Mutations In Ligand-binding Domain Research Articles

Androgen Signaling in Prostate Cancer: When a Friend Turns Foe.

Androgen (AR) signaling is the main signaling for the development of the prostate and its normal functioning. AR is highly specific for testosterone and dihydrotestosterone, significantly contributing to prostate development, physiology, and cancer. All these receptors have emerged as crucial therapeutic targets for PCa. In the year 1966, the Noble prize was awarded to Huggins and Hodge for their groundbreaking discovery of AR. As it is a pioneer transcription factor, it belongs to the steroid hormone receptor family and consists of domains, including DNA binding domain (DBD), hormone response elements (HRE), C-terminal ligand binding domain (LBD), and N-terminal regulatory domains. Structural variations in AR, such as AR gene amplification, LBD mutations, alternative splicing of exons, hypermethylation of AR, and co- regulators, are major contributors to PCa. It's signaling is crucial for the development and functioning of the prostate gland, with the AR being the key player. The specificity of AR for testosterone and dihydrotestosterone is important in prostate physiology. However, when it is dysregulated, AR contributes significantly to PCa. However, the structural variations in AR, such as gene amplification, mutations, alternative splicing, and epigenetic modifications, drive the PCa progression. Therefore, understanding AR function and dysregulation is essential for developing effective therapeutic strategies. Thus, the aim of this review was to examine how AR was initially pivotal for prostate development and how it turned out to show both positive and detrimental implications for the prostate.

Prevalence and Spectrum of AR Ligand-Binding Domain Mutations Detected in Circulating-Tumor DNA Across Disease States in Men With Metastatic Castration-Resistant Prostate Cancer.

Metastatic castration-resistant prostate cancer (mCRPC) is typically treated with agents directly or indirectly targeting the androgen receptor (AR) pathway. However, such treatment is limited by resistance mechanisms, including the development of activating mutations in the AR ligand-binding domain (AR-LBD). This study evaluated a database of over 15,000 patients with advanced prostate cancer (PC) undergoing comprehensive circulating-tumor DNA analysis (Guardant360, Redwood City, CA) between 2014 and 2021, with associated clinical information from administrative claims (GuardantINFORM database). Of 15,705 patients with PC included, 54% had mCRPC at the time of their blood draw. Of those, 49% had previous treatment with an AR pathway inhibitor (ARPi). AR-LBD mutation prevalence was 15% in patients with mCRPC who were untreated with a next-generation ARPi, 22% in those after one line of ARPi therapy, and 24% in those after two lines of ARPi treatment. Next-generation ARPi treatment yielded an increase in AR L702H and T878A/S mutations after abiraterone, and an increase in AR L702H and F877L mutations after enzalutamide. AR-LBD+ patients demonstrated unique biology, including increased concurrent mutations in the cell-cycle, wingless-related integration site, homologous recombination repair, and phospho-inositide 3-kinase pathways (all P < .0005), and greater low-level (copy number <10) AR amplifications (P = .0041). AR-LBD+ patients exhibited worse overall survival (OS) relative to a matched cohort of AR-LBD- patients (50.1 v 60.7 months, unadjusted log-rank P = .013). This large database analysis demonstrates that AR-LBD mutation prevalence increases after next-generation ARPi use. AR-LBD+ tumors demonstrate unique biology (more oncogenic pathway mutations and low-level AR amplification) and reduced OS. These findings inform the development of novel therapies designed to circumvent AR-mediated therapeutic resistance.

Abstract 3544: OncoKB™, MSK’s precision oncology knowledge base: 2023 updates

Abstract OncoKBTM is Memorial Sloan Kettering Cancer Center’s (MSK) FDA-recognized precision oncology knowledge base that contains detailed, evidence-based information about the oncogenic effect and therapeutic implications of individual somatic mutations and structural alterations present in patient tumors. Since its public release in 2016, OncoKBTM has expanded to include annotation for &amp;gt;7,500 alterations in ~820 cancer-associated genes. OncoKB supports variant interpretation by the cBioPortal for Cancer Genomics, is used to annotate &amp;gt;15,000 MSK patient sequencing reports annually and its data is publicly available through the website (www.oncokb.org). Programmatic access to OncoKBTM data via its web-based API is freely available to users in an academic setting while users from commercial and hospital settings require a fee-based license. OncoKBTM utilizes its Therapeutic Levels of Evidence system to classify variants based on their tumor type-specific sensitivity or resistance to matched standard care or investigational targeted therapies. To date, OncoKB includes 49 Level 1 genes as well as MSI-H and TMB-H (included in the FDA drug label), 24 Level 2 genes (included in professional guidelines), 35 Level 3A genes (predictive of drug response in well-powered clinical studies), 27 Level 4 genes (predictive of drug response based on compelling biological evidence), and 11 R1/R2 resistance genes. In 2023, OncoKBTM captured the following notable changes in precision oncology drug development: Level 1 annotation of ESR1 ligand-binding domain mutations in breast cancer and promotion from Level 2 to Level 1 of ERBB2 amplification in colorectal cancer following the FDA drug approvals of elacestrant and tucatinib + trastuzumab, respectively. Additionally, KRAS G12C became a Level 2 biomarker in pancreatic and colon cancers with the listing of adagrasib and sotorasib as systemic therapy options for KRAS G12C-mutant disease in the NCCN pancreatic and colon cancer guidelines. IDH1/2 mutations in low grade glioma were annotated as Level 3A based on compelling clinical evidence demonstrating response to the IDH-specific inhibitor vorasidenib. Lastly, noting emerging data with the KRAS G12X-specific inhibitor, RMC-6236, OncoKBTM included all alleles at KRAS position G12 as Level 4. In sum, six novel clinically actionable biomarkers (all Level 1) and 14 follow-on precision oncology therapies for existing leveled biomarkers were added to OncoKBTM in the past year. OncoKBTM’s current focus includes coverage of additional cancer-associated genes, annotation of germline alterations and incorporation of OncoKBTM data into an electronic health record system. Citation Format: Sarah P. Suehnholz, Moriah Nissan, Hongxin Zhang, Ritika Kundra, Calvin Lu, Amanda Dhaneshwar, Nicole Fernandez, Benjamin Preiser, Maria E. Arcila, Marc Ladanyi, Michael F. Berger, Aijazuddin Syed, A. Rose Brannon, Ross Levine, Ahmet Dogan, Alexander Drilon, David B. Solit, Nikolaus Schultz, Debyani Chakravarty. OncoKB™, MSK’s precision oncology knowledge base: 2023 updates [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3544.

Real-world outcomes of patients with metastatic castration-resistant prostate cancer (mCRPC) and tumors harboring androgen receptor (AR) ligand-binding domain (LBD) mutations.

77 Background: In mCRPC, AR T878 and/or H875 and L702H mutations have been associated with disease progression and poor prognosis. This study characterized real-world overall survival (rwOS), testing practices, mutation prevalence, and treatment patterns in patients with mCRPC with and without tumors with missense mutations in the AR LBD (amino acids 671–920). Methods: In this retrospective analysis of the Guardant Inform Database (Mar 11, 2014–Jun 30, 2022), men (aged ≥18 y) with mCRPC who were tested with Guardant360 (G360) next-generation sequencing and diagnosed/treated at a clinical site in the US with a first-line (1L) treatment were included. Matched rwOS was assessed in 2 subpopulations: patients with any missense mutation in AR LBD, excluding those with AR L702H alone ( AR LBD group), and patients with AR T878 and/or H875 without L702H ( AR 878/875 group) and compared with patients without any AR LBD missense mutations (control group). Patients were matched (1:5) on key prognostic variables, including age, Elixhauser Comorbidity Index weighted score, smoking status, prior novel hormonal agent (NHA) use, and 1L treatment year. Mutation prevalence, patient demographics, frequency and timing of G360 tests, and treatment patterns were assessed in patients treated from 2018 to 2022 to reflect more recent trends. Results: In the matched rwOS analysis, patients in the AR LBD group (n=275) had statistically significantly shorter 1L median rwOS compared with the control group (n=1375): 27.3 vs 47.8 months; P&lt;0.0001. Similarly, patients in the AR 878/875 group (n=154) had shortened median rwOS compared with the control group (n=770): 28.9 vs 44.4 months; P=0.0004. Of 4833 patients treated for mCRPC from 2018 to 2022, 20% had AR LBD mutations, 4% had AR 878/875 with L702H, and 6% had AR 878/875 without L702H. Most patients (59%) were first tested at some point after 1L therapy, with only 15% of patients receiving &gt;1 G360 test. Mutation prevalence was higher in patients tested in fourth-line (4L) compared with 1L, particularly for AR L702H mutations (17% vs 10%) and all AR LBD mutations (27% vs 20%). Use of NHAs was lower in 4L compared with 1L (20% vs 33%), whereas use of taxanes was higher (31% vs 22%). Conclusions: Patients with AR LBD–mutated mCRPC had notably shorter rwOS than those whose tumors did not harbor AR LBD mutations, indicating an unmet need for this population. The prevalence of AR LBD mutations was higher in later lines of therapy, but repeat testing rates were low, suggesting that AR LBD mutations may go undetected.

A phase 1/2 study of ONCT-534, a dual-action androgen receptor inhibitor (DAARI), in patients with metastatic castration-resistant prostate cancer.

TPS241 Background: Current therapeutic strategies for metastatic castration-resistant prostate cancer (mCRPC) include treatment with next-generation androgen receptor signaling inhibitors (ARSIs), such as enzalutamide, darolutamide, and apalutamide, which all target the ligand-binding domain (LBD) of the AR, and abiraterone, an androgen biosynthesis inhibitor. Approximately 5% to 10% of patients will have primary resistance to ARSIs and most initial responders will develop resistance within 1 to 3 years. Eventually, nearly all men with prostate cancer treated with ARSIs develop resistance via several mechanisms including activation of AR genomic alterations, epigenetic alterations, and expression of truncated constitutively active AR splice variants. Hence, there is substantial unmet need for treatment of men with mCRPC resistant to at least one next-generation ARSI. ONCT-534 is a DAARI with a novel mechanism of action that combines inhibition of AR function with degradation of the AR protein. Importantly, this activity includes interaction with the N-terminal domain (NTD), rendering it effective against splice variants and LBD mutants, unlike existing ARSIs. ONCT-534 has demonstrated preclinical activity in prostate cancer models against unmutated AR and multiple forms of AR alteration, including amplification, mutations in the LBD, and splice variants with loss of LBD. Methods: ONCT-534-101 is a phase 1/2, multi-center study to evaluate the safety, tolerability, antitumor activity, and pharmacokinetics of ONCT-534 in subjects with mCRPC without neuroendocrine differentiation or small cell features who have relapsed or are refractory (R/R) to at least one next-generation ARSI. The study will be separated into a Phase 1 Dose Escalation and a Phase 2 Dose Expansion. The Phase 1 portion will evaluate approximately 27 subjects in 5 dose levels using an adaptive Bayesian Optimal Interval (BOIN) design to assess safety, tolerability and DLT at escalating doses and to determine the MTD and inform the 2 dose levels or schedules to be tested in Phase 2. DLTs will be assessed during the first 28 days of treatment. The AR phenotype and AR levels of each subject’s disease will be evaluated pre- and post-treatment. The Phase 2 portion will evaluate approximately 32 subjects in 2 randomized cohorts to assess safety and tolerability of ONCT-534, compare the 2 different dose levels or schedules to select the optimal dose for further study, and assess the preliminary antitumor activity of ONCT-534. In both phases, after a screening period, eligible subjects with mCRPC will receive their assigned dose regimen of ONCT-534, which will be administered orally daily for 2 years or until disease progression and no longer clinically benefiting from treatment or development of unacceptable toxicity. The study was opened for enrollment September 2023. Clinical trial information: NCT05917470 .

Targeted Inhibition of CYP11A1 in Castration-Resistant Prostate Cancer

BackgroundProstate cancer is regulated by steroid hormones, even in castration-resistant disease. ODM-208, a novel inhibitor of cytochrome P450 11A1 (which catalyzes the first step of steroid-hormone biosynthesis), was investigated in patients with heavily pretreated metastatic castration-resistant prostate cancer (mCRPC).MethodsCYPIDES is a first-in-human phase 1 (3 + 3 design) and phase 2 study. We administered ODM-208 twice daily with glucocorticoid/mineralocorticoid replacement and ongoing androgen deprivation therapy to adults with previously treated mCRPC, regardless of androgen receptor gene (AR) ligand-binding domain mutations (phase 1) and with activating AR ligand-binding domain mutations (ARmut; phase 2). Safety, pharmacokinetics, steroid-hormone pharmacodynamics, and preliminary efficacy were the key outcomes.ResultsNinety-two patients received one or more doses of ODM-208: 47 in phase 1 (20 [42.6%] with ARmut) and 45 in phase 2 (all ARmut). A dose of ODM-208 of 5 mg twice a day with dexamethasone 1 mg/fludrocortisone 0.1 mg provided a balance between decreased steroidogenesis and toxicity. Treatment-related adrenal insufficiency was the most common toxicity in phase 1 (n=17, 36.2%; necessitating ODM-208 discontinuation in one patient); this toxicity occurred in six patients (13.3%) at 5 mg twice a day in phase 2. Median circulating testosterone levels declined from 3.0 ng/dl (interquartile range, 1.3 to 6.2 ng/dl) at baseline to undetectable levels within the first week of ODM-208 5 mg twice a day treatment in 46 of 53 (87%) patients. A decrease in prostate-specific antigen levels of 50% or more occurred in 14 of 19 (73.7%) patients with ARmut and 2 of 23 (8.7%) patients with AR wild type in phase 1 and in 24 of 45 (53.3%) patients with ARmut in phase 2.ConclusionsODM-208 potently inhibited steroid-hormone biosynthesis with the expected toxicity of adrenal insufficiency. Evidence of antitumor activity was observed in this heavily pretreated mCRPC population, especially in those with ARmut. (Funded by Orion Pharma; ClinicalTrials.gov number, NCT03436485.)

Recurring EPHB1 mutations in human cancers alter receptor signalling and compartmentalisation of colorectal cancer cells

BackgroundEphrin (EPH) receptors have been implicated in tumorigenesis and metastasis, but the functional understanding of mutations observed in human cancers is limited. We previously demonstrated reduced cell compartmentalisation for somatic EPHB1 mutations found in metastatic colorectal cancer cases. We therefore integrated pan-cancer and pan-EPH mutational data to prioritise recurrent EPHB1 mutations for functional studies to understand their contribution to cancer development and metastasis.MethodsHere, 79,151 somatic mutations in 9,898 samples of 33 different tumour types were analysed with a bioinformatic pipeline to find 3D-mutated cluster pairs and hotspot mutations in EPH receptors. From these, 15 recurring EPHB1 mutations were stably expressed in colorectal cancer followed by confocal microscopy based in vitro compartmentalisation assays and phospho-proteome analysis.ResultsThe 3D-protein structure-based bioinformatics analysis resulted in 63% EPHB1 mutants with compartmentalisation phenotypes vs 43% for hotspot mutations. Whereas the ligand-binding domain mutations C61Y, R90C, and R170W, the fibronectin domain mutation R351L, and the kinase domain mutation D762N displayed reduced to strongly compromised cell compartmentalisation, the kinase domain mutations R743W and G821R enhanced this phenotype. While mutants with reduced compartmentalisation also had reduced ligand induced receptor phosphorylation, the enhanced compartmentalisation was not linked to receptor phosphorylation level. Phosphoproteome mapping pinpointed the PI3K pathway and PIK3C2B phosphorylation in cells harbouring mutants with reduced compartmentalisation.ConclusionsThis is the first integrative study of pan-cancer EPH receptor mutations followed by in vitro validation, a robust way to identify cancer-causing mutations, uncovering EPHB1 mutation phenotypes and demonstrating the utility of protein structure-based mutation analysis in characterization of novel cancer genes.BS_YKV7jikZ2WYCZfZ4ZjJVideo

Partner Gene Associated Trinary Fusion and Orchestration of Multiple Molecular Events in Acute Promyelocytic Leukemia with Variant Rara Fusion Genes

Background All-trans retinoic acid (ATRA) has been used with great success in acute promyelocytic leukemia (APL) cases with PML::RARA fusion gene (FG). There are still PML::RARA negative cases manifest as APL. So far, 19 RARA fusion partner genes have been reported, with all reported as 5′ partners ( X::RARA). Intriguingly, a significant proportion of X::RARA cases were resistant to ATRA, and mechanisms are yet to be elucidated. Here we report the finding of a novel type of trinary fusion and the orchestrating of multiple molecular events that confer leukemogenesis and ATRA resistance in X::RARA-APL. Methods A total of 23 cases (Fig. 1A) with RNA-seq and WGS data from 10 centers were enrolled. We used Arriba for routine FG calling and manually investigated the nonconventional fusion sequence. RT-PCR, Sanger sequencing, and optical genome mapping (OGM) were used to validate the FGs and to determine whether the RARA 5′ and 3′ fusion events were located on the same cistron. The responsiveness of the fusion proteins to ATRA was evaluated using an optimized UAS/GAL4 reporter system. Results Among the cases enrolled, there were 17 males and 6 females, aged 3-66 (median 32) years. Morphological and immunophenotype analyses all showed features of APL. We first identified the STAT3::RARA fusion event in an ATRA-resistant case (the index case, #A1). Moreover, we noticed another fusion event of truncated RARA exon 9 and a 3′ partner sequence that did not map to any annotated protein-coding genes. Further analysis determined it was the RC sequence of the transposable element (TE) LTR40a with the annotated loci at 17p11.2. Both OGM (at the genome level) and RT-PCR (Fig. 1A) confirmed that the two RARA fusion events were in the same cistron. Thus, the fusion transcript being STAT3::RARA::LTR40a-RC spliced in tandem, but not separate transcripts of STAT3::RARA and RARA::LTR40a-RC. We named this novel form of tandem splicingtrinary fusion. We, therefore, recruit X::RARA-APL cases to investigate the recurrence of RARA 3′ fusion. We identified 9 RARA 5′ partner genes in 23 cases (Fig. 1B), with TTMV -ORF2, STAT5B, STAT3, IRF2BP1, THRAP3, and TNRC18 in multiple cases. Ten (43.5%) cases also harbor RARA 3′ fusion partners. The partner of case #A1_8 were TEs, and the partner of case #A9_10 was the same gene as their 5′ one. Thirteen cases did not harbor 3′ fusions. Notably, both TNRC18::RARA cases (#15_16) carry mutations on their RARA ligand binding domain (LBD) at the primary onset. Gene expression analysis confirmed the aberrant activation of the involved TEs. All TEs involved in the RARA 3' fusions confer a poly_A signal sequence, which was essential for a mRNA. In-depth analysis indicated that TEs participate in forming RARA-FGs through a transposition mechanism rather than a translocation mechanism. The molecular events addition to RARA 5′ splicing showed a 5′ partner associated pattern: all of the 4 STAT3::RARA and 4 STAT5B::RARA cases harbor TEs as their 3′ partner, both TNRC18::RARA cases carry LBD mutation, while all 7 TTMV -ORF2::RARA cases did not harbor 3′ splicing or LBD mutation. Remarkably, although the RARA 3' splicing sites varied, they all resulted in partial or complete exon 9 truncation. RARA exon 9 encodes helix 11_12 (H11_12) of its LBD, which is pivotal in allosteric response to ATRA. The RARA trinary fusion will result in LBD-H12 or H11_12 castration due to the 3' splicing, then lead to losing responsiveness to ATRA via an allosteric disability mechanism (Fig. 1C, 1D). Experiments confirmed that cells transfected with STAT3::RARA fusion with intact LBD responded well to ATRA, while both the fusion in case #A1 and artificial fusion with LBD-H12 castration showed no response to physiological and pharmacological concentrations of ATRA (Fig. 1E). Conclusions This is the first report of pathological trinary fusion, the TEs and transposition mechanism participating in composing pathological fusion genes, and the RARA LBD castration confers ATRA resistance in X::RARA-APL. Also, there might be another synergistic mechanism of RARA 5' fusion and LBD mutation. The formation of trinary fusion and gain of LBD mutation, in addition to X::RARA fusion, requires more genome splicing or transposition or mutation events, which explains the rarity of X::RARA-APL. This study highlights mechanisms of partner gene-associated trinary fusion and the orchestrating of multiple molecular events in the leukemogenesis in X::RARA-APL cases.

Synthesis and Evaluation of Small Molecule Inhibitors of the Androgen Receptor N-Terminal Domain.

The androgen receptor (AR) is central to prostate cancer pathogenesis and has been extensively validated as a drug target. However, small-molecule anti-androgen therapies remain limited due to resistance and will eventually fail to suppress tumor growth, resulting in progression to castration-resistant prostate cancer (CRPC). The intrinsically disordered N-terminal domain (NTD) is crucial for AR transactivation and has been investigated as a suitable target in the presence of ligand binding domain mutations. A screening campaign identified biaryl isoxazole compound 7 as a weak inhibitor of the AR NTD. A library of biaryl analogues were synthesized, and their biological activities were assessed in a VCaP cell-based luciferase reporter gene assay. A structure-activity relationship (SAR) study revealed that indazole analogue 16 exhibited increased potency and favorable physicochemical properties with a benchmarked pharmacokinetic profile, providing a suitable starting point for further optimization of 16 as a CRPC therapeutic in the presence of AR mutations.

RuX: A Novel, Flexible, and Sensitive Mifepristone-Induced Transcriptional Regulation System.

Inducible gene regulation methods are indispensable in diverse biological applications, yet many of them have severe limitations in their applicability. These include inducer toxicity, a limited variety of organisms the given system can be used in, and side effects of the induction method. In this study, a novel inducible system, the RuX system, was created using a mutant ligand-binding domain of the glucocorticoid receptor (CS1/CD), used together with various genetic elements such as the Gal4 DNA-binding domain or Cre recombinase. The RuX system is shown to be capable of over 1000-fold inducibility, has flexible applications, and is offered for use in cell cultures.

SAT023 The Role Of Cysteine Residues In The Binding Of Inhibitors Of The N-Terminal Domain Of The Androgen Receptor

Abstract Disclosure: L. Deneault: None. A. Tien: None. A. Banuelos: Stock Owner; Self; ESSA. T. Tam: None. R. Andersen: Stock Owner; Self; ESSA. M.D. Sadar: Stock Owner; Self; ESSA. Background: Therapies for prostate cancer (PC) involve pharmaceutical castration by targeting the androgen receptor (AR) ligand-binding domain (LBD). These therapies are not curative with the malignancy progressing to castration-resistant PC (CRPC). Most CRPC remains driven by AR through multiple resistances, e.g. constitutively active AR splice variants that lack LBD (AR-Vs), or gain-of-function mutations in LBD. This has led to clinical development of EPI analogues (“EPI”) which target the AR N-terminal domain (NTD) to block the transcriptional activities of, AR-Vs and mutated ARs. The EPI-binding pocket involves region 341-446 amino acid residues of the NTD. AR NTD is enriched in cysteine residues with C404 suggested to be important in the binding mechanism of EPI. Free cysteine residues are able interact forming disulfide bonds, which are important in intra- and intermolecular protein-protein interactions. Here we begin to elucidate the role of cysteines in the inhibition of AR activity by EPI. Methods: Binding assays assess the role of cysteines in the covalent binding mechanism used a fluorescein-labelled EPI with fragments of recombinant AR-NTD (rNTD) with or without the addition of iodoacetamide (caps cysteines). Protein-protein interactions requiring disulfide bonds with AR examined by non-reducing SDS-PAGE and diagonal SDS-PAGE of cells treated with inhibitors. A role of specific cysteines in the activity of EPI to block AR transcriptional activity was assessed by cysteine mutants using a reporter gene construct driven by AR. Results: Capping cysteine residues on rNTD with iodoacetamide reduced the amount of covalent binding of EPI. This data suggests that cysteines are essential for covalent binding of EPI to AR-NTD. EPI bound preferentially to the oxidized form of rNTD suggesting the importance of disulfide bridges. Diagonal SDS-PAGE revealed differential alterations in intramolecular and intermolecular disulfide bonds in response to EPI compared to AR LBD inhibitors. These gels also revealed unique differences in protein-protein interactions with AR depending on the inhibitor. AR-driven reporter gene assays revealed mutations of cysteine residues in the AR- NTD did not adversely affect AR transcriptional activity in response to androgen. Conclusions: Although cysteines residing in the AR-NTD were required for covalent binding of EPI, mutation of C404 in the EPI-binding site was not essential for the efficacy of EPI to block AR transcriptional activity. There were differential patterns in protein-protein interactions with AR depending upon which domain the inhibitor targeted. Our future goal is to identify these unique proteins to provide insight into the mechanisms and develop more potent and more specific therapeutics for the treatment of CRPC. Presentation: Saturday, June 17, 2023

RETooling the RET Inhibitor Pralsetinib for ESR1 Fusion-Positive Breast Cancer and Beyond.

Transcriptionally active fusions of ESR1 (ESR1-TAF) and somatic mutations in the estrogen receptor alpha (ERα) ligand-binding domain (LBD) cause endocrine therapy resistance in breast cancer. In searching for therapeutic target kinase(s) in these breast cancers, Gou and colleagues identified FLT4, RET, JAK1, and IGF1R as the top upregulated kinases induced by ESR1-TAFs and ERα LBD mutants in breast cancer cells. Among them, inhibition of RET by pralsetinib suppressed ESR1-TAF-driven and ERα LBD mutant-driven cell proliferation and patient-derived xenograft growth. Pralsetinib is an inhibitor of the RET protein tyrosine kinase that is approved for treating oncogenic RET mutation-positive and RET fusion-positive thyroid cancers and non-small cell lung cancer. The work by Gou and colleagues reinforces the knowledge of RET as an ESR1 target gene and highlights that RET interacts with ERα to promote breast cancer tumorigenesis and antiestrogen resistance. It also raises the prospect of repositioning pralsetinib to target wildtype RET in ER-positive breast cancer. See related article by Gou et al., p. 3237.

1803P Phase I/II study of bavdegalutamide, a PROTAC androgen receptor (AR) degrader in metastatic castration-resistant prostate cancer (mCRPC): Radiographic progression-free survival (rPFS) in patients (pts) with AR ligand-binding domain (LBD) mutations

1803P Phase I/II study of bavdegalutamide, a PROTAC androgen receptor (AR) degrader in metastatic castration-resistant prostate cancer (mCRPC): Radiographic progression-free survival (rPFS) in patients (pts) with AR ligand-binding domain (LBD) mutations

Novel Inhibitors of androgen receptor's DNA binding domain identified using an ultra-large virtual screening.

Androgen receptor (AR) inhibition remains the primary strategy to combat the progression of prostate cancer (PC). However, all clinically used AR inhibitors target the ligand-binding domain (LBD), which is highly susceptible to truncations through splicing or mutations that confer drug resistance. Thus, there exists an urgent need for AR inhibitors with novel modes of action. We thus launched a virtual screening of an ultra-large chemical library to find novel inhibitors of the AR DNA-binding domain (DBD) at two sites: protein-DNA interface (P-box) and dimerization site (D-box). The compounds selected through vigorous computational filtering were then experimentally validated. We identified several novel chemotypes that effectively suppress transcriptional activity of AR and its splice variant V7. The identified compounds represent previously unexplored chemical scaffolds with a mechanism of action that evades the conventional drug resistance manifested through LBD mutations. Additionally, we describe the binding features required to inhibit AR DBD at both P-box and D-box target sites.

Tricyclic Diterpenoids Selectively Suppress Androgen Receptor-Positive Prostate Cancer Cells.

Androgen receptor (AR) is a viable therapeutic target for lethal castration-resistant prostate cancer (CRPC), because the continued progression of CRPC is mainly driven by the reactivation of AR transcriptional activity. The current FDA-approved AR antagonists binding to ligand binding domain (LBD) become ineffective in CRPC with AR gene amplification, LBD mutation, and the evolution of LBD-truncated AR splice variants. Encouraged by the fact that tricyclic aromatic diterpenoid QW07 has recently been established as a potential N-terminal AR antagonist, this study aims to explore the structure-activity relationship of tricyclic diterpenoids and their potential to suppress AR-positive cell proliferation. Dehydroabietylamine, abietic acid, dehydroabietic acid, and their derivatives were selected, since they have a similar core structure as QW07. Twenty diterpenoids were prepared for the evaluation of their antiproliferative potency on AR-positive prostate cancer cell models (LNCaP and 22Rv1) using AR-null cell models (PC-3 and DU145) as comparisons. Our data indicated that six tricyclic diterpenoids possess greater potency than enzalutamide (FDA-approved AR antagonist) towards LNCaP and 22Rv1 AR-positive cells, and four diterpenoids are more potent than enzalutamide against 22Rv1 AR-positive cells. The optimal derivative possesses greater potency (IC50 = 0.27 µM) and selectivity than QW07 towards AR-positive 22Rv1 cells.

Discovery of a Small-Molecule Inhibitor Targeting the Androgen Receptor N-Terminal Domain for Castration-Resistant Prostate Cancer.

The current mainstay therapeutic strategy for advanced prostate cancer is to suppress androgen receptor (AR) signaling. However, castration-resistant prostate cancer (CRPC) invariably arises with restored AR signaling activity. To date, the AR ligand-binding domain (LBD) is the only targeted region for all clinically available AR signaling antagonists, such as enzalutamide (ENZ). Major resistance mechanisms have been uncovered to sustain the AR signaling in CRPC despite these treatments, including AR amplification, AR LBD mutants, and the emergence of AR splice variants (AR-Vs) such as AR-V7. AR-V7 is a constitutively active truncated form of AR that lacks the LBD; thus, it can not be inhibited by AR LBD-targeting drugs. Therefore, an approach to inhibit AR through the regions outside of LBD is urgently needed. In this study, we have discovered a novel small molecule SC428, which directly binds to the AR N-terminal domain (NTD) and exhibits pan-AR inhibitory effect. SC428 potently decreased the transactivation of AR-V7, ARv567es, as well as full-length AR (AR-FL) and its LBD mutants. SC428 substantially suppressed androgen-stimulated AR-FL nuclear translocation, chromatin binding, and AR-regulated gene transcription. Moreover, SC428 also significantly attenuated AR-V7-mediated AR signaling that does not rely on androgen, hampered AR-V7 nuclear localization, and disrupted AR-V7 homodimerization. SC428 inhibited in vitro proliferation and in vivo tumor growth of cells that expressed a high level of AR-V7 and were unresponsive to ENZ treatment. Together, these results indicated the potential therapeutic benefits of AR-NTD targeting for overcoming drug resistance in CRPC.

Abstract 4354: An exosome-based ESR1 monitoring RT-qPCR technology that rapidly and accurately detects circulating tumor acquired resistance variants at ≤0.1% frequency in liquid biopsy samples

Abstract Introduction: Hormone receptor-positive/human epidermal growth factor receptor 2-negative (HR+/HER2-) breast cancer is the most common type of breast cancer. Patients with HR+/HER2- metastatic breast cancer (mBC) often become resistant to aromatase inhibitors commonly used in endocrine therapy (ET). Estrogen receptor (ESR1) ligand binding domain mutations are frequently detected in HR+ mBC and have been reported to be associated with ET resistance, noting up to 60% patients with mBC will develop resistance to treatment via acquired resistance in ESR1. Recent studies have shown that monitoring of ESR1 mutations in plasma may serve as a predictive biomarker of acquired resistance to ET, showcasing a strong need for sensitive nucleic acid-based assays. We describe a comprehensive methodology for targeted clinical RT-qPCR monitoring of ESR1 mutations in plasma that utilizes both exosomal nucleic acids and cfDNA to report multiple mutations, provide a streamlined workflow, and accommodate a range of inputs from clinically relevant samples. Methods: Exosomal nucleic acids (exosomal DNA and RNA) and circulating cell-free DNA (cfDNA) were co-isolated using the ExoLution Plus Isolation Kit (Exosome Diagnostics). RT-qPCR-based target enrichment was performed using modified QuantideX reagents (Asuragen) and mutations were confirmed on the QuantStudio qPCR Platform (ThermoFisher). Bioinformatic analyses were conducted using custom software. Results: We developed novel technologies and tested greater than samples across key ET resistance mutations, utilizing contrived samples as needed due to availability. Preliminary studies demonstrated that ESR1 exosomal RNA + cfDNA ranged from approximately 9,500 copies to 64,000 copies in a set of 2mL female presumed normal plasma samples; these results suggest that even 2mL plasma could provide sufficient exosomal RNA/DNA + cfDNA for mutation detection at variant frequencies less than or equal to 0.01%. The ESR1 RT-qPCR showed greater than 90% analytical sensitivity (at less than or equal to 1% mutant allele frequency) and greater than90% analytical specificity. Further, the technology detected less than 10 mutant copies for most tested mutants, showing an LOD of at least 0.1% (5 mutant copies in a background of 5,000 WT copies). Conclusion: A fast, efficient, and sensitive ESR1 RT-qPCR panel approach was developed and evaluated, demonstrating the reliable and specific detection of rare variants in liquid biopsy specimens. Importantly, this prototype technology has potential to address several challenges associated with mutation monitoring in liquid biopsies by expanding detection of mutant analytes (exosomal RNA and DNA), improving analytical sensitivity (novel reagents and software), and increasing accessibility (qPCR instrument install base). Citation Format: Sarah Statt, Julie Thibert, Kurt Franzen, Aquiles Sanchez, Liangjing Chen, Megan Yociss, Elliot Hallmark, Melissa Church, Stela Filipovic-Sadic, Gary Latham, Johan Skog. An exosome-based ESR1 monitoring RT-qPCR technology that rapidly and accurately detects circulating tumor acquired resistance variants at ≤0.1% frequency in liquid biopsy samples. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4354.

Abstract 5011: Targeting kinome reprogramming in ESR1 fusion-driven breast cancer

Abstract Background: Transcriptionally active ESR1 gene fusions (ESR1-TAF) are a potent cause of estrogen receptor alpha-positive (ERα+) breast cancer endocrine therapy (ET) resistance. These ESR1-TAF are gain-of-function mutations, exhibiting estrogen-independent cell growth, motility and ET resistance. They are not directly druggable because the ERα C-terminal ligand binding domain (LBD) encoding sequence is replaced with a translocated in-frame partner gene sequence. Herein we utilized proteomic approaches to develop novel targeted therapies against ESR1-TAF driven tumorigenesis. Methods: ESR1 fusion cDNA constructs were expressed in ERα+ breast cancer cell lines (T47D and MCF7). Cell growth was assayed by an Alamar blue assay. A mass spectrometry (MS)-based Kinase Inhibitor Pulldown Assay (KIPA) was employed to identify druggable kinases that are commonly upregulated by diverse ESR1-TAFs. A panel of 22 ERα+ patient-derived xenograft (PDX) models were profiled using proteomics and phosphoproteomics to identify models with sensitivity to RET kinase inhibition. Results: KIPA detected an increased abundance of a receptor tyrosine kinase, RET, in T47D cells expressing ESR1-TAFs in an estrogen-independent manner, compared to stable cell lines expressing transcriptionally inactive fusions as well as wild-type ERα protein. Interestingly, RET was also increased when constitutive activating ERα LBD point mutants, Y537S and D538G, were expressed in breast cancer cells. Inhibition of the RET kinase in vitro by repurposing pralsetinib, an FDA-approved RET inhibitor for advanced thyroid and non-small-cell lung cancers, demonstrated a significant reduction in the growth of cells expressing ESR1-TAFs and ERα LBD mutants. These data nominate RET kinase as a common therapeutic vulnerability for ESR1-TAF expressing breast cancers. Proteomic profiling of 22 biologically heterogenous ERα+ PDX tumors defined targetable pathways and predicted tumor subsets that were responsive to RET inhibition therapy. Organoids and xenografts from the pan-ET resistant WHIM18 PDX (that expresses the ESR1-YAP1 TAF) were inhibited by pralsetinib to a similar extent as the CDK4/6 inhibitor palbociclib. These data provide key preclinical rationale for the consideration of RET inhibition for the treatment of ESR1-TAF-driven ET-resistant breast cancer. Interestingly, the growth of WHIM37 PDX (that expresses ERα D538G) that had low level of RET and high level of GFRα-1, the co-receptor of RET, was also suppressed by pralsetinib. This data suggests that either RET or GFRα-1 is a predictive biomarker for RET inhibitor efficacy. Conclusions: Kinome analysis of ESR1 translocated breast tumors using KIPA followed by drug sensitivity studies nominated RET as a new therapeutic target for ET-resistant ERα+ breast cancer. Citation Format: Xuxu Gou, Beom-Jun Kim, Meenakshi Anurag, Jonathan T. Lei, Meggie N. Young, Matthew V. Holt, Diana Fandino, Craig T. Vollert, Purba Singh, Mohammad A. Alzubi, Anna Malovannaya, Lacey E. Dobrolecki, Michael T. Lewis, Shunqiang Li, Matthew J. Ellis, Charles E. Foulds. Targeting kinome reprogramming in ESR1 fusion-driven breast cancer. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5011.

Androgen receptor (AR) mutations in men with metastatic castration-resistant prostate cancer (mCRPC): Incidence and natural history.

221 Background: AR ligand binding domain (LBD) activating somatic point mutation is a known mechanism of resistance to androgen-receptor signaling inhibitor (ARSI) treatment in mCRPC, leading to a persistent addiction to steroid hormones. Two early phase trials investigating CYP11A1 inhibitors - CYPIDES (ODM208, NCT03436485) and STESIDES (ODM209, NCT03878823) - recruited mCRPC patients with and without pre-specified AR LBD mutation. In CYPIDES Part 1, AR LBD mutations (ARm) strongly predicted for PSA decline with ODM-208 (Fizazi, ASCO GU 2021). Here, we evaluated clinical and genomic data associated with ARm in patients with mCRPC. Methods: We reviewed clinical and molecular data from patients with mCRPC who progressed after ≥1 ARSI and ≥1 line of taxane-based chemotherapy or were ineligible to chemotherapy, pre-screened in CYPIDES and STESIDES trials at our center. Circulating tumor DNA (ctDNA) was tested for AR LBD mutation (Guardant360 CDx and Sysmex OncoBEAM) and for genomic analysis (FoundationOne Liquid CDx, STING trial, NCT04932525) to screen for actionable targets. Results: From March 2020 to January 2022, 272 men were screened. Median age was 62y. Overall, 212 (78%), 202 (74%), and 240 (88%) patients had received previous abiraterone, enzalutamide, and docetaxel respectively. Median PSA at screening was 59 ng/mL (IQR 11; 246). ARm was found in 69 patients (25%). The most frequent mutations were L702H (15%), T878A (12%), H875Y (7%), V716M (2%), F877L (2%), W742C (1%), globally consistent with previous reports. Associations of ARm with relevant baseline characteristics of patients are summarized. Duration of enzalutamide exposure was correlated with the number of AR mutations per patient (p = 0.006) whereas no correlation was found for abiraterone exposure duration (p=0.71). In 173 (64%) patients with available genomic testing, no alterations were found to be mutually exclusive with ARm, including in DNA repair alteration. Conclusions: AR LBD mutations were detected in ctDNA in 25% of men with advanced mCRPC treated with at least one ARSI. Their incidence was associated with a longer duration of treatment with enzalutamide and a longer time from CRPC and prostate cancer diagnosis. [Table: see text]

Real-world assessment of AR-LBD mutations in metastatic castration-resistant prostate cancer.

204 Background: Treatment of men with metastatic castration-resistant prostate cancer (mCRPC) using drugs targeting the AR pathway is hampered by development of resistance including activating AR ligand-binding domain (LBD) point mutations. AR LBD mutations are found in approximately 10-20% of patients (pts) with mCRPC progressing on AR pathway inhibition (ARPi) therapy and may be associated with AR activation by alternative steroid hormones. We utilized the Guardant INFORM real-world database to explore the detection rate and clinical features associated with AR-LBD mutations in men with mCRPC. Methods: This observational study was conducted in a nationally representative clinical-genomic database of 200,000+ advanced cancer pts undergoing comprehensive ctDNA analysis (Guardant360, Redwood City, CA) between 2014 and 2021, with associated clinical information from administrative claims. ctDNA samples of men with mCRPC were assessed for presence or absence of an activating AR-LBD mutation. The incidence of co-occurring genetic alterations was also investigated. Clinical outcomes were studied for matched cohorts comparing pts following first line (1L) or second line (2L) mCRPC therapy with or without AR-LBD mutations. Results: Of 15,705 prostate cancer pts included in the database, 8420 (53.6%) had mCRPC at the time of sample collection. 4122 (49.0%) of these mCRPC pts had prior treatment with an ARPi, of which 854 (21%) had an AR-LBD mutation. AR-LBD mutation prevalence was 15% in mCRPC pts untreated with an ARPi, 23% in those after 1 line, and 24% in those after 2 lines of ARPi treatment. AR L702H and T878A/S showed the greatest increase in prevalence post abiraterone, whereas AR L702H, T878S and F877L had the greatest increase in prevalence post enzalutamide. Prevalence of AR L702H and H875Y doubled (from 8.8% to 17.5%) for pts that received chemotherapy post ARPi, while AR F877L incidence declined by half (3.5% to 1.7%). Compared to pts without AR-LBD mutations, AR-LBD+ mCRPC pts were enriched for co-alterations in APC, CTNNB1, PTEN, BRCA2, and ARID1A. Rates of TP53 and RB1 alterations were comparable in the AR-LBD+ and ARLBD- cohorts. Matched mCRPC pts had similar time-to-treatment-discontinuation (TTD) and time-to-next-treatment (TTNT) between AR-LBD+ and AR-LBD- mCRPC pts in the 1L and 2L settings; however, AR-LBD+ pts exhibited worse overall survival from 1L mCRPC therapy (50.1 vs 60.7 months, log-rank P=0.013). Conclusions: In this large database, the overall detection rate of activating AR-LBD mutations in men with mCRPC undergoing ctDNA analysis was 21%, and increased with prior use of 2nd generation ARPi (abiraterone and enzalutamide). Men with AR-LBD mutations were more likely to harbor other alterations relevant to tumor progression. These findings inform on the ctDNA prevalence and impact of AR-LBD mutations in a real-world dataset of mCRPC.