PARP inhibitors in prostate cancer: practical guidance for busy clinicians.

The management of prostate cancer entered a new era of biomarker-driven therapy in May of 2020, when the US Food and Drug Administration (FDA) approved the poly (ADP-ribose) polymerase (PARP) inhibitors rucaparib and olaparib as the first targeted therapies in biomarker-preselected patients with metastatic castration-resistant prostate cancer. This approval provided new options for patients with deleterious BRCA1 or BRCA2 mutations (olaparib and rucaparib), or with deleterious mutations in one of a number of homologous recombination repair genes (olaparib). Compared with either enzalutamide or abiraterone, olaparib demonstrated an overall survival benefit in men with metastatic castration-resistant prostate cancer who had disease progression while receiving enzalutamide and/or abiraterone. Additional PARP inhibitors are currently being evaluated as monotherapy. The data are strongest for alterations in BRCA2; alterations in other genes are associated with less benefit or occur less frequently. To date, tissue DNA remains the gold standard for identifying predictive mutations, but sequencing from tissue DNA fails to provide a result in approximately 30 percent of cases. Biopsies of metastatic sites are more likely to yield results and more likely to identify predictive alterations. Plasma-based sequencing platforms are also approved by the FDA, and they appear to provide a result in most patients with late-stage disease. The best way and time to evaluate for the presence of selection biomarkers are not firmly established, but patients whose disease has progressed on androgen deprivation therapy should be evaluated. PARP inhibitors are also being studied in combination with other therapies, such as AR-targeted therapies, immunotherapies, and radiation, among others, in unselected patients.

Circulating tumor cells capture disease evolution in advanced prostate cancer.

BACKGROUND: Genetic analysis of advanced cancer is limited by availability of representative tissue. Biopsies of prostate cancer metastasized to bone are invasive with low quantity of tumor tissue. The prostate cancer genome is dynamic, however, with temporal heterogeneity requiring repeated evaluation as the disease evolves. Circulating tumor cells (CTCs) offer an alternative, "liquid biopsy", though single CTC sequencing efforts are laborious with high failure rates. METHODS: We performed exome sequencing of matched treatment-naïve tumor tissue, castrate resistant tumor tissue, and pooled CTC samples, and compared mutations identified in each. RESULTS: Thirty-seven percent of CTC mutations were private to CTCs, one mutation was shared with treatment-naïve disease alone, and 62% of mutations were shared with castrate-resistant disease, either alone or with treatment-naïve disease. An acquired nonsense mutation in the Retinoblastoma gene, which is associated with progression to small cell cancer, was identified in castrate resistant and CTC samples, but not treatment-naïve disease. This timecourse correlated with the tumor acquiring neuroendocrine features and a change to neuroendocrine-specific therapy. CONCLUSIONS: These data support the use of pooled CTCs to facilitate the genetic analysis of late stage prostate cancer.

Low-grade prostate cancer diverges early from high grade and metastatic disease.

Understanding the developmental relationship between indolent and aggressive tumors is central to understanding disease progression and making treatment decisions. For example, most men diagnosed with prostate cancer have clinically indolent disease and die from other causes. Overtreatment of prostate cancer remains a concern. Here we use laser microdissection followed by exome sequencing of low- and high-grade prostate cancer foci from four subjects, and metastatic disease from two of those subjects, to evaluate the molecular relationship of coincident cancer foci. Seventy of 79 (87%) high-confidence somatic mutations in low-grade disease were private to low-grade foci. In contrast, high-grade foci and metastases harbored many of the same mutations. In cases in which there was a metastatic focus, 15 of 80 (19%) high-confidence somatic mutations in high-grade foci were private. Seven of the 80 (9%) were shared with low-grade foci and 65 (82%) were shared with metastatic foci. Notably, mutations in cancer-associated genes and the p53 signaling pathway were found exclusively in high-grade foci and metastases. The pattern of mutations is consistent with early divergence between low- and high-grade foci and late divergence between high-grade foci and metastases. These data provide insights into the development of high-grade and metastatic prostate cancer.

Quantitative characterization of androgen receptor protein expression and cellular localization in circulating tumor cells from patients with metastatic castration-resistant prostate cancer.

BACKGROUND: Many current therapies for metastatic castration-resistant prostate cancer (mCRPC) are aimed at AR signaling; however, resistance to these therapies is inevitable. To personalize CRPC therapy in an individual with clinical progression despite maximal AR signaling blockade, it is important to characterize the status of AR activity within their cancer. Biopsies of bone metastases are invasive and frequently fail to yield sufficient tissue for further study. Evaluation of circulating tumor cells (CTCs) offers an alternative, minimally invasive mechanism to characterize and study late-stage disease. The goal of this study was to evaluate the utility of CTC interrogation with respect to the AR as a potential novel therapeutic biomarker in patients with mCRPC. METHODS: Fifteen mL of whole blood was collected from patients with progressive, metastatic mCRPC, the mononuclear cell portion was isolated, and fluorescence-activated cell sorting (FACS) was used to isolate and evaluate CTCs. A novel protocol was optimized to use ImageStreamX to quantitatively analyze AR expression and subcellular localization within CTCs. Co-expression of AR and the proliferation marker Ki67 was also determined using ImageStreamX. RESULTS: We found inter-patient and intra-patient heterogeneity in expression and localization of AR. Increased AR expression and nuclear localization are associated with elevated co-expression of Ki-67, consistent with the continued role for AR in castration-resistant disease. Despite intra-patient heterogeneity, CTCs from patients with prior exposure to abiraterone had increased AR expression compared to CTCs from patients who were abiraterone-naïve. CONCLUSIONS: As our toolbox for targeting AR function expands, our ability to evaluate AR expression and function within tumor samples from patients with late-stage disease will likely be a critical component of the personalized management of advanced prostate cancer. AR expression and nuclear localization varies within patients and between patients; however it remains associated with markers of proliferation. This supports a molecularly diverse AR-centric pathobiology imparting castration-resistance.

Should We "PROpel" Olaparib Forward for Metastatic Castration-Resistant Prostate Cancer?

Poly(adenosine diphosphate-ribose) polymerase (PARP) inhibitors have shown therapeutic success for patients with metastatic castration-resistant prostate cancer (mCRPC) with homologous recombination repair (HRR) deficiency.1,2 Preclinical data suggest that PARP inhibitors may have efficacy in a wider population if combined with androgen receptor inhibition.3,4 One phase 2 trial for late-stage mCRPC supports this notion, finding that olaparib added to abiraterone/prednisone improved radiographic progression-free survival (PFS) versus abiraterone/prednisone alone in a population that was not biomarker preselected.5 However, another trial with abiraterone and veliparib did not show benefit.6.

Antibody-drug conjugates and predictive biomarkers in advanced urothelial carcinoma.

The use of antibody-drug conjugates (ADCs) is expanding in several malignancies, including urothelial carcinoma where two of these medications have been approved for use and several others remain under study. ADCs act by binding to specific cell surface proteins, delivering anticancer agents directly to the target cells. Preclinical studies suggest that loss of these surface proteins alters sensitivity to therapy and expression of target proteins vary significantly based on the tumor subtype, prior therapies and other characteristics. However, use of biomarkers to predict treatment response have not been regularly included in clinical trials and clinician practice. In this review we summarize what is known about potential predictive biomarkers for ADCs in UC and discuss potential areas where use of biomarkers may improve patient care.

A Phase II Study of sEphB4-HSA in Metastatic Castration-Resistant Prostate Cancer.

INTRODUCTION: Ephrin receptors and their membrane-localized ligands induce bidirectional signaling and facilitate tumor-stroma interactions. Blocking the EphB4-EphrinB2 pathway, which can be accomplished by soluble EphB4 conjugated to human serum albumin (sEphB4-HSA), promotes cell death in preclinical models of aggressive prostate cancer. We hypothesized that targeting the EphB4-EphrinB2 pathway may serve as a therapeutic target in the treatment of metastatic castration resistant prostate cancer (mCRPC). PATIENTS AND METHODS: We conducted a single arm, phase II trial in patients with progressive mCRPC who had received no more than 3 prior therapies for mCRPC. sEphB4-HSA 1000 mg IV was administered every 2 weeks, extending to 3 weeks starting from cycle 7. The primary endpoint was confirmed prostate specific antigen (PSA) response rate. We employed a Simon 2-stage Minimax design with 15 patients in the first stage and 10 additional patients in the second stage. RESULTS: Fourteen eligible patients enrolled in the study with median age of 73.5 years (range: 52-83) and median baseline PSA of 65.11 ng/mL (range: 7.77-2850 ng/mL). Most patients received 3 prior therapies for mCRPC. The median treatment duration with sEphB4-HSA was 6.5 weeks (range: 2-35 weeks). Three patients experienced a serious adverse event potentially related to therapy, including 1 patient with a grade 5 event (cerebral vascular accident) possibly related to the study drug. No patient had a confirmed PSA response, and the study was stopped for futility. Thirteen patients had PSA progression. The median time to PSA progression was 28 days (90% CI: 28-42 days), and median time to radiologic progression was 55 days (90% CI: 54-72 days). Of 3 patients with measurable disease, 2 had stable disease and one had progressive disease. CONCLUSION: In patients with mCRPC who progressed on prior second generation AR-targeted therapy, sEphB4-HSA monotherapy had no discernable anti-tumor activity.

Inhibition of glycolysis modulates prednisolone resistance in acute lymphoblastic leukemia cells.

Treatment failure in pediatric acute lymphoblastic leukemia (ALL) is related to cellular resistance to glucocorticoids (eg, prednisolone). Recently, we demonstrated that genes associated with glucose metabolism are differentially expressed between prednisolone-sensitive and prednisolone-resistant precursor B-lineage leukemic patients. Here, we show that prednisolone resistance is associated with increased glucose consumption and that inhibition of glycolysis sensitizes prednisolone-resistant ALL cell lines to glucocorticoids. Treatment of prednisolone-resistant Jurkat and Molt4 cells with 2-deoxy-D-glucose (2-DG), lonidamine (LND), or 3-bromopyruvate (3-BrPA) increased the in vitro sensitivity to glucocorticoids, while treatment of the prednisolone-sensitive cell lines Tom-1 and RS4; 11 did not influence drug cytotoxicity. This sensitizing effect of the glycolysis inhibitors in glucocorticoid-resistant ALL cells was not found for other classes of antileukemic drugs (ie, vincristine and daunorubicin). Moreover, down-regulation of the expression of GAPDH by RNA interference also sensitized to prednisolone, comparable with treatment with glycolytic inhibitors. Importantly, the ability of 2-DG to reverse glucocorticoid resistance was not limited to cell lines, but was also observed in isolated primary ALL cells from patients. Together, these findings indicate the importance of the glycolytic pathway in glucocorticoid resistance in ALL and suggest that targeting glycolysis is a viable strategy for modulating prednisolone resistance in ALL.

Nascent Prostate Cancer Heterogeneity Drives Evolution and Resistance to Intense Hormonal Therapy.

BACKGROUND: Patients diagnosed with high risk localized prostate cancer have variable outcomes following surgery. Trials of intense neoadjuvant androgen deprivation therapy (NADT) have shown lower rates of recurrence among patients with minimal residual disease after treatment. The molecular features that distinguish exceptional responders from poor responders are not known. OBJECTIVE: To identify genomic and histologic features associated with treatment resistance at baseline. DESIGN, SETTING, AND PARTICIPANTS: Targeted biopsies were obtained from 37 men with intermediate- to high-risk prostate cancer before receiving 6 mo of ADT plus enzalutamide. Biopsy tissues were used for whole-exome sequencing and immunohistochemistry (IHC). OUTCOME MEASUREMENTS AND STATISTICAL ANALYSIS: We assessed the relationship of molecular features with final pathologic response using a cutpoint of 0.05 cm3 for residual cancer burden to compare exceptional responders to incomplete and nonresponders. We assessed intratumoral heterogeneity at the tissue and genomic level, and compared the volume of residual disease to the Shannon diversity index for each tumor. We generated multivariate models of resistance based on three molecular features and one histologic feature, with and without multiparametric magnetic resonance imaging estimates of baseline tumor volume. RESULTS AND LIMITATIONS: Loss of chromosome 10q (containing PTEN) and alterations to TP53 were predictive of poor response, as were the expression of nuclear ERG on IHC and the presence of intraductal carcinoma of the prostate. Patients with incompletely and nonresponding tumors harbored greater tumor diversity as estimated via phylogenetic tree reconstruction from DNA sequencing and analysis of IHC staining. Our four-factor binary model (area under the receiver operating characteristic curve [AUC] 0.89) to predict poor response correlated with greater diversity in our cohort and a validation cohort of 57 Gleason score 8-10 prostate cancers from The Cancer Genome Atlas. When baseline tumor volume was added to the model, it distinguished poor response to NADT with an AUC of 0.98. Prospective use of this model requires further retrospective validation with biopsies from additional trials. CONCLUSIONS: A subset of prostate cancers exhibit greater histologic and genomic diversity at the time of diagnosis, and these localized tumors have greater fitness to resist therapy. PATIENT SUMMARY: Some prostate cancer tumors do not respond well to a hormonal treatment called androgen deprivation therapy (ADT). We used tumor volume and four other parameters to develop a model to identify tumors that will not respond well to ADT. Treatments other than ADT should be considered for these patients.

Sequential Prostate Magnetic Resonance Imaging in Newly Diagnosed High-risk Prostate Cancer Treated with Neoadjuvant Enzalutamide is Predictive of Therapeutic Response.

PURPOSE: For high-risk prostate cancer, standard treatment options include radical prostatectomy (RP) or radiotherapy plus androgen deprivation therapy (ADT). Despite definitive therapy, many patients will have disease recurrence. Imaging has the potential to better define characteristics of response and resistance. In this study, we evaluated prostate multiparametric MRI (mpMRI) before and after neoadjuvant enzalutamide plus ADT. PATIENTS AND METHODS: Men with localized intermediate- or high-risk prostate cancer underwent a baseline mpMRI and mpMRI-targeted biopsy followed by a second mpMRI after 6 months of enzalutamide and ADT prior to RP. Specimens were sectioned in the same plane as mpMRI using patient-specific 3D-printed molds to permit mpMRI-targeted biopsies to be compared with the same lesion from the RP. Specimens were analyzed for imaging and histologic correlates of response. RESULTS: Of 39 patients enrolled, 36 completed imaging and RP. Most patients (92%) had high-risk disease. Fifty-eight lesions were detected on baseline mpMRI, of which 40 (69%) remained measurable at 6-month follow-up imaging. Fifty-five of 59 lesions (93%) demonstrated >50% volume reduction on posttreatment mpMRI. Three of 59 lesions (5%) demonstrated growth in size at follow-up imaging, with two lesions increasing more than 3-fold in volume. On whole-mount pathology, 15 patients demonstrated minimal residual disease (MRD) of <0.05 cc or pathologic complete response. Low initial mpMRI relative tumor burden was most predictive of MRD on final pathology. CONCLUSIONS: Low relative lesion volume at baseline mpMRI was predictive of pathologic response. A subset of patients had limited response. Selection of patients based on these metrics may improve outcomes in high-risk disease.

Targeting the PI3K/AKT Pathway Overcomes Enzalutamide Resistance by Inhibiting Induction of the Glucocorticoid Receptor.

The PI3K-AKT pathway has pleiotropic effects and its inhibition has long been of interest in the management of prostate cancer, where a compensatory increase in PI3K signaling has been reported following androgen receptor (AR) blockade. Prostate cancer cells can also bypass AR blockade through induction of other hormone receptors, in particular the glucocorticoid receptor (GR). Here we demonstrate that AKT inhibition significantly decreases cell proliferation through both cytostatic and cytotoxic effects. The cytotoxic effect is enhanced by AR inhibition and is most pronounced in models that induce compensatory GR expression. AKT inhibition increases canonical AR activity and remodels the chromatin landscape, decreasing enhancer interaction at the GR gene (NR3C1) locus. Importantly, it blocks induction of GR expression and activity following AR blockade. This is confirmed in multiple in vivo models, where AKT inhibition of established xenografts leads to increased canonical AR activity, decreased GR expression, and marked antitumor activity. Overall, our results demonstrate that inhibition of the PI3K/AKT pathway can block GR activity and overcome GR-mediated resistance to AR-targeted therapy. Ipatasertib is currently in clinical development, and GR induction may be a biomarker to identify responsive patients or a responsive disease state.

A case report of multiple primary prostate tumors with differential drug sensitivity.

Localized prostate cancers are genetically variable and frequently multifocal, comprising spatially distinct regions with multiple independently-evolving clones. To date there is no understanding of whether this variability can influence management decisions for patients with prostate tumors. Here, we present a single case from a clinical trial of neoadjuvant intense androgen deprivation therapy. A patient was diagnosed with a large semi-contiguous tumor by imaging, histologically composed of a large Gleason score 9 tumor with an adjacent Gleason score 7 nodule. DNA sequencing demonstrates these are two independent tumors, as only the Gleason 9 tumor harbors single-copy losses of PTEN and TP53. The PTEN/TP53-deficient tumor demonstrates treatment resistance, selecting for subclones with mutations to the remaining copies of PTEN and TP53, while the Gleason 7 PTEN-intact tumor is almost entirely ablated. These findings indicate that spatiogenetic variability is a major confounder for personalized treatment of patients with prostate cancer.

Past, Current, and Future of Immunotherapies for Prostate Cancer.

Prostate cancer (PCa) is the most common cancer in men, and the second leading cause of cancer related death in men in Western countries. The standard therapy for metastatic PCa is androgen suppression therapy (AST). Men undergoing AST eventually develop metastatic castration-resistant prostate cancer (mCRPC), of which there are limited treatment options available. Immunotherapy has presented substantial benefits for many types of cancer, but only a marginal benefit for mCRPC, at least in part, due to the immunosuppressive tumor microenvironment (TME). Current clinical trials are investigating monotherapies or combination therapies involving adoptive cellular therapy, viral, DNA vaccines, oncolytic viruses, and immune checkpoint inhibitors (ICI). Immunotherapies are also being combined with chemotherapy, radiation, and AST. Additionally, preclinical investigations show promise with the recent description of alternative ways to circumvent the immunosuppressive nature of the prostate tumor microenvironment, including harnessing the immune stimulatory NKG2D pathway, inhibiting myeloid derived suppressor cells, and utilizing immunomodulatory oncolytic viruses. Herein we provide an overview of recent preclinical and clinical developments in cancer immunotherapies and discuss the perspectives for future immunotherapies in PCa.

Genomic Heterogeneity Within Individual Prostate Cancer Foci Impacts Predictive Biomarkers of Targeted Therapy.

BACKGROUND: Most lethal prostate cancers progress from relapse of aggressive primary disease. Recently, the most significant advances in survival benefit from systemic therapy have come from moving the administration of therapy to an earlier disease state. There is movement toward using biomarkers from the intraprostatic index lesion to guide early systemic therapy. OBJECTIVE: To determine the genomic heterogeneity, including the heterogeneity of predictive biomarkers, within the index focus of treatment-naïve prostate cancer. DESIGN, SETTING, AND PARTICIPANTS: Ten patients with treatment-naïve prostate cancer underwent prostatectomy. DNA was extracted from 70 spatially distinct regions of the 10 index foci. OUTCOME MEASUREMENTS AND STATISTICAL ANALYSIS: Single nucleotide mutations, small indels, and copy number changes were identified. Intrafocal genomic heterogeneity and heterogeneity of alterations that predict response to therapy was determined. RESULTS AND LIMITATIONS: Exome sequencing and copy number estimates demonstrate branched evolution with >75% of point mutations being subclonal, including numerous pathways associated with castrate-resistant prostate cancer. Seven of 10 patients harbor alterations in one of five genes that predict response to targeted therapies with survival benefit in prostate cancer. Within biomarker-positive cases, 25% of intraprostatic regions are biomarker negative, with discordance between intraprostatic regions and lymph node metastases. CONCLUSIONS: Treatment-naïve, nonmetastatic prostate cancer has marked intrafocal heterogeneity. Numerous alterations in pathways associated with castration-resistant prostate cancer are present in subclonal populations, including biomarkers predictive of response to targeted therapy. PATIENT SUMMARY: Untreated patients' tumors have alterations that predict response to targeted therapies, but the presence of a biomarker is dependent on what region of the tumor was evaluated.

mpMRI preoperative staging in men treated with antiandrogen and androgen deprivation therapy before robotic prostatectomy.

INTRODUCTION: Using multiparametric magnetic resonance imaging (mpMRI), we sought to preoperatively characterize prostate cancer (PCa) in the setting of antiandrogen plus androgen deprivation therapy (AA-ADT) prior to robotic-assisted radical prostatectomy (RARP). We present our preliminary findings regarding mpMRI depiction of changes of disease staging features and lesion appearance in treated prostate. METHODS: Prior to RARP, men received 6 months of enzalutamide and goserelin. mpMRI consisting of T2 weighted, b = 2,000 diffusion weighted imaging, apparent diffusion coefficient mapping, and dynamic contrast enhancement sequences was acquired before and after neoadjuvant therapy. Custom MRI-based prostate molds were printed to directly compare mpMRI findings to H&E whole-mount pathology as part of a phase II clinical trial (NCT02430480). RESULTS: Twenty men underwent imaging and RARP after a regimen of AA-ADT. Positive predictive values for post-AA-ADT mpMRI diagnosis of extraprostatic extension, seminal vesicle invasion, organ-confined disease, and biopsy-confirmed PCa lesions were 71%, 80%, 80%, and 85%, respectively. Post-treatment mpMRI correctly staged disease in 15/20 (75%) cases with 17/20 (85%) correctly identified as organ-confined or not. Of those incorrectly staged, 2 were falsely positive for higher stage features and 1 was falsely negative. Post-AA-ADT T2 weighted sequences best depicted presence of PCa lesions as compared to diffusion weighted imaging and dynamic contrast enhancement sequences. CONCLUSION: mpMRI proved reliable in detecting lesion changes after antiandrogen therapy corresponding to PCa pathology. Therefore, mpMRI of treated prostates may be helpful for assessing men for surgical planning and staging.

Mammalian target of rapamycin promotes vincristine resistance through multiple mechanisms independent of maintained glycolytic rate.

Deregulation of the phosphoinositide 3-kinase-Akt pathway is a major contributor to oncogenesis and resistance to cancer therapy. Recent work has shown mammalian target of rapamycin (mTOR) to be a major target downstream of Akt that contributes to both transformation and therapeutic resistance. Although inhibitors of Akt are not yet clinically available, rapamycin, a mTOR-specific inhibitor, has long been used as an immunosuppressant, and several rapamycin analogues are now in clinical trials in oncology. Recent data indicate that a mTOR complex phosphorylates Akt, and this complex is insensitive to rapamycin. We show that dominant-negative mTOR diminishes phosphorylation of endogenous Akt and exogenous myristoylated Akt (mAkt), that prolonged exposure to rapamycin also inhibits Akt activation, and that this inhibition is dependent on new protein synthesis. These data suggest that mTOR facilitates Akt activation through mechanisms other than direct phosphorylation. A constitutively active mTOR mutant that fails to enhance Akt phosphorylation nevertheless promotes resistance to multiple antimicrotubule agents, indicating that mTOR also mediates survival independent of Akt. Although Akt- and mTOR-mediated survival has been linked to regulation of cellular metabolism, we also show that survival and metabolic control are separable. The hexokinase inhibitor 5-thioglucose markedly inhibits glycolytic rate but does not diminish vincristine resistance mediated by mAkt or mTOR, and it has only a minor effect on mTOR- or mAkt-mediated resistance to growth factor withdrawal, suggesting that Akt-mTOR-mediated resistance is largely independent of maintenance of glycolytic rate. We conclude that mTOR activity can promote resistance through multiple mechanisms independent of maintained glycolytic rate.

Inhibition of glutathione synthesis reverses Bcl-2-mediated cisplatin resistance.

Cisplatin is a potent cytotoxic agent that functions as a bivalent electrophile, forming both interstrand and intrastrand DNA cross-links. Cisplatin-mediated DNA damage results in cell cycle arrest and initiation of apoptotic cell death. Increased cellular glutathione concentrations have been closely correlated with cisplatin resistance but do not reduce the extent of cisplatin-DNA adduct formation. One hypothesis to explain the ability of glutathione to inhibit cisplatin cytotoxicity is that glutathione, through its antioxidant function, plays a role in apoptotic regulatory pathways. We tested this hypothesis using MCF-7 breast cancer cells transfected with the apoptotic inhibitor Bcl-2. Bcl-2 overexpression in MCF-7 cells was associated with a nearly 3-fold increase in cellular glutathione levels and with increased resistance to cell death after cisplatin exposure. Treatment of MCF-7 lines with buthionine sulfoximine, an inhibitor of glutathione synthesis, normalized glutathione levels in Bcl-2 and control transfectants and completely abrogated Bcl-2-mediated cisplatin resistance without affecting Bcl-2 expression. Bcl-2 overexpression and up-regulation of glutathione were not associated with a change in either cisplatin-DNA adduct formation or repair over time. These results suggest that Bcl-2-mediated cisplatin resistance in MCF-7 cells is dependent on up-regulation of glutathione production, which contributes to cell survival by mechanisms independent of cisplatin inactivation or inhibition of DNA adduct formation. A similar dependence on glutathione for Bcl-2-mediated inhibition of cisplatin toxicity was confirmed in a second cell line, the lymphocytic precursor FL5.12. Taken together, these data suggest that apoptotic signaling after genotoxic exposure can be inhibited by the antioxidant activity of glutathione. Inhibition of glutathione synthesis or modulation of glutathione stores in tumors that overexpress Bcl-2 may comprise a novel anticancer strategy.