucaparib treatment compared with untreated mouse liver, and a significant decrease in NAD+ levels in the temozolomide-treated group. The combination of rucaparib and temozolomide did not prevent the NAD+ depletion caused by temozolomide treatment. The 1 H-MRS results show that NAD+ levels can be used as a biomarker of PARP inhibitor and methylating agent treatments, and suggest that in vivo measurement of NAD+ would be valuable.

Functional characterisation of a novel ovarian cancer cell line, NUOC-1.

BACKGROUND: Cell lines provide a powerful model to study cancer and here we describe a new spontaneously immortalised epithelial ovarian cancer cell line (NUOC-1) derived from the ascites collected at a time of primary debulking surgery for a mixed endometrioid / clear cell / High Grade Serous (HGS) histology. RESULTS: This spontaneously immortalised cell line was found to maintain morphology and epithelial markers throughout long-term culture. NUOC-1 cells grow as an adherent monolayer with a doubling time of 58 hours. The cells are TP53 wildtype, positive for PTEN, HER2 and HER3 expression but negative for oestrogen, progesterone and androgen receptor expression. NUOC-1 cells are competent in homologous recombination and non-homologous end joining, but base excision repair defective. Karyotype analysis demonstrated a complex tetraploid karyotype. SNP array analysis of parent and derived subpopulations (NUOC-1-A1 and NUOC-1-A2) cells demonstrated heterogeneous cell populations with numerous copy number alterations and a pro-amplification phenotype. The characteristics of this new cell line lends it to be an excellent model for investigation of a number of the identified targets. MATERIALS AND METHODS: The cell line has been characterised for growth, drug sensitivity, expression of common ovarian markers and mutations, clonogenic potential and ability to form xenografts in SCID mice. Copy number changes and clonal evolution were assessed by SNP arrays.

Enhanced anti-tumour activity of the combination of the novel MEK inhibitor WX-554 and the novel PI3K inhibitor WX-037.

PURPOSE: Tumours frequently have defects in multiple oncogenic pathways, e.g. MAPK and PI3K signalling pathways, and combinations of targeted therapies may be required for optimal activity. This study evaluated the novel MEK inhibitor WX-554 and the novel PI3K inhibitor WX-037, as single agents and in combination, in colorectal carcinoma cell lines and tumour xenograft-bearing mice. METHODS: In vitro growth inhibition, survival and signal transduction were measured using the Sulforhodamine B, clonogenic and Western blotting assays, respectively, in HCT116 and HT29 cell lines. In vivo anti-tumour efficacy and pharmacokinetic properties were assessed in HCT116 and HT29 human colorectal cancer xenograft tumour-bearing mice. RESULTS: The combination of WX-554 and WX-037 exhibited marked synergistic growth inhibition in vitro, which was associated with increased cytotoxicity and enhanced inhibition of ERK and S6 phosphorylation, compared to either agent alone. Pharmacokinetic analyses indicated that there was no PK interaction between the two drugs at low doses, but that at higher doses, WX-037 may delay the tumour uptake of WX-554. In vivo efficacy studies revealed that the combination of WX-037 and WX-554 was non-toxic and exhibited marked tumour growth inhibition greater than observed with either agent alone. CONCLUSION: These studies show for the first time that combination treatment with the novel MEK inhibitor WX-554 and the novel PI3K inhibitor WX-037 can induce synergistic growth inhibition in vitro, which translates into enhanced anti-tumour efficacy in vivo.

Combined PI3K and CDK2 inhibition induces cell death and enhances in vivo antitumour activity in colorectal cancer.

BACKGROUND: The phosphatidylinositol-3-kinase/mammalian target of rapamycin (PI3K/mTOR) pathway is commonly deregulated in human cancer, hence many PI3K and mTOR inhibitors have been developed and have now reached clinical trials. Similarly, CDKs have been investigated as cancer drug targets. METHODS: We have synthesised and characterised a series of 6-aminopyrimidines identified from a kinase screen that inhibit PI3K and/or mTOR and/or CDK2. Kinase inhibition, tumour cell growth, cell cycle distribution, cytotoxicity and signalling experiments were undertaken in HCT116 and HT29 colorectal cancer cell lines, and in vivo HT29 efficacy studies. RESULTS: 2,6-Diaminopyrimidines with an O(4)-cyclohexylmethyl substituent and a C-5-nitroso or cyano group (1,2,5) induced cell cycle phase alterations and were growth inhibitory (GI50<20 µM). Compound 1, but not 2 or 5, potently inhibits CDK2 (IC50=0.1 nM) as well as PI3K, and was cytotoxic at growth inhibitory concentrations. Consistent with kinase inhibition data, compound 1 reduced phospho-Rb and phospho-rS6 at GI50 concentrations. Combination of NU6102 (CDK2 inhibitor) and pictilisib (GDC-0941; pan-PI3K inhibitor) resulted in synergistic growth inhibition, and enhanced cytotoxicity in HT29 cells in vitro and HT29 tumour growth inhibition in vivo. CONCLUSIONS: These studies identified a novel series of mixed CDK2/PI3K inhibitors and demonstrate that dual targeting of CDK2 and PI3K can result in enhanced antitumour activity.

Pre-clinical use of isogenic cell lines and tumours in vitro and in vivo for predictive biomarker discovery; impact of KRAS and PI3KCA mutation status on MEK inhibitor activity is model dependent.

Studies to identify predictive biomarkers can be carried out in isogenic cancer cell lines, which enable interrogation of the effect of a specific mutation. We assessed the effects of four drugs, the PI3K-mammalian target of rapamycin inhibitor dactolisib, the PI3K inhibitor pictrelisib, and the MEK (MAPK/ERK Kinase) inhibitors PD 0325901 and selumetinib, in isogenic DLD1 parental, KRAS(+/-), KRAS(G13D/-), PIK3CA(+/-) and PIK3CA(E545K/-) colorectal carcinoma cell lines. Importantly, we found substantial differences in the growth of these cells and in their drug sensitivity depending on whether they were studied under 2D (standard tissue culture on plastic) or 3D (in vitro soft agar and in vivo xenograft) conditions. DLD1 KRAS(+/-) and DLD1 PIK3CA(+/-) cells were more sensitive to MEK inhibitors than parental, DLD1 KRAS(G13D/-) and DLD1 PIK3CA(E545K/-) cells under 2D conditions, whereas DLD1 KRAS(G13D/-) and DLD1 PIK3CA(E545K/-) xenografts were sensitive to 10 mg/kg daily ×14 PD 0325901 in vivo (p ≤ 0.02) but tumours derived from parental DLD1 cells were not. These findings indicate that KRAS and PIK3CA mutations can influence the response of DLD1 colorectal cancer cell lines to MEK and PI3K inhibitors, but that the effect is dependent on the experimental model used to assess drug sensitivity.

Vasoactivity of rucaparib, a PARP-1 inhibitor, is a complex process that involves myosin light chain kinase, P2 receptors, and PARP itself.

Therapeutic inhibition of poly(ADP-ribose) polymerase (PARP), as monotherapy or to supplement the potencies of other agents, is a promising strategy in cancer treatment. We previously reported that the first PARP inhibitor to enter clinical trial, rucaparib (AG014699), induced vasodilation in vivo in xenografts, potentiating response to temozolomide. We now report that rucaparib inhibits the activity of the muscle contraction mediator myosin light chain kinase (MLCK) 10-fold more potently than its commercially available inhibitor ML-9. Moreover, rucaparib produces additive relaxation above the maximal degree achievable with ML-9, suggesting that MLCK inhibition is not solely responsible for dilation. Inhibition of nitric oxide synthesis using L-NMMA also failed to impact rucaparib's activity. Rucaparib contains the nicotinamide pharmacophore, suggesting it may inhibit other NAD+-dependent processes. NAD+ exerts P2 purinergic receptor-dependent inhibition of smooth muscle contraction. Indiscriminate blockade of the P2 purinergic receptors with suramin abrogated rucaparib-induced vasodilation in rat arterial tissue without affecting ML-9-evoked dilation, although the specific receptor subtypes responsible have not been unequivocally identified. Furthermore, dorsal window chamber and real time tumor vessel perfusion analyses in PARP-1-/- mice indicate a potential role for PARP in dilation of tumor-recruited vessels. Finally, rucaparib provoked relaxation in 70% of patient-derived tumor-associated vessels. These data provide tantalising evidence of the complexity of the mechanism underlying rucaparib-mediated vasodilation.

DNA-PK-A candidate driver of hepatocarcinogenesis and tissue biomarker that predicts response to treatment and survival.

PURPOSE: Therapy resistance and associated liver disease make hepatocellular carcinomas (HCC) difficult to treat with traditional cytotoxic therapies, whereas newer targeted approaches offer only modest survival benefit. We focused on DNA-dependent protein kinase, DNA-PKcs, encoded by PRKDC and central to DNA damage repair by nonhomologous end joining. Our aim was to explore its roles in hepatocarcinogenesis and as a novel therapeutic candidate. EXPERIMENTAL DESIGN: PRKDC was characterized in liver tissues from of 132 patients [normal liver (n = 10), cirrhotic liver (n = 13), dysplastic nodules (n = 18), HCC (n = 91)] using Affymetrix U133 Plus 2.0 and 500 K Human Mapping SNP arrays (cohort 1). In addition, we studied a case series of 45 patients with HCC undergoing diagnostic biopsy (cohort 2). Histological grading, response to treatment, and survival were correlated with DNA-PKcs quantified immunohistochemically. Parallel in vitro studies determined the impact of DNA-PK on DNA repair and response to cytotoxic therapy. RESULTS: Increased PRKDC expression in HCC was associated with amplification of its genetic locus in cohort 1. In cohort 2, elevated DNA-PKcs identified patients with treatment-resistant HCC, progressing at a median of 4.5 months compared with 16.9 months, whereas elevation of activated pDNA-PK independently predicted poorer survival. DNA-PKcs was high in HCC cell lines, where its inhibition with NU7441 potentiated irradiation and doxorubicin-induced cytotoxicity, whereas the combination suppressed HCC growth in vitro and in vivo. CONCLUSIONS: These data identify PRKDC/DNA-PKcs as a candidate driver of hepatocarcinogenesis, whose biopsy characterization at diagnosis may impact stratification of current therapies, and whose specific future targeting may overcome resistance.

The conditioned place preference test for assessing welfare consequences and potential refinements in a mouse bladder cancer model.

Most pre-clinical analgesic efficacy assays still involve nociceptive testing in rodents. This is despite concerns as to the relevance of these tests for evaluating the pain-preventative properties of drugs. More appropriate methods would target pain rather than nociception, but these are currently not available, so it remains unknown whether animal pain equates to the negatively affective and subjective/emotional state it causes in humans. Mouse cancer models are common despite the likelihood of substantial pain. We used Conditioned Place Preference (CPP) testing, assessments of thermal hyperalgesia and behaviour to determine the likelihood that MBT-2 bladder cancer impacts negatively on mouse welfare, such as by causing pain. There was no CPP to saline, but morphine preference in tumour bearing mice exceeded that seen in tumour-free controls. This occurred up to 10 days before the study end-point alongside reduced body weight, development of hyperalgesia and behaviour changes. These effects indicated mice experienced a negative welfare state caused by malaise (if not pain) before euthanasia. Due to the complexity of the assessments needed to demonstrate this, it is unlikely that this approach could be used for routine welfare assessment on a study-by-study basis. However, our results show mice in sufficiently similar studies are likely to benefit from more intensive severity assessment and re-evaluation of end-points with a view to implementing appropriate refinements. In this particular case, a refinement would have been to have euthanased mice at least 7 days earlier or possibly by provision of end-stage pain relief. CPP testing was found to be a helpful method to investigate the responses of mice to analgesics, possibly on a subjective level. These findings and those of other recent studies show it could be a valuable method of screening candidate analgesics for efficacy against cancer pain and possibly other pain or disease models.

The enhanced in vivo activity of the combination of a MEK and a PI3K inhibitor correlates with [18F]-FLT PET in human colorectal cancer xenograft tumour-bearing mice.

Combined targeting of the MAPK and PI3K signalling pathways in cancer may be necessary for optimal therapeutic activity. To support clinical studies of combination therapy, 3'-deoxy-3'-[(18)F]-fluorothymidine ([(18)F]-FLT) uptake measured by Positron Emission Tomography (PET) was evaluated as a non-invasive surrogate response biomarker in pre-clinical models. The in vivo anti-tumour efficacy and PK-PD properties of the MEK inhibitor PD 0325901 and the PI3K inhibitor GDC-0941, alone and in combination, were evaluated in HCT116 and HT29 human colorectal cancer xenograft tumour-bearing mice, and [(18)F]-FLT PET investigated in mice bearing HCT116 xenografts. Dual targeting of PI3K and MEK induced marked tumour growth inhibition in vivo, and enhanced anti-tumour activity was predicted by [(18)F]-FLT PET scanning after 2 days of treatment. Pharmacodynamic analyses using the combination of the PI3K inhibitor GDC-0941 and the MEK inhibitor PD 0325901 revealed that increased efficacy is associated with an enhanced inhibition of the phosphorylation of ERK1/2, S6 and 4EBP1, compared to that observed with either single agent, and maintained inhibition of AKT phosphorylation. Pharmacokinetic studies indicated that there was no marked PK interaction between the two drugs. Together these results indicate that the combination of PI3K and MEK inhibitors can result in significant efficacy, and demonstrate for the first time that [(18)F]-FLT PET can be correlated to the improved efficacy of combined PI3K and MEK inhibitor treatment.

Characterisation of the roles of ABCB1, ABCC1, ABCC2 and ABCG2 in the transport and pharmacokinetics of actinomycin D in vitro and in vivo.

Actinomycin D plays a key role in the successful treatment of Wilms tumour. However, associated liver toxicities remain a drawback to potentially curative treatment. We have used MDCKII cells over-expressing ABCB1, ABCC1, ABCC2 and ABCG2, alongside knockout mouse models to characterise actinomycin D transport and its impact on pharmacokinetics. Growth inhibition, intracellular accumulation and cellular efflux assays were utilised. A 59-fold difference in GI(50) was observed between MDCKII-WT and MDCKII-ABCB1 cells (12.7 nM vs. 745 nM, p<0.0001). Reduced sensitivity was also seen in MDCKII-ABCC1 and ABCC2 cells (GI(50) 25.7 and 40.4 nM respectively, p<0.0001). Lower intracellular accumulation of actinomycin D was observed in MDCKII-ABCB1 cells as compared to MDCKII-WT (0.98 nM vs. 0.1 nM, p<0.0001), which was reversed upon ABCB1 inhibition. Lower accumulation was also seen in MDCKII-ABCC1 and ABCC2 cells. Actinomycin D efflux over 2 h was most pronounced in MDCKII-ABCB1 cells, with 5.5-fold lower intracellular levels compared to WT. In vivo studies showed that actinomycin D plasma concentrations were significantly higher in Abcb1a/1b(-/-) as compared to WT mice following administration of 0.5 mg/kg actinomycin D (AUC(0-6 h) 242 vs. 152 µg/Lh respectively). While comparable actinomycin D concentrations were observed in the kidneys and livers of Abcb1a/1b(-/-) and Abcc2(-/-) mice, concentrations in the brain were significantly higher at 6h following drug administration in Abcb1a/1b(-/-) mice compared to WT. Results confirm actinomycin D as a substrate for ABCB1, ABCC1 and ABCC2, and indicate that Abcb1a/1b and Abcc2 can influence the in vivo disposition of actinomycin D. These data have implications for ongoing clinical pharmacology trials involving children treated with actinomycin D.

Side population in human non-muscle invasive bladder cancer enriches for cancer stem cells that are maintained by MAPK signalling.

Side population (SP) and ABC transporter expression enrich for stem cells in numerous tissues. We explored if this phenotype characterised human bladder cancer stem cells (CSCs) and attempted to identify regulatory mechanisms. Focusing on non-muscle invasive bladder cancer (NMIBC), multiple human cell lines were used to characterise SP and ABC transporter expression. In vitro and in vivo phenotypic and functional assessments of CSC behaviour were undertaken. Expression of putative CSC marker ABCG2 was assessed in clinical NMIBC samples (n = 148), and a role for MAPK signalling, a central mechanism of bladder tumourigenesis, was investigated. Results showed that the ABCG2 transporter was predominantly expressed and was up-regulated in the SP fraction by 3-fold (ABCG2(hi)) relative to the non-SP (NSP) fraction (ABCG2(low)). ABCG2(hi) SP cells displayed enrichment of stem cell markers (Nanog, Notch1 and SOX2) and a three-fold increase in colony forming efficiency (CFE) in comparison to ABCG2(low) NSP cells. In vivo, ABCG2(hi) SP cells enriched for tumour growth compared with ABCG2(low) NSP cells, consistent with CSCs. pERK was constitutively active in ABCG2(hi) SP cells and MEK inhibition also inhibited the ABCG2(hi) SP phenotype and significantly suppressed CFE. Furthermore, on examining clinical NMIBC samples, ABCG2 expression correlated with increased recurrence and decreased progression free survival. Additionally, pERK expression also correlated with decreased progression free survival, whilst a positive correlation was further demonstrated between ABCG2 and pERK expression. In conclusion, we confirm ABCG2(hi) SP enriches for CSCs in human NMIBC and MAPK/ERK pathway is a suitable therapeutic target.

Therapy-induced carboplatin-DNA adduct levels in human ovarian tumours in relation to assessment of adduct measurement in mouse tissues.

Despite an increasing understanding of the molecular mechanisms by which platinum drug DNA adducts interact with cellular processes, the relationship between adduct formation in tumours and clinical response remains unclear. We have determined carboplatin-DNA adduct levels in biopsies removed from ovarian cancer patients following treatment. Reliability of DNA adduct measurements in tissues samples were assessed using experimental animals. Platinum-DNA adduct levels were measured using inductively coupled plasma mass spectrometry (ICP-MS) and plasma drug concentrations determined by atomic absorption spectrometry (AAS). Adduct levels in tissues and plasma pharmacokinetics were determined in Balb/c mice exposed to platinum drugs. Comparisons of adduct levels in tumour and normal tissue were made in nu/nu mice carrying human neuroblastoma xenografts. At 30 min post-cisplatin administration, adduct levels in DNA from kidney and liver were approximately 10- and 6-fold higher than spleen or tumour. By 60 min, levels in liver and kidney, but not spleen or tumour, had fallen considerably. Carboplatin showed high adduct levels only in kidney. Adduct levels in tumour xenografts were comparable to those induced in vitro with similar drug exposures. In clinical samples removed 6h after drug administration, adduct levels ranged from 1.9 to 4.3 and 0.2 to 3.6 nmol Pt/g DNA for tumour biopsies and peripheral blood mononuclear cells, respectively. No correlation was apparent between these two data sets. The present results demonstrate that reliable measurements of adducts in clinical tumours are feasible. Future results should provide insight into drug resistance.

The clinically active PARP inhibitor AG014699 ameliorates cardiotoxicity but does not enhance the efficacy of doxorubicin, despite improving tumor perfusion and radiation response in mice.

AG014699 was the first inhibitor of the DNA repair enzyme PARP-1 to enter clinical trial in cancer patients. In addition to enhancing the cytotoxic effect of DNA-damaging chemotherapies, we have previously shown that AG014699 is vasoactive, thereby having the potential to improve drug biodistribution. The effectiveness of the clinical agent doxorubicin is confounded both by poor tumor penetration and cardiotoxicity elicited via PARP hyperactivation. In this study, we analyzed the impact of AG014699 on doxorubicin tolerance and response in breast (MDA-MB-231) and colorectal (SW620, LoVo) tumor models in vitro and in vivo. As anticipated, AG014699 did not potentiate the response to doxorubicin in vitro. In vivo, AG014699 did not influence the pharmacokinetics of doxorubicin; however, it did ameliorate cardiotoxicity. Both toxicity and extent of amelioration were more pronounced in male than in female mice. AG014699 improved vessel perfusion in both MDA-MB-231 and SW620 tumors; however, this neither led to improved tumor-accumulation of doxorubicin nor enhanced therapeutic response. In contrast, when combined with radiotherapy, AG014699 significantly enhanced response both in vitro and in vivo. Real-time assessment of tumor vessel function and companion histologic studies indicate that doxorubicin causes a profound antivascular effect that counters the positive effect of AG014699 on perfusion. These data indicate that although AG014699 can enhance response to some chemotherapeutic drugs via improved delivery, this does not apply to doxorubicin. PARP inhibitors may still be of use to counter doxorubicin toxicity, and if the gender effect translates from rodents to humans, this would have greater effect in males.