Samuel R. Denmeade

AR-V7 and Resistance to Enzalutamide and Abiraterone in Prostate Cancer

BACKGROUND The androgen-receptor isoform encoded by splice variant 7 lacks the ligand-binding domain, which is the target of enzalutamide and abiraterone, but remains constitutively active as a transcription factor. We hypothesized that detection of androgen-receptor splice variant 7 messenger RNA (AR-V7) in circulating tumor cells from men with advanced prostate cancer would be associated with resistance to enzalutamide and abiraterone. METHODS We used a quantitative reverse-transcriptase-polymerase-chain-reaction assay to evaluate AR-V7 in circulating tumor cells from prospectively enrolled patients with metastatic castration-resistant prostate cancer who were initiating treatment with either enzalutamide or abiraterone. We examined associations between AR-V7 status (positive vs. negative) and prostate-specific antigen (PSA) response rates (the primary end point), freedom from PSA progression (PSA progression-free survival), clinical or radiographic progression-free survival, and overall survival. RESULTS A total of 31 enzalutamide-treated patients and 31 abiraterone-treated patients were enrolled, of whom 39% and 19%, respectively, had detectable AR-V7 in circulating tumor cells. Among men receiving enzalutamide, AR-V7-positive patients had lower PSA response rates than AR-V7-negative patients (0% vs. 53%, P=0.004) and shorter PSA progression-free survival (median, 1.4 months vs. 6.0 months; P<0.001), clinical or radiographic progression-free survival (median, 2.1 months vs. 6.1 months; P<0.001), and overall survival (median, 5.5 months vs. not reached; P=0.002). Similarly, among men receiving abiraterone, AR-V7-positive patients had lower PSA response rates than AR-V7-negative patients (0% vs. 68%, P=0.004) and shorter PSA progression-free survival (median, 1.3 months vs. not reached; P<0.001), clinical or radiographic progression-free survival (median, 2.3 months vs. not reached; P<0.001), and overall survival (median, 10.6 months vs. not reached, P=0.006). The association between AR-V7 detection and therapeutic resistance was maintained after adjustment for expression of full-length androgen receptor messenger RNA. CONCLUSIONS Detection of AR-V7 in circulating tumor cells from patients with castration-resistant prostate cancer may be associated with resistance to enzalutamide and abiraterone. These findings require large-scale prospective validation. (Funded by the Prostate Cancer Foundation and others.).

Role of programmed (apoptotic) cell death during the progression and therapy for prostate cancer

Cells possess within their epigenetic repertoire the ability to undergo an active process of cellular suicide termed programmed (or apoptotic) cell death. This programmed cell death process involves an epigenetic reprogramming of the cell that results in an energy‐dependent cascade of biochemical and morphologic changes (also termed apoptosis) within the cell, resulting in its death and elimination. Although the final steps (i.e., DNA and cellular fragmentation) are common to cells undergoing programmed cell death, the activation of this death process is initiated either by sufficient injury to the cell induced by various exogenous damaging agents (e.g., radiation, chemicals, viruses) or by changes in the levels of a series of endogenous signals (e.g., hormones and growth/survival factors). Within the prostate, androgens are capable of both stimulating proliferation as well as inhibiting the rate of the glandular epithelial cell death. Androgen withdrawal triggers the programmed cell death pathway in both normal prostate glandular epithelia and androgen‐dependent prostate cancer cells. Androgen‐independent prostate cancer cells do not initiate the programmed cell death pathway upon androgen ablation; however, they do retain the cellular machinery necessary to activate the programmed cell death cascade when sufficiently damaged by exogenous agents. In the normal prostate epithelium, cell proliferation is balanced by an equal rate of programmed cell death, such that neither involution nor overgrowth normal occurs. In prostatic cancer, however, this balance is lost, such that there is greater proliferation than death producing continuous net growth. Thus, an imbalance in programmed cell death must occur during prostatic cancer progression. The goal of effective therapy for prostatic cancer, therefore, is to correct this imbalance. Unfortunately, this has not been achieved and metastatic prostatic cancer is still a lethal disease for which no curative therapy is currently available. In order to develop such effective therapy, an understanding of the programmed death pathway, and what controls it, is critical. Thus, a review of the present state of knowledge concerning programmed cell death of normal and malignant prostatic cells will be presented.

A history of prostate cancer treatment

The increased incidence of prostate cancer has led to remarkable changes in diagnosis and treatment over the past century. What were the first ways in which prostate cancer was treated, and how did these evolve into the variety of therapeutic strategies from which patients have to choose today?

Phase II Multicenter Study of Abiraterone Acetate Plus Prednisone Therapy in Patients With Docetaxel-Treated Castration-Resistant Prostate Cancer

PURPOSE Persistence of ligand-mediated androgen receptor signaling has been documented in castration-resistant prostate cancers (CRPCs). Abiraterone acetate (AA) is a potent and selective inhibitor of CYP17, which is required for androgen biosynthesis in the testes, adrenal glands, and prostate tissue. This trial evaluated the efficacy and safety of AA in combination with prednisone to reduce the symptoms of secondary hyperaldosteronism that can occur with AA monotherapy. PATIENTS AND METHODS Fifty-eight men with progressive metastatic CRPC who experienced treatment failure with docetaxel-based chemotherapy received AA (1,000 mg daily) with prednisone (5 mg twice daily). Twenty-seven (47%) patients had received prior ketoconazole. The primary outcome was > or = 50% prostate-specific antigen (PSA) decline, with objective response by Response Evaluation Criteria in Solid Tumors (RECIST) criteria, and changes in Eastern Cooperative Oncology Group (ECOG) performance status (PS) and circulating tumor cell (CTC) numbers. Safety was also evaluated. RESULTS A > or = 50% decline in PSA was confirmed in 22 (36%) patients, including 14 (45%) of 31 ketoconazole-naïve and seven (26%) of 27 ketoconazole-pretreated patients. Partial responses were seen in four (18%) of 22 patients with RECIST-evaluable target lesions. Improved ECOG PS was seen in 28% of patients. Median time to PSA progression was 169 days (95% CI, 82 to 200 days). CTC conversions with treatment from > or = 5 to < 5 were noted in 10 (34%) of 29 patients. The majority of AA-related adverse events were grade 1 to 2, and no AA-related grade 4 events were seen. CONCLUSION AA plus prednisone was well tolerated, with encouraging antitumor activity in heavily pretreated CRPC patients. The incidence of mineralocorticoid-related toxicities (hypertension or hypokalemia) was reduced by adding low-dose prednisone. The combination of AA plus prednisone is recommended for phase III investigations.

Androgen Receptor Splice Variant 7 and Efficacy of Taxane Chemotherapy in Patients With Metastatic Castration-Resistant Prostate Cancer

IMPORTANCE We previously showed that detection of androgen receptor splice variant 7 (AR-V7) in circulating tumor cells (CTCs) from men with castration-resistant prostate cancer (CRPC) was associated with primary resistance to enzalutamide and abiraterone therapy, but the relevance of AR-V7 status in the context of chemotherapy is unknown. OBJECTIVE To investigate whether AR-V7-positive patients would retain sensitivity to taxane chemotherapy and whether AR-V7 status would have a differential impact on taxane-treated men compared with enzalutamide- or abiraterone-treated men. DESIGN, SETTING, AND PARTICIPANTS We examined CTCs for AR-V7 mRNA using a reverse-transcription polymerase chain reaction assay. From January 2013 to July 2014, we prospectively enrolled patients with metastatic CRPC initiating taxane chemotherapy (docetaxel or cabazitaxel) at a single academic institution (Johns Hopkins). Our prespecified statistical plan required a sample size of 36 taxane-treated men. MAIN OUTCOMES AND MEASURES We evaluated associations between AR-V7 status and prostate-specific antigen (PSA) response rates, PSA progression-free survival (PSA PFS), and clinical and/or radiographic progression-free survival (PFS). After incorporating updated data from our prior study of 62 patients treated with enzalutamide or abiraterone, we also investigated the interaction between AR-V7 status (positive or negative) and treatment type (taxane vs enzalutamide or abiraterone). RESULTS Of 37 taxane-treated patients enrolled, 17 (46%) had detectable AR-V7 in CTCs. Prostate-specific antigen responses were achieved in both AR-V7-positive and AR-V7-negative men (41% vs 65%; P = .19). Similarly, PSA PFS (hazard ratio [HR], 1.7, 95% CI, 0.6-5.0; P = .32) and PFS (HR, 2.7, 95% CI, 0.8-8.8; P = .11) were comparable in AR-V7-positive and AR-V7-negative patients. A significant interaction was observed between AR-V7 status and treatment type (P < .001). Clinical outcomes were superior with taxanes compared with enzalutamide or abiraterone therapy in AR-V7-positive men, whereas outcomes did not differ by treatment type in AR-V7-negative men. In AR-V7-positive patients, PSA responses were higher in taxane-treated vs enzalutamide- or abiraterone-treated men (41% vs 0%; P < .001), and PSA PFS and PFS were significantly longer in taxane-treated men (HR, 0.19 [95% CI, 0.07-0.52] for PSA PFS, P = .001; HR, 0.21 [95% CI, 0.07-0.59] for PFS, P = .003). CONCLUSIONS AND RELEVANCE Detection of AR-V7 in CTCs from men with metastatic CRPC is not associated with primary resistance to taxane chemotherapy. In AR-V7-positive men, taxanes appear to be more efficacious than enzalutamide or abiraterone therapy, whereas in AR-V7-negative men, taxanes and enzalutamide or abiraterone may have comparable efficacy. Circulating tumor cell-based AR-V7 detection may serve as a treatment selection biomarker in CRPC.

The SERCA pump as a therapeutic target: making a "smart bomb" for prostate cancer.

Prostate cancer is uniformly fatal once it has spread outside of the prostate gland. Prostate cancers have a remarkably low proliferative rate, which may in part explain their relative unresponsiveness to conventional antiproliferative chemotherapy. New therapies for prostate cancer that activate proliferation independent cell death are therefore needed. The endoplasmic reticulum (ER) has emerged as an organelle that plays a major role in cell signaling pathways, cellular response to stress and cellular activation of apoptosis. In this review, the SERCA pump is identified as an ER protein whose normal function is required by all cells and represents a potential therapeutic target for cancer therapy. Sustained SERCA inhibition by agents such as thapsigargin results in activation of ER-stress response and simultaneous activation of apoptotic pathways within the ER and the mitochondria. Due to the SERCA pumps critical role in normal cellular metabolism, agents like thapsigargin directed toward inhibiting SERCA function would likely produce significant toxicity to normal cells and, therefore, must be selectively targeted to cancer sites. The cytotoxicity of thapsigargin can be attenuated, however by coupling to a targeting peptide to produce an inactive prodrug that is only activated by prostate cancer specific proteases such as the serine protease prostate-specific antigen (PSA). PSA-activated thapsigargin prodrugs have been characterized that are selectively toxic to PSA-producing prostate cancer cells in vitro and in vivo. These prodrugs are currently undergoing preclinical evaluation as potential targeted therapy for prostate cancer.

Engineering a prostate-specific membrane antigen-activated tumor endothelial cell prodrug for cancer therapy

A prostate-specific membrane antigen–activated prodrug selectively kills cancer cells and is being tested in patients with advanced cancer. An Old Approach Is New Again In the 1995 film The Last Supper, a group of graduate students invite a diverse cast of characters for a series of Sunday dinners. After one guest threatens the lives of several of the students, subsequent dinners turn deadly. If the guest holds views that the group considers toxic to society, then the house wine is made poisonous and served only to the unwanted houseguest, who promptly dies. In a related scenario, Denmeade et al. use a prodrug to seek out and selectively poison unsavory guests that are toxic to the body—namely, cancer cells. The new work describes the development of a thapsigargin (TG) prodrug that is activated in the vasculature of solid tumors by tumor endothelial cells. The carboxypeptidase prostate-specific membrane antigen (PSMA)—which is selectively expressed on the surface of prostate cancer cells, including metastatic ones, and tumor, but not normal, endothelial cells—cleaves and activates the prodrug extracellularly in the tumor microenvironment. The activated cytotoxic moiety then poisons neighboring cancer cells within sites of metastases by entering the cells and inhibiting the sarcoplasmic/endoplasmic reticulum calcium ATPase (SERCA) pump, which is essential to the function of all normal and tumor cell types. The authors showed that treatment with the prodrug caused significant tumor regression in two mouse xenograft models of human prostate cancer and one model of human breast cancer with relatively little toxicity—less than that of the maximally tolerated dose of the widely used cancer drug docetaxel. Although the targeted prodrug concept is not new, the current approach has several features that make it superior to many previous ones. First, unlike most cytotoxic cancer drugs, TG is not cell cycle–dependent and thus can kill nondividing cancer cells. Furthermore, drug toxicity is expected to be low, because the PSMA substrate in the prodrug is cleaved primarily by prostate cancer cells and in the vicinity of tumor endothelial cells. In fact, the authors report that studies in cynomolgus monkeys showed minimal toxic effects except in the kidney, and even that renal toxicity was minimal to mild and reversible at the low drug dose. As with all cancer drugs, the new findings will require clinical validation in ongoing studies. However, this unusual therapeutic approach has the potential to be an effective and selective ouster of unwanted invaders that threaten their hosts. Heterogeneous expression of drug target proteins within tumor sites is a major mechanism of resistance to anticancer therapies. We describe a strategy to selectively inhibit, within tumor sites, the function of a critical intracellular protein, the sarcoplasmic/endoplasmic reticulum calcium adenosine triphosphatase (SERCA) pump, whose proper function is required by all cell types for viability. To achieve targeted inhibition, we took advantage of the unique expression of the carboxypeptidase prostate-specific membrane antigen (PSMA) by tumor endothelial cells within the microenvironment of solid tumors. We generated a prodrug, G202, consisting of a PSMA-specific peptide coupled to an analog of the potent SERCA pump inhibitor thapsigargin. G202 produced substantial tumor regression against a panel of human cancer xenografts in vivo at doses that were minimally toxic to the host. On the basis of these data, a phase 1 dose-escalation clinical trial has been initiated with G202 in patients with advanced cancer.

COMPLETE ANDROGEN BLOCKADE FOR PROSTATE CANCER: WHAT WENT WRONG?

PURPOSE We summarized and critically assessed all available data from phase III clinical trials on complete androgen blockade versus surgical or medical castration alone. MATERIALS AND METHODS Published results in journals and abstracts of phase III trials, and published meta-analyses were reviewed. We also reviewed quality of life and toxicity issues associated with the addition of antiandrogens to medical or surgical castration. Finally, we discuss the original rationale for complete androgen blockade in the context of current knowledge. RESULTS A total of 27 clinical trials using various combinations of androgen deprivation were identified, of which 3 showed a statistically significant benefit for the complete androgen blockade arm. There were 5 publications of meta-analyses that each used different selection criteria for the inclusion of studies in the final analysis. Toxicity and quality of life have not been widely investigated in prospective fashion but the available data suggest a higher toxicity rate and decreased quality of life with complete androgen blockade. CONCLUSIONS The extensive body of data does not support routine use of antiandrogens in combination with medical or surgical castration as first line hormonal therapy in patients with metastatic prostate cancer.

Targeting the cancer stroma with a fibroblast activation protein-activated promelittin protoxin.

Fibroblast-Activation Protein-α (FAP) is a membrane-bound serine protease that is expressed on the surface of reactive stromal fibroblasts present within the majority of human epithelial tumors but is not expressed by normal tissues. FAP is a postprolyl peptidase that differs from other dipeptidyl prolyl peptidases such as diprolylpeptidase 4 in that it also has gelatinase and collagenase endopeptidase activity. Therefore, FAP represents a potential pan-tumor target whose enzymatic activity can be exploited for the intratumoral activation of prodrugs and protoxins. To evaluate FAP as a tumor-specific target, putative FAP-selective peptide protoxins were constructed through modification of the prodomain of melittin, a 26 amino acid amphipathic cytolytic peptide that is the main toxic component in the venom of the common European honeybee Apis milefera. Melittin is synthesized as promelittin, containing a 22 amino acid NH2-terminal prodomain rich in the amino acids proline and alanine. In this study, peptides containing truncated melittin prodomain sequences were tested on erythrocytes to determine the optimal prodomain length for inhibiting cytolytic activity. Once optimized, modified promelittin peptides were generated in which previously identified FAP substrate sequences were introduced into the prodomain. Peptide protoxins were identified that were efficiently activated by FAP and selectively toxic to FAP-expressing cell lines with an IC50 value in the low micromolar range that is similar to melittin. Intratumoral injection of an FAP-activated protoxin produced significant lysis and growth inhibition of human breast and prostate cancer xenografts with minimal toxicity to the host animal. [Mol Cancer Ther 2009;8(5):1378–86]

Knockin of mutant PIK3CA activates multiple oncogenic pathways

The phosphatidylinositol 3-kinase subunit PIK3CA is frequently mutated in human cancers. Here we used gene targeting to “knock in” PIK3CA mutations into human breast epithelial cells to identify new therapeutic targets associated with oncogenic PIK3CA. Mutant PIK3CA knockin cells were capable of epidermal growth factor and mTOR-independent cell proliferation that was associated with AKT, ERK, and GSK3β phosphorylation. Paradoxically, the GSK3β inhibitors lithium chloride and SB216763 selectively decreased the proliferation of human breast and colorectal cancer cell lines with oncogenic PIK3CA mutations and led to a decrease in the GSK3β target gene CYCLIN D1. Oral treatment with lithium preferentially inhibited the growth of nude mouse xenografts of HCT-116 colon cancer cells with mutant PIK3CA compared with isogenic HCT-116 knockout cells containing only wild-type PIK3CA. Our findings suggest GSK3β is an important effector of mutant PIK3CA, and that lithium, an FDA-approved therapy for bipolar disorders, has selective antineoplastic properties against cancers that harbor these mutations.