Maximizing ER-α Degradation Maximizes Activity in a Tamoxifen-Resistant Breast Cancer Model: Identification of GDC-0927.

The further optimization of ER-α degradation efficacy of a series of ER modulators by refining side-chain substitution led to efficacious selective estrogen receptor degraders (SERDs). A fluoromethyl azetidine group was found to be preferred and resulted in the identification of bis-phenol chromene 17ha. In a tamoxifen-resistant breast cancer xenograft model, 17ha (ER-α degradation efficacy = 97%) demonstrated tumor regression, together with robust reduction of intratumoral ER-α levels. However, despite superior oral exposure, 5a (ER-α degradation efficacy = 91%) had inferior activity. This result suggests that optimizing ER-α degradation efficacy leads to compounds with robust effects in a model of tamoxifen-resistant breast cancer. Compound 17ha (GDC-0927) was evaluated in clinical trials in women with metastatic estrogen receptor-positive breast cancer.

Selective estrogen receptor degraders with novel structural motifs induce regression in a tamoxifen-resistant breast cancer xenograft.

Potent estrogen receptor ligands typically contain a phenolic hydrogen-bond donor. The indazole of the selective estrogen receptor degrader (SERD) ARN-810 is believed to mimic this. Disclosed herein is the discovery of ARN-810 analogs which lack this hydrogen-bond donor. These SERDs induced tumor regression in a tamoxifen-resistant breast cancer xenograft, demonstrating that the indazole NH is not necessary for robust ER-modulation and anti-tumor activity.

Identification of an Orally Bioavailable Chromene-Based Selective Estrogen Receptor Degrader (SERD) That Demonstrates Robust Activity in a Model of Tamoxifen-Resistant Breast Cancer.

About 75% of breast cancers are estrogen receptor alpha (ER-α) positive, and women typically initially respond well to antihormonal therapies such as tamoxifen and aromatase inhibitors, but resistance often emerges. Fulvestrant is a steroid-based, selective estrogen receptor degrader (SERD) that both antagonizes and degrades ER-α and shows some activity in patients who have progressed on antihormonal agents. However, fulvestrant must be administered by intramuscular injections that limit its efficacy. We describe the optimization of ER-α degradation efficacy of a chromene series of ER modulators resulting in highly potent and efficacious SERDs such as 14n. When examined in a xenograft model of tamoxifen-resistant breast cancer, 14n (ER-α degradation efficacy = 91%) demonstrated robust activity, while, despite superior oral exposure, 15g (ER-α degradation efficacy = 82%) was essentially inactive. This result suggests that optimizing ER-α degradation efficacy in the MCF-7 cell line leads to compounds with robust effects in models of tamoxifen-resistant breast cancer derived from an MCF-7 background.

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.

Optimization of an indazole series of selective estrogen receptor degraders: Tumor regression in a tamoxifen-resistant breast cancer xenograft.

Selective estrogen receptor degraders (SERDs) have shown promise for the treatment of ER+ breast cancer. Disclosed herein is the continued optimization of our indazole series of SERDs. Exploration of ER degradation and antagonism in vitro followed by in vivo antagonism and oral exposure culminated in the discovery of indazoles 47 and 56, which induce tumor regression in a tamoxifen-resistant breast cancer xenograft.

Identification of GDC-0810 (ARN-810), an Orally Bioavailable Selective Estrogen Receptor Degrader (SERD) that Demonstrates Robust Activity in Tamoxifen-Resistant Breast Cancer Xenografts.

Approximately 80% of breast cancers are estrogen receptor alpha (ER-α) positive, and although women typically initially respond well to antihormonal therapies such as tamoxifen and aromatase inhibitors, resistance often emerges. Although a variety of resistance mechanism may be at play in this state, there is evidence that in many cases the ER still plays a central role, including mutations in the ER leading to constitutively active receptor. Fulvestrant is a steroid-based, selective estrogen receptor degrader (SERD) that both antagonizes and degrades ER-α and is active in patients who have progressed on antihormonal agents. However, fulvestrant suffers from poor pharmaceutical properties and must be administered by intramuscular injections that limit the total amount of drug that can be administered and hence lead to the potential for incomplete receptor blockade. We describe the identification and characterization of a series of small-molecule, orally bioavailable SERDs which are potent antagonists and degraders of ER-α and in which the ER-α degrading properties were prospectively optimized. The lead compound 11l (GDC-0810 or ARN-810) demonstrates robust activity in models of tamoxifen-sensitive and tamoxifen-resistant breast cancer, and is currently in clinical trials in women with locally advanced or metastatic estrogen receptor-positive breast cancer.

A clinically relevant androgen receptor mutation confers resistance to second-generation antiandrogens enzalutamide and ARN-509.

UNLABELLED: Despite the impressive clinical activity of the second-generation antiandrogens enzalutamide and ARN-509 in patients with prostate cancer, acquired resistance invariably emerges. To identify the molecular mechanisms underlying acquired resistance, we developed and characterized cell lines resistant to ARN-509 and enzalutamide. In a subset of cell lines, ARN-509 and enzalutamide exhibit agonist activity due to a missense mutation (F876L) in the ligand-binding domain of the androgen receptor (AR). AR F876L is sufficient to confer resistance to ARN-509 and enzalutamide in in vitro and in vivo models of castration-resistant prostate cancer (CRPC). Importantly, the AR F876L mutant is detected in plasma DNA from ARN-509-treated patients with progressive CRPC. Thus, selective outgrowth of AR F876L is a clinically relevant mechanism of second-generation antiandrogen resistance that can potentially be targeted with next-generation antiandrogens. SIGNIFICANCE: A missense mutation in the ligand-binding domain of the androgen receptor F876L confers resistance to the second-generation antiandrogens enzalutamide and ARN-509 in preclinical models of AR function and prostate cancer and is detected in plasma DNA from ARN-509-treated patients with progressive disease. These results chart a new path for the discovery and development of next-generation antiandrogens that could be coupled with a blood-based companion diagnostic to guide treatment decisions.

ARN-509: a novel antiandrogen for prostate cancer treatment.

Continued reliance on the androgen receptor (AR) is now understood as a core mechanism in castration-resistant prostate cancer (CRPC), the most advanced form of this disease. While established and novel AR pathway-targeting agents display clinical efficacy in metastatic CRPC, dose-limiting side effects remain problematic for all current agents. In this study, we report the discovery and development of ARN-509, a competitive AR inhibitor that is fully antagonistic to AR overexpression, a common and important feature of CRPC. ARN-509 was optimized for inhibition of AR transcriptional activity and prostate cancer cell proliferation, pharmacokinetics, and in vivo efficacy. In contrast to bicalutamide, ARN-509 lacked significant agonist activity in preclinical models of CRPC. Moreover, ARN-509 lacked inducing activity for AR nuclear localization or DNA binding. In a clinically valid murine xenograft model of human CRPC, ARN-509 showed greater efficacy than MDV3100. Maximal therapeutic response in this model was achieved at 30 mg/kg/d of ARN-509, whereas the same response required 100 mg/kg/d of MDV3100 and higher steady-state plasma concentrations. Thus, ARN-509 exhibits characteristics predicting a higher therapeutic index with a greater potential to reach maximally efficacious doses in man than current AR antagonists. Our findings offer preclinical proof of principle for ARN-509 as a promising therapeutic in both castration-sensitive and castration-resistant forms of prostate cancer.

An interdomain interaction of the androgen receptor is required for its aggregation and toxicity in spinal and bulbar muscular atrophy.

Polyglutamine expansion within the androgen receptor (AR) causes spinal and bulbar muscular atrophy (SBMA) and is associated with misfolded and aggregated species of the mutant AR. We showed previously that nuclear localization of the mutant AR was necessary but not sufficient for SBMA. Here we show that an interdomain interaction of the AR that is central to its function within the nucleus is required for AR aggregation and toxicity. Ligands that prevent the interaction between the amino-terminal FXXLF motif and carboxyl-terminal AF-2 domain (N/C interaction) prevented toxicity and AR aggregation in an SBMA cell model and rescued primary SBMA motor neurons from 5α-dihydrotestosterone-induced toxicity. Moreover, genetic mutation of the FXXLF motif prevented AR aggregation and 5α-dihydrotestosterone toxicity. Finally, selective androgen receptor modulators, which prevent the N/C interaction, ameliorated AR aggregation and toxicity while maintaining AR function, highlighting a novel therapeutic strategy to prevent the SBMA phenotype while retaining AR transcriptional function.

WNT11 expression is induced by estrogen-related receptor alpha and beta-catenin and acts in an autocrine manner to increase cancer cell migration.

Elevated expression of the orphan nuclear receptor estrogen-related receptor α (ERRα) has been associated with a negative outcome in several cancers, although the mechanism(s) by which this receptor influences the pathophysiology of this disease and how its activity is regulated remain unknown. Using a chemical biology approach, it was determined that compounds, previously shown to inhibit canonical Wnt signaling, also inhibited the transcriptional activity of ERRα. The significance of this association was revealed in a series of biochemical and genetic experiments that show that (a) ERRα, ß-catenin (ß-cat), and lymphoid enhancer-binding factor-1 form macromolecular complexes in cells, (b) ERRα transcriptional activity is enhanced by ß-cat expression and vice versa, and (c) there is a high level of overlap among genes previously shown to be regulated by ERRα or ß-cat. Furthermore, silencing of ERRα and ß-cat expression individually or together dramatically reduced the migratory capacity of breast, prostate, and colon cancer cells in vitro. This increased migration could be attributed to the ERRα/ß-cat-dependent induction of WNT11. Specifically, using (a) conditioned medium from cells overexpressing recombinant WNT11 or (b) WNT11 neutralizing antibodies, we were able to show that this protein was the key mediator of the promigratory activities of ERRα/ß-cat. Together, these data provide evidence for an autocrine regulatory loop involving transcriptional upregulation of WNT11 by ERRα and ß-cat that influences the migratory capacity of cancer cells.

Pharmacological targeting of the mitochondrial phosphatase PTPMT1.

The dual-specificity protein tyrosine phosphatases (PTPs) play integral roles in the regulation of cell signaling. There is a need for new tools to study these phosphatases, and the identification of inhibitors potentially affords not only new means for their study, but also possible therapeutics for the treatment of diseases caused by their dysregulation. However, the identification of selective inhibitors of the protein phosphatases has proven somewhat difficult. PTP localized to mitochondrion 1 (PTPMT1) is a recently discovered dual-specificity phosphatase that has been implicated in the regulation of insulin secretion. Screening of a commercially available small-molecule library yielded alexidine dihydrochloride, a dibiguanide compound, as an effective and selective inhibitor of PTPMT1 with an in vitro concentration that inhibits response by 50% of 1.08 microM. A related dibiguanide analog, chlorhexidine dihydrochloride, also significantly inhibited PTPMT1, albeit with lower potency, while a monobiguanide analog showed very weak inhibition. Treatment of isolated rat pancreatic islets with alexidine dihydrochloride resulted in a dose-dependent increase in insulin secretion, whereas treatment of a pancreatic beta-cell line with the drug affected the phosphorylation of mitochondrial proteins in a manner similar to genetic inhibition of PTPMT1. Furthermore, knockdown of PTPMT1 in rat islets rendered them insensitive to alexidine dihydrochloride treatment, providing evidence for mechanism-based activity of the inhibitor. Taken together, these studies establish alexidine dihydrochloride as an effective inhibitor of PTPMT1, both in vitro and in cells, and support the notion that PTPMT1 could serve as a pharmacological target in the treatment of type II diabetes.

The homeodomain protein HOXB13 regulates the cellular response to androgens.

HOXB13 is a member of the homeodomain family of sequence-specific transcription factors and, together with the androgen receptor (AR), plays a critical role in the normal development of the prostate gland. We demonstrate here that, in prostate cancer cells, HOXB13 is a key determinant of the response to androgens. Specifically, it was determined that HOXB13 interacts with the DNA-binding domain of AR and inhibits the transcription of genes that contain an androgen-response element (ARE). In contrast, the AR:HOXB13 complex confers androgen responsiveness to promoters that contain a specific HOXB13-response element. Further, HOXB13 and AR synergize to enhance the transcription of genes that contain a HOX element juxtaposed to an ARE. The profound effects of HOXB13 knockdown on androgen-regulated proliferation, migration, and lipogenesis in prostate cancer cells highlight the importance of the observed changes in gene expression.

Inhibition of prostate cancer cell growth by second-site androgen receptor antagonists.

The impact of ligand binding on nuclear receptor (NR) structure and the ability of target cells to distinguish between different receptor-ligand complexes are key determinants of the pharmacological activity of NR ligands. However, until relatively recently, these mechanistic insights have not been used in a prospective manner to develop screens for NR modulators with specific therapeutic activities. Driven by the need for unique androgen receptor (AR) antagonists that retain activity in hormone-refractory prostate cancer, we developed and applied a conformation-based screen to identify AR antagonists that were mechanistically distinct from existing drugs of this class. Two molecules were identified by using this approach, D36 and D80, which interact with AR in a unique manner and allosterically inhibit AR agonist activity. Unlike the clinically important antiandrogens, casodex and hydroxyflutamide, both D36 and D80 block androgen action in cellular models of hormone-refractory prostate cancer. Mechanistically, these compounds further distinguish themselves from classical AR antagonists in that they do not promote AR nuclear translocation and quantitatively inhibit the association of AR with DNA even under conditions of overexpression. Although the therapeutic potential of these antiandrogens is apparent, it is the demonstration that it is possible, to modulate the interaction of cofactors with agonist-activated AR, using second-site modulators, that has the greatest potential with respect to the therapeutic exploitation of AR and other NRs.

Induction of Kruppel-like factor 5 expression by androgens results in increased CXCR4-dependent migration of prostate cancer cells in vitro.

Advanced prostate cancers preferentially metastasize to bone, suggesting that this tissue produces factors that provide a suitable microenvironment for prostate cancer cells. Recently, it has become clear that even in antiandrogen-resistant cancers, the androgen receptor (AR)-signaling axis is required for prostate cancer progression. Therefore, we hypothesized that AR may be involved in the regulation of pathways that are responsible for the homing of prostate cancer cells to select microenvironments. In support of this hypothesis, we have determined that chemokine (C-X-C motif) receptor 4 (CXCR4), the receptor for the chemokine CXCL12, is up-regulated in prostate cancer cells in response to androgens. Given that the levels of CXCL12 are elevated at sites of known prostate cancer metastases such as bone, these results suggest that androgens may influence prostate cancer metastasis. Specifically, we demonstrate that androgens increase the levels of both CXCR4 mRNA and functional protein in LNCaP prostate cancer cells. Importantly, androgens enhanced the migration of LNCaP cells toward a CXCL12 gradient, an effect that could be blocked by the specific CXCR4 antagonist AMD3100. Interestingly, CXCR4 is not directly regulated by androgens but rather is positively up-regulated by Krüppel-like factor 5 (KLF5), a transcription factor that we have shown to be an early, direct target of AR. Further, KLF5 is both required and sufficient for androgen-mediated CXCR4 expression and migration toward CXCL12. Taken together, these findings demonstrate that AR can utilize the CXCL12/CXCR4 axis through induction of KLF5 expression to promote prostate cancer progression and highlight the potential utility of CXCR4 antagonists as prostate cancer therapeutics.

PINA is essential for growth and positively influences NIMA function in Aspergillus nidulans.

The phospho-Ser/Thr-directed prolyl-isomerase Pin1 was originally identified in vertebrate systems as a negative regulator of NIMA, a Ser/Thr protein kinase that regulates the G(2)/M transition in Aspergillus nidulans. Here we explore the physiological roles of the Pin1 orthologue, PINA, in A. nidulans and evaluate the relevance of the interaction of PINA with NIMA in this fungus. We find pinA to be an essential gene in A. nidulans. In addition, when PINA levels are reduced 50-fold the cells grow at a reduced rate. Upon germination under conditions that repress PINA expression, the cells are delayed in the interphase activation of NIMX(cdc2), whereas they traverse the other phases of the cell cycle at a similar rate to controls. These results indicate that a marked reduction of PINA results in a lengthening of G(1). Additionally, PINA repression increases the rate at which the cells enter mitosis following release from a hydroxyurea arrest without altering the sensitivity of the fungus to agents that activate the replication or DNA damage checkpoints. In contrast to predictions based on Pin1, the physical interaction between PINA and NIMA is primarily dependent upon the prolylisomerase domain of PINA and the C-terminal 303 amino acids of NIMA. Finally, reduction of PINA levels exacerbates the nimA5 temperature-sensitive mutant, whereas overexpression of PINA decreases the severity of this mutation, results that are consistent with a positive genetic interaction between PINA and NIMA. Thus, although PINA is essential and positively regulates NIMA function, A. nidulans is most sensitive to a reduction in PINA concentration in G(1) rather than in G(2)/M.

Catalytic activity is required for calcium/calmodulin-dependent protein kinase IV to enter the nucleus.

Calcium/calmodulin-dependent protein kinase IV (CaMKIV) is a nuclear protein kinase that responds to acute rises in intracellular calcium by phosphorylating and activating proteins involved in transcription. Consistent with these roles, CaMKIV is found predominantly in the nucleus of cells in which it is expressed. Here we evaluate nuclear entry of CaMKIV and demonstrate that the protein kinase homology domain is both necessary and sufficient for nuclear localization. Unexpectedly, although catalytic activity is required for nuclear translocation, it is not required for CaMKIV to interact with the nuclear adaptor protein, importin-alpha. Because the catalytically inactive molecules remain in the cytoplasm, these data suggest that this interaction is not sufficient for nuclear entry. We evaluated a role for other proteins known to interact with CaMKIV in regulation of its nuclear entry. Although our data do not support a role for calmodulin or protein phosphatase 2A, the catalytically inactive CaMKIV proteins interact more avidly with CaM-dependent protein kinase kinase (CaMKK), which is restricted to the cytoplasm. We find that the catalytically inactive proteins do not inhibit nuclear entry of wild-type CaMKIV but do inhibit the ability of the wild-type protein kinase to stimulate cyclic AMP response element-binding protein-mediated transcription. Because activation loop phosphorylation is required for the transcriptional roles of CaMKIV, these data suggest that CaMKK phosphorylation of CaMKIV may occur in the cytoplasm. We propose that sequestration of CaMKK may be the molecular mechanism by which catalytically inactive mutants of CaMKIV exert their "dominant-negative" functions within the cell.

The peptidyl-prolyl isomerase Pin1.

The phospho-Ser/Thr-Pro specific prolyl-isomerase Pin1 has been implicated in multiple aspects of cell cycle regulation. It has been suggested that Pin1 function is required for both normal mitotic progression and reentry into the cell cycle from quiescence. In support of this hypothesis, numerous key regulators of G1 and mitosis have been identified as Pin1 interacting proteins. However, the cellular consequence of Pin1 binding to these proteins has rarely been rigorously characterized. In this review we focus on the role of Pin1 and its binding proteins in cell cycle regulation and the potential value of Pin1 as a therapeutic target.

Proteomic analysis of calcium/calmodulin-dependent protein kinase I and IV in vitro substrates reveals distinct catalytic preferences.

The multifunctional calcium/calmodulin-dependent protein kinases I and IV (CaMKI and CaMKIV) are closely related by primary sequence and predicted to have similar substrate specificities based on peptide studies. We identified a fragment of p300-(1-117) that is a substrate of both kinases, and through both mutagenesis and Edman phosphate ((32)P) release sequencing, established that CaMKI and CaMKIV phosphorylate completely different sites. The CaMKI site, Ser(89) ((84)LLRSGSSPNL(93)), fits the expected consensus whereas the CaMKIV site, Ser(24) ((19)SSPALSASAS(28)), is novel. To compare kinase substrate preferences more generally, we employed a proteomic display technique that allowed comparison of complex cell extracts phosphorylated by each kinase in a rapid in vitro assay, thereby demonstrating substrate preferences that overlapped but were clearly distinct. To validate this approach, one of the proteins labeled in this assay was identified by microsequencing as HSP25, purified as a recombinant protein, and examined as a substrate for both CaMKI and CaMKIV. Again, CaMKI and CaMKIV were different, this time in kinetics and stoichiometry of the phosphorylation sites, with CaMKI preferring Ser(15) ((10)LLRTPSWGPF(19)) to Ser(85) ((80)LNRQLSSGVS(89)) 3:1, but CaMKIV phosphorylating the two sites equally. These differences in substrate specificities emphasize the need to consider these protein kinases independently despite their close homology.

Calcium binding is required for calmodulin function in Aspergillus nidulans.

To explore the structural basis for the essential role of calmodulin (CaM) in Aspergillus nidulans, we have compared the biochemical and in vivo properties of A. nidulans CaM (AnCaM) with those of heterologous CaMs. Neither Saccharomyces cerevisiae CaM (ScCaM) nor a Ca2+ binding mutant of A. nidulans CaM (1234) interacts appreciably with A. nidulans CaM binding proteins by an overlay assay or activates two essential CaMKs, CMKA and CMKB. In contrast, although vertebrate CaM (VCaM) binds a spectrum of proteins similar to that for AnCaM, it is unable to fully activate CMKA and CMKB, displaying a higher K(CaM) and reduced Vmax for both enzymes. In correlation with the biochemical analysis, neither ScCaM nor 1234 can support A. nidulans growth in the absence of the endogenous protein, whereas VCaM only partially complements the absence of wild-type CaM. Analysis of VCaM and AnCaM chimeras demonstrates that amino acid variations in both N- and C-terminal domains contribute to the inability of VCaM to activate CMKB, but differences in the N terminus are largely responsible for the reduced activity towards CMKA. In vivo, the chimeric molecules support growth equivalently, but only to levels intermediate between those of VCaM and AnCaM, suggesting that the reduced ability to activate the CaMKs is not solely responsible for the inability of VCaM to complement the absence of the wild-type protein. Thus, not only is Ca2+ binding required for CaM function in A. nidulans, but the essential in vivo functions of A. nidulans CaM are uniquely sensitive to the subtle amino acid variations present in vertebrate CaM.