Lyndsey Hanson

Inhibition of PI3Kβ Signaling with AZD8186 Inhibits Growth of PTEN-Deficient Breast and Prostate Tumors Alone and in Combination with Docetaxel

Loss of PTEN protein results in upregulation of the PI3K/AKT pathway, which appears dependent on the PI3Kβ isoform. Inhibitors of PI3Kβ have potential to reduce growth of tumors in which loss of PTEN drives tumor progression. We have developed a small-molecule inhibitor of PI3Kβ and PI3Kδ (AZD8186) and assessed its antitumor activity across a panel of cell lines. We have then explored the antitumor effects as single agent and in combination with docetaxel in triple-negative breast (TNBC) and prostate cancer models. In vitro, AZD8186 inhibited growth of a range of cell lines. Sensitivity was associated with inhibition of the AKT pathway. Cells sensitive to AZD8186 (GI50 < 1 μmol/L) are enriched for, but not exclusively associated with, PTEN deficiency. In vivo, AZD8186 inhibits PI3K pathway biomarkers in prostate and TNBC tumors. Scheduling treatment with AZD8186 shows antitumor activity required only intermittent exposure, and that increased tumor control is achieved when AZD8186 is used in combination with docetaxel. AZD8186 is a potent inhibitor of PI3Kβ with activity against PI3Kδ signaling, and has potential to reduce growth of tumors dependent on dysregulated PTEN for growth. Moreover, AZD8186 can be combined with docetaxel, a chemotherapy commonly used to treat advanced TBNC and prostate tumors. The ability to schedule AZD8186 and maintain efficacy offers opportunity to combine AZD8186 more effectively with other drugs. Mol Cancer Ther; 14(1); 48–58. ©2014 AACR.

Tumors with AKT1E17K Mutations Are Rational Targets for Single Agent or Combination Therapy with AKT Inhibitors

AKT1E17K mutations occur at low frequency in a variety of solid tumors, including those of the breast and urinary bladder. Although this mutation has been shown to transform rodent cells in culture, it was found to be less oncogenic than PIK3CA mutations in breast epithelial cells. Moreover, the therapeutic potential of AKT inhibitors in human tumors with an endogenous AKT1E17K mutation is not known. Expression of exogenous copies of AKT1E17K in MCF10A breast epithelial cells increased phosphorylation of AKT and its substrates, induced colony formation in soft agar, and formation of lesions in the mammary fat pad of immunodeficient mice. These effects were inhibited by the allosteric and catalytic AKT inhibitors MK-2206 and AZD5363, respectively. Both AKT inhibitors caused highly significant growth inhibition of breast cancer explant models with AKT1E17K mutation. Furthermore, in a phase I clinical study, the catalytic Akt inhibitor AZD5363 induced partial responses in patients with breast and ovarian cancer with tumors containing AKT1E17K mutations. In MGH-U3 bladder cancer xenografts, which contain both AKT1E17K and FGFR3Y373C mutations, AZD5363 monotherapy did not significantly reduce tumor growth, but tumor regression was observed in combination with the FGFR inhibitor AZD4547. The data show that tumors with AKT1E17K mutations are rational therapeutic targets for AKT inhibitors, although combinations with other targeted agents may be required where activating oncogenic mutations of other proteins are present in the same tumor. Mol Cancer Ther; 14(11); 2441–51. ©2015 AACR.

Targeting KRAS-dependent tumors with AZD4785, a high-affinity therapeutic antisense oligonucleotide inhibitor of KRAS

AZD4785 is a therapeutic antisense oligonucleotide targeting KRAS mRNA with promising preclinical antitumor activity and a favorable safety profile. An antisensible approach to targeting KRAS Mutations that cause activation of the KRAS oncogene are common in human cancer, including treatment-resistant tumor types such as lung and pancreatic cancer. KRAS has also proven to be notoriously difficult to target with small molecules. To overcome this issue, Ross et al. have turned to genetic technology, demonstrating an antisense oligonucleotide–based therapy for inhibiting KRAS. The antisense oligonucleotide used in this study was chemically modified, allowing systemic delivery through subcutaneous injection and avoiding the need for a specialized delivery vehicle. The authors tested the efficacy of this therapy in multiple mouse models of non–small cell lung cancer and evaluated its safety in primates, demonstrating its potential suitability for translation to humans. Activating mutations in KRAS underlie the pathogenesis of up to 20% of human tumors, and KRAS is one of the most frequently mutated genes in cancer. Developing therapeutics to block KRAS activity has proven difficult, and no direct inhibitor of KRAS function has entered clinical trials. We describe the preclinical evaluation of AZD4785, a high-affinity constrained ethyl–containing therapeutic antisense oligonucleotide (ASO) targeting KRAS mRNA. AZD4785 potently and selectively depleted cellular KRAS mRNA and protein, resulting in inhibition of downstream effector pathways and antiproliferative effects selectively in KRAS mutant cells. AZD4785-mediated depletion of KRAS was not associated with feedback activation of the mitogen-activated protein kinase (MAPK) pathway, which is seen with RAS-MAPK pathway inhibitors. Systemic delivery of AZD4785 to mice bearing KRAS mutant non–small cell lung cancer cell line xenografts or patient-derived xenografts resulted in inhibition of KRAS expression in tumors and antitumor activity. The safety of this approach was demonstrated in mice and monkeys with KRAS ASOs that produced robust target knockdown in a broad set of tissues without any adverse effects. Together, these data suggest that AZD4785 is an attractive therapeutic for the treatment of KRAS-driven human cancers and warrants further development.

Intermittent High-Dose Scheduling of AZD8835, a Novel Selective Inhibitor of PI3Kα and PI3Kδ, Demonstrates Treatment Strategies for PIK3CA-Dependent Breast Cancers

The PIK3CA gene, encoding the p110α catalytic unit of PI3Kα, is one of the most frequently mutated oncogenes in human cancer. Hence, PI3Kα is a target subject to intensive efforts in identifying inhibitors and evaluating their therapeutic potential. Here, we report studies with a novel PI3K inhibitor, AZD8835, currently in phase I clinical evaluation. AZD8835 is a potent inhibitor of PI3Kα and PI3Kδ with selectivity versus PI3Kβ, PI3Kγ, and other kinases that preferentially inhibited growth in cells with mutant PIK3CA status, such as in estrogen receptor–positive (ER+) breast cancer cell lines BT474, MCF7, and T47D (sub-μmol/L GI50s). Consistent with this, AZD8835 demonstrated antitumor efficacy in corresponding breast cancer xenograft models when dosed continuously. In addition, an alternative approach of intermittent high-dose scheduling (IHDS) was explored given our observations that higher exposures achieved greater pathway inhibition and induced apoptosis. Indeed, using IHDS, monotherapy AZD8835 was able to induce tumor xenograft regression. Furthermore, AZD8835 IHDS in combination with other targeted therapeutic agents further enhanced antitumor activity (up to 92% regression). Combination partners were prioritized on the basis of our mechanistic insights demonstrating signaling pathway cross-talk, with a focus on targeting interdependent ER and/or CDK4/6 pathways or alternatively a node (mTOR) in the PI3K-pathway, approaches with demonstrated clinical benefit in ER+ breast cancer patients. In summary, AZD8835 IHDS delivers strong antitumor efficacy in a range of combination settings and provides a promising alternative to continuous dosing to optimize the therapeutic index in patients. Such schedules merit clinical evaluation. Mol Cancer Ther; 15(5); 877–89. ©2016 AACR.

Structure-Guided Discovery of Potent and Selective Inhibitors of ERK1/2 from a Modestly Active and Promiscuous Chemical Start Point.

There are a number of small-molecule inhibitors targeting the RAS/RAF/MEK/ERK signaling pathway that have either been approved or are in clinical development for oncology across a range of disease indications. The inhibition of ERK1/2 is of significant current interest, as cell lines with acquired resistance to BRAF and MEK inhibitors have been shown to maintain sensitivity to ERK1/2 inhibition in preclinical models. This article reports on our recent work to identify novel, potent, and selective reversible ERK1/2 inhibitors from a low-molecular-weight, modestly active, and highly promiscuous chemical start point, compound 4. To guide and inform the evolution of this series, inhibitor binding mode information from X-ray crystal structures was critical in the rapid exploration of this template to compound 35, which was active when tested in in vivo antitumor efficacy experiments.

Discovery of a novel aminopyrazine series as selective PI3Kα inhibitors

We report the discovery of a novel aminopyrazine series of PI3Kα inhibitors, designed by hybridizing two known scaffolds of PI3K inhibitors. We describe the progress achieved from the first compounds plagued with poor general kinase selectivity to compounds showing high selectivity for PI3Kα over PI3Kβ and excellent general kinase selectivity. This effort culminated with the identification of compound 5 displaying high potency and selectivity, and suitable physiochemical and pharmacokinetic properties for oral administration. In vivo, compound 5 showed good inhibition of tumour growth (86% tumour growth inhibition at 50mg/kg twice daily orally) in the MCF7 xenograft model in mice.

Abstract PR12: Discovery and preclinical evaluation of cEt-modified KRAS antisense oligonucleotide inhibitors

KRAS is one of the most frequently mutated genes in cancer and its activation is thought to underlie the pathogenesis of up to 30% of all human tumors. However, to date KRAS has proven difficult to target with traditional pharmacologic approaches. Antisense technology is particularly attractive for such difficult drug targets as antisense oligonucleotide (ASO) inhibitors can be designed based on a targets RNA sequence alone. cEt ASOs have been recently described and demonstrated to have significantly increased potency over previous generation ASO chemistries. Moreover, STAT3Rx/AZD9150, a cEt modified ASO targeting STAT3 mRNA was recently shown to produce robust STAT3 depletion in a broad range of xenograft models (AACR 2013) and importantly has also demonstrated promising single-agent antitumor activity in patients with advanced treatment-refractory cancers in phase I studies (ASCO 2013, EORTC 2014). Here, we describe the preclinical evaluation of potent and selective cEt ASOs targeted to human or mouse KRAS mRNA. A human-specific KRAS cEt ASO which potently and selectively down-regulated KRAS demonstrated anti-proliferative effects and the expected down-stream pathway inhibition in a panel of KRAS mutant NSCLC, CRC and PDAC cells. The KRAS ASO was differentiated from MAPK pathway inhibitors selectively inhibiting the proliferation of KRAS mutant not KRAS wild type cells and not causing feedback reactivation of the MAPK or PI3K pathways. Systemic delivery of the KRAS ASO to mice bearing KRAS mutant NSCLC or CRC xenografts resulted in significant inhibition of KRAS tumour expression and antitumor activity. Importantly, the KRAS ASO also showed significant target knockdown and antitumor effects in KRAS mutant NSCLC patient-derived xenografts (PDX). Finally, potent and selective murine-specific KRAS ASO produced robust target knockdown in a broad set of tissues without detectable tolerability signals associated with ASO-mediated systemic KRAS inhibition. Taken together these data suggest that KRAS ASOs are an attractive therapeutic approach to target KRAS for the treatment of human cancers. Citation Format: Alexey S. Revenko, Sarah J. Ross, Lyndsey L. Hanson, Rebecca Ellston, Chris May, Sanjay K. Pandey, Linda K. Buckett, Stephanie K. Klein, Mitchell Revill, Kevin Hudson, Brett P. Monia, David C. Blakey, Paul Lyne, Allan R. MacLeod. Discovery and preclinical evaluation of cEt-modified KRAS antisense oligonucleotide inhibitors. [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 PR12.

Abstract 2665: High dose intermittent scheduling of AZD8835, a novel potent and selective inhibitor of PI3Kα and PI3Kδ, identifies potential treatment strategies for PIK3CA-dependent cancers

The PIK3CA gene, encoding the p110 catalytic unit of PI3Kα, is one of the most frequently mutated oncogenes described in human cancer. Hence PI3Kα is a target subject to intensive efforts in identifying inhibitors and evaluating their therapeutic potential. To date most studies with PI3K inhibitors have used a continuous (daily) dosing schedule and although clinical responses have been reported the overall activity observed has been moderate. This may in part be due to suboptimal pathway inhibition which is capped by normal tissue toxicities such as rash, diarrhoea and hyperglycaemia. Furthermore, additional dose reduction may be required when such agents are used in combination with other therapies. Therefore in our efforts to optimise inhibition of PI3K pathway signalling, we have explored high dose intermittent scheduling as an alternative to continuous dosing. Here we describe pre-clinical studies that exemplify such concepts, centred around use of AZD8835, a PI3K inhibitor currently in Phase 1 clinical evaluation. AZD8835 is a novel and potent inhibitor of PI3Kα and PI3Kδ, with selectivity vs. PI3Kβ, PI3Kγ(IC50s of 6nM, 6nM, 431nM and 90nM respectively in enzyme assays) and other kinases. AZD8835 preferentially displays activity in tumour models with a mutant PIK3CA background, such as ER+ve breast cancer models. Such models were used in our investigations, in both cell culture and in mouse xenograft contexts. We demonstrate that a high dose intermittent schedule of single agent AZD8835 achieves greater pathway inhibition yielding significant anti-tumour responses. In the sensitive BT474 xenograft model, a dose of 100mg/kg AZD8835 BID on days 1 and 4 in a weekly schedule delivered -36% tumour regression, accompanied by a strongly elevated rapid onset apoptosis signal with 4-16% cells staining positively for cleaved-caspase3. We also evaluated AZD8835 in combination with other targeted therapeutic agents, in MCF7, BT474 and T47D breast models, observing increased sensitivity relative to single agent AZD8835; firstly with agents that target other nodes in the PI3K pathway; secondly with agents targeting parallel but interconnected driver pathways in breast disease (ER, CDK4/6). Overall the data indicate that high dose intermittent scheduling can deliver strong anti-tumour efficacy in a range of combination settings and provides a promising alternative to continuous dosing. Such schedules merit clinical evaluation. Citation Format: Kevin Hudson, Urs Hancox, Cath Trigwell, Phillippa Dudley, Lyndsey Hanson, Robert McEwen, Alys Jones, Marie Cumberbatch, Urszula Polanska, Rebecca Ellston, Oona Delpuech, Pablo Morentin Gutierrez, Lara Ward, Francisco Cruzalegui, Stephen Green. High dose intermittent scheduling of AZD8835, a novel potent and selective inhibitor of PI3Kα and PI3Kδ, identifies potential treatment strategies for PIK3CA-dependent cancers. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2665. doi:10.1158/1538-7445.AM2015-2665

Abstract 4249: The Pi3Kβ/δ inhibitor AZD8186 has potential to treat tumours in combination with key signalling pathway inhibitors

AZD8186 inhibits Pi3K isoforms Pi3Kβ and δ, with selectivity over Pi3Kα and γ. In solid tumours Pi3Kβ drives tumour growth when the tumour suppressor PTEN is deleted, mutated or downregulated. It also mediates signals from specific GPCR receptors. Pi3Kδ signals downstream of the B-cell receptor, creating potential for targeted treatment of haematological malignancies such as CLL, MCL & indolent NHL and possibly DLBCL. AZD8186 is differentiated from many other agents that target Pi3K family members as it isn9t likely to impact glucose control. AZD8186 has single agent activity in a range of models, although maximal benefit is anticipated when used in combination. Loss of PTEN mediated control, and hence dependency on PI3Kβ occurs in many solid tumour types, and is altered in up to 40-50% of tumours in some individual disease states. Commonly PTEN dysregulation is associated with other activation of other signaling pathways. To explore this we have combined AZD8186 with a number of different agents. In HCC70 (triple negative breast cancer) and PC3 (prostate cancer) xenografts combination of AZD8186 (25mg/kg bid) with single dose docetaxel (15mg/kg) gives >90% tumour growth inhibition (TGI), compared to 40-50% with docetaxel alone. AZD8186 also combines with other targeted agents. In HCC70, AZD8186 (25mg/kg bid) combined with selumetinib (10mg/kg) gave 94% TGI compared to 66%, and 47% with each single agent. In combination with AZD2014 (mTORC1/2 inhibitor) (15mg/kg qd) in HCC70 xenografts AZD8186 (25mg/kg bid) gave regressions (-23%) compared to TGI of 87% and 77% for each agent alone. In 786-0 (renal cancer) xenografts AZD8186 (12.5mg/kg bid) and AZD2014 (15mg/kg qd) gave regression of -82% compared to TGI of 33% and regression of -39% with each agent alone. This data establishes the potential for AZD8186 to be used in combination with a number of different agents including the ability to customise dose and schedule to optimise both tolerability as well as anti-tumour effects. Further exploration of the combination opportunities for AZD8186 with other molecular targeted agents would inform on the potential for inhibitors of Pi3Kβ and δ to give benefit in different tumour types. Citation Format: Simon T. Barry, Kathryn Cronin, Marie Cumberbatch, Rebecca Ellston, Emily Foster, Urs Hancox, Lyndsey Hanson, Liz Harrington, Carol Lenaghan, Stefan Symeonides, Cath Trigwell, Lara Ward. The Pi3Kβ/δ inhibitor AZD8186 has potential to treat tumours in combination with key signalling pathway inhibitors. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4249. doi:10.1158/1538-7445.AM2014-4249