Targeting myeloid chemotaxis to reverse prostate cancer therapy resistance.

Inflammation is a hallmark of cancer1. In patients with cancer, peripheral blood myeloid expansion, indicated by a high neutrophil-to-lymphocyte ratio, associates with shorter survival and treatment resistance across malignancies and therapeutic modalities2-5. Whether myeloid inflammation drives progression of prostate cancer in humans remain unclear. Here we show that inhibition of myeloid chemotaxis can reduce tumour-elicited myeloid inflammation and reverse therapy resistance in a subset of patients with metastatic castration-resistant prostate cancer (CRPC). We show that a higher blood neutrophil-to-lymphocyte ratio reflects tumour myeloid infiltration and tumour expression of senescence-associated mRNA species, including those that encode myeloid-chemoattracting CXCR2 ligands. To determine whether myeloid cells fuel resistance to androgen receptor signalling inhibitors, and whether inhibiting CXCR2 to block myeloid chemotaxis reverses this, we conducted an investigator-initiated, proof-of-concept clinical trial of a CXCR2 inhibitor (AZD5069) plus enzalutamide in patients with metastatic CRPC that is resistant to androgen receptor signalling inhibitors. This combination was well tolerated without dose-limiting toxicity and it decreased circulating neutrophil levels, reduced intratumour CD11b+HLA-DRloCD15+CD14- myeloid cell infiltration and imparted durable clinical benefit with biochemical and radiological responses in a subset of patients with metastatic CRPC. This study provides clinical evidence that senescence-associated myeloid inflammation can fuel metastatic CRPC progression and resistance to androgen receptor blockade. Targeting myeloid chemotaxis merits broader evaluation in other cancers.

Durable responses to ATR inhibition with ceralasertib in tumors with genomic defects and high inflammation.

BACKGROUNDPhase 1 study of ATRinhibition alone or with radiation therapy (PATRIOT) was a first-in-human phase I study of the oral ATR (ataxia telangiectasia and Rad3-related) inhibitor ceralasertib (AZD6738) in advanced solid tumors.METHODSThe primary objective was safety. Secondary objectives included assessment of antitumor responses and pharmacokinetic (PK) and pharmacodynamic (PD) studies. Sixty-seven patients received 20-240 mg ceralasertib BD continuously or intermittently (14 of a 28-day cycle).RESULTSIntermittent dosing was better tolerated than continuous, which was associated with dose-limiting hematological toxicity. The recommended phase 2 dose of ceralasertib was 160 mg twice daily for 2 weeks in a 4-weekly cycle. Modulation of target and increased DNA damage were identified in tumor and surrogate PD. There were 5 (8%) confirmed partial responses (PRs) (40-240 mg BD), 34 (52%) stable disease (SD), including 1 unconfirmed PR, and 27 (41%) progressive disease. Durable responses were seen in tumors with loss of AT-rich interactive domain-containing protein 1A (ARID1A) and DNA damage-response defects. Treatment-modulated tumor and systemic immune markers and responding tumors were more immune inflamed than nonresponding.CONCLUSIONCeralasertib monotherapy was tolerated at 160 mg BD intermittently and associated with antitumor activity.TRIAL REGISTRATIONClinicaltrials.gov: NCT02223923, EudraCT: 2013-003994-84.FUNDINGCancer Research UK, AstraZeneca, UK Department of Health (National Institute for Health Research), Rosetrees Trust, Experimental Cancer Medicine Centre.

Triplet Therapy with Palbociclib, Taselisib, and Fulvestrant in PIK3CA-Mutant Breast Cancer and Doublet Palbociclib and Taselisib in Pathway-Mutant Solid Cancers.

Cyclin-dependent kinase 4/6 (CDK4/6) and PI3K inhibitors synergize in PIK3CA-mutant ER-positive HER2-negative breast cancer models. We conducted a phase Ib trial investigating the safety and efficacy of doublet CDK4/6 inhibitor palbociclib plus selective PI3K inhibitor taselisib in advanced solid tumors, and triplet palbociclib plus taselisib plus fulvestrant in 25 patients with PIK3CA-mutant, ER-positive HER2-negative advanced breast cancer. The triplet therapy response rate in PIK3CA-mutant, ER-positive HER2-negative cancer was 37.5% [95% confidence interval (CI), 18.8-59.4]. Durable disease control was observed in PIK3CA-mutant ER-negative breast cancer and other solid tumors with doublet therapy. Both combinations were well tolerated at pharmacodynamically active doses. In the triplet group, high baseline cyclin E1 expression associated with shorter progression-free survival (PFS; HR = 4.2; 95% CI, 1.3-13.1; P = 0.02). Early circulating tumor DNA (ctDNA) dynamics demonstrated high on-treatment ctDNA association with shorter PFS (HR = 5.2; 95% CI, 1.4-19.4; P = 0.04). Longitudinal plasma ctDNA sequencing provided genomic evolution evidence during triplet therapy. SIGNIFICANCE: The triplet of palbociclib, taselisib, and fulvestrant has promising efficacy in patients with heavily pretreated PIK3CA-mutant ER-positive HER2-negative advanced breast cancer. A subset of patients with PIK3CA-mutant triple-negative breast cancer derived clinical benefit from palbociclib and taselisib doublet, suggesting a potential nonchemotherapy targeted approach for this population.This article is highlighted in the In This Issue feature, p. 1.

First-in-Human Study of AT13148, a Dual ROCK-AKT Inhibitor in Patients with Solid Tumors.

PURPOSE: AT13148 is an oral AGC kinase inhibitor, which potently inhibits ROCK and AKT kinases. In preclinical models, AT13148 has been shown to have antimetastatic and antiproliferative activity. PATIENTS AND METHODS: The trial followed a rolling six design during dose escalation. An intrapatient dose escalation arm to evaluate tolerability and a biopsy cohort to study pharmacodynamic effects were later added. AT13148 was administered orally three days a week (Mon-Wed-Fri) in 28-day cycles. Pharmacokinetic profiles were assessed using mass spectrometry and pharmacodynamic studies included quantifying p-GSK3ß levels in platelet-rich plasma (PRP) and p-cofilin and p-MLC2 levels in tumor biopsies. RESULTS: Fifty-one patients were treated on study. The safety of 5-300 mg of AT13148 was studied. Further, the doses of 120-180-240 mg were studied in an intrapatient dose escalation cohort. The dose-limiting toxicities included hypotension (300 mg), pneumonitis, and elevated liver enzymes (240 mg), and skin rash (180 mg). The most common side effects were fatigue, nausea, headaches, and hypotension. On the basis of tolerability, 180 mg was considered the maximally tolerated dose. At 180 mg, mean C max and AUC were 400 nmol/L and 13,000 nmol/L/hour, respectively. At 180 mg, ≥50% reduction of p-cofilin was observed in 3 of 8 posttreatment biopsies. CONCLUSIONS: AT13148 was the first dual potent ROCK-AKT inhibitor to be investigated for the treatment of solid tumors. The narrow therapeutic index and the pharmacokinetic profile led to recommend not developing this compound further. There are significant lessons learned in designing and testing agents that simultaneously inhibit multiple kinases including AGC kinases in cancer.

Phase I Trial of the PARP Inhibitor Olaparib and AKT Inhibitor Capivasertib in Patients with BRCA1/2- and Non-BRCA1/2-Mutant Cancers.

Preclinical studies have demonstrated synergy between PARP and PI3K/AKT pathway inhibitors in BRCA1 and BRCA2 (BRCA1/2)-deficient and BRCA1/2-proficient tumors. We conducted an investigator-initiated phase I trial utilizing a prospective intrapatient dose- escalation design to assess two schedules of capivasertib (AKT inhibitor) with olaparib (PARP inhibitor) in 64 patients with advanced solid tumors. Dose expansions enrolled germline BRCA1/2-mutant tumors, or BRCA1/2 wild-type cancers harboring somatic DNA damage response (DDR) or PI3K-AKT pathway alterations. The combination was well tolerated. Recommended phase II doses for the two schedules were: olaparib 300 mg twice a day with either capivasertib 400 mg twice a day 4 days on, 3 days off, or capivasertib 640 mg twice a day 2 days on, 5 days off. Pharmacokinetics were dose proportional. Pharmacodynamic studies confirmed phosphorylated (p) GSK3ß suppression, increased pERK, and decreased BRCA1 expression. Twenty-five (44.6%) of 56 evaluable patients achieved clinical benefit (RECIST complete response/partial response or stable disease ≥ 4 months), including patients with tumors harboring germline BRCA1/2 mutations and BRCA1/2 wild-type cancers with or without DDR and PI3K-AKT pathway alterations. SIGNIFICANCE: In the first trial to combine PARP and AKT inhibitors, a prospective intrapatient dose- escalation design demonstrated safety, tolerability, and pharmacokinetic-pharmacodynamic activity and assessed predictive biomarkers of response/resistance. Antitumor activity was observed in patients harboring tumors with germline BRCA1/2 mutations and BRCA1/2 wild-type cancers with or without somatic DDR and/or PI3K-AKT pathway alterations.This article is highlighted in the In This Issue feature, p. 1426.

Farnesoid x receptor ligands inhibit vascular smooth muscle cell inflammation and migration.

OBJECTIVE: The farnesoid X receptor/bile acid receptor (FXR; NR1H4) is a ligand-activated transcription factor that regulates bile acid and lipid homeostasis, and is highly expressed in enterohepatic tissue. FXR is also expressed in vascular tissue. We have investigated whether FXR regulates inflammation and migration in vascular smooth muscle cells. METHODS AND RESULTS: The FXR target gene, small heterodimer partner (SHP), was induced in vascular smooth muscle cells after treatment with synthetic FXR ligands, GW4064, or 6alpha-ethyl-chenodeoxycholic acid. FXR ligands induced smooth muscle cell death and downregulated interleukin (IL)-1beta-induced inducible nitric oxide synthase and cyclooxygenase-2 expression. In addition, FXR ligands suppressed smooth muscle cell migration stimulated by platelet-derived growth factor-BB. Reporter gene assays showed that FXR ligands activated an FXR reporter gene and suppressed IL-1beta-induced nuclear factor (NF)-kappaB activation and iNOS in a manner that required functional FXR and SHP. CONCLUSIONS: Our observations suggest that a FXR-SHP pathway may be a novel therapeutic target for vascular inflammation, remodeling, and atherosclerotic plaque stability.

The farnesoid X receptor is expressed in breast cancer and regulates apoptosis and aromatase expression.

Bile acids are present at high concentrations in breast cysts and in the plasma of postmenopausal women with breast cancer. The farnesoid X receptor (FXR) is a member of the nuclear receptor superfamily that regulates bile acid homeostasis. FXR was detected in normal and tumor breast tissue, with a high level of expression in ductal epithelial cells of normal breast and infiltrating ductal carcinoma cells. FXR was also present in the human breast carcinoma cells, MCF-7 and MDA-MB-468. Activation of FXR by high concentrations of ligands induced MCF-7 and MDA-MB-468 apoptosis. At lower concentrations that had no direct effect on viability, the FXR agonist GW4064 induced expression of mRNA for the FXR target genes, small heterodimer partner (SHP), intestinal bile acid binding protein, and multidrug resistance-associated protein 2 (MRP-2), and repressed the expression of the SHP target gene aromatase. In contrast to MRP-2, mRNA for the breast cancer target genes MDR-3, MRP-1, and solute carrier transporter 7A5 were decreased. Although multidrug resistance transporters were regulated and are known FXR target genes, GW4064 had no effect on the cell death induced by the anticancer drug paclitaxel. Our findings show for the first time that FXR is expressed in breast cancer tissue and has multiple properties that could be used for the treatment of breast cancer.

Evaluation of the combination of the dual m-TORC1/2 inhibitor vistusertib (AZD2014) and paclitaxel in ovarian cancer models.

Activation of the PI3K/mTOR pathway has been shown to be correlated with resistance to chemotherapy in ovarian cancer. We aimed to investigate the effects of combining inhibition of mTORC1 and 2 using the mTOR kinase inhibitor vistusertib (AZD2014) with paclitaxel in in vitro and in vivo ovarian cancer models. The combination of vistusertib and paclitaxel on cell growth was additive in a majority of cell lines in the panel (n = 12) studied. A cisplatin- resistant model (A2780Cis) was studied in vitro and in vivo. We demonstrated inhibition of mTORC1 and mTORC2 by vistusertib and the combination by showing reduction in p-S6 and p-AKT levels, respectively. In the A2780CisR xenograft model compared to control, there was a significant reduction in tumor volumes (p = 0.03) caused by the combination and not paclitaxel or vistusertib alone. In vivo, we observed a significant increase in apoptosis (cleaved PARP measured by immunohistochemistry; p = 0.0003). Decreases in phospholipid and bioenergetic metabolites were studied using magnetic resonance spectroscopy and significant changes in phosphocholine (p = 0.01), and ATP (p = 0.04) were seen in tumors treated with the combination when compared to vehicle-control. Based on this data, a clinical trial evaluating the combination of paclitaxel and vistusertib has been initiated (NCT02193633). Interestingly, treatment of ovarian cancer patients with paclitaxel caused an increase in p-AKT levels in platelet-rich plasma and it was possible to abrogate this increase with the co-treatment with vistusertib in 4/5 patients: we believe this combination will benefit patients with ovarian cancer.

Activation of the Farnesoid X-receptor in breast cancer cell lines results in cytotoxicity but not increased migration potential.

Breast cancer is the commonest form of cancer in women, but successful treatment is confounded by the heterogeneous nature of breast tumours: Effective treatments exist for hormone-sensitive tumours, but triple-negative breast cancer results in poor survival. An area of increasing interest is metabolic reprogramming, whereby drug-induced alterations in the metabolic landscape of a tumour slow tumour growth and/or increase sensitivity to existing therapeutics. Nuclear receptors are transcription factors central to the expression of metabolic and transport proteins, and thus represent potential targets for metabolic reprogramming. We show that activation of the nuclear receptor FXR, either by its endogenous ligand CDCA or the synthetic GW4064, leads to cell death in four breast cancer cell lines with distinct phenotypes: MCF-10A (normal), MCF-7 (receptor positive), MDA-MB-231 and MDA-MB-468 (triple negative). Furthermore, we show that the mechanism of cell death is predominantly through the intrinsic apoptotic pathway. Finally, we demonstrate that FXR agonists do not stimulate migration in breast cancer cell lines, an important potential adverse effect. Together, our data support the continued examination of FXR agonists as a novel class of therapeutics for the treatment of breast cancer.

Pregnane X receptor regulates drug metabolism and transport in the vasculature and protects from oxidative stress.

AIMS: Circulating endogenous, dietary, and foreign chemicals can contribute to vascular dysfunction. The mechanism by which the vasculature protects itself from these chemicals is unknown. This study investigates whether the pregnane X receptor (PXR), the major transcriptional regulator of hepatic drug metabolism and transport that responds to such xenobiotics, mediates vascular protection by co-ordinating a defence gene programme in the vasculature. METHODS AND RESULTS: PXR was detected in primary human and rat aortic endothelial and smooth muscle cells (SMC) and blood vessels including the human and rat aorta. Metabolic PXR target genes cytochrome P450 3A, 2B, 2C, and glutathione S-transferase mRNA and activity were induced by PXR ligands in rodent and human vascular cells and absent in the aortas from PXR-null mice stimulated in vivo or in rat aortic SMC expressing dominant-negative PXR. Activation of aortic PXR by classical agonists had several protective effects: increased xenobiotic metabolism demonstrated by bioactivation of the pro-drug clopidogrel, which reduced adenosine diphosphate-induced platelet aggregation; increased expression of multidrug resistance protein 1, mediating chemical efflux from the vasculature; and protection from reactive oxygen species-mediated cell death. CONCLUSION: PXR co-ordinately up-regulates drug metabolism, transport, and antioxidant genes to protect the vasculature from endogenous and exogenous insults, thus representing a novel gatekeeper for vascular defence.

Endogenous epoxygenases are modulators of monocyte/macrophage activity.

BACKGROUND: Arachidonic acid is metabolized through three major metabolic pathways, the cyclooxygenase, lipoxygenase and CYP450 enzyme systems. Unlike cyclooxygenase and lipoxygenases, the role of CYP450 epoxygenases in monocyte/macrophage-mediated responses is not known. METHODOLOGY/PRINCIPAL FINDINGS: When transfected in vitro, CYP2J2 is an efficient activator of anti-inflammatory pathways through the nuclear receptor peroxisome proliferator-activated receptor (PPAR) α. Human monocytes and macrophages contain PPARα and here we show they express the epoxygenases CYP2J2 and CYP2C8. Inhibition of constitutive monocyte epoxygenases using the epoxygenase inhibitor SKF525A induces cyclooxygenase (COX)-2 expression and activity, and the release of TNFα, and can be reversed by either add back of the endogenous epoxygenase products and PPARα ligand 11,12- epoxyeicosatrienoic acid (EET) or the addition of the selective synthetic PPARα ligand GW7647. In alternatively activated (IL-4-treated) monocytes, in contrast to classically activated cells, epoxygenase inhibition decreased TNFα release. Epoxygenases can be pro-inflammatory via superoxide anion production. The suppression of TNFα by SKF525A in the presence of IL-4 was associated with a reduction in superoxide anion generation and reproduced by the superoxide dismutase MnCl(2). Similar to these acute activation studies, in monocyte derived macrophages, epoxygenase inhibition elevates M1 macrophage TNFα mRNA and further decreases M2 macrophage TNFα. CONCLUSIONS/SIGNIFICANCE: In conclusion, epoxygenase activity represents an important endogenous pathway which limits monocyte activation. Moreover endogenous epoxygenases are immuno-modulators regulating monocyte/macrophage activation depending on the underlying activation state.

The Role of PPARs in the Endothelium: Implications for Cancer Therapy.

The growth and metastasis of cancers intimately involve the vasculature and in particular the endothelial cell layer. Tumours require new blood vessel formation via angiogenesis to support growth. In addition, inflammation, coagulation, and platelet activation are common signals in the growth and metastasis of tumour cells. The endothelium plays a central role in the homeostatic control of inflammatory cell recruitment, regulating platelet activation and coagulation pathways. PPARalpha, -beta/delta, and -gamma are all expressed in endothelial cells. This review will discuss the roles of PPARs in endothelial cells in relation to angiogenesis, inflammation, coagulation, and platelet control pathways. In particular, we will discuss the recent evidence that supports the hypothesis that PPARalpha and PPARgamma are antiangiogenic receptors, while PPARbeta/delta is proangiogenic.

Nongenomic signaling of the retinoid X receptor through binding and inhibiting Gq in human platelets.

Retinoid X receptors (RXRs) are important transcriptional nuclear hormone receptors, acting as either homodimers or the binding partner for at least one fourth of all the known human nuclear receptors. Functional nongenomic effects of nuclear receptors are poorly understood; however, recently peroxisome proliferator-activated receptor (PPAR) gamma, PPARbeta, and the glucocorticoid receptor have all been found active in human platelets. Human platelets express RXRalpha and RXRbeta. RXR ligands inhibit platelet aggregation and TXA(2) release to ADP and the TXA(2) receptors, but only weakly to collagen. ADP and TXA(2) both signal via the G protein, Gq. RXR rapidly binds Gq but not Gi/z/o/t/gust in a ligand-dependent manner and inhibits Gq-induced Rac activation and intracellular calcium release. We propose that RXR ligands may have beneficial clinical actions through inhibition of platelet activation. Furthermore, our results demonstrate a novel nongenomic mode for nuclear receptor action and a functional cross-talk between G-protein and nuclear receptor signaling families.