Maciej Kaliszczak

A novel small molecule hydroxamate preferentially inhibits HDAC6 activity and tumour growth

Background:This study investigates whether a histone deacetylase subtype 6 (HDAC6) inhibitor could be used in the treatment of solid tumours.Methods:We evaluated the effect of a novel inhibitor, C1A, on HDAC6 biochemical activity and cell growth. We further examined potential of early noninvasive imaging of cell proliferation by [18F]fluorothymidine positron emission tomography ([18F]FLT-PET) to detect therapy response.Results:C1A induced sustained acetylation of HDAC6 substrates, α-tubulin and HSP90, compared with current clinically approved HDAC inhibitor SAHA. C1A induced apoptosis and inhibited proliferation of a panel of human tumour cell lines from different origins in the low micromolar range. Systemic administration of the drug inhibited the growth of colon tumours in vivo by 78%. The drug showed restricted activity on gene expression with <0.065% of genes modulated during 24 h of treatment. C1A treatment reduced tumour [18F]FLT uptake by 1.7-fold at 48 h, suggesting that molecular imaging could provide value in future studies of this compound.Conclusion:C1A preferentially inhibits HDAC6 and modulates HDAC6 downstream targets leading to growth inhibition of a diverse set of cancer cell lines. This property together with the favourable pharmacokinetics and efficacy in vivo makes it a candidate for further pre-clinical and clinical development.

Positron Emission Tomography Imaging with 18F-Labeled ZHER2:2891 Affibody for Detection of HER2 Expression and Pharmacodynamic Response to HER2-Modulating Therapies

Purpose: Expression of HER2 has profound implications on treatment strategies in various types of cancer. We investigated the specificity of radiolabeled HER2-targeting ZHER2:2891 Affibody, [18F]GE-226, for positron emission tomography (PET) imaging. Experimental Design: Intrinsic cellular [18F]GE-226 uptake and tumor-specific tracer binding were assessed in cells and xenografts with and without drug treatment. Specificity was further determined by comparing tumor localization of a fluorescently labeled analogue with DAKO HercepTest. Results: [18F]GE-226 uptake was 11- to 67-fold higher in 10 HER2-positive versus HER2-negative cell lines in vitro independent of lineage. Uptake in HER2-positive xenografts was rapid with net irreversible binding kinetics making possible the distinction of HER2-negative [MCF7 and MCF7-p95HER2: NUV60 (%ID/mL) 6.1 ± 0.7; Ki (mL/cm3/min) 0.0069 ± 0.0014] from HER2-positive tumors (NUV60 and Ki: MCF7-HER2, 10.9 ± 1.5 and 0.015 ± 0.0035; MDA-MB-361, 18.2 ± 3.4 and 0.025 ± 0.0052; SKOV-3, 18.7 ± 2.4 and 0.036 ± 0.0065) within 1 hour. Tumor uptake correlated with HER2 expression determined by ELISA (r2 = 0.78), and a fluorophore-labeled tracer analogue colocalized with HER2 expression. Tracer uptake was not influenced by short-term or continuous treatment with trastuzumab in keeping with differential epitope binding, but reflected HER2 degradation by short-term NVP-AUY922 treatment in SKOV-3 xenografts (NUV60: 13.5 ± 2.1 %ID/mL vs. 9.0 ± 0.9 %ID/mL for vehicle or drug, respectively). Conclusions: [18F]GE-226 binds with high specificity to HER2 independent of cell lineage. The tracer has potential utility for HER2 detection, irrespective of prior trastuzumab treatment, and to discern HSP90 inhibitor-mediated HER2 degradation. Clin Cancer Res; 20(6); 1632–43. ©2014 AACR.

Development of a cyclin-dependent kinase inhibitor devoid of ABC transporter-dependent drug resistance.

Background:Cyclin-dependent kinases (CDKs) control cell cycle progression, RNA transcription and apoptosis, making them attractive targets for anticancer drug development. Unfortunately, CDK inhibitors developed to date have demonstrated variable efficacy.Methods:We generated drug-resistant cells by continuous low-dose exposure to a model pyrazolo[1,5-a]pyrimidine CDK inhibitor and investigated potential structural alterations for optimal efficacy.Results:We identified induction of the ATP-binding cassette (ABC) transporters, ABCB1 and ABCG2, in resistant cells. Assessment of features involved in the ABC transporter substrate specificity from a compound library revealed high polar surface area (>100 Å2) as a key determinant of transporter interaction. We developed ICEC-0782 that preferentially inhibited CDK2, CDK7 and CDK9 in the nanomolar range. The compound inhibited phosphorylation of CDK substrates and downregulated the short-lived proteins, Mcl-1 and cyclin D1. ICEC-0782 induced G2/M arrest and apoptosis. The permeability and cytotoxicity of ICEC-0782 were unaffected by ABC transporter expression. Following daily oral dosing, the compound inhibited growth of human colon HCT-116 and human breast MCF7 tumour xenografts in vivo by 84% and 94%, respectively.Conclusion:We identified a promising pyrazolo[1,5-a]pyrimidine compound devoid of ABC transporter interaction, highly suitable for further preclinical and clinical evaluation for the treatment of cancer.

Preclinical Evaluation of 3-18F-Fluoro-2,2-Dimethylpropionic Acid as an Imaging Agent for Tumor Detection

Deregulated cellular metabolism is a hallmark of many cancers. In addition to increased glycolytic flux, exploited for cancer imaging with 18F-FDG, tumor cells display aberrant lipid metabolism. Pivalic acid is a short-chain, branched carboxylic acid used to increase oral bioavailability of prodrugs. After prodrug hydrolysis, pivalic acid undergoes intracellular metabolism via the fatty acid oxidation pathway. We have designed a new probe, 3-18F-fluoro-2,2-dimethylpropionic acid, also called 18F-fluoro-pivalic acid (18F-FPIA), for the imaging of aberrant lipid metabolism and cancer detection. Methods: Cell intrinsic uptake of 18F-FPIA was measured in murine EMT6 breast adenocarcinoma cells. In vivo dynamic imaging, time course biodistribution, and radiotracer stability testing were performed. 18F-FPIA tumor retention was further compared in vivo to 18F-FDG uptake in several xenograft models and inflammatory tissue. Results: 18F-FPIA rapidly accumulated in EMT6 breast cancer cells, with retention of intracellular radioactivity predicted to occur via a putative 18F-FPIA carnitine-ester. The radiotracer was metabolically stable to degradation in mice. In vivo imaging of implanted EMT6 murine and BT474 human breast adenocarcinoma cells by 18F-FPIA PET showed rapid and extensive tumor localization, reaching 9.1% ± 0.5% and 7.6% ± 1.2% injected dose/g, respectively, at 60 min after injection. Substantial uptake in the cortex of the kidney was seen, with clearance primarily via urinary excretion. Regarding diagnostic utility, uptake of 18F-FPIA was comparable to that of 18F-FDG in EMT6 tumors but superior in the DU145 human prostate cancer model (54% higher uptake; P = 0.002). Furthermore, compared with 18F-FDG, 18F-FPIA had lower normal-brain uptake resulting in a superior tumor-to-brain ratio (2.5 vs. 1.3 in subcutaneously implanted U87 human glioma tumors; P = 0.001), predicting higher contrast for brain cancer imaging. Both radiotracers showed increased localization in inflammatory tissue. Conclusion: 18F-FPIA shows promise as an imaging agent for cancer detection and warrants further investigation.

Design of symmetrical and nonsymmetrical N,N-dimethylaminopyridine derivatives as highly potent choline kinase alpha inhibitors

Choline kinase alpha is hyperactivated in many solid tumours and regulates malignant progression, making it a promising cancer drug target. The successful design and synthesis of novel inhibitors with high cellular activity are described.

The novel choline kinase inhibitor ICL-CCIC-0019 reprograms cellular metabolism and inhibits cancer cell growth

The glycerophospholipid phosphatidylcholine is the most abundant phospholipid species of eukaryotic membranes and essential for structural integrity and signaling function of cell membranes required for cancer cell growth. Inhibition of choline kinase alpha (CHKA), the first committed step to phosphatidylcholine synthesis, by the selective small-molecule ICL-CCIC-0019, potently suppressed growth of a panel of 60 cancer cell lines with median GI50 of 1.12 μM and inhibited tumor xenograft growth in mice. ICL-CCIC-0019 decreased phosphocholine levels and the fraction of labeled choline in lipids, and induced G1 arrest, endoplasmic reticulum stress and apoptosis. Changes in phosphocholine cellular levels following treatment could be detected non-invasively in tumor xenografts by [18F]-fluoromethyl-[1,2–2H4]-choline positron emission tomography. Herein, we reveal a previously unappreciated effect of choline metabolism on mitochondria function. Comparative metabolomics demonstrated that phosphatidylcholine pathway inhibition leads to a metabolically stressed phenotype analogous to mitochondria toxin treatment but without reactive oxygen species activation. Drug treatment decreased mitochondria function with associated reduction of citrate synthase expression and AMPK activation. Glucose and acetate uptake were increased in an attempt to overcome the metabolic stress. This study indicates that choline pathway pharmacological inhibition critically affects the metabolic function of the cell beyond reduced synthesis of phospholipids.

Targeting autophagy sensitises lung cancer cells to Src family kinase inhibitors

Lung cancer is the main cancer killer in both men and women, mostly due to the rapid development of drug resistant metastatic disease. Here, we evaluate the potential involvement of SRC family kinases (SFK) in lung cancer biology and assess the possible benefits of their inhibition as a therapeutic approach. We demonstrated that various SRC family members, including LYN and LCK, normally expressed solely in hematopoietic cells and neural tissues, are overexpressed and activated in a panel of SCLC and NSCLC cell lines. This was clinically relevant as LYN and FYN are also overexpressed in lung cancer clinical specimens. Moreover, LYN overexpression correlated with decreased patient survival on univariate and multivariate analysis. Dasatinib (BMS-354825), a SRC/ABL inhibitor, effectively blocked SFK activation at nanomolar concentrations which correlated with a significant decrease in cell numbers of multiple lung cancer cell lines. This effect was matched by a decrease in DNA synthesis, but only moderate induction of apoptosis. Indeed, dasatinib as well as PP2, another SFK inhibitor, strongly induced autophagy that likely prevented apoptosis. However, inhibition of this autophagic response induced robust apoptosis and sensitised lung cancer cells to dasatinib in vitro and in vivo. Our results provide an explanation for why dasatinib failed in NSCLC clinical trials. Furthermore, our data suggest that combining SFK inhibitors with autophagy inhibitors could provide a novel therapeutic approach in this disease.

Identification of ABC Transporter Interaction of a Novel Cyanoquinoline Radiotracer and Implications for Tumour Imaging by Positron Emission Tomography.

Background The epidermal growth factor receptor (EGFR) is overexpressed in many cancers including lung, ovarian, breast, head and neck and brain. Mutation of this receptor has been shown to play a crucial role in the response of non-small cell lung carcinoma (NSCLC) to EGFR-targeted therapies. It is envisaged that imaging of EGFR using positron emission tomography (PET) could aid in selection of patients for treatment with novel inhibitors. We recognised multi-drug resistant phenotype as a threat to development of successful imaging agents. In this report, we describe discovery of a novel cyanoquinoline radiotracer that lacks ABC transporter activity. Methods Cellular retention of the prototype cyanoquinoline [18F](2E)-N-{4-[(3-chloro-4-fluorophenyl)amino]-3-cyano-7-ethoxyquinolin-6-yl}-4-({[1-(2-fluoroethyl)-1H-1,2,3-triazol-4-yl]methyl}amino)-but-2-enamide ([18F]FED6) and [18F](2E)-N-{4-[(3-chloro-4-fluorophenyl)amino]-3-cyano-7-ethoxyquinolin-6-yl}-4-[({1-[(2R,5S)-3-fluoro-4,5-dihydroxy-6-(hydroxymethyl)oxan-2-yl]-1H-1,2,3-triazol-4-yl}methyl)amino]but-2-enamide ([18F]FED20) were evaluated to establish potential for imaging specificity. The substrate specificity of a number of cyanoquinolines towards ABC transporters was investigated in cell lines proficient or deficient in ABCB1 or ABCG2. Results FED6 demonstrated substrate specificity for both ABCG2 and ABCB1, a property that was not observed for all cyanoquinolines tested, suggesting scope for designing novel probes. ABC transporter activity was confirmed by attenuating the activity of transporters with drug inhibitors or siRNA. We synthesized a more hydrophilic compound [18F]FED20 to overcome ABC transporter activity. FED20 lacked substrate specificity for both ABCB1 and ABCG2, and maintained a strong affinity for EGFR. Furthermore, FED20 showed higher inhibitory affinity for active mutant EGFR versus wild-type or resistant mutant EGFR; this property resulted in higher [18F]FED20 cellular retention in active mutant EGFR expressing NSCLC. Conclusion [18F]FED20 binds EGFR but is devoid of ABC transporter activity, thus, has potential for EGFR imaging.

Abstract B140: Positron emission tomography imaging of HER2 expression and pharmacodynamic response to HSP90 inhibition with the next-generation ZHER2:2891 Affibody molecule [18F]GE-226.

Accurate assessment of HER2 status remains a clinical challenge, with up to 20% of patients being potentially withdrawn from therapy or exposed to unnecessary toxicity. Non-invasive imaging is widely seen as a viable alternative to current methods, in particular in the setting of locoregional and distant recurrences not amenable to biopsy. A next-generation HER2-targeting Affibody-based radiotracer has been developed, [18F]GE-226, with enhanced pharmacokinetic characteristics and improved properties for large-scale and GMP grade synthesis. Kinetic modeling gave insights into Affibody-HER2 interactions. Intrinsic affinity to HER2 (KD = 76 pM) resulted in 11 to 67-fold higher [18F]GE-226 uptake in ten HER2 positive versus negative cell lines in vitro independent of lineage. Uptake correlated with HER2 protein expression but was independent of presence of other targets like EGFR. Blocking with [19F]GE-226 and HER2 siRNA treatment reduced uptake by 96.8 ± 2.6% and 81.7 ± 9.2%, respectively. Uptake in HER2 positive xenografts was rapid with steady state net irreversible binding kinetics making possible the distinction of HER2 negative (MCF7 (n = 6) and MCF7-p95HER2 (n = 3): NUV60 (normalized uptake value at 60 min; %ID/mL) 6.1 ± 0.7; Ki (irreversible uptake rate; mL/cm3/min) 0.0069 ± 0.0014) from HER2 positive tumors (NUV60 and Ki: MCF7-HER2, 10.9 ± 1.5 and 0.015 ± 0.0035; MDA-MB-361, 18.2 ± 3.4 and 0.025 ± 0.0052; SKOV-3, 18.7 ± 2.4 and 0.036 ± 0.0065; all n = 6) within 1 h. Tumor uptake correlated with HER2 expression determined by ELISA (r2=0.78). Specificity was further determined by comparing tumor localization of a fluorescently labeled tracer analogue with DAKO HercepTest. Affibody signal co-localized with HER2 expression at the cellular level independent of spatial heterogeneity. Tracer binding was not influenced by short-term or continuous exposure to trastuzumab in SKOV-3 xenografts (n=6) in keeping with differential epitope binding. Inhibition of the chaperone HSP90– of which HER2 is a client protein– by the therapeutic development candidate NVP-AUY922 caused dose-dependent HER2 degradation and consequently reduced tracer uptake in SKOV-3 cells in vitro and xenografts in vivo (area under the curve, AUC0-60: 618.4±90.1 and 446.7±42.8 %ID/mL*min for vehicle (n=4) and drug (n=5), respectively; P=0.043). In conclusion, [18F]GE-226 differentiates HER2 negative from HER2 expressing tumors. The tracer has potential utility for HER2 detection, irrespective of prior trastuzumab treatment and to monitor response to HSP90 inhibition. Lineage-independence of these results extends application beyond breast cancer. Due to the specific annotation to HER2, enhanced pharmacokinetic properties and completion of initial preclinical toxicology testing, [18F]GE-226 is now transitioning into clinical development. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):B140. Citation Format: Sebastian Trousil, Susan Hoppmann, Quang-De Nguyen, Maciej Kaliszczak, Giampaolo Tomasi, Peter Iveson, Duncan Hiscock, Eric O. Aboagye. Positron emission tomography imaging of HER2 expression and pharmacodynamic response to HSP90 inhibition with the next-generation ZHER2:2891 Affibody molecule [18F]GE-226. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr B140.

Abstract 2608: HDAC-C1A: An irreversible HDAC inhibitor with significant anti-tumor activity

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL Histone deacetylase (HDAC) enzymes exert control over gene transcription and cell cycle progression and their inhibition has recently emerged as an efficacious strategy to treat cancer. However, current HDAC inhibitors have been linked to a shared undesirable toxicological profile that prompts the development of new entities. The present study evaluates the in vitro and in vivo PK/PD of a newly developed HDAC inhibitor, HDAC-C1A. HDAC-C1A inhibited class I, II and sirtuins, with highest affinity for HDAC6 (IC50 = 63 ng/mL), an HDAC subtype thought to be associated with low toxicity; HDAC6 knockout does not lead to embryonic lethality. The drug irreversibly inhibited HDAC from HeLa cell extract; in HCT116 cells inhibition of enzyme activity as assessed by levels of acetyl-histone H3, H4 and acetyl-tubulin was maintained after washout demonstrating an irreversible mechanism. HDAC-C1A treatment was associated with a dose and time dependent increase of histone and non-histone targets that was maintained at 4 hours after washout; acetylation was lost by 4 h with clinically licensed HDAC inhibitor SAHA. HDAC-C1A inhibited the growth of a panel of 7 cancer cell lines with a mean GI50 of 1.6 ± 0.6 µg/mL. The drug was relatively stable after parenteral administration for 4 h. The Cmax and AUC0-4h following i.p. injection (160 mg/kg) were 20.4 µg/mL and 50 µg/mL*h, respectively. Regarding efficacy, HDAC-C1A treatment was associated with a Tumor Growth Delay (TGD2x) of 5.7 ± 1.4 days and a Tumor Growth Inhibition (TGI) of 78% compared with vehicle when given i.p. at 20 mg/kg b.i.d. in a HCT116 human colon cancer xenograft model. This dose was non-toxic (no reduction in body weight). We also assessed the potential of [18F]fluorothymidine positron emission tomography ([18F]FLT-PET) to measure early response to HDAC-C1A treatment in HCT116 xenograft bearing mice. There was a 2-fold decrease in tumor [18F]FLT uptake compared to vehicle treated animals at 48 h post-treatment; the area under the normalized [18F]FLT time versus activity curve was 117 ± 6.9 at before treatment and decreased to 106 ± 5.5 (P = 0.02) at 24 hours and 54 ± 5 (P = 0.0001) at 48 h after initiating treatment. In summary, HDAC-C1A combines irreversible HDAC inhibition with a favourable pharmacokinetic profile leading to significant anti-tumor activity in the HCT116 tumor model. Early response to the drug was detectable by [18F]FLT-PET. These preclinical data support further development of HDAC-C1A. This work was supported in part by Cancer Research U.K.-Engineering and Physical Sciences Research Council Grant C2536/A10337. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2608. doi:10.1158/1538-7445.AM2011-2608