BID expression determines the apoptotic fate of cancer cells after abrogation of the spindle assembly checkpoint by AURKB or TTK inhibitors.

BACKGROUND: Drugs targeting the spindle assembly checkpoint (SAC), such as inhibitors of Aurora kinase B (AURKB) and dual specific protein kinase TTK, are in different stages of clinical development. However, cell response to SAC abrogation is poorly understood and there are no markers for patient selection. METHODS: A panel of 53 tumor cell lines of different origins was used. The effects of drugs were analyzed by MTT and flow cytometry. Copy number status was determined by FISH and Q-PCR; mRNA expression by nCounter and RT-Q-PCR and protein expression by Western blotting. CRISPR-Cas9 technology was used for gene knock-out (KO) and a doxycycline-inducible pTRIPZ vector for ectopic expression. Finally, in vivo experiments were performed by implanting cultured cells or fragments of tumors into immunodeficient mice. RESULTS: Tumor cells and patient-derived xenografts (PDXs) sensitive to AURKB and TTK inhibitors consistently showed high expression levels of BH3-interacting domain death agonist (BID), while cell lines and PDXs with low BID were uniformly resistant. Gene silencing rendered BID-overexpressing cells insensitive to SAC abrogation while ectopic BID expression in BID-low cells significantly increased sensitivity. SAC abrogation induced activation of CASP-2, leading to cleavage of CASP-3 and extensive cell death only in presence of high levels of BID. Finally, a prevalence study revealed high BID mRNA in 6% of human solid tumors. CONCLUSIONS: The fate of tumor cells after SAC abrogation is driven by an AURKB/ CASP-2 signaling mechanism, regulated by BID levels. Our results pave the way to clinically explore SAC-targeting drugs in tumors with high BID expression.

Targeting Bfl-1 via acute CDK9 inhibition overcomes intrinsic BH3-mimetic resistance in lymphomas.

BH3 mimetics like venetoclax target prosurvival Bcl-2 family proteins and are important therapeutics in the treatment of hematological malignancies. We demonstrate that endogenous Bfl-1 expression can render preclinical lymphoma tumor models insensitive to Mcl-1 and Bcl-2 inhibitors. However, suppression of Bfl-1 alone was insufficient to fully induce apoptosis in Bfl-1-expressing lymphomas, highlighting the need for targeting additional prosurvival proteins in this context. Importantly, we demonstrated that cyclin-dependent kinase 9 (CDK9) inhibitors rapidly downregulate both Bfl-1 and Mcl-1, inducing apoptosis in BH3-mimetic-resistant lymphoma cell lines in vitro and driving in vivo tumor regressions in diffuse large B-cell lymphoma patient-derived xenograft models expressing Bfl-1. These data underscore the need to clinically develop CDK9 inhibitors, like AZD4573, for the treatment of lymphomas using Bfl-1 as a selection biomarker.

Discovery of heterocyclic sulfonamides as sphingosine 1-phosphate receptor 1 (S1P1) antagonists.

We have discovered a novel class of heterocyclic sulfonamides that act as antagonists of the S1P1 receptor. While members of this series identified from a high-throughput screen showed promising levels of potency in a cell-based assay measuring the inhibition of receptor internalization, most compounds were excessively lipophilic and contained an oxidation-prone thioether moiety. As a result, such compounds suffered from poor physical properties and metabolic stability, limiting their utility as in vivo probes. By removing the thioether group and systematically developing an understanding of structure-activity relationships and the effects of lipophilicity on potency within this series, we have been able to identify potent compounds with vastly improved physical properties. A representative enantiopure triazole sulfonamide (33) has measurable bioavailability following a low (3mg/kg) oral dose in rat, highlighting an achievement of the early hit-to-lead efforts for this series.

Essential role of TAK1 in regulating mantle cell lymphoma survival.

TGF-ß-activated kinase 1 (TAK1), a member of the MAPK kinase family, plays a key role in B-cell growth and development. In the present study, we examined the potential role of TAK1 as a therapeutic target for lymphoma. Here, we show that the active phosphorylated form of TAK1 is abundantly expressed in a panel of lymphoma cell lines, including mantle cell, anaplastic large cell, and Hodgkin lymphoma cell lines. Silencing TAK1 expression via the use of siRNA inhibited the activation of NF-κB and p38 and induced apoptosis in lymphoma cell lines. Moreover, submicromolar concentrations of AZ-TAK1, a novel ATP-competitive small molecule inhibitor of TAK1, dephosphorylated TAK1, p38, and IκB-α in lymphoma cell lines. These molecular events were associated with the release of cytochrome c into the cytosol, down-regulation of X-linked inhibitor of apoptosis, activation of caspase 9, and induction of apoptosis. We also demonstrate that primary lymphoma cells express TAK1 and pTAK1 and were sensitive to AZ-TAK1-mediated cell death. Collectively, our data demonstrate an essential role for TAK1 in regulating critical survival mechanisms in lymphoma and suggest that it may serve as a therapeutic target.

Structure-based design and synthesis of tricyclic IAP (Inhibitors of Apoptosis Proteins) inhibitors.

The design and synthesis of a series of novel tricyclic IAP inhibitors is reported. Rapid assembly of the core tricycle involved two key steps: Rh-catalyzed hydrogenation of an unsaturated bicyclic ring system and a Ru-catalyzed ring closing alkene metathesis reaction. The final Smac mimetics bind to cIAP1 and XIAP BIR3 domains and elicit the desired phenotype in cellular proliferation assays. Dimeric IAP inhibitors were found to possess nanomolar potency in a cellular proliferation assay and favourable in vitro drug-like properties.

Towards the next generation of dual Bcl-2/Bcl-xL inhibitors.

Structural modifications of the left-hand side of compound 1 were identified which retained or improved potent binding to Bcl-2 and Bcl-xL in in vitro biochemical assays and had strong activity in an RS4;11 apoptotic cellular assay. For example, sulfoxide diastereomer 13 maintained good binding affinity and comparable cellular potency to 1 while improving aqueous solubility. The corresponding diastereomer (14) was significantly less potent in the cell, and docking studies suggest that this is due to a stereochemical preference for the RS versus SS sulfoxide. Appending a dimethylaminoethoxy side chain (27) adjacent to the benzylic position of the biphenyl moiety of 1 improved cellular activity by approximately three-fold, and this activity was corroborated in cell lines overexpressing Bcl-2 and Bcl-xL.

Development of a Series of Pyrrolopyridone MAT2A Inhibitors.

The optimization of an allosteric fragment, discovered by differential scanning fluorimetry, to an in vivo MAT2a tool inhibitor is discussed. The structure-based drug discovery approach, aided by relative binding free energy calculations, resulted in AZ'9567 (21), a potent inhibitor in vitro with excellent preclinical pharmacokinetic properties. This tool showed a selective antiproliferative effect on methylthioadenosine phosphorylase (MTAP) KO cells, both in vitro and in vivo, providing further evidence to support the utility of MAT2a inhibitors as potential anticancer therapies for MTAP-deficient tumors.

First-in-human Study of AZD5153, A Small-molecule Inhibitor of Bromodomain Protein 4, in Patients with Relapsed/Refractory Malignant Solid Tumors and Lymphoma.

AZD5153, a reversible, bivalent inhibitor of the bromodomain and extraterminal family protein BRD4, has preclinical activity in multiple tumors. This first-in-human, phase I study investigated AZD5153 alone or with olaparib in patients with relapsed/refractory solid tumors or lymphoma. Adults with relapsed tumors intolerant of, or refractory to, prior therapies received escalating doses of oral AZD5153 once daily or twice daily continuously (21-day cycles), or AZD5153 once daily/twice daily continuously or intermittently plus olaparib 300 mg twice daily, until disease progression or unacceptable toxicity. Between June 30, 2017 and April 19, 2021, 34 patients received monotherapy and 15 received combination therapy. Dose-limiting toxicities were thrombocytopenia/platelet count decreased (n = 4/n = 2) and diarrhea (n = 1). The recommended phase II doses (RP2D) were AZD5153 30 mg once daily or 15 mg twice daily (monotherapy) and 10 mg once daily (intermittent schedule) with olaparib. With AZD5153 monotherapy, common treatment-emergent adverse events (TEAE) included fatigue (38.2%), thrombocytopenia, and diarrhea (each 32.4%); common grade ≥ 3 TEAEs were thrombocytopenia (14.7%) and anemia (8.8%). With the combination, common TEAEs included nausea (66.7%) and fatigue (53.3%); the most common grade ≥ 3 TEAE was thrombocytopenia (26.7%). AZD5153 had dose-dependent pharmacokinetics, with minimal accumulation, and demonstrated dose-dependent modulation of peripheral biomarkers, including upregulation of HEXIM1. One patient with metastatic pancreatic cancer receiving combination treatment had a partial response lasting 4.2 months. These results show AZD5153 was tolerable as monotherapy and in combination at the RP2Ds; common toxicities were fatigue, hematologic AEs, and gastrointestinal AEs. Strong evidence of peripheral target engagement was observed.

Targeting BCL2 Overcomes Resistance and Augments Response to Aurora Kinase B Inhibition by AZD2811 in Small Cell Lung Cancer.

PURPOSE: Therapeutic resistance to frontline therapy develops rapidly in small cell lung cancer (SCLC). Treatment options are also limited by the lack of targetable driver mutations. Therefore, there is an unmet need for developing better therapeutic strategies and biomarkers of response. Aurora kinase B (AURKB) inhibition exploits an inherent genomic vulnerability in SCLC and is a promising therapeutic approach. Here, we identify biomarkers of response and develop rational combinations with AURKB inhibition to improve treatment efficacy. EXPERIMENTAL DESIGN: Selective AURKB inhibitor AZD2811 was profiled in a large panel of SCLC cell lines (n = 57) and patient-derived xenograft (PDX) models. Proteomic and transcriptomic profiles were analyzed to identify candidate biomarkers of response and resistance. Effects on polyploidy, DNA damage, and apoptosis were measured by flow cytometry and Western blotting. Rational drug combinations were validated in SCLC cell lines and PDX models. RESULTS: AZD2811 showed potent growth inhibitory activity in a subset of SCLC, often characterized by, but not limited to, high cMYC expression. Importantly, high BCL2 expression predicted resistance to AURKB inhibitor response in SCLC, independent of cMYC status. AZD2811-induced DNA damage and apoptosis were suppressed by high BCL2 levels, while combining AZD2811 with a BCL2 inhibitor significantly sensitized resistant models. In vivo, sustained tumor growth reduction and regression was achieved even with intermittent dosing of AZD2811 and venetoclax, an FDA-approved BCL2 inhibitor. CONCLUSIONS: BCL2 inhibition overcomes intrinsic resistance and enhances sensitivity to AURKB inhibition in SCLC preclinical models.

Discovery of aminopiperidine-based Smac mimetics as IAP antagonists.

A series of structurally unique Smac mimetics that act as antagonists of inhibitor of apoptosis proteins (IAPs) has been discovered. While most previously described Smac mimetics contain the proline ring (or a similar cyclic motif) found in Smac, a key feature of the compounds described herein is that this ring has been removed. Despite this, compounds in this series potently bind to cIAP1 and elicit the expected phenotype of cIAP1 inhibition in cancer cells. Marked selectivity for cIAP1 over XIAP is observed for these compounds, which is attributed to a slight difference in the binding groove between the two proteins and the resulting steric interactions with the inhibitors. XIAP binding can be improved by constraining the inhibitor so that these unfavorable steric interactions are minimized.

Design and optimisation of dendrimer-conjugated Bcl-2/xL inhibitor, AZD0466, with improved therapeutic index for cancer therapy.

Dual Bcl-2/Bcl-xL inhibitors are expected to deliver therapeutic benefit in many haematological and solid malignancies, however, their use is limited by tolerability issues. AZD4320, a potent dual Bcl-2/Bcl-xL inhibitor, has shown good efficacy however had dose limiting cardiovascular toxicity in preclinical species, coupled with challenging physicochemical properties, which prevented its clinical development. Here, we describe the design and development of AZD0466, a drug-dendrimer conjugate, where AZD4320 is chemically conjugated to a PEGylated poly-lysine dendrimer. Mathematical modelling was employed to determine the optimal release rate of the drug from the dendrimer for maximal therapeutic index in terms of preclinical anti-tumour efficacy and cardiovascular tolerability. The optimised candidate is shown to be efficacious and better tolerated in preclinical models compared with AZD4320 alone. The AZD4320-dendrimer conjugate (AZD0466) identified, through mathematical modelling, has resulted in an improved therapeutic index and thus enabled progression of this promising dual Bcl-2/Bcl-xL inhibitor into clinical development.

AZD4573 Is a Highly Selective CDK9 Inhibitor That Suppresses MCL-1 and Induces Apoptosis in Hematologic Cancer Cells.

PURPOSE: Cyclin-dependent kinase 9 (CDK9) is a transcriptional regulator and potential therapeutic target for many cancers. Multiple nonselective CDK9 inhibitors have progressed clinically but were limited by a narrow therapeutic window. This work describes a novel, potent, and highly selective CDK9 inhibitor, AZD4573. EXPERIMENTAL DESIGN: The antitumor activity of AZD4573 was determined across broad cancer cell line panels in vitro as well as cell line- and patient-derived xenograft models in vivo. Multiple approaches, including integrated transcriptomic and proteomic analyses, loss-of-function pathway interrogation, and pharmacologic comparisons, were employed to further understand the major mechanism driving AZD4573 activity and to establish an exposure/effect relationship. RESULTS: AZD4573 is a highly selective and potent CDK9 inhibitor. It demonstrated rapid induction of apoptosis and subsequent cell death broadly across hematologic cancer models in vitro, and MCL-1 depletion in a dose- and time-dependent manner was identified as a major mechanism through which AZD4573 induces cell death in tumor cells. This pharmacodynamic (PD) response was also observed in vivo, which led to regressions in both subcutaneous tumor xenografts and disseminated models at tolerated doses both as monotherapy or in combination with venetoclax. This understanding of the mechanism, exposure, and antitumor activity of AZD4573 facilitated development of a robust pharmacokinetic/PD/efficacy model used to inform the clinical trial design. CONCLUSIONS: Selective targeting of CDK9 enables the indirect inhibition of MCL-1, providing a therapeutic option for MCL-1-dependent diseases. Accordingly, AZD4573 is currently being evaluated in a phase I clinical trial for patients with hematologic malignancies (clinicaltrials.gov identifier: NCT03263637).See related commentary by Alcon et al., p. 761.

AZD4320, A Dual Inhibitor of Bcl-2 and Bcl-xL, Induces Tumor Regression in Hematologic Cancer Models without Dose-limiting Thrombocytopenia.

PURPOSE: Targeting Bcl-2 family members upregulated in multiple cancers has emerged as an important area of cancer therapeutics. While venetoclax, a Bcl-2-selective inhibitor, has had success in the clinic, another family member, Bcl-xL, has also emerged as an important target and as a mechanism of resistance. Therefore, we developed a dual Bcl-2/Bcl-xL inhibitor that broadens the therapeutic activity while minimizing Bcl-xL-mediated thrombocytopenia. EXPERIMENTAL DESIGN: We used structure-based chemistry to design a small-molecule inhibitor of Bcl-2 and Bcl-xL and assessed the activity against in vitro cell lines, patient samples, and in vivo models. We applied pharmacokinetic/pharmacodynamic (PK/PD) modeling to integrate our understanding of on-target activity of the dual inhibitor in tumors and platelets across dose levels and over time. RESULTS: We discovered AZD4320, which has nanomolar affinity for Bcl-2 and Bcl-xL, and mechanistically drives cell death through the mitochondrial apoptotic pathway. AZD4320 demonstrates activity in both Bcl-2- and Bcl-xL-dependent hematologic cancer cell lines and enhanced activity in acute myeloid leukemia (AML) patient samples compared with the Bcl-2-selective agent venetoclax. A single intravenous bolus dose of AZD4320 induces tumor regression with transient thrombocytopenia, which recovers in less than a week, suggesting a clinical weekly schedule would enable targeting of Bcl-2/Bcl-xL-dependent tumors without incurring dose-limiting thrombocytopenia. AZD4320 demonstrates monotherapy activity in patient-derived AML and venetoclax-resistant xenograft models. CONCLUSIONS: AZD4320 is a potent molecule with manageable thrombocytopenia risk to explore the utility of a dual Bcl-2/Bcl-xL inhibitor across a broad range of tumor types with dysregulation of Bcl-2 prosurvival proteins.

Selective Androgen Receptor Modulator RAD140 Inhibits the Growth of Androgen/Estrogen Receptor-Positive Breast Cancer Models with a Distinct Mechanism of Action.

Purpose: Steroidal androgens suppress androgen receptor and estrogen receptor positive (AR/ER+) breast cancer cells and were used to treat breast cancer, eliciting favorable response. The current study evaluates the activity and efficacy of the oral selective AR modulator RAD140 in in vivo and in vitro models of AR/ER+ breast cancer.Experimental Design: A series of in vitro assays were used to determine the affinity of RAD140 to 4 nuclear receptors and evaluate its tissue-selective AR activity. The efficacy and pharmacodynamics of RAD140 as monotherapy or in combination with palbociclib were evaluated in AR/ER+ breast cancer xenograft models.Results: RAD140 bound AR with high affinity and specificity and activated AR in breast cancer but not prostate cancer cells. Oral administration of RAD140 substantially inhibited the growth of AR/ER+ breast cancer patient-derived xenografts (PDX). Activation of AR and suppression of ER pathway, including the ESR1 gene, were seen with RAD140 treatment. Coadministration of RAD140 and palbociclib showed improved efficacy in the AR/ER+ PDX models. In line with efficacy, a subset of AR-repressed genes associated with DNA replication was suppressed with RAD140 treatment, an effect apparently enhanced by concurrent administration of palbociclib.Conclusions: RAD140 is a potent AR agonist in breast cancer cells with a distinct mechanism of action, including the AR-mediated repression of ESR1 It inhibits the growth of multiple AR/ER+ breast cancer PDX models as a single agent, and in combination with palbociclib. The preclinical data presented here support further clinical investigation of RAD140 in AR/ER+ breast cancer patients. Clin Cancer Res; 23(24); 7608-20. ©2017 AACR.

Accurate prediction of the relative potencies of members of a series of kinase inhibitors using molecular docking and MM-GBSA scoring.

The ability of molecular docking, using the program Glide and an MM-GBSA postdocking scoring protocol, to correctly rank a number of congeneric kinase inhibitors was assessed. The approach was successful for the cases considered and suggests that this may be useful for the design of inhibitors in the lead optimization phase of drug discovery.

Identification and Optimization of Benzimidazole Sulfonamides as Orally Bioavailable Sphingosine 1-Phosphate Receptor 1 Antagonists with in Vivo Activity.

We report here a novel series of benzimidazole sulfonamides that act as antagonists of the S1P1 receptor, identified by exploiting an understanding of the pharmacophore of a high throughput screening (HTS)-derived series of compounds described previously. Lead compound 2 potently inhibits S1P-induced receptor internalization in a cell-based assay (EC50 = 0.05 µM), but has poor physical properties and metabolic stability. Evolution of this compound through structure-activity relationship development and property optimization led to in vivo probes such as 4. However, this compound was unexpectedly found to be a potent CYP3A inducer in human hepatocytes, and thus further chemistry efforts were directed at addressing this liability. By employing a pregnane X receptor (PXR) reporter gene assay to prioritize compounds for further testing in human hepatocytes, we identified lipophilicity as a key molecular property influencing the likelihood of P450 induction. Ultimately, we have identified compounds such as 46 and 47, which demonstrate the desired S1P1 antagonist activity while having greatly reduced risk of CYP3A induction in humans. These compounds have excellent oral bioavailability in preclinical species and exhibit pharmacodynamic effects of S1P1 antagonism in several in vivo models following oral dosing. Relatively modest antitumor activity was observed in multiple xenograft models, however, suggesting that selective S1P1 antagonists would have limited utility as anticancer therapeutics as single agents.

Mechanism and in vitro pharmacology of TAK1 inhibition by (5Z)-7-Oxozeaenol.

Transforming growth factor-ß activated kinase-1 (TAK1) is a member of the mitogen-activated protein kinase kinase kinase (MAP3K) family that regulates several signaling pathways including NF-κB signal transduction and p38 activation. TAK1 deregulation has been implicated in human diseases including cancer and inflammation. Here, we show that, in addition to its kinase activity, TAK1 has intrinsic ATPase activity, that (5Z)-7-Oxozeaenol irreversibly inhibits TAK1, and that sensitivity to (5Z)-7-Oxozeaenol inhibition in hematological cancer cell lines is NRAS mutation status and TAK1 pathway dependent. X-ray crystallographic and mass spectrometric studies showed that (5Z)-7-Oxozeaenol forms a covalent complex with TAK1. Detailed biochemical characterization revealed that (5Z)-7-Oxozeaenol inhibited both the kinase and the ATPase activity of TAK1 following a bi-phase kinetics, consistent with the irreversible inhibition mechanism. In DoHH2 cells, (5Z)-7-Oxozeaenol potently inhibited the p38 phosphorylation driven by TAK1, and the inhibition lasted over 6 h after withdrawal of (5Z)-7-Oxozeaenol. Profiling (5Z)-7-Oxozeaenol in a panel of hematological cancer cells showed that sensitive cell lines tended to carry NRAS mutations and that genes in TAK1 regulated pathways were enriched in sensitive cell lines. Taken together, we have elucidated the molecular mechanism of a TAK1 irreversible inhibitor and laid the foundation for designing next generation TAK1 irreversible inhibitors. The NRAS-TAK1-Wnt signaling network discerned in our study may prove to be useful in patient selection for TAK1 targeted agents in hematological cancers.

Discovery of a novel class of dimeric Smac mimetics as potent IAP antagonists resulting in a clinical candidate for the treatment of cancer (AZD5582).

A series of dimeric compounds based on the AVPI motif of Smac were designed and prepared as antagonists of the inhibitor of apoptosis proteins (IAPs). Optimization of cellular potency, physical properties, and pharmacokinetic parameters led to the identification of compound 14 (AZD5582), which binds potently to the BIR3 domains of cIAP1, cIAP2, and XIAP (IC50 = 15, 21, and 15 nM, respectively). This compound causes cIAP1 degradation and induces apoptosis in the MDA-MB-231 breast cancer cell line at subnanomolar concentrations in vitro. When administered intravenously to MDA-MB-231 xenograft-bearing mice, 14 results in cIAP1 degradation and caspase-3 cleavage within tumor cells and causes substantial tumor regressions following two weekly doses of 3.0 mg/kg. Antiproliferative effects are observed with 14 in only a small subset of the over 200 cancer cell lines examined, consistent with other published IAP inhibitors. As a result of its in vitro and in vivo profile, 14 was nominated as a candidate for clinical development.

Structure and Property Based Design of Pyrazolo[1,5-a]pyrimidine Inhibitors of CK2 Kinase with Activity in Vivo.

In this letter, we describe the design, synthesis, and structure-activity relationship of 5-anilinopyrazolo[1,5-a]pyrimidine inhibitors of CK2 kinase. Property-based optimization of early leads using the 7-oxetan-3-yl amino group led to a series of matched molecular pairs with lower lipophilicity, decreased affinity for human plasma proteins, and reduced binding to the hERG ion channel. Agents in this study were shown to modulate pAKT(S129), a direct substrate of CK2, in vitro and in vivo, and exhibited tumor growth inhibition when administered orally in a murine DLD-1 xenograft.

Mechanisms of acquired crizotinib resistance in ALK-rearranged lung Cancers.

Most anaplastic lymphoma kinase (ALK)-positive non-small cell lung cancers (NSCLCs) are highly responsive to treatment with ALK tyrosine kinase inhibitors (TKIs). However, patients with these cancers invariably relapse, typically within 1 year, because of the development of drug resistance. Herein, we report findings from a series of lung cancer patients (n = 18) with acquired resistance to the ALK TKI crizotinib. In about one-fourth of patients, we identified a diverse array of secondary mutations distributed throughout the ALK TK domain, including new resistance mutations located in the solvent-exposed region of the adenosine triphosphate-binding pocket, as well as amplification of the ALK fusion gene. Next-generation ALK inhibitors, developed to overcome crizotinib resistance, had differing potencies against specific resistance mutations. In addition to secondary ALK mutations and ALK gene amplification, we also identified aberrant activation of other kinases including marked amplification of KIT and increased autophosphorylation of epidermal growth factor receptor in drug-resistant tumors from patients. In a subset of patients, we found evidence of multiple resistance mechanisms developing simultaneously. These results highlight the unique features of TKI resistance in ALK-positive NSCLCs and provide the rationale for pursuing combinatorial therapeutics that are tailored to the precise resistance mechanisms identified in patients who relapse on crizotinib treatment.