Activating mutations in underlie the pathogenesis of up to 20% of human tumors, and 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 mRNA. AZD4785 potently and selectively depleted cellular mRNA and protein, resulting in inhibition of downstream effector pathways and antiproliferative effects selectively in 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 mutant non-small cell lung cancer cell line xenografts or patient-derived xenografts resulted in inhibition of 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-driven human cancers and warrants further development.

Attempts to directly drug the important oncogene KRAS have met with limited success despite numerous efforts across industry and academia. The KRASG12C mutant represents an “Achilles heel” and has recently yielded to covalent targeting with small molecules that bind the mutant cysteine and create an allosteric pocket on GDP-bound RAS, locking it in an inactive state. A weak inhibitor at this site was optimized through conformational locking of a piperazine–quinazoline motif and linker modification. Subsequent introduction of a key methyl group to the piperazine resulted in enhancements in potency, permeability, clearance, and reactivity, leading to identification of a potent KRASG12C inhibitor with high selectivity and excellent cross-species pharmacokinetic parameters and in vivo efficacy.

Loss of PTEN protein results in upregulation of the PI3K/AKT pathway, which appears dependent on the PI3Kb isoform. Inhibitors of PI3Kb have potential to reduce growth of tumors in which loss of PTEN drives tumor progression. We have developed a small-molecule inhibitor of PI3Kb and PI3Kd (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 (GI 50 < 1 mmol/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 PI3Kb with activity against PI3Kd 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.

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 AKT1 E17K mutation is not known. Expression of exogenous copies of AKT1 E17K 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 AKT1 E17K 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 AKT1 E17K mutations. In MGH-U3 bladder cancer xenografts, which contain both AKT1 E17K and FGFR3 Y373C 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 AKT1 E17K 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.

The PIK3CA gene, encoding the p110a catalytic unit of PI3Ka, is one of the most frequently mutated oncogenes in human cancer. Hence, PI3Ka 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 PI3Ka and PI3Kd with selectivity versus PI3Kb, PI3Kg, and other kinases that preferentially inhibited growth in cells with mutant PIK3CA status, such as in estrogen receptor-positive (ER þ ) breast cancer cell lines BT474, MCF7, and T47D (submmol/L GI 50 s). 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 þ 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. Ó2016 AACR.

The RAS/MAPK pathway is a major driver of oncogenesis and is dysregulated in approximately 30% of human cancers, primarily by mutations in the BRAF or RAS genes. The extracellular-signal-regulated kinases (ERK1 and ERK2) serve as central nodes within this pathway. The feasibility of targeting the RAS/MAPK pathway has been demonstrated by the clinical responses observed through the use of BRAF and MEK inhibitors in BRAF V600E/K metastatic melanoma; however, resistance frequently develops. Importantly, ERK1/2 inhibition may have clinical utility in overcoming acquired resistance to RAF and MEK inhibitors, where RAS/MAPK pathway reactivation has occurred, such as relapsed BRAF V600E/K melanoma. We describe our structure-based design approach leading to the discovery of AZD0364, a potent and selective inhibitor of ERK1 and ERK2. AZD0364 exhibits high cellular potency (IC50 = 6 nM) as well as excellent physicochemical and absorption, distribution, metabolism, and excretion (ADME) properties and has demonstrated encouraging antitumor activity in preclinical models.

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.

KRAS is an archetypal high-value intractable oncology drug target. The glycine to cysteine mutation at codon 12 represents an Achilles heel that has now rendered this important GTPase druggable. Herein, we report our structure-based drug design approach that led to the identification of 21, AZD4625, a clinical development candidate for the treatment of KRASG12C positive tumors. Highlights include a quinazoline tethering strategy to lock out a bio-relevant binding conformation and an optimization strategy focused on the reduction of extrahepatic clearance mechanisms seen in preclinical species. Crystallographic analysis was also key in helping to rationalize unusual structure–activity relationship in terms of ring size and enantio-preference. AZD4625 is a highly potent and selective inhibitor of KRASG12C with an anticipated low clearance and high oral bioavailability profile in humans.