Immunosuppressive adenosine (ADO) is catabolized from adenosine monophosphate (AMP) by CD73 in the tumor microenvironment and corresponds to poor patient prognosis in many cancers. Reducing levels of ADO via inhibition of CD73 may reverse this immunosuppression. Herein we describe the discovery of ORIC-533 (6), an inhibitor of CD73 with subnanomolar biochemical potency and potent cellular activity in both human and mouse tumor cell lines. Compound 6 rescues T-cell activation and cytokine production at low nanomolar concentrations, showing robust immunomodulatory activity. Notably, in high AMP environments compound 6 also promotes CD8+ T-cell proliferation. Oral dosing of 6 reduces the concentration of ADO in the tumor microenvironment with a concomitant increase in CD8+ cells, resulting in tumor growth inhibition in a syngeneic mouse model of cancer. The strong potency and oral bioavailability support a potential best-in-class profile for 6, a CD73 inhibitor that entered phase 1b in patients with multiple myeloma.

Supplemental Figure 3. Body weights of mice in xenograft efficacy studies

Supplemental Figure 2. AMG 232 treatment causes p53 stabilization, and increased p21 and MDM2 proteins in p53 wild-type cells.

Supplemental Figure 1. AMG 232 chemical structure, and 3-D model of AMG 232 bound to MDM2 protein.

PDF file - 132KB, Figure S1. Plasma concentration-time profiles of AMG 925 in rat, dog, and cynomolgus monkey following oral administration. Figure S2. Inhibition of STAT5 and Rb phosphorylation by AMG 925 in MOLM13-Luc tumor cells in bone marrow. Table S1. Selectivity profile of AMG 925 (Kd for kinases with POC < 20 at 1 microM in KenomScan).

KRAS is one of the most frequently mutated genes in cancer with alterations occurring in > 14% of all tumors. Recent advances have led to the discovery and development of inhibitors that bind the inactive (GDP-bound) form of KRASG12C. The most advanced of these first-generation molecules demonstrated clinical response rates of 30-45% and approximately 6-month progression-free survival in lung cancer patients. While significant, a majority of patients failed to achieve a clinical response and acquired resistance can be rapid. One hypothesis to explain tumor resistance is the failure of existing inhibitors to recognize the activated (GTP-bound) form of KRASG12C that can be upregulated in response to these first-generation inhibitors. Here we report the discovery of a series of novel inhibitors that effectively inhibit both the GTP- and GDP-bound forms of KRASG12C. These “dual-acting” inhibitors bind in the switch II pocket of both GTP-bound and GDP-bound KRASG12C and rapidly form a covalent bond with cysteine 12. This results in significantly increased inhibition of RAF1 and PI3Kα effector interactions (IC50 < 5 nM at 2 hrs.) in comparison to inactive state inhibitors. Dual targeting of both GTP- and GDP-bound KRASG12C results in potent cellular activity in models that are both sensitive (NCI-H358 and MIA PaCa-2) and resistant (NCI-H2122) to adagrasib and sotorasib. In contrast to adagrasib and sotorasib which are less effective in the NCI-H2122 cell line model, dual-acting inhibitors of GTP- and GDP-bound KRASG12C elicit rapid inhibition of pERK in < 1 hour with sustained inhibition of MAPK signaling through 48 hours. To model resistance to first generation inhibitors, an A59G mutation was introduced into KRASG12C, abrogating GTPase activity. This decreases the activity of both adagrasib and sotorasib in tumor cell viability assays by more than an order of magnitude whereas dual-acting inhibitors of GTP- and GDP-bound KRASG12C are equally effective in the G12C/A59G and parental G12C cell lines. Evaluation of dual-acting inhibitors of KRASG12C in vivo demonstrated rapid and > 90% KRASG12C target occupancy, resulting in regression of MIA PaCa-2 tumors. Dual-acting inhibitors of both the active and inactive states of KRASG12C may provide the potential for broader and more durable responses in the clinic. Citation Format: Philamer Calses, Sam Clark, Jacob Corpuz, Susan Fong, Phil Gerkin, Mohammad Hekmatnejad, Evan McMahon, Megan Murray, Truc Nguyen, Tony Phan, Allison Roberts, Phillip Schwartz, Mikayla Shanafelt, Hiroko Tanaka, Jennifer Tomczyk, John Widen, Monika Williams, John Eksterowicz, Daniel Erlanson, Marie Evangelista, Johannes Hermann, Richard M. Neve, Snahel Patel, Kevin R. Webster. Discovery of novel dual-acting KRASG12C inhibitors that target both the active and inactive forms of the protein [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3601.

The adenosinergic pathway represents an attractive new therapeutic approach in cancer immunotherapy. In this pathway, ecto-5-nucleotidase CD73 has the unique function of regulating production of immunosuppressive adenosine (ADO) through the hydrolysis of AMP. CD73 is overexpressed in many cancers, resulting in elevated levels of ADO that correspond to poor patient prognosis. Therefore, reducing the level of ADO via inhibition of CD73 is a potential strategy for treating cancers. Based on the binding mode of adenosine 5'-(α,β-methylene)diphosphate (AOPCP) with human CD73, we designed a series of novel monophosphonate small-molecule CD73 inhibitors. Among them, OP-5244 (35) proved to be a highly potent and orally bioavailable CD73 inhibitor. In preclinical studies, 35 completely inhibited ADO production in both human cancer cells and CD8+ T cells. Furthermore, 35 lowered the ratio of ADO/AMP significantly and reversed immunosuppression in mouse models, indicating its potential as an in vivo tool compound for further development.

Structure-based modification of mifepristone (1) led to the discovery of novel mifepristone derivatives with improved selectivity profile. Addition of a methyl group at the C10 position of the steroid has a significant impact on progesterone receptor (PR) and androgen receptor (AR) activity. Within this series, OP-3633 (15) emerged as a glucocorticoid receptor (GR) antagonist with increased selectivity against PR and AR, improved cytochrome P450 inhibition profile, and significantly improved pharmacokinetic properties compared to 1. Furthermore, 15 demonstrated substantial inhibition of GR transcriptional activity in the GR positive HCC1806 triple negative breast cancer xenograft model. Overall, compound 15 is a promising GR antagonist candidate to clinically evaluate the impact of GR inhibition in reversal or prevention of therapy resistance.

The Glucocorticoid Receptor (GR) is a member of the superfamily of nuclear hormone receptors that is activated by human cortisol and synthetic glucocorticoids such as dexamethasone (Dex). Upon ligand binding, GR translocates to the nucleus and regulates the expression of a wide spectrum of genes involved in diverse biological processes, including inflammation, immunity, metabolism, cell cycle, and differentiation. Dysregulated cortisol levels are associated with poor prognosis, drug resistance, and increased cancer recurrence. Multiple studies have shown that GR inhibition reverses resistance to chemotherapy in cancers of epithelial origin including prostate, bladder, renal, ovarian, pancreatic, and triple negative breast cancer (TNBC). We have recently reported the discovery of ORIC-101, a potent GR antagonist with a unique cytochrome P450 inhibition profile that makes this compound particularly suitable for combination with taxanes such as paclitaxel (Rew Y et al, 2018). Consistent with previous reports, our data showed that activation of GR promoted growth of TNBC cells in 3D culture conditions and protected TNBC cells from paclitaxel. Treatment with ORIC-101 fully reversed these effects. To understand the molecular basis of the observed GR-mediated chemotherapy resistance, we set out to isolate the pool of TNBC cells that escaped from paclitaxel treatment in the presence of Dex. Molecular profiling of these “chemotherapy escapees” pointed to a number of glucocorticoid-regulated biological pathways, including basal stem cell lineage genes and mesenchymal markers, suggesting the acquisition of an EMT-like phenotype in chemo-resistant cells. In support of this finding, we found using ChIP-seq analysis that GR directly bound within the promoter/enhancer regions of well-established EMT genes such as SNAI2 and FN1, and regulated their expression in response to Dex treatment. Functionally, RNAi-mediated knockdown of SNAI2 partially restored sensitivity to paclitaxel, suggesting that the GR-driven EMT phenotype contributes to paclitaxel resistance in TNBC cells. Consistent with the in vitro observations, immunohistochemical analysis showed that GR activation upregulated the levels of both basal stem cell and mesenchymal markers in “chemotherapy escapees” from paclitaxel-treated TNBC xenografts. Importantly oral administration of ORIC-101 fully blocked these effects. Altogether, we found that activation of GR drove an EMT-like phenotype in TNBC cells in vitro and in vivo. ORIC-101 reversed these effects and sensitized TNBC cells to chemotherapy. Our findings thus provide mechanistic insights into the role of GR as a mediator of therapy resistance in TNBC. Clinical evaluation is being planned to assess the therapeutic potential of ORIC-101 in combination with standard-of-care chemotherapeutic agents. Citation Format: Haiying Zhou, Jessica Sun, Wayne Kong, Yosup Rew, Xiaohui Du, John Eksterowicz, Daqing Sun, Qiuping Ye, Omar Kabbarah, Valeria R. Fantin. ORIC-101 reverses a GR-driven EMT-like phenotype and sensitizes TNBC cells to chemotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3822.

The glucocorticoid receptor (GR) has been linked to therapy resistance across a wide range of cancer types. Preclinical data suggest that antagonists of this nuclear receptor may enhance the activity of anticancer therapy. The first-generation GR antagonist mifepristone is currently undergoing clinical evaluation in various oncology settings. Structure-based modification of mifepristone led to the discovery of ORIC-101 (28), a highly potent steroidal GR antagonist with reduced androgen receptor (AR) agonistic activity amenable for dosing in androgen receptor positive tumors and with improved CYP2C8 and CYP2C9 inhibition profile to minimize drug-drug interaction potential. Unlike mifepristone, 28 could be codosed with chemotherapeutic agents readily metabolized by CYP2C8 such as paclitaxel. Furthermore, 28 demonstrated in vivo antitumor activity by enhancing response to chemotherapy in the GR+ OVCAR5 ovarian cancer xenograft model. Clinical evaluation of safety and therapeutic potential of 28 is underway.

Abstract Androgen receptor (AR) signaling is crucial for normal development and homeostasis of the prostate, and is a key driver of prostate cancer initiation and progression. Hormone therapies that deprive the cancer of androgen have long been a mainstay of prostate cancer treatment. More recently, anti-androgens, such as abiraterone and enzalutamide, have been approved for use in metastatic castration resistant prostate cancer (mCRPC). Evidence also suggests that AR may play an oncogenic role in certain breast cancers. Several recent publications have demonstrated that activation of the Glucocorticoid Receptor (GR) can confer resistance to enzalutamide, and GR has also been shown to provide protection from conventional chemotherapies in other solid tumor indications. Mifepristone, is a synthetic steroidal antagonist of progesterone receptor, and to a lesser extent of GR and AR. It is currently being tested in clinical trials in combination with enzalutamide in mCRPC, and in combination with chemotherapy in triple negative breast cancer (TNBC). We sought to characterize the effect of mifepristone in pre-clinical models of prostate and breast cancer. Here we show that in the absence of androgen, mifepristone acts as a partial AR agonist, and this agonism can only be partially overcome by enzalutamide. We find that in low androgen conditions, mifepristone promotes the growth of prostate cancer cells in vitro and accelerates the growth of prostate tumors in xenograft models. Moreover, when given in combination, mifepristone significantly reduces the efficacy of enzalutamide in the LN-AR xenograft model. We are currently assessing the effects of mifepristone treatment in TNBC. Our findings suggest that partial AR agonist activity of mifepristone may have a negative impact in prostate and other AR positive cancers. We have developed a GR antagonist that is devoid of AR agonism to circumvent undesired effects on proliferation and drug response. Citation Format: Haiying Zhou, Nadine Jachan, Mallika Singh, Chris Tran, Dan McWeeney, Minna Balbas, Emily Schenkein, Tatiana Zovorotinskaya, Erica L. Jackson, Julio Medina, Daqing Sun, Yosup Rew, Xiaohui Du, John Eksterowicz, Xuelei Yan, Liusheng Zhu, Qiuping Ye, Valeria Fantin. Activation of AR signaling by mifepristone enhances prostate cancer growth and impairs enzalutamide response [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4172. doi:10.1158/1538-7445.AM2017-4172

Abstract The p53 tumor suppressor is controlled by MDM2, which binds p53 and negatively regulates its transcriptional activity and stability. Many tumors overproduce MDM2 to impair p53 function. Therefore, restoration of p53 activity by inhibiting p53-MDM2 binding represents an attractive, novel approach to cancer therapy. We previously reported the discovery of AM-8553, a potent and selective piperidinone inhibitor of the MDM2-p53 interaction (Rew et al. J. Med. Chem. 2012, 55, 4936). We report here continued optimization of the N-alkyl substituent of this series, focused in particular on a previously underutilized interaction in a shallow cleft on the MDM2 surface that led to the discovery of a variety of extremely potent sulfonamides such as 14 with an IC50 of 5.3 nM in the cell proliferation assay. The compound 14 interacts specifically with the p53-binding pocket of MDM2 and releases the p53 protein from negative control. Treatment of cancer cells expressing wild-type p53 with sulfonamide 14 stabilizes p53 and activates the p53 pathway, leading to cell cycle arrest and apoptosis. The compound 14 showed excellent efficacy and caused tumor regression in the SJSA-1 tumor xenograft model. Citation Format: Zhihong Li, Jiasheng Fu, Yosup Rew, Michael W. Gribble, Jude Canon, Ada Chen, John Eksterowicz, Xin Huang, Lixia Jin, Mei-Chu Lo, Lawrence R. McGee, Tao Osgood, Anne Y. Saiki, Paul Shaffer, Daqing Sun, Sarah Wortman, Qiuping Ye, Dongyin Yu, Xiaoning Zhao, Jing Zhou, Jonathan D. Oliner, Steve H. Olson, Julio C. Medina. Discovery of sulfonamide-piperidinones as potent inhibitors of the MDM2-p53 protein-protein interaction. [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 3663. doi:10.1158/1538-7445.AM2015-3663

Background: JAK JH2s (pseudokinase domains) mediate important regulatory functions; it is unclear whether TYK2 JH2 binds ATP and possesses enzymatic activity.Results: TYK2 JH2 binds ATP, but is catalytically inactive; ATP stabilizes JH2 and modulates TYK2 activity.Conclusion: ATP binding to JH2 is functionally important; the rigid activation loop probably hinders substrate phosphorylation.Significance: The TYK2 JH2 domain can be targeted with ATP-competitive compounds for therapeutics.JAK (Janus family of cytoplasmic tyrosine kinases) family tyrosine kinase 2 (TYK2) participates in signaling through cytokine receptors involved in immune responses and inflammation. JAKs are characterized by dual kinase domain: a tyrosine kinase domain (JH1) that is preceded by a pseudokinase domain (JH2). The majority of disease-associated mutations in JAKs map to JH2, demonstrating its central regulatory function. JH2s were considered catalytically inactive, but JAK2 JH2 was found to have low autoregulatory catalytic activity. Whether the other JAK JH2s share ATP binding and enzymatic activity has been unclear. Here we report the crystal structure of TYK2 JH2 in complex with adenosine 5'-O-(thiotriphosphate) (ATP-gamma S) and characterize its nucleotide binding by biochemical and biophysical methods. TYK2 JH2 did not show phosphotransfer activity, but it binds ATP and the nucleotide binding stabilizes the protein without inducing major conformational changes. Mutation of the JH2 ATP-binding pocket increased basal TYK2 phosphorylation and downstream signaling. The overall structural characteristics of TYK2 JH2 resemble JAK2 JH2, but distinct stabilizing molecular interactions around helix alpha AL in the activation loop provide a structural basis for differences in substrate access and catalytic activities among JAK family JH2s. The structural and biochemical data suggest that ATP binding is functionally important for both TYK2 and JAK2 JH2s, whereas the regulatory phosphorylation appears to be a unique property of JAK2. Finally, the co-crystal structure of TYK2 JH2 complexed with a small molecule inhibitor demonstrates that JH2 is accessible to ATP-competitive compounds, which offers novel approaches for targeting cytokine signaling as well as potential therapeutic applications.

Abstract 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. Mol Cancer Ther; 14(3); 649–58. ©2015 AACR.

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.