Elaine Cadogan

Aurora kinase inhibitor nanoparticles target tumors with favorable therapeutic index in vivo

A nanoparticle formulation of an Aurora B kinase inhibitor uses ion pairing to achieve controlled release and efficacious, nontoxic target inhibition in tumors. Accurin nanoparticles dutifully deliver drug A class of drugs, called kinase inhibitors, could stop cancer in its tracks…if only these drugs could reach the tumors, stay for a while, and not be toxic. Hypothesizing that a nanoparticle formulation would solve the inhibitors’ woes, Ashton and colleagues investigated several different compositions of so-called Accurins—polymeric particles that encapsulate charged drugs through ion pairing. An Aurora B kinase, once formulated in Accurins, demonstrated a much-improved therapeutic index and preclinical efficacy compared with its parent molecule, when administered to rats and mice bearing human tumors. The Accurins allowed for sustained release of the drug over days, and did not have the same blood toxicity seen with the parent drug. A phase 1 trial is the next step for this nanomedicine, and additional preclinical studies will reveal whether such nanoformulations can improve the tolerability and efficacy of the broader class of molecularly targeted cancer therapeutics, including cell cycle inhibitors. Efforts to apply nanotechnology in cancer have focused almost exclusively on the delivery of cytotoxic drugs to improve therapeutic index. There has been little consideration of molecularly targeted agents, in particular kinase inhibitors, which can also present considerable therapeutic index limitations. We describe the development of Accurin polymeric nanoparticles that encapsulate the clinical candidate AZD2811, an Aurora B kinase inhibitor, using an ion pairing approach. Accurins increase biodistribution to tumor sites and provide extended release of encapsulated drug payloads. AZD2811 nanoparticles containing pharmaceutically acceptable organic acids as ion pairing agents displayed continuous drug release for more than 1 week in vitro and a corresponding extended pharmacodynamic reduction of tumor phosphorylated histone H3 levels in vivo for up to 96 hours after a single administration. A specific AZD2811 nanoparticle formulation profile showed accumulation and retention in tumors with minimal impact on bone marrow pathology, and resulted in lower toxicity and increased efficacy in multiple tumor models at half the dose intensity of AZD1152, a water-soluble prodrug of AZD2811. These studies demonstrate that AZD2811 can be formulated in nanoparticles using ion pairing agents to give improved efficacy and tolerability in preclinical models with less frequent dosing. Accurins specifically, and nanotechnology in general, can increase the therapeutic index of molecularly targeted agents, including kinase inhibitors targeting cell cycle and oncogenic signal transduction pathways, which have to date proved toxic in humans.

Targeting the kinase activities of ATR and ATM exhibits antitumoral activity in mouse models of MLL-rearranged AML

Chemotherapy-resistant acute myeloid leukemia may respond to inhibition of ATR or ATM. New hope for AML patients A pair of papers provides new hope for patients with acute myeloid leukemia (AML) by showing that the DNA replication checkpoint pathway is a viable target for therapeutic intervention. By integrating survival data from 198 treated AML patients with gene expression data for genes encoding proteins involved in the regulation of DNA replication, David et al. identified the CHEK1 gene and its product, the DNA replication checkpoint kinase CHK1, as both a prognostic indicator of survival and a therapeutic target to overcome resistance to the current standard of chemotherapy. The patients had all received standard-of-care chemotherapy. Patients with high expression of CHEK1 in their AML cells had reduced survival, and AML patient cells with high CHK1 abundance were resistant to the toxic effects of the DNA replication inhibitor cytarabine. CHK1 is activated by the kinase ATR in response to DNA replication stress arising from DNA damage. The identification of CHEK1 expression as high in lymphomas and leukemias, including AML, prompted Morgado-Palacin et al. to investigate targeting ATR and ATM, the most upstream kinases in the DNA damage response, as possible AML therapies. AML cells with oncogenic rearrangements in MLL are particularly resistant to genotoxic therapies that form the backbone of AML treatment. Inhibiting ATR resulted in death of AMLMLL cells in culture and exhibited antitumoral activity in AMLMLL mouse models. Inhibiting ATM also prolonged survival of the allograft mouse model, indicating that targeting the DNA damage response pathways alone or in combination with other chemotherapeutic agents may be beneficial in patients with AML. Among the various subtypes of acute myeloid leukemia (AML), those with chromosomal rearrangements of the MLL oncogene (AML-MLL) have a poor prognosis. AML-MLL tumor cells are resistant to current genotoxic therapies because of an attenuated response by p53, a protein that induces cell cycle arrest and apoptosis in response to DNA damage. In addition to chemicals that damage DNA, efforts have focused on targeting DNA repair enzymes as a general chemotherapeutic approach to cancer treatment. Here, we found that inhibition of the kinase ATR, which is the primary sensor of DNA replication stress, induced chromosomal breakage and death of mouse AMLMLL cells (with an MLL-ENL fusion and a constitutively active N-RAS) independently of p53. Moreover, ATR inhibition as a single agent exhibited antitumoral activity, both reducing tumor burden after establishment and preventing tumors from growing, in an immunocompetent allograft mouse model of AMLMLL and in xenografts of a human AML-MLL cell line. We also found that inhibition of ATM, a kinase that senses DNA double-strand breaks, also promoted the survival of the AMLMLL mice. Collectively, these data indicated that ATR or ATM inhibition represent potential therapeutic strategies for the treatment of AML, especially MLL-driven leukemias.

Discovery of Novel 3-Quinoline Carboxamides as Potent, Selective and Orally Bioavailable Inhibitors of Ataxia Telangiectasia Mutated (Atm) Kinase.

A novel series of 3-quinoline carboxamides has been discovered and optimized as selective inhibitors of the ataxia telangiectasia mutated (ATM) kinase. From a modestly potent HTS hit (4), we identified molecules such as 6-[6-(methoxymethyl)-3-pyridinyl]-4-{[(1R)-1-(tetrahydro-2H-pyran-4-yl)ethyl]amino}-3-quinolinecarboxamide (72) and 7-fluoro-6-[6-(methoxymethyl)pyridin-3-yl]-4-{[(1S)-1-(1-methyl-1H-pyrazol-3-yl)ethyl]amino}quinoline-3-carboxamide (74) as potent and highly selective ATM inhibitors with overall ADME properties suitable for oral administration. 72 and 74 constitute excellent oral tools to probe ATM inhibition in vivo. Efficacy in combination with the DSB-inducing agent irinotecan was observed in a disease relevant model.

The Identification of Potent, Selective, and Orally Available Inhibitors of Ataxia Telangiectasia Mutated (ATM) Kinase: The Discovery of AZD0156 (8-{6-[3-(Dimethylamino)propoxy]pyridin-3-yl}-3-methyl-1-(tetrahydro-2H-pyran-4-yl)-1,3-dihydro-2H-imidazo[4,5-c]quinolin-2-one)

ATM inhibitors, such as 7, have demonstrated the antitumor potential of ATM inhibition when combined with DNA double-strand break-inducing agents in mouse xenograft models. However, the properties of 7 result in a relatively high predicted clinically efficacious dose. In an attempt to minimize attrition during clinical development, we sought to identify ATM inhibitors with a low predicted clinical dose (<50 mg) and focused on strategies to increase both ATM potency and predicted human pharmacokinetic half-life (predominantly through the increase of volume of distribution). These efforts resulted in the discovery of 64 (AZD0156), an exceptionally potent and selective inhibitor of ATM based on an imidazo[4,5- c]quinolin-2-one core. 64 has good preclinical phamacokinetics, a low predicted clinical dose, and a high maximum absorbable dose. 64 has been shown to potentiate the efficacy of the approved drugs irinotecan and olaparib in disease relevant mouse models and is currently undergoing clinical evaluation with these agents.

The Combination of the PARP Inhibitor Olaparib and the WEE1 Inhibitor AZD1775 as a New Therapeutic Option for Small Cell Lung Cancer

Purpose: Introduced in 1987, platinum-based chemotherapy remains standard of care for small cell lung cancer (SCLC), a most aggressive, recalcitrant tumor. Prominent barriers to progress are paucity of tumor tissue to identify drug targets and patient-relevant models to interrogate novel therapies. Following our development of circulating tumor cell patient–derived explants (CDX) as models that faithfully mirror patient disease, here we exploit CDX to examine new therapeutic options for SCLC. Experimental Design: We investigated the efficacy of the PARP inhibitor olaparib alone or in combination with the WEE1 kinase inhibitor AZD1775 in 10 phenotypically distinct SCLC CDX in vivo and/or ex vivo. These CDX represent chemosensitive and chemorefractory disease including the first reported paired CDX generated longitudinally before treatment and upon disease progression. Results: There was a heterogeneous depth and duration of response to olaparib/AZD1775 that diminished when tested at disease progression. However, efficacy of this combination consistently exceeded that of cisplatin/etoposide, with cures in one CDX model. Genomic and protein analyses revealed defects in homologous recombination repair genes and oncogenes that induce replication stress (such as MYC family members), predisposed CDX to combined olaparib/AZD1775 sensitivity, although universal predictors of response were not noted. Conclusions: These preclinical data provide a strong rationale to trial this combination in the clinic informed by prevalent, readily accessed circulating tumor cell–based biomarkers. New therapies will be evaluated in SCLC patients after first-line chemotherapy, and our data suggest that the combination of olaparib/AZD1775 should be used as early as possible and before disease relapse. Clin Cancer Res; 24(20); 5153–64. ©2018 AACR.

Abstract 3041: Blood-brain barrier penetrating ATM inhibitor (AZ32) radiosensitises intracranial gliomas in mice

Ataxia-telangiectasia mutated (ATM) kinase is a central DNA damage response (DDR) component signalling the presence of DNA double strand breaks (DSBs) to DNA repair, checkpoint and survival pathways. Clinical doses of fractionated radiotherapy directed at tumours kill cells by inducing single strand breaks and DSBs, the latter being particularly lethal to all cells if not repaired. Poor survival rates of glioblastoma multiforme (GBM) patients is attributed to an inability to excise all invasive tumor tissue (if operable) and an intrinsic tumour chemo/radioresistance, which has been linked to elevated ATM activity in glioma stem cells. ATM inhibition (ATMi) radiosensitises cancer cell lines in vitro and in vivo. ATMi also acutely radiosensitises patient-derived glioma stem cells more effectively than by inhibiting other DDR or cell cycle checkpoint components (PARP, ATR, Chk1). Checkpoint-defective glioblastoma multiforme (GBM) cell lines seem to be particularly radio-sensitised by ATMi. In addition, several studies suggest that normal brain is radioprotected when ATM activity is deficient, suggesting ATM is required for apoptosis in neurons. ATMi in brain may therefore provide a wide therapeutic margin. Furthermore, ATM9s role in signalling DNA damage independent redox stress has been linked to promoting neoangiogenesis in tumour vasculature. Taken together, these studies suggest ATM is an extremely attractive target to inhibit during and potentially following radiotherapy. However, one impediment to preclinical and clinical studies is that current ATMi9s have limited CNS bioavailability. We report AZ32 as the first known selective, orally bioavailable blood-brain barrier (BBB) penetrating ATMi probe. AZ32 inhibits the DDR and radiosensitizes p53/checkpoint-defective GBM cells in vitro. Oral daily dosing providing sufficient mouse free brain pharmacokinetic exposures over AZ329s ATM IC50, during just four daily doses of whole brain irradiation results in significant improvement in median overall survival of syngeneic orthotopic mouse glioma models (log-rank AZ32/radiation vs. radiation p = 0.0194). Tumor eradication was confirmed by tumor imaging. This result was recapitulated with a human orthotopic model. These findings support the development of clinical grade BBB-penetrating ATMi as a potential treatment for GBM and potentially other intracranial tumours dosed in combination with fractionated radiotherapy used in standard clinical practice. Citation Format: Steve T. Durant, Jeremy Karlin, Kurt Pike, Nicola Colclough, N Mukhopadhyay, S F. Ahmad, J M. Bekta, M Tokarz, Catherine Bardelle, Gareth Hughes, Bhavika Patel, Andrew Thomason, Elaine Cadogan, Ian Barrett, Alan Lau, Martin Pass, Kristoffer Valerie. Blood-brain barrier penetrating ATM inhibitor (AZ32) radiosensitises intracranial gliomas in mice. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3041.

Abstract 4420: Generating preclinical models to assess bone marrow toxicity induced by the PARP inhibitor olaparib in combination with chemotherapy.

Inhibitors of the DNA damage response (DDR) provide an exciting opportunity as new anti-cancer therapies. A prominent example is olaparib, an inhibitor of poly (ADP-ribose) polymerase-1 (PARP-1), that has shown potential as a monotherapy for tumours bearing BRCA1/2 defects in clinical trials. Apart from monotherapy based on the concept of synthetic lethality, PARP inhibitors also have the potential to provide additional benefit when used in combination with DNA damage-inducing chemotherapies. In the clinic, however, this approach has been challenging due to enhanced bone marrow toxicity. Our aim is to generate preclinical models to provide a better understanding of the mechanisms for this limiting bone marrow toxicity. We characterized in vitro responses to DNA damage induced by IR and chemotherapy in human hematopoietic multipotent progenitors and mesenchymal stem cells, both components of bone marrow. In parallel, we modelled the effects of a platinum chemotherapy (carboplatin) in combination with olaparib in wild type rats with the aim of reproducing the clinical combination-induced bone marrow toxicity. We will present data that provide initial insights into the suitability of the rat pre-clinical model to guide combination dose and schedules with olaparib and an assessment of the ability of different bone marrow and peripheral blood cell populations to act as useful biomarkers to guide better tolerated combination schedules. Citation Format: Mark J. O9Connor, Helen Mason, Steve Horner, Ian Slater, Alan Lau, Elaine Cadogan, Lenka Oplustilova. Generating preclinical models to assess bone marrow toxicity induced by the PARP inhibitor olaparib in combination with chemotherapy. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4420. doi:10.1158/1538-7445.AM2013-4420

Abstract C60: Pre-clinical efficacy of the ATR inhibitor AZD6738 in combination with the PARP inhibitor olaparib

The PARP inhibitor olaparib acts through both inhibition of DNA single-strand-break repair and trapping of PARP-DNA complexes creating DNA lesions which cause replication fork stalling and collapsed fork DNA breaks. Cells which have lost BRCA-dependent homologous recombination repair are highly sensitive to olaparib treatment and this has led to its approval in patients with tumors carrying BRCA mutations. In addition, ATM (Ataxia telangiectasia mutated) and ATR (Ataxia telangiectasia mutated and Rad3 related) dependent DNA repair processes are hypothesized to be important survival pathways to PARP inhibitor treatment. In pre-clinical studies cancer cells which have defects in either ATM or ATR have been shown to be sensitive to PARP inhibitors. Here we present data that the orally bioavailable ATR inhibitor AZD6738 (in Phase-I clinial trials) combines synergistically with olaparib leading to cell death and anti-tumour activity in pre-clinical models. The combinations are effective across a panel of gastric and lung cancer cell lines in vitro . In addition results from isogenic ATM −/-/− knockout versus ATM +/+/+ FaDu head and neck cancer cell line pairs show enhanced combination activity in ATM knockout cells versus ATM wild-type cells. Studies in vivo show that through intermittent dosing the combination is tolerated while demonstrating significant anti-tumour efficacy and regressions across multiple human patient derived primary explant models. Together, these data support the notion of development of AZD6738 and olaparib combinations for the treatment of ATM-deficient cancers. Citation Format: Alan Lau, Elaine Brown, Andrew Thomason, Rajesh Odedra, Victoria Sheridan, Elaine Cadogan, Shirlian Xu, Andy Cui, Paul R. Gavine, Mark O9Connor. Pre-clinical efficacy of the ATR inhibitor AZD6738 in combination with the PARP inhibitor olaparib. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr C60.

Orally Bioavailable and Blood–Brain Barrier-Penetrating ATM Inhibitor (AZ32) Radiosensitizes Intracranial Gliomas in Mice

Inhibition of ataxia-telangiectasia mutated (ATM) during radiotherapy of glioblastoma multiforme (GBM) may improve tumor control by short-circuiting the response to radiation-induced DNA damage. A major impediment for clinical implementation is that current inhibitors have limited central nervous system (CNS) bioavailability; thus, the goal was to identify ATM inhibitors (ATMi) with improved CNS penetration. Drug screens and refinement of lead compounds identified AZ31 and AZ32. The compounds were then tested in vivo for efficacy and impact on tumor and healthy brain. Both AZ31 and AZ32 blocked the DNA damage response and radiosensitized GBM cells in vitro. AZ32, with enhanced blood–brain barrier (BBB) penetration, was highly efficient in vivo as radiosensitizer in syngeneic and human, orthotopic mouse glioma model compared with AZ31. Furthermore, human glioma cell lines expressing mutant p53 or having checkpoint-defective mutations were particularly sensitive to ATMi radiosensitization. The mechanism for this p53 effect involves a propensity to undergo mitotic catastrophe relative to cells with wild-type p53. In vivo, apoptosis was >6-fold higher in tumor relative to healthy brain after exposure to AZ32 and low-dose radiation. AZ32 is the first ATMi with oral bioavailability shown to radiosensitize glioma and improve survival in orthotopic mouse models. These findings support the development of a clinical-grade, BBB-penetrating ATMi for the treatment of GBM. Importantly, because many GBMs have defective p53 signaling, the use of an ATMi concurrent with standard radiotherapy is expected to be cancer-specific, increase the therapeutic ratio, and maintain full therapeutic effect at lower radiation doses. Mol Cancer Ther; 17(8); 1637–47. ©2018 AACR.

Discovery of a Series of 3-Cinnoline Carboxamides as Orally Bioavailable, Highly Potent, and Selective ATM Inhibitors

We report the discovery of a novel series of 3-cinnoline carboxamides as highly potent and selective ataxia telangiectasia mutated (ATM) kinase inhibitors. Optimization of this series focusing on potency and physicochemical properties (especially permeability) led to the identification of compound 21, a highly potent ATM inhibitor (ATM cell IC50 0.0028 μM) with excellent kinase selectivity and favorable physicochemical and pharmacokinetics properties. In vivo, 21 in combination with irinotecan showed tumor regression in the SW620 colorectal tumor xenograft model, superior inhibition to irinotecan alone. Compound 21 was selected for preclinical evaluation alongside AZD0156.