Small-molecule inhibition of kinesin KIF18A reveals a mitotic vulnerability enriched in chromosomally unstable cancers.

Chromosomal instability (CIN) is a hallmark of cancer, caused by persistent errors in chromosome segregation during mitosis. Aggressive cancers like high-grade serous ovarian cancer (HGSOC) and triple-negative breast cancer (TNBC) have a high frequency of CIN and TP53 mutations. Here, we show that inhibitors of the KIF18A motor protein activate the mitotic checkpoint and selectively kill chromosomally unstable cancer cells. Sensitivity to KIF18A inhibition is enriched in TP53-mutant HGSOC and TNBC cell lines with CIN features, including in a subset of CCNE1-amplified, CDK4-CDK6-inhibitor-resistant and BRCA1-altered cell line models. Our KIF18A inhibitors have minimal detrimental effects on human bone marrow cells in culture, distinct from other anti-mitotic agents. In mice, inhibition of KIF18A leads to robust anti-cancer effects with tumor regression observed in human HGSOC and TNBC models at well-tolerated doses. Collectively, our results provide a rational therapeutic strategy for selective targeting of CIN cancers via KIF18A inhibition.

Liver Metastasis Modulate Responses of Suppressive Macrophages and Exhausted T Cells to Immunotherapy Revealed by Single Cell Sequencing.

Liver metastasis is associated with immunotherapy resistance, although the underlying mechanisms remain incompletely understood. By applying single cell RNA-sequencing to a concurrent subcutaneous and liver tumor murine model to recapitulate liver metastases, it is identified that subsets within tumor-infiltrating exhausted CD8+ T (Tex) cells and immunosuppressive tumor-associated macrophages (TAMs) display opposite responses to concurrent liver tumors and anti-PD-1 treatment, suggesting a complex immune regulating network. Both angiogenic and interferon-reactive TAMs show increased frequencies in implanted liver tumors, and anti-PD-1 treatment further elevates the frequencies of angiogenic TAMs. Such TAMs frequencies negatively correlate with the proportions of cytotoxic T cell subsets. Further, expression of interferon-stimulated genes in TAMs is dramatically reduced under effective anti-PD-1 treatment, while such tendencies are diminished in mice with implanted liver tumors. Therefore, the study indicates that liver metastases could increase immunosuppressive TAMs frequencies and inhibit Tex responses to PD-1 blockade, resulting in compromised systemic antitumor immunity and limited immunotherapy efficacy.

Denosumab compared with zoledronic acid on PFS in multiple myeloma: exploratory results of an international phase 3 study.

An exploratory end point from a recent trial in patients with newly diagnosed multiple myeloma showed that median progression-free survival (PFS) was increased by 10.7 months with denosumab vs zoledronic acid. We performed additional analyses to identify factors that may have contributed to the favorable PFS with denosumab. Ad hoc analyses were performed for patients intending to undergo autologous stem cell transplantation (ASCT; ASCT intent), not intending to undergo ASCT (ASCT no intent), and intent-to-treat according to age (<70 or ≥70 years) and baseline renal function (≤60 mL/min or >60 mL/min creatinine clearance [CrCl]). Of 1718 patients, 930 (54.1%) were in the ASCT-intent subgroup, and 788 (45.9%) were in the ASCT-no-intent subgroup. In the ASCT-intent subgroup, frontline triplet (median PFS, not estimable vs 35.7 months; hazard ratio [HR] [95% confidence interval (CI)], 0.65 [0.47-0.90]; descriptive P = .009) or bortezomib-only (median PFS, not estimable vs not estimable; HR [95% CI], 0.61 [0.39-0.95]; descriptive P = .029) induction regimens demonstrated the strongest PFS benefit favoring denosumab vs zoledronic acid. In the ASCT-no-intent subgroup, no benefit with denosumab vs zoledronic acid was observed. PFS favored denosumab vs zoledronic acid in patients with CrCl >60 mL/min and in patients <70 years old, but no difference was observed in patients with CrCl ≤60 mL/min or patients ≥70 years old. The PFS difference observed with denosumab is one of the notable benefits reported in newly diagnosed multiple myeloma and was most pronounced in patients intending to undergo ASCT and those who received proteasome inhibitor (PI)-based triplet regimens. This study was registered at www.clinicaltrials.gov as #NCT01345019.

AMG 757, a Half-Life Extended, DLL3-Targeted Bispecific T-Cell Engager, Shows High Potency and Sensitivity in Preclinical Models of Small-Cell Lung Cancer.

PURPOSE: Small-cell lung cancer (SCLC) is an aggressive neuroendocrine tumor with a high relapse rate, limited therapeutic options, and poor prognosis. We investigated the antitumor activity of AMG 757, a half-life extended bispecific T-cell engager molecule targeting delta-like ligand 3 (DLL3)-a target that is selectively expressed in SCLC tumors, but with minimal normal tissue expression. EXPERIMENTAL DESIGN: AMG 757 efficacy was evaluated in SCLC cell lines and in orthotopic and patient-derived xenograft (PDX) mouse SCLC models. Following AMG 757 administration, changes in tumor volume, pharmacodynamic changes in tumor-infiltrating T cells (TILs), and the spatial relationship between the appearance of TILs and tumor histology were examined. Tolerability was assessed in nonhuman primates (NHPs). RESULTS: AMG 757 showed potent and specific killing of even those SCLC cell lines with very low DLL3 expression (<1,000 molecules per cell). AMG 757 effectively engaged systemically administered human T cells, induced T-cell activation, and redirected T cells to lyse tumor cells to promote significant tumor regression and complete responses in PDX models of SCLC and in orthotopic models of established primary lung SCLC and metastatic liver lesions. AMG 757 was well tolerated with no AMG 757-related adverse findings up to the highest tested dose (4.5 mg/kg weekly) in NHP. AMG 757 exhibits an extended half-life in NHP, which is projected to enable intermittent administration in patients. CONCLUSIONS: AMG 757 has a compelling safety and efficacy profile in preclinical studies making it a viable option for targeting DLL3-expressing SCLC tumors in the clinical setting.

KRASG12C Inhibition with Sotorasib in Advanced Solid Tumors.

BACKGROUND: No therapies for targeting KRAS mutations in cancer have been approved. The KRAS p.G12C mutation occurs in 13% of non-small-cell lung cancers (NSCLCs) and in 1 to 3% of colorectal cancers and other cancers. Sotorasib is a small molecule that selectively and irreversibly targets KRASG12C. METHODS: We conducted a phase 1 trial of sotorasib in patients with advanced solid tumors harboring the KRAS p.G12C mutation. Patients received sotorasib orally once daily. The primary end point was safety. Key secondary end points were pharmacokinetics and objective response, as assessed according to Response Evaluation Criteria in Solid Tumors (RECIST), version 1.1. RESULTS: A total of 129 patients (59 with NSCLC, 42 with colorectal cancer, and 28 with other tumors) were included in dose escalation and expansion cohorts. Patients had received a median of 3 (range, 0 to 11) previous lines of anticancer therapies for metastatic disease. No dose-limiting toxic effects or treatment-related deaths were observed. A total of 73 patients (56.6%) had treatment-related adverse events; 15 patients (11.6%) had grade 3 or 4 events. In the subgroup with NSCLC, 32.2% (19 patients) had a confirmed objective response (complete or partial response) and 88.1% (52 patients) had disease control (objective response or stable disease); the median progression-free survival was 6.3 months (range, 0.0+ to 14.9 [with + indicating that the value includes patient data that were censored at data cutoff]). In the subgroup with colorectal cancer, 7.1% (3 patients) had a confirmed response, and 73.8% (31 patients) had disease control; the median progression-free survival was 4.0 months (range, 0.0+ to 11.1+). Responses were also observed in patients with pancreatic, endometrial, and appendiceal cancers and melanoma. CONCLUSIONS: Sotorasib showed encouraging anticancer activity in patients with heavily pretreated advanced solid tumors harboring the KRAS p.G12C mutation. Grade 3 or 4 treatment-related toxic effects occurred in 11.6% of the patients. (Funded by Amgen and others; CodeBreaK100 ClinicalTrials.gov number, NCT03600883.).

Discovery of a Covalent Inhibitor of KRASG12C (AMG 510) for the Treatment of Solid Tumors.

KRASG12C has emerged as a promising target in the treatment of solid tumors. Covalent inhibitors targeting the mutant cysteine-12 residue have been shown to disrupt signaling by this long-"undruggable" target; however clinically viable inhibitors have yet to be identified. Here, we report efforts to exploit a cryptic pocket (H95/Y96/Q99) we identified in KRASG12C to identify inhibitors suitable for clinical development. Structure-based design efforts leading to the identification of a novel quinazolinone scaffold are described, along with optimization efforts that overcame a configurational stability issue arising from restricted rotation about an axially chiral biaryl bond. Biopharmaceutical optimization of the resulting leads culminated in the identification of AMG 510, a highly potent, selective, and well-tolerated KRASG12C inhibitor currently in phase I clinical trials (NCT03600883).

The clinical KRAS(G12C) inhibitor AMG 510 drives anti-tumour immunity.

KRAS is the most frequently mutated oncogene in cancer and encodes a key signalling protein in tumours1,2. The KRAS(G12C) mutant has a cysteine residue that has been exploited to design covalent inhibitors that have promising preclinical activity3-5. Here we optimized a series of inhibitors, using novel binding interactions to markedly enhance their potency and selectivity. Our efforts have led to the discovery of AMG 510, which is, to our knowledge, the first KRAS(G12C) inhibitor in clinical development. In preclinical analyses, treatment with AMG 510 led to the regression of KRASG12C tumours and improved the anti-tumour efficacy of chemotherapy and targeted agents. In immune-competent mice, treatment with AMG 510 resulted in a pro-inflammatory tumour microenvironment and produced durable cures alone as well as in combination with immune-checkpoint inhibitors. Cured mice rejected the growth of isogenic KRASG12D tumours, which suggests adaptive immunity against shared antigens. Furthermore, in clinical trials, AMG 510 demonstrated anti-tumour activity in the first dosing cohorts and represents a potentially transformative therapy for patients for whom effective treatments are lacking.

Targeting Multiple Myeloma with AMG 424, a Novel Anti-CD38/CD3 Bispecific T-cell-recruiting Antibody Optimized for Cytotoxicity and Cytokine Release.

PURPOSE: Despite advances in the treatment of multiple myeloma, new therapies are needed to induce more profound clinical responses. T-cell-redirected lysis triggered by bispecific antibodies recruiting T cells to cancer cells is a clinically validated mechanism of action against hematologic malignancies and CD38 is a tumor-associated antigen with near-universal expression in multiple myeloma. Thus, an anti-CD38/CD3 bispecific T-cell-recruiting antibody has the potential to be an effective new therapeutic for multiple myeloma. EXPERIMENTAL DESIGN: Anti-CD38/CD3 XmAb T-cell-recruiting antibodies with different affinities for CD38 and CD3 were assessed in vitro and in vivo for their redirected T-cell lysis activity against cancer cell lines, their lower levels of cytokine release, and their potency in the presence of high levels of soluble CD38. Select candidates were further tested in cynomolgus monkeys for B-cell depletion and cytokine release properties. RESULTS: AMG 424 was selected on the basis of its ability to kill cancer cells expressing high and low levels of CD38 in vitro and trigger T-cell proliferation, but with attenuated cytokine release. In vivo, AMG 424 induces tumor growth inhibition in bone marrow-invasive mouse cancer models and the depletion of peripheral B cells in cynomolgus monkeys, without triggering excessive cytokine release. The activity of AMG 424 against normal immune cells expressing CD38 is also presented. CONCLUSIONS: These findings support the clinical development of AMG 424, an affinity-optimized T-cell-recruiting antibody with the potential to elicit significant clinical activity in patients with multiple myeloma.

Discovery of ( R)-8-(6-Methyl-4-oxo-1,4,5,6-tetrahydropyrrolo[3,4- b]pyrrol-2-yl)-3-(1-methylcyclopropyl)-2-((1-methylcyclopropyl)amino)quinazolin-4(3 H)-one, a Potent and Selective Pim-1/2 Kinase Inhibitor for Hematological Malignancies.

Pim kinases are a family of constitutively active serine/threonine kinases that are partially redundant and regulate multiple pathways important for cell growth and survival. In human disease, high expression of the three Pim isoforms has been implicated in the progression of hematopoietic and solid tumor cancers, which suggests that Pim kinase inhibitors could provide patients with therapeutic benefit. Herein, we describe the structure-guided optimization of a series of quinazolinone-pyrrolodihydropyrrolone analogs leading to the identification of potent pan-Pim inhibitor 28 with improved potency, solubility, and drug-like properties. Compound 28 demonstrated on-target Pim activity in an in vivo pharmacodynamic assay with significant inhibition of BAD phosphorylation in KMS-12-BM multiple myeloma tumors for 16 h postdose. In a 2-week mouse xenograft model, daily dosing of compound 28 resulted in 33% tumor regression at 100 mg/kg.

AMG 176, a Selective MCL1 Inhibitor, Is Effective in Hematologic Cancer Models Alone and in Combination with Established Therapies.

The prosurvival BCL2 family member MCL1 is frequently dysregulated in cancer. To overcome the significant challenges associated with inhibition of MCL1 protein-protein interactions, we rigorously applied small-molecule conformational restriction, which culminated in the discovery of AMG 176, the first selective MCL1 inhibitor to be studied in humans. We demonstrate that MCL1 inhibition induces a rapid and committed step toward apoptosis in subsets of hematologic cancer cell lines, tumor xenograft models, and primary patient samples. With the use of a human MCL1 knock-in mouse, we demonstrate that MCL1 inhibition at active doses of AMG 176 is tolerated and correlates with clear pharmacodynamic effects, demonstrated by reductions in B cells, monocytes, and neutrophils. Furthermore, the combination of AMG 176 and venetoclax is synergistic in acute myeloid leukemia (AML) tumor models and in primary patient samples at tolerated doses. These results highlight the therapeutic promise of AMG 176 and the potential for combinations with other BH3 mimetics. SIGNIFICANCE: AMG 176 is a potent, selective, and orally bioavailable MCL1 inhibitor that induces a rapid commitment to apoptosis in models of hematologic malignancies. The synergistic combination of AMG 176 and venetoclax demonstrates robust activity in models of AML at tolerated doses, highlighting the promise of BH3-mimetic combinations in hematologic cancers.See related commentary by Leber et al., p. 1511.This article is highlighted in the In This Issue feature, p. 1494.

RANK rewires energy homeostasis in lung cancer cells and drives primary lung cancer.

Lung cancer is the leading cause of cancer deaths. Besides smoking, epidemiological studies have linked female sex hormones to lung cancer in women; however, the underlying mechanisms remain unclear. Here we report that the receptor activator of nuclear factor-kB (RANK), the key regulator of osteoclastogenesis, is frequently expressed in primary lung tumors, an active RANK pathway correlates with decreased survival, and pharmacologic RANK inhibition reduces tumor growth in patient-derived lung cancer xenografts. Clonal genetic inactivation of KRasG12D in mouse lung epithelial cells markedly impairs the progression of KRasG12D -driven lung cancer, resulting in a significant survival advantage. Mechanistically, RANK rewires energy homeostasis in human and murine lung cancer cells and promotes expansion of lung cancer stem-like cells, which is blocked by inhibiting mitochondrial respiration. Our data also indicate survival differences in KRasG12D -driven lung cancer between male and female mice, and we show that female sex hormones can promote lung cancer progression via the RANK pathway. These data uncover a direct role for RANK in lung cancer and may explain why female sex hormones accelerate lung cancer development. Inhibition of RANK using the approved drug denosumab may be a therapeutic drug candidate for primary lung cancer.

Radiosensitization of Adenoid Cystic Carcinoma with MDM2 Inhibition.

Purpose: Adenoid cystic carcinoma (ACC) is a rare cancer arising from the major or minor salivary gland tissues of the head and neck. There are currently no approved systemic agents or known radiosensitizers for ACC. Unlike the more common head and neck squamous cell carcinomas that frequently harbor TP53 mutations, ACCs contain TP53 mutations at a rate of <5%, rendering them an attractive target for MDM2 inhibition.Experimental Design: We report the successful establishment and detailed characterization of a TP53-WT ACC patient-derived xenograft (PDX), which retained the histologic features of the original patient tumor. We evaluated this model for response to the MDM2 inhibitor AMG 232 as monotherapy and in combination with radiotherapy.Results: AMG 232 monotherapy induced modest tumor growth inhibition, and radiation monotherapy induced a transient tumor growth delay in a dose-dependent fashion. Strikingly, combination treatment of AMG 232 with radiotherapy (including low-dose radiotherapy of 2 Gy/fraction) induced dramatic tumor response and high local tumor control rates 3 months following treatment. Posttreatment analysis revealed that although both AMG 232 and radiotherapy alone induced TP53 tumor-suppressive activities, combination therapy amplified this response with potent induction of apoptosis after combination treatment.Conclusions: These data identify that MDM2 inhibition can provide potent radiosensitization in TP53-WT ACC. In light of the absence of effective systemic agents for ACC, the powerful response profile observed here suggests that clinical trial evaluation of this drug/radiotherapy combination may be warranted to improve local control in this challenging malignancy. Clin Cancer Res; 23(20); 6044-53. ©2017 AACR.

Small Molecule Inhibition of MDM2-p53 Interaction Augments Radiation Response in Human Tumors.

MDM2-p53 interaction and downstream signaling affect cellular response to DNA damage. AMG 232 is a potent small molecule inhibitor that blocks the interaction of MDM2 and p53. We examined the capacity of AMG 232 to augment radiation response across a spectrum of human tumor cell lines and xenografts. AMG 232 effectively inhibited proliferation and enhanced radiosensitivity via inhibition of damage repair signaling. Combined AMG 232 and radiation treatment resulted in the accumulation of γH2AX-related DNA damage and induction of senescence with promotion of apoptotic and/or autophagic cell death. Several molecules involved in senescence, autophagy, and apoptosis were specifically modulated following the combined AMG 232/radiation treatment, including FoxM1, ULK-1, DRAM, and BAX. In vivo xenograft studies confirmed more potent antitumor and antiangiogenesis efficacy with combined AMG 232/radiation treatment than treatment with drug or radiation alone. Taken together, these data identify the capacity of AMG 232 to augment radiation response across a variety of tumor types harboring functional p53.

The MDM2 Inhibitor AMG 232 Demonstrates Robust Antitumor Efficacy and Potentiates the Activity of p53-Inducing Cytotoxic Agents.

p53 is a critical tumor suppressor and is the most frequently inactivated gene in human cancer. Inhibition of the interaction of p53 with its negative regulator MDM2 represents a promising clinical strategy to treat p53 wild-type tumors. AMG 232 is a potential best-in-class inhibitor of the MDM2-p53 interaction and is currently in clinical trials. We characterized the activity of AMG 232 and its effect on p53 signaling in several preclinical tumor models. AMG 232 binds the MDM2 protein with picomolar affinity and robustly induces p53 activity, leading to cell-cycle arrest and inhibition of tumor cell proliferation. AMG 232 treatment inhibited the in vivo growth of several tumor xenografts and led to complete and durable regression of MDM2-amplified SJSA-1 tumors via growth arrest and induction of apoptosis. Therapeutic combination studies of AMG 232 with chemotherapies that induce DNA damage and p53 activity resulted in significantly superior antitumor efficacy and regression, and markedly increased activation of p53 signaling in tumors. These preclinical data support the further evaluation of AMG 232 in clinical trials as both a monotherapy and in combination with standard-of-care cytotoxics.

Discovery of AM-7209, a potent and selective 4-amidobenzoic acid inhibitor of the MDM2-p53 interaction.

Structure-based rational design and extensive structure-activity relationship studies led to the discovery of AMG 232 (1), a potent piperidinone inhibitor of the MDM2-p53 association, which is currently being evaluated in human clinical trials for the treatment of cancer. Further modifications of 1, including replacing the carboxylic acid with a 4-amidobenzoic acid, afforded AM-7209 (25), featuring improved potency (KD from ITC competition was 38 pM, SJSA-1 EdU IC50 = 1.6 nM), remarkable pharmacokinetic properties, and in vivo antitumor activity in both the SJSA-1 osteosarcoma xenograft model (ED50 = 2.6 mg/kg QD) and the HCT-116 colorectal carcinoma xenograft model (ED50 = 10 mg/kg QD). In addition, 25 possesses distinct mechanisms of elimination compared to 1.

Discovery of Potent and Simplified Piperidinone-Based Inhibitors of the MDM2-p53 Interaction.

Continued optimization of the N-substituent in the piperidinone series provided potent piperidinone-pyridine inhibitors 6, 7, 14, and 15 with improved pharmacokinetic properties in rats. Reducing structure complexity of the N-alkyl substituent led to the discovery of 23, a potent and simplified inhibitor of MDM2. Compound 23 exhibits excellent pharmacokinetic properties and substantial in vivo antitumor activity in the SJSA-1 osteosarcoma xenograft mouse model.

MDM2 antagonists synergize broadly and robustly with compounds targeting fundamental oncogenic signaling pathways.

While MDM2 inhibitors hold great promise as cancer therapeutics, drug resistance will likely limit their efficacy as single agents. To identify drug combinations that might circumvent resistance, we screened for agents that could synergize with MDM2 inhibition in the suppression of cell viability. We observed broad and robust synergy when combining MDM2 antagonists with either MEK or PI3K inhibitors. Synergy was not limited to cell lines harboring MAPK or PI3K pathway mutations, nor did it depend on which node of the PI3K axis was targeted. MDM2 inhibitors also synergized strongly with BH3 mimetics, BCR-ABL antagonists, and HDAC inhibitors. MDM2 inhibitor-mediated synergy with agents targeting these mechanisms was much more prevalent than previously appreciated, implying that clinical translation of these combinations could have far-reaching implications for public health. These findings highlight the importance of combinatorial drug targeting and provide a framework for the rational design of MDM2 inhibitor clinical trials.

Novel inhibitors of the MDM2-p53 interaction featuring hydrogen bond acceptors as carboxylic acid isosteres.

We previously reported the discovery of potent and selective morpholinone and piperidinone inhibitors of the MDM2-p53 interaction. These inhibitors have in common a carboxylic acid moiety that engages in an electrostatic interaction with MDM2-His96. Our continued search for potent and diverse inhibitors led to the discovery of novel replacements for these acids uncovering new interactions with the MDM2 protein. In particular, using pyridine or thiazole as isosteres of the carboxylic acid moiety resulted in very potent analogues. From these, AM-6761 (4) emerged as a potent inhibitor with remarkable biochemical (HTRF IC50 = 0.1 nM) and cellular potency (SJSA-1 EdU IC50 = 16 nM), as well as favorable pharmacokinetic properties. Compound 4 also shows excellent antitumor activity in the SJSA-1 osteosarcoma xenograft model with an ED50 of 11 mg/kg. Optimization efforts toward the discovery of these inhibitors as well as the new interactions observed with the MDM2 protein are described herein.

Selective and potent morpholinone inhibitors of the MDM2-p53 protein-protein interaction.

We previously reported the discovery of AMG 232, a highly potent and selective piperidinone inhibitor of the MDM2-p53 interaction. Our continued search for potent and diverse analogues led to the discovery of novel morpholinone MDM2 inhibitors. This change to a morpholinone core has a significant impact on both potency and metabolic stability compared to the piperidinone series. Within this morpholinone series, AM-8735 emerged as an inhibitor with remarkable biochemical potency (HTRF IC50 = 0.4 nM) and cellular potency (SJSA-1 EdU IC50 = 25 nM), as well as pharmacokinetic properties. Compound 4 also shows excellent antitumor activity in the SJSA-1 osteosarcoma xenograft model with an ED50 of 41 mg/kg. Lead optimization toward the discovery of this inhibitor as well as key differences between the morpholinone and the piperidinone series will be described herein.

Discovery of AMG 232, a potent, selective, and orally bioavailable MDM2-p53 inhibitor in clinical development.

We recently reported the discovery of AM-8553 (1), a potent and selective piperidinone inhibitor of the MDM2-p53 interaction. Continued research investigation of the N-alkyl substituent of this series, focused in particular on a previously underutilized interaction in a shallow cleft on the MDM2 surface, led to the discovery of a one-carbon tethered sulfone which gave rise to substantial improvements in biochemical and cellular potency. Further investigation produced AMG 232 (2), which is currently being evaluated in human clinical trials for the treatment of cancer. Compound 2 is an extremely potent MDM2 inhibitor (SPR KD = 0.045 nM, SJSA-1 EdU IC50 = 9.1 nM), with remarkable pharmacokinetic properties and in vivo antitumor activity in the SJSA-1 osteosarcoma xenograft model (ED50 = 9.1 mg/kg).