Discovery of GDC-0077 (Inavolisib), a Highly Selective Inhibitor and Degrader of Mutant PI3Kα.

Small molecule inhibitors that target the phosphatidylinositol 3-kinase (PI3K) signaling pathway have received significant interest for the treatment of cancers. The class I isoform PI3Kα is most commonly associated with solid tumors via gene amplification or activating mutations. However, inhibitors demonstrating both PI3K isoform and mutant specificity have remained elusive. Herein, we describe the optimization and characterization of a series of benzoxazepin-oxazolidinone ATP-competitive inhibitors of PI3Kα which also induce the selective degradation of the mutant p110α protein, the catalytic subunit of PI3Kα. Structure-based design informed isoform-specific interactions within the binding site, leading to potent inhibitors with greater than 300-fold selectivity over the other Class I PI3K isoforms. Further optimization of pharmacokinetic properties led to excellent in vivo exposure and efficacy and the identification of clinical candidate GDC-0077 (inavolisib, 32), which is now under evaluation in a Phase III clinical trial as a treatment for patients with PIK3CA-mutant breast cancer.

RTK-Dependent Inducible Degradation of Mutant PI3Kα Drives GDC-0077 (Inavolisib) Efficacy.

PIK3CA is one of the most frequently mutated oncogenes; the p110a protein it encodes plays a central role in tumor cell proliferation. Small-molecule inhibitors targeting the PI3K p110a catalytic subunit have entered clinical trials, with early-phase GDC-0077 studies showing antitumor activity and a manageable safety profile in patients with PIK3CA-mutant breast cancer. However, preclinical studies have shown that PI3K pathway inhibition releases negative feedback and activates receptor tyrosine kinase signaling, reengaging the pathway and attenuating drug activity. Here we discover that GDC-0077 and taselisib more potently inhibit mutant PI3K pathway signaling and cell viability through unique HER2-dependent mutant p110a degradation. Both are more effective than other PI3K inhibitors at maintaining prolonged pathway suppression. This study establishes a new strategy for identifying inhibitors that specifically target mutant tumors by selective degradation of the mutant oncoprotein and provide a strong rationale for pursuing PI3Kα degraders in patients with HER2-positive breast cancer. SIGNIFICANCE: The PI3K inhibitors GDC-0077 and taselisib have a unique mechanism of action; both inhibitors lead to degradation of mutant p110a protein. The inhibitors that have the ability to trigger specific degradation of mutant p110a without significant change in wild-type p110a protein may result in improved therapeutic index in PIK3CA-mutant tumors.See related commentary by Vanhaesebroeck et al., p. 20.This article is highlighted in the In This Issue feature, p. 1.

Discovery of GNE-502 as an orally bioavailable and potent degrader for estrogen receptor positive breast cancer.

Fulvestrant is an FDA-approved drug with a dual mechanism of action (MOA), acting as a full antagonist and degrader of the estrogen receptor protein. A significant limitation of fulvestrant is the dosing regimen required for efficacy. Due to its high lipophilicity and poor pharmacokinetic profile, fulvestrant needs to be administered through intramuscular injections which leads to injection site soreness. This route of administration also limits the dose and target occupancy in patients. We envisioned a best-in-class molecule that would function with the same dual MOA as fulvestrant, but with improved physicochemical properties and would be orally bioavailable. Herein we report our progress toward that goal, resulting in a new lead GNE-502 which addressed some of the liabilities of our previously reported lead molecule GNE-149.

GDC-9545 (Giredestrant): A Potent and Orally Bioavailable Selective Estrogen Receptor Antagonist and Degrader with an Exceptional Preclinical Profile for ER+ Breast Cancer.

Breast cancer remains a leading cause of cancer death in women, representing a significant unmet medical need. Here, we disclose our discovery efforts culminating in a clinical candidate, 35 (GDC-9545 or giredestrant). 35 is an efficient and potent selective estrogen receptor degrader (SERD) and a full antagonist, which translates into better antiproliferation activity than known SERDs (1, 6, 7, and 9) across multiple cell lines. Fine-tuning the physiochemical properties enabled once daily oral dosing of 35 in preclinical species and humans. 35 exhibits low drug-drug interaction liability and demonstrates excellent in vitro and in vivo safety profiles. At low doses, 35 induces tumor regressions either as a single agent or in combination with a CDK4/6 inhibitor in an ESR1Y537S mutant PDX or a wild-type ERα tumor model. Currently, 35 is being evaluated in Phase III clinical trials.

Discovery of GNE-149 as a Full Antagonist and Efficient Degrader of Estrogen Receptor alpha for ER+ Breast Cancer.

Estrogen receptor alpha (ERα) is a well-validated drug target for ER-positive (ER+) breast cancer. Fulvestrant is FDA-approved to treat ER+ breast cancer and works through two mechanisms-as a full antagonist and selective estrogen receptor degrader (SERD)-but lacks oral bioavailability. Thus, we envisioned a "best-in-class" molecule with the same dual mechanisms as fulvestrant, but with significant oral exposure. Through lead optimization, we discovered a tool molecule 12 (GNE-149) with improved degradation and antiproliferative activity in both MCF7 and T47D cells. To illustrate the binding mode and key interactions of this scaffold with ERα, we obtained a cocrystal structure of 6 that showed ionic interaction of azetidine with Asp351 residue. Importantly, 12 showed favorable metabolic stability and good oral exposure. 12 exhibited antagonist effect in the uterus and demonstrated robust dose-dependent efficacy in xenograft models.

Therapeutic Ligands Antagonize Estrogen Receptor Function by Impairing Its Mobility.

Estrogen receptor-positive (ER+) breast cancers frequently remain dependent on ER signaling even after acquiring resistance to endocrine agents, prompting the development of optimized ER antagonists. Fulvestrant is unique among approved ER therapeutics due to its capacity for full ER antagonism, thought to be achieved through ER degradation. The clinical potential of fulvestrant is limited by poor physicochemical features, spurring attempts to generate ER degraders with improved drug-like properties. We show that optimization of ER degradation does not guarantee full ER antagonism in breast cancer cells; ER "degraders" exhibit a spectrum of transcriptional activities and anti-proliferative potential. Mechanistically, we find that fulvestrant-like antagonists suppress ER transcriptional activity not by ER elimination, but by markedly slowing the intra-nuclear mobility of ER. Increased ER turnover occurs as a consequence of ER immobilization. These findings provide proof-of-concept that small molecule perturbation of transcription factor mobility may enable therapeutic targeting of this challenging target class.

Unexpected equivalent potency of a constrained chromene enantiomeric pair rationalized by co-crystal structures in complex with estrogen receptor alpha.

Despite tremendous progress made in the understanding of the ERα signaling pathway and the approval of many therapeutic agents, ER+ breast cancer continues to be a leading cause of cancer death in women. We set out to discover compounds with a dual mechanism of action in which they not only compete with estradiol for binding with ERα, but also can induce the degradation of the ERα protein itself. We were attracted to the constrained chromenes containing a tetracyclic benzopyranobenzoxepine scaffold, which were reported as potent selective estrogen receptor modulators (SERMs). Incorporation of a fluoromethyl azetidine side chain yielded highly potent and efficacious selective estrogen receptor degraders (SERDs), such as 16aa and surprisingly, also its enantiomeric pair 16ab. Co-crystal structures of the enantiomeric pair 16aa and 16ab in complex with ERα revealed default (mimics the A-D rings of endogenous ligand estradiol) and core-flipped binding modes, rationalizing the equivalent potency observed for these enantiomers in the ERα degradation and MCF-7 anti-proliferation assays.

The Kinase Activity of Hematopoietic Progenitor Kinase 1 Is Essential for the Regulation of T Cell Function.

We examined hematopoietic protein kinase 1 (HPK1), whose reliance on scaffold versus kinase functions for negative immune cell regulation is poorly understood and critical to its assessment as a viable drug target. We identify kinase-dependent roles for HPK1 in CD8 T cells that restrict their anti-viral and anti-tumor responses by using HPK1 kinase-dead (HPK1.kd) knockin mice. Loss of HPK1 kinase function enhanced T cell receptor signaling and cytokine secretion in a T-cell-intrinsic manner. In response to chronic lymphocytic choriomeningitis virus (LCMV) infection or tumor challenge, viral clearance and tumor growth inhibition were enhanced in HPK1.kd mice, accompanied by an increase in effector CD8 T cell function. Co-blockade of PD-L1 further enhanced T effector cell function, resulting in superior anti-viral and anti-tumor immunity over single target blockade. These results identify the importance of HPK1 kinase activity in the negative regulation of CD8 effector functions, implicating its potential as a cancer immunotherapy target.

Pan-Cancer Metabolic Signature Predicts Co-Dependency on Glutaminase and De Novo Glutathione Synthesis Linked to a High-Mesenchymal Cell State.

The enzyme glutaminase (GLS1) is currently in clinical trials for oncology, yet there are no clear diagnostic criteria to identify responders. The evaluation of 25 basal breast lines expressing GLS1, predominantly through its splice isoform GAC, demonstrated that only GLS1-dependent basal B lines required it for maintaining de novo glutathione synthesis in addition to mitochondrial bioenergetics. Drug sensitivity profiling of 407 tumor lines with GLS1 and gamma-glutamylcysteine synthetase (GCS) inhibitors revealed a high degree of co-dependency on both enzymes across indications, suggesting that redox balance is a key function of GLS1 in tumors. To leverage these findings, we derived a pan-cancer metabolic signature predictive of GLS1/GCS co-dependency and validated it in vivo using four lung patient-derived xenograft models, revealing the additional requirement for expression of GAC above a threshold (log2RPKM + 1 ≥ 4.5, where RPKM is reads per kilobase per million mapped reads). Analysis of the pan-TCGA dataset with our signature identified multiple indications, including mesenchymal tumors, as putative responders to GLS1 inhibitors.

Conjugation of Indoles to Antibodies through a Novel Self-Immolating Linker.

A novel strategy to attach indole-containing payloads to antibodies through a carbamate moiety and a self-immolating, disulfide-based linker is described. This new strategy was employed to connect a selective estrogen receptor down-regulator (SERD) to various antibodies in a site-selective manner. The resulting conjugates displayed potent, antigen-dependent down-regulation of estrogen receptor levels in MCF7-neo/HER2 and MCF7-hB7H4 cells. They also exhibited similar antigen-dependent modulation of the estrogen receptor in tumors when administered intravenously to mice bearing MCF7-neo/HER2 tumor xenografts. The indole-carbamate moiety present in the new linker was stable in whole blood from various species and also exhibited good in vivo stability properties in mice.

A First-in-Human Phase I Study of the ATP-Competitive AKT Inhibitor Ipatasertib Demonstrates Robust and Safe Targeting of AKT in Patients with Solid Tumors.

Activation of AKT signaling by PTEN loss or PIK3CA mutations occurs frequently in human cancers, but targeting AKT has been difficult due to the mechanism-based toxicities of inhibitors that target the inactive conformation of AKT. Ipatasertib (GDC-0068) is a novel selective ATP-competitive small-molecule inhibitor of AKT that preferentially targets active phosphorylated AKT (pAKT) and is potent in cell lines with evidence of AKT activation. In this phase I study, ipatasertib was well tolerated; most adverse events were gastrointestinal and grade 1-2 in severity. The exposures of ipatasertib ≥200 mg daily in patients correlated with preclinical TGI90, and pharmacodynamic studies confirmed that multiple targets (i.e., PRAS40, GSK3ß, and mTOR) were inhibited in paired on-treatment biopsies. Preliminary antitumor activity was observed; 16 of 52 patients (30%), with diverse solid tumors and who progressed on prior therapies, had radiographic stable disease, and many of their tumors had activation of AKT. SIGNIFICANCE: Potent inhibition of AKT signaling with ipatasertib was associated with a tolerable safety profile and meaningful disease control in a subgroup of patients. Targeting pAKT with an ATP-competitive inhibitor provides a greater therapeutic window than allosteric inhibitors. Further investigation with ipatasertib is ongoing in phase II studies. Cancer Discov; 7(1); 102-13. ©2016 AACR.This article is highlighted in the In This Issue feature, p. 1.

The selective estrogen receptor downregulator GDC-0810 is efficacious in diverse models of ER+ breast cancer.

ER-targeted therapeutics provide valuable treatment options for patients with ER+ breast cancer, however, current relapse and mortality rates emphasize the need for improved therapeutic strategies. The recent discovery of prevalent ESR1 mutations in relapsed tumors underscores a sustained reliance of advanced tumors on ERα signaling, and provides a strong rationale for continued targeting of ERα. Here we describe GDC-0810, a novel, non-steroidal, orally bioavailable selective ER downregulator (SERD), which was identified by prospectively optimizing ERα degradation, antagonism and pharmacokinetic properties. GDC-0810 induces a distinct ERα conformation, relative to that induced by currently approved therapeutics, suggesting a unique mechanism of action. GDC-0810 has robust in vitro and in vivo activity against a variety of human breast cancer cell lines and patient derived xenografts, including a tamoxifen-resistant model and those that harbor ERα mutations. GDC-0810 is currently being evaluated in Phase II clinical studies in women with ER+ breast cancer.

Strategically Timing Inhibition of Phosphatidylinositol 3-Kinase to Maximize Therapeutic Index in Estrogen Receptor Alpha-Positive, PIK3CA-Mutant Breast Cancer.

PURPOSE: Phosphatidylinositol 3-kinase (PI3K) inhibitors are being developed for the treatment of estrogen receptor α (ER)-positive breast cancer in combination with antiestrogens. Understanding the temporal response and pharmacodynamic effects of PI3K inhibition in ER(+) breast cancer will provide a rationale for treatment scheduling to maximize therapeutic index. EXPERIMENTAL DESIGN: Antiestrogen-sensitive and antiestrogen-resistant ER(+) human breast cancer cell lines and mice bearing PIK3CA-mutant xenografts were treated with the antiestrogen fulvestrant, the PI3K inhibitor GDC-0941 (pictilisib; varied doses/schedules that provided similar amounts of drug each week), or combinations. Cell viability, signaling pathway inhibition, proliferation, apoptosis, tumor volume, and GDC-0941 concentrations in plasma and tumors were temporally measured. RESULTS: Treatment with the combination of fulvestrant and GDC-0941, regardless of dose/schedule, was significantly more effective than that with single-agent treatments in fulvestrant-resistant tumors. Short-term, complete PI3K inhibition blocked cell growth in vitro more effectively than chronic, incomplete inhibition. Longer-term PI3K inhibition hypersensitized cells to growth factor signaling upon drug withdrawal. Different schedules of GDC-0941 elicited similar tumor responses. While weekly high-dose GDC-0941 with fulvestrant continuously suppressed PI3K signaling for 72 hours, inducing a bolus of apoptosis and inhibiting proliferation, PI3K reactivation upon GDC-0941 washout induced a proliferative burst. Fulvestrant with daily low-dose GDC-0941 metronomically suppressed PI3K for 6 to 9 hours/day, repeatedly inducing small amounts of apoptosis and temporarily inhibiting proliferation, followed by proliferative rebound compared with fulvestrant alone. CONCLUSIONS: Continuous and metronomic PI3K inhibition elicits robust anticancer effects in ER(+), PIK3CA-mutant breast cancer. Clinical exploration of alternate treatment schedules of PI3K inhibitors with antiestrogens is warranted. Clin Cancer Res; 22(9); 2250-60. ©2016 AACRSee related commentary by Toska and Baselga, p. 2099.

Modeling targeted inhibition of MEK and PI3 kinase in human pancreatic cancer.

Activating mutations in the KRAS oncogene occur in approximately 90% of pancreatic cancers, resulting in aberrant activation of the MAPK and the PI3K pathways, driving malignant progression. Significant efforts to develop targeted inhibitors of nodes within these pathways are underway and several are currently in clinical trials for patients with KRAS-mutant tumors, including patients with pancreatic cancer. To model MEK and PI3K inhibition in late-stage pancreatic cancer, we conducted preclinical trials with a mutant Kras-driven genetically engineered mouse model that faithfully recapitulates human pancreatic ductal adenocarcinoma development. Treatment of advanced disease with either a MEK (GDC-0973) or PI3K inhibitor (GDC-0941) alone showed modest tumor growth inhibition and did not significantly enhance overall survival. However, combination of the two agents resulted in a significant survival advantage as compared with control tumor-bearing mice. To model the clinical scenario, we also evaluated the combination of these targeted agents with gemcitabine, the current standard-of-care chemotherapy for pancreatic cancer. The addition of MEK or PI3K inhibition to gemcitabine, or the triple combination regimen, incrementally enhanced overall survival as compared with gemcitabine alone. These results are reminiscent of the survival advantage conferred in this model and in patients by the combination of gemcitabine and erlotinib, an approved therapeutic regimen for advanced nonresectable pancreatic cancer. Taken together, these data indicate that inhibition of MEK and PI3K alone or in combination with chemotherapy do not confer a dramatic improvement as compared with currently available therapies for patients with pancreatic cancer.

Nanosuspension delivery of paclitaxel to xenograft mice can alter drug disposition and anti-tumor activity.

Paclitaxel is a common chemotherapeutic agent that is effective against various cancers. The poor aqueous solubility of paclitaxel necessitates a large percentage of Cremophor EL:ethanol (USP) in its commercial formulation which leads to hypersensitivity reactions in patients. We evaluate the use of a crystalline nanosuspension versus the USP formulation to deliver paclitaxel to tumor-bearing xenograft mice. Anti-tumor efficacy was assessed following intravenous administration of three 20 mg/kg doses of paclitaxel. Paclitaxel pharmacokinetics and tissue distribution were evaluated, and differences were observed between the two formulations. Plasma clearance and tissue to plasma ratio of mice that were dosed with the nanosuspension are approximately 33- and 11-fold higher compared to those of mice that were given the USP formulation. Despite a higher tumor to plasma ratio for the nanosuspension treatment group, absolute paclitaxel tumor exposure was higher for the USP group. Accordingly, a higher anti-tumor effect was observed in the xenograft mice that were dosed with the USP formulation (90% versus 42% tumor growth inhibition). This reduction in activity of nanoparticle formulation appeared to result from a slower than anticipated dissolution in vivo. This study illustrates a need for careful consideration of both dose and systemic solubility prior utilizing nanosuspension as a mode of intravenous delivery.

MAPK pathway inhibition enhances the efficacy of an anti-endothelin B receptor drug conjugate by inducing target expression in melanoma.

Therapies targeting the mitogen-activated protein (MAP) kinase pathway in melanoma have produced significant clinical responses; however, duration of response is limited by acquisition of drug resistance. Rational drug combinations may improve outcomes in this setting. We assessed the therapeutic combination of an antibody-drug conjugate (ADC) targeting the endothelin B receptor (EDNRB) with small-molecule inhibitors of the MAP kinase signaling pathway in melanoma. Cell lines and tumor models containing either mutant BRAF or NRAS, or wild-type for both, were exposed to small-molecule inhibitors of BRAF and MEK. Expression of EDNRB was analyzed and the therapeutic impact of combining the anti-EDNRB ADC with the BRAF and MEK inhibitors was assessed. Increased expression of EDNRB in response to inhibition of BRAF and/or MEK was observed and augmented the antitumor activity of the ADC. Enhanced target expression and ADC antitumor activity were realized irrespective of the response of the tumor model to the BRAF or MEK inhibitors alone and could be achieved in melanoma with mutant NRAS, BRAF, or neither mutation. Cells that acquired resistance to BRAF inhibition through long-term culture retained drug-induced elevated levels of EDNRB expression. Expression of EDNRB was not enhanced in normal human melanocytes by inhibition of BRAF and the combination of the ADC with MAPK inhibitors was well-tolerated in mice. The anti-EDNRB ADC combines well with BRAF and MEK inhibitors and could have therapeutic use in the majority of human melanoma cases.

Evaluation and clinical analyses of downstream targets of the Akt inhibitor GDC-0068.

PURPOSE: The oncogenic PI3K/Akt/mTOR pathway is an attractive therapeutic target in cancer. However, it is unknown whether the pathway blockade required for tumor growth inhibition is clinically achievable. Therefore, we conducted pharmacodynamic studies with GDC-0068, an ATP competitive, selective Akt1/2/3 inhibitor, in preclinical models and in patients treated with this compound. EXPERIMENTAL DESIGN: We used a reverse phase protein array (RPPA) platform to identify a biomarker set indicative of Akt inhibition in cell lines and human-tumor xenografts, and correlated the degree of pathway inhibition with antitumor activity. Akt pathway activity was measured using this biomarker set in pre- and post-dose tumor biopsies from patients treated with GDC-0068 in the dose escalation clinical trial. RESULTS: The set of biomarkers of Akt inhibition is composed of 10 phosphoproteins, including Akt and PRAS40, and is modulated in a dose-dependent fashion, both in vitro and in vivo. In human-tumor xenografts, this dose dependency significantly correlated with tumor growth inhibition. Tumor biopsies from patients treated with GDC-0068 at clinically achievable doses attained a degree of biomarker inhibition that correlated with tumor growth inhibition in preclinical models. In these clinical samples, compensatory feedback activation of ERK and HER3 was observed, consistent with preclinical observations. CONCLUSION: This study identified a set of biomarkers of Akt inhibition that can be used in the clinical setting to assess target engagement. Here, it was used to show that robust Akt inhibition in tumors from patients treated with GDC-0068 is achievable, supporting the clinical development of this compound in defined patient populations.

Anti-EGFL7 antibodies enhance stress-induced endothelial cell death and anti-VEGF efficacy.

Many oncology drugs are administered at their maximally tolerated dose without the knowledge of their optimal efficacious dose range. In this study, we describe a multifaceted approach that integrated preclinical and clinical data to identify the optimal dose for an antiangiogenesis agent, anti-EGFL7. EGFL7 is an extracellular matrix-associated protein expressed in activated endothelium. Recombinant EGFL7 protein supported EC adhesion and protected ECs from stress-induced apoptosis. Anti-EGFL7 antibodies inhibited both of these key processes and augmented anti-VEGF-mediated vascular damage in various murine tumor models. In a genetically engineered mouse model of advanced non-small cell lung cancer, we found that anti-EGFL7 enhanced both the progression-free and overall survival benefits derived from anti-VEGF therapy in a dose-dependent manner. In addition, we identified a circulating progenitor cell type that was regulated by EGFL7 and evaluated the response of these cells to anti-EGFL7 treatment in both tumor-bearing mice and cancer patients from a phase I clinical trial. Importantly, these preclinical efficacy and clinical biomarker results enabled rational selection of the anti-EGFL7 dose currently being tested in phase II clinical trials.

Discovery of 2-{3-[2-(1-isopropyl-3-methyl-1H-1,2-4-triazol-5-yl)-5,6-dihydrobenzo[f]imidazo[1,2-d][1,4]oxazepin-9-yl]-1H-pyrazol-1-yl}-2-methylpropanamide (GDC-0032): a ß-sparing phosphoinositide 3-kinase inhibitor with high unbound exposure and robust in vivo antitumor activity.

Dysfunctional signaling through the phosphoinositide 3-kinase (PI3K)/AKT/mTOR pathway leads to uncontrolled tumor proliferation. In the course of the discovery of novel benzoxepin PI3K inhibitors, we observed a strong dependency of in vivo antitumor activity on the free-drug exposure. By lowering the intrinsic clearance, we derived a set of imidazobenzoxazepin compounds that showed improved unbound drug exposure and effectively suppressed growth of tumors in a mouse xenograft model at low drug dose levels. One of these compounds, GDC-0032 (11l), was progressed to clinical trials and is currently under phase I evaluation as a potential treatment for human malignancies.