Randomized Phase II Multicenter Trial of Abiraterone Acetate With or Without Cabazitaxel in the Treatment of Metastatic Castration-Resistant Prostate Cancer.

PURPOSE: Improving clinical outcomes with novel drug combinations to treat metastatic castration-resistant prostate cancer (mCRPC) is challenging. Preclinical studies showed cabazitaxel had superior antitumor efficacy compared with docetaxel. Gene expression profiling revealed divergent effects of these taxanes in cycling cells. mCRPC are RB deficient rendering them hypersensitive to taxanes. These data suggested that upfront treatment with cabazitaxel with abiraterone may affect therapeutic response. We designed a phase II randomized noncomparative trial of abiraterone acetate/prednisone (AAP) or AAP and cabazitaxel (AAP + C) in men with mCRPC to address this hypothesis. METHODS: This trial of 81 men with mCRPC determined the radiographic progression-free survival (rPFS), prostate-specific antigen (PSA) progression-free survival, overall objective response, and safety of AAP or AAP + C. Equally allocated patients received AAP followed by switching to cabazitaxel upon radiographic progression (arm 1) or upfront with AAP + C (arm 2). Patients were stratified into high-/low-risk groups by the Halabi nomogram. Real-time assessment of RB status and circulating tumor cell (CTC) analysis to correlate with clinical outcomes was exploratory. RESULTS: Both treatment arms were well-tolerated. Median rPFS in AAP was 6.4 months (95% CI, 3.8 to 10.6) and median overall survival (OS) 18.3 months (95% CI, 14.4 to 37.6), respectively. Fifty-six percent of patients showed ≥50% decline in PSA. Median rPFS in AAP + C was 14.8 months (95% CI, 10.6 to 16.4), and median OS 24.5 months (95% CI, 20.4 to 35.0). There was a ≥50% decline in PSA in 92.1% of men. Neither RB expression in pretherapy tumor biopsy, CTC, or tissue explants identified those who may benefit from AAP + C. CONCLUSION: AAP + C was safe with improved rPFS, OS duration, and a higher proportion of PSA declines. This suggests that AAP + C given earlier rather than sequentially may benefit some men. Further work is needed to identify this population.

Mutant p53 elicits context-dependent pro-tumorigenic phenotypes.

The tumor suppressor gene TP53 is the most frequently mutated gene in numerous cancer types, including prostate cancer (PCa). Specifically, missense mutations in TP53 are selectively enriched in PCa, and cluster to particular "hot spots" in the p53 DNA binding domain with mutation at the R273 residue occurring most frequently. While this residue is similarly mutated to R273C-p53 or R273H-p53 in all cancer types examined, in PCa selective enrichment of R273C-p53 is observed. Importantly, examination of clinical datasets indicated that TP53 heterozygosity can either be maintained or loss of heterozygosity (LOH) occurs. Thus, to mimic tumor-associated mutant p53, R273C-p53 and R273H-p53 isogenic PCa models were developed in the presence or absence of wild-type p53. In the absence of wild-type p53, both R273C-p53 and R273H-p53 exhibited similar loss of DNA binding, transcriptional profiles, and loss of canonical tumor suppressor functions associated with wild-type p53. In the presence of wild-type p53 expression, both R273C-p53 and R273H-p53 supported canonical p53 target gene expression yet elicited distinct cistromic and transcriptional profiles when compared to each other. Moreover, heterozygous modeling of R273C-p53 or R273H-p53 expression resulted in distinct phenotypic outcomes in vitro and in vivo. Thus, mutant p53 acts in a context-dependent manner to elicit pro-tumorigenic transcriptional profiles, providing critical insight into mutant p53-mediated prostate cancer progression.

SLC36A1-mTORC1 signaling drives acquired resistance to CDK4/6 inhibitors.

The cyclin-dependent kinase 4/6 (CDK4/6) kinase is dysregulated in melanoma, highlighting it as a potential therapeutic target. CDK4/6 inhibitors are being evaluated in trials for melanoma and additional cancers. While beneficial, resistance to therapy is a concern, and the molecular mechanisms of such resistance remain undefined. We demonstrate that reactivation of mammalian target of rapamycin 1 (mTORC1) signaling through increased expression of the amino acid transporter, solute carrier family 36 member 1 (SLC36A1), drives resistance to CDK4/6 inhibitors. Increased expression of SLC36A1 reflects two distinct mechanisms: (i) Rb loss, which drives SLC36A1 via reduced suppression of E2f; (ii) fragile X mental retardation syndrome-associated protein 1 overexpression, which promotes SLC36A1 translation and subsequently mTORC1. Last, we demonstrate that a combination of a CDK4/6 inhibitor with an mTORC1 inhibitor has increased therapeutic efficacy in vivo, providing an important avenue for improved therapeutic intervention in aggressive melanoma.

Pleiotropic Impact of DNA-PK in Cancer and Implications for Therapeutic Strategies.

PURPOSE: DNA-dependent protein kinase catalytic subunit (DNA-PK) is a pleiotropic kinase involved in DNA repair and transcriptional regulation. DNA-PK is deregulated in selected cancer types and is strongly associated with poor outcome. The underlying mechanisms by which DNA-PK promotes aggressive tumor phenotypes are not well understood. Here, unbiased molecular investigation in clinically relevant tumor models reveals novel functions of DNA-PK in cancer.Experimental Design: DNA-PK function was modulated using both genetic and pharmacologic methods in a series of in vitro models, in vivo xenografts, and patient-derived explants (PDE), and the impact on the downstream signaling and cellular cancer phenotypes was discerned. Data obtained were used to develop novel strategies for combinatorial targeting of DNA-PK and hormone signaling pathways. RESULTS: Key findings reveal that (i) DNA-PK regulates tumor cell proliferation; (ii) pharmacologic targeting of DNA-PK suppresses tumor growth both in vitro, in vivo, and ex vivo; (iii) DNA-PK transcriptionally regulates the known DNA-PK-mediated functions as well as novel cancer-related pathways that promote tumor growth; (iv) dual targeting of DNA-PK/TOR kinase (TORK) transcriptionally upregulates androgen signaling, which can be mitigated using the androgen receptor (AR) antagonist enzalutamide; (v) cotargeting AR and DNA-PK/TORK leads to the expansion of antitumor effects, uncovering the modulation of novel, highly relevant protumorigenic cancer pathways; and (viii) cotargeting DNA-PK/TORK and AR has cooperative growth inhibitory effects in vitro and in vivo. CONCLUSIONS: These findings uncovered novel DNA-PK transcriptional regulatory functions and led to the development of a combinatorial therapeutic strategy for patients with advanced prostate cancer, currently being tested in the clinical setting.

Tumor cell heterogeneity and resistance; report from the 2018 Coffey-Holden Prostate Cancer Academy Meeting.

INTRODUCTION: The 2018 Coffey-Holden Prostate Cancer Academy (CHPCA) Meeting, "Tumor Cell Heterogeneity and Resistance," was held in Los Angeles, California from June 21 to 24, 2018. METHODS: The CHPCA Meeting is a unique, discussion-oriented scientific conference convened annually by the Prostate Cancer Foundation (PCF), which focuses on the most critical topics in need of further study to advance the treatment of lethal prostate cancer. The 6th Annual CHPCA Meeting was attended by 70 investigators and concentrated on prostate cancer heterogeneity and treatment resistance. RESULTS: The meeting focused on topics including: recognition of tumor heterogeneity, molecular drivers of heterogeneity, the role of the tumor microenvironment, the role of heterogeneity in disease progression, metastasis and treatment resistance, clinical trials designed to target resistance and tumor heterogeneity, and immunotherapeutic approaches to target and overcome tumor heterogeneity. DISCUSSION: This review article summarizes the presentations and discussions from the 2018 CHPCA Meeting in order to share this knowledge with the scientific community and encourage new studies that will lead to improved treatments and outcomes for men with prostate cancer.

PARP-1 regulates DNA repair factor availability.

PARP-1 holds major functions on chromatin, DNA damage repair and transcriptional regulation, both of which are relevant in the context of cancer. Here, unbiased transcriptional profiling revealed the downstream transcriptional profile of PARP-1 enzymatic activity. Further investigation of the PARP-1-regulated transcriptome and secondary strategies for assessing PARP-1 activity in patient tissues revealed that PARP-1 activity was unexpectedly enriched as a function of disease progression and was associated with poor outcome independent of DNA double-strand breaks, suggesting that enhanced PARP-1 activity may promote aggressive phenotypes. Mechanistic investigation revealed that active PARP-1 served to enhance E2F1 transcription factor activity, and specifically promoted E2F1-mediated induction of DNA repair factors involved in homologous recombination (HR). Conversely, PARP-1 inhibition reduced HR factor availability and thus acted to induce or enhance "BRCA-ness". These observations bring new understanding of PARP-1 function in cancer and have significant ramifications on predicting PARP-1 inhibitor function in the clinical setting.

Patient-derived Models Reveal Impact of the Tumor Microenvironment on Therapeutic Response.

BACKGROUND: Androgen deprivation therapy is a first-line treatment for disseminated prostate cancer (PCa). However, virtually all tumors become resistant and recur as castration-resistant PCa, which has no durable cure. One major hurdle in the development of more effective therapies is the lack of preclinical models that adequately recapitulate the heterogeneity of PCa, significantly hindering the ability to accurately predict therapeutic response. OBJECTIVE: To leverage the ex vivo culture method termed patient-derived explant (PDE) to examine the impact of PCa therapeutics on a patient-by-patient basis. DESIGN SETTING AND PARTICIPANTS: Fresh PCa tissue from patients who underwent radical prostatectomy was cultured as PDEs to examine therapeutic response. OUTCOME MEASUREMENTS AND STATISTICAL ANALYSIS: The impact of genomic and chemical perturbations in PDEs was assessed using various parameters (eg, AR levels, Ki67 staining, and desmoplastic indices). RESULTS AND LIMITATIONS: PDE maintained the integrity of the native tumor microenvironment (TME), tumor tissue morphology, viability, and endogenous hormone signaling. Tumor cells in this model system exhibited de novo proliferative capacity. Examination of the native TME in the PDE revealed a first-in-field insight into patient-specific desmoplastic stromal indices and predicted responsiveness to AR-directed therapeutics. CONCLUSIONS: The PDE model allows for a comprehensive evaluation of individual tumors in their native TME to ultimately develop more effective therapeutic regimens tailored to individuals. Discernment of novel stromal markers may provide a basis for applying precision medicine in treating advanced PCa, which would have a transformative effect on patient outcomes. PATIENT SUMMARY: In this study, an innovative model system was used to more effectively mimic human disease. The patient-derived explant (PDE) system can be used to predict therapeutic response and identify novel targets in advanced disease. Thus, the PDE will be an asset for the development of novel metrics for the implementation of precision medicine in prostate cancer.The patient-derived explant (PDE) model allows for a comprehensive evaluation of individual human tumors in their native tumor microenvironment (TME). TME analysis revealed first-in-field insight into predicted tumor responsiveness to AR-directed therapeutics through evaluation of patient-specific desmoplastic stromal indices.

MAPK Reliance via Acquired CDK4/6 Inhibitor Resistance in Cancer.

Purpose: Loss of cell-cycle control is a hallmark of cancer, which can be targeted with agents, including cyclin-dependent kinase-4/6 (CDK4/6) kinase inhibitors that impinge upon the G1-S cell-cycle checkpoint via maintaining activity of the retinoblastoma tumor suppressor (RB). This class of drugs is under clinical investigation for various solid tumor types and has recently been FDA-approved for treatment of breast cancer. However, development of therapeutic resistance is not uncommon.Experimental Design: In this study, palbociclib (a CDK4/6 inhibitor) resistance was established in models of early stage, RB-positive cancer.Results: This study demonstrates that acquired palbociclib resistance renders cancer cells broadly resistant to CDK4/6 inhibitors. Acquired resistance was associated with aggressive in vitro and in vivo phenotypes, including proliferation, migration, and invasion. Integration of RNA sequencing analysis and phosphoproteomics profiling revealed rewiring of the kinome, with a strong enrichment for enhanced MAPK signaling across all resistance models, which resulted in aggressive in vitro and in vivo phenotypes and prometastatic signaling. However, CDK4/6 inhibitor-resistant models were sensitized to MEK inhibitors, revealing reliance on active MAPK signaling to promote tumor cell growth and invasion.Conclusions: In sum, these studies identify MAPK reliance in acquired CDK4/6 inhibitor resistance that promotes aggressive disease, while nominating MEK inhibition as putative novel therapeutic strategy to treat or prevent CDK4/6 inhibitor resistance in cancer. Clin Cancer Res; 24(17); 4201-14. ©2018 AACR.

Novel actions of next-generation taxanes benefit advanced stages of prostate cancer.

PURPOSE: To improve the outcomes of patients with castration-resistant prostate cancer (CRPC), there is an urgent need for more effective therapies and approaches that individualize specific treatments for patients with CRPC. These studies compared the novel taxane cabazitaxel with the previous generation docetaxel, and aimed to determine which tumors are most likely to respond. EXPERIMENTAL DESIGN: Cabazitaxel and docetaxel were compared via in vitro modeling to determine the molecular mechanism, biochemical and cell biologic impact, and cell proliferation, which was further assessed ex vivo in human tumor explants. Isogenic pairs of RB knockdown and control cells were interrogated in vitro and in xenograft tumors for cabazitaxel response. RESULTS: The data herein show that (i) cabazitaxel exerts stronger cytostatic and cytotoxic response compared with docetaxel, especially in CRPC; (ii) cabazitaxel induces aberrant mitosis, leading to pyknotic and multinucleated cells; (iii) taxanes do not act through the androgen receptor (AR); (iv) gene-expression profiling reveals distinct molecular actions for cabazitaxel; and (v) tumors that have progressed to castration resistance via loss of RB show enhanced sensitivity to cabazitaxel. CONCLUSIONS: Cabazitaxel not only induces improved cytostatic and cytotoxic effects, but also affects distinct molecular pathways, compared with docetaxel, which could underlie its efficacy after docetaxel treatment has failed in patients with CRPC. Finally, RB is identified as the first potential biomarker that could define the therapeutic response to taxanes in metastatic CRPC. This would suggest that loss of RB function induces sensitization to taxanes, which could benefit up to 50% of CRPC cases.

A hormone-DNA repair circuit governs the response to genotoxic insult.

UNLABELLED: Alterations in DNA repair promote tumor development, but the impact on tumor progression is poorly understood. Here, discovery of a biochemical circuit linking hormone signaling to DNA repair and therapeutic resistance is reported. Findings show that androgen receptor (AR) activity is induced by DNA damage and promotes expression and activation of a gene expression program governing DNA repair. Subsequent investigation revealed that activated AR promotes resolution of double-strand breaks and resistance to DNA damage both in vitro and in vivo. Mechanistically, DNA-dependent protein kinase catalytic subunit (DNAPKcs) was identified as a key target of AR after damage, controlling AR-mediated DNA repair and cell survival after genotoxic insult. Finally, DNAPKcs was shown to potentiate AR function, consistent with a dual role in both DNA repair and transcriptional regulation. Combined, these studies identify the AR-DNAPKcs circuit as a major effector of DNA repair and therapeutic resistance and establish a new node for therapeutic intervention in advanced disease. SIGNIFICANCE: The present study identifies for the fi rst time a positive feedback circuit linking hormone action to the DNA damage response and shows the significant impact of this process on tumor progression and therapeutic response. These provocative findings provide the foundation for development of novel nodes of therapeutic intervention for advanced disease.

Serine-305 phosphorylation modulates estrogen receptor alpha binding to a coregulator peptide array, with potential application in predicting responses to tamoxifen.

With current techniques, it remains a challenge to assess coregulator binding of nuclear receptors, for example, the estrogen receptor alpha (ERα). ERα is critical in many breast tumors and is inhibited by antiestrogens such as tamoxifen in cancer therapy. ERα is also modified by acetylation and phosphorylation that affect responses to the antiestrogens as well as interactions with coregulators. Phosphorylation of ERα at Ser305 is one of the mechanisms causing tamoxifen resistance. Detection of resistance in patient samples would greatly facilitate clinical decisions on treatment, in which such patients would receive other treatments such as aromatase inhibitors or fulvestrant. Here we describe a coregulator peptide array that can be used for high-throughput analysis of full-length estrogen receptor binding. The peptide chip can detect ERα binding in cell and tumor lysates. We show that ERα phosphorylated at Ser305 associates stronger to various coregulator peptides on the chip. This implies that ERαSer305 phosphorylation increases estrogen receptor function. As this is also detected in a breast tumor sample of a tamoxifen-insensitive patient, the peptide array, as described here, may be applicable to detect tamoxifen resistance in breast tumor samples at an early stage of disease and contribute to personalized medicine.

A role for estrogen receptor phosphorylation in the resistance to tamoxifen.

About two thirds of all human breast cancer cases are estrogen receptor positive. The drug of first choice for these patients is tamoxifen. However, about half of the recurrences after removal of the primary tumor are or become resistant to this drug. While many mechanisms have been identified for tamoxifen resistance in the lab, at present only a few have been translated to the clinic. This paper highlights the role in tamoxifen resistance of phosphorylation by different kinases on different sites of the estrogen receptor. We will discuss the molecular pathways and kinases that are involved in phosphorylation of ERα and how these affect tamoxifen resistance. Finally, we will elaborate on the clinical translation of these observations and the possibility to predict tamoxifen responses in patient tumor samples before treatment onset. The findings made originally on the bench may translate into a better and personalized treatment of breast cancer patients using an old and safe anticancer drug: tamoxifen.

The hinge region of the human estrogen receptor determines functional synergy between AF-1 and AF-2 in the quantitative response to estradiol and tamoxifen.

Human estrogen receptors alpha and beta (ERalpha and ERbeta) greatly differ in their target genes, transcriptional potency and cofactor-binding capacity, and are differentially expressed in various tissues. In classical estrogen response element (ERE)-mediated transactivation, ERbeta has a markedly reduced activation potential compared with ERalpha; the mechanism underlying this difference is unclear. Here, we report that the binding of steroid receptor coactivator-1 (SRC-1) to the AF-1 domain of ERalpha is essential but not sufficient to facilitate synergy between the AF-1 and AF-2 domains, which is required for a full agonistic response to estradiol (E2). Complete synergy is achieved through the distinct hinge domain of ERalpha, which enables combined action of the AF-1 and AF-2 domains. AF-1 of ERbeta lacks the capacity to interact with SRC-1, which prevents hinge-mediated synergy between AF-1 and AF-2, thereby explaining the reduced E2-mediated transactivation of ERbeta. Transactivation of ERbeta by E2 requires only the AF-2 domain. A weak agonistic response to tamoxifen occurs for ERalpha, but not for ERbeta, and depends on AF-1 and the hinge-region domain of ERalpha.