Activation of a cGAS-STING-mediated immune response predicts response to neoadjuvant chemotherapy in early breast cancer.

BACKGROUND: The DNA-damage immune-response (DDIR) signature is an immune-driven gene expression signature retrospectively validated as predicting response to anthracycline-based therapy. This feasibility study prospectively evaluates the use of this assay to predict neoadjuvant chemotherapy response in early breast cancer. METHODS: This feasibility study assessed the integration of a novel biomarker into clinical workflows. Tumour samples were collected from patients receiving standard of care neoadjuvant chemotherapy (FEC + /-taxane and anti-HER2 therapy as appropriate) at baseline, mid- and post-chemotherapy. Baseline DDIR signature scores were correlated with pathological treatment response. RNA sequencing was used to assess chemotherapy/response-related changes in biologically linked gene signatures. RESULTS: DDIR signature reports were available within 14 days for 97.8% of 46 patients (13 TNBC, 16 HER2 + ve, 27 ER + HER2-ve). Positive scores predicted response to treatment (odds ratio 4.67 for RCB 0-1 disease (95% CI 1.13-15.09, P = 0.032)). DDIR positivity correlated with immune infiltration and upregulated immune-checkpoint gene expression. CONCLUSIONS: This study validates the DDIR signature as predictive of response to neoadjuvant chemotherapy which can be integrated into clinical workflows, potentially identifying a subgroup with high sensitivity to anthracycline chemotherapy. Transcriptomic data suggest induction with anthracycline-containing regimens in immune restricted, "cold" tumours may be effective for immune priming. TRIAL REGISTRATION: Not applicable (non-interventional study). CRUK Internal Database Number 14232.

In-depth Clinical and Biological Exploration of DNA Damage Immune Response as a Biomarker for Oxaliplatin Use in Colorectal Cancer.

PURPOSE: The DNA damage immune response (DDIR) assay was developed in breast cancer based on biology associated with deficiencies in homologous recombination and Fanconi anemia pathways. A positive DDIR call identifies patients likely to respond to platinum-based chemotherapies in breast and esophageal cancers. In colorectal cancer, there is currently no biomarker to predict response to oxaliplatin. We tested the ability of the DDIR assay to predict response to oxaliplatin-based chemotherapy in colorectal cancer and characterized the biology in DDIR-positive colorectal cancer. EXPERIMENTAL DESIGN: Samples and clinical data were assessed according to DDIR status from patients who received either 5-fluorouracil (5-FU) or 5FUFA (bolus and infusion 5-FU with folinic acid) plus oxaliplatin (FOLFOX) within the FOCUS trial (n = 361, stage IV), or neoadjuvant FOLFOX in the FOxTROT trial (n = 97, stage II/III). Whole transcriptome, mutation, and IHC data of these samples were used to interrogate the biology of DDIR in colorectal cancer. RESULTS: Contrary to our hypothesis, DDIR-negative patients displayed a trend toward improved outcome for oxaliplatin-based chemotherapy compared with DDIR-positive patients. DDIR positivity was associated with microsatellite instability (MSI) and colorectal molecular subtype 1. Refinement of the DDIR signature, based on overlapping IFN-related chemokine signaling associated with DDIR positivity across colorectal cancer and breast cancer cohorts, further confirmed that the DDIR assay did not have predictive value for oxaliplatin-based chemotherapy in colorectal cancer. CONCLUSIONS: DDIR positivity does not predict improved response following oxaliplatin treatment in colorectal cancer. However, data presented here suggest the potential of the DDIR assay in identifying immune-rich tumors that may benefit from immune checkpoint blockade, beyond current use of MSI status.

Image-based consensus molecular subtype (imCMS) classification of colorectal cancer using deep learning.

OBJECTIVE: Complex phenotypes captured on histological slides represent the biological processes at play in individual cancers, but the link to underlying molecular classification has not been clarified or systematised. In colorectal cancer (CRC), histological grading is a poor predictor of disease progression, and consensus molecular subtypes (CMSs) cannot be distinguished without gene expression profiling. We hypothesise that image analysis is a cost-effective tool to associate complex features of tissue organisation with molecular and outcome data and to resolve unclassifiable or heterogeneous cases. In this study, we present an image-based approach to predict CRC CMS from standard H&E sections using deep learning. DESIGN: Training and evaluation of a neural network were performed using a total of n=1206 tissue sections with comprehensive multi-omic data from three independent datasets (training on FOCUS trial, n=278 patients; test on rectal cancer biopsies, GRAMPIAN cohort, n=144 patients; and The Cancer Genome Atlas (TCGA), n=430 patients). Ground truth CMS calls were ascertained by matching random forest and single sample predictions from CMS classifier. RESULTS: Image-based CMS (imCMS) accurately classified slides in unseen datasets from TCGA (n=431 slides, AUC)=0.84) and rectal cancer biopsies (n=265 slides, AUC=0.85). imCMS spatially resolved intratumoural heterogeneity and provided secondary calls correlating with bioinformatic prediction from molecular data. imCMS classified samples previously unclassifiable by RNA expression profiling, reproduced the expected correlations with genomic and epigenetic alterations and showed similar prognostic associations as transcriptomic CMS. CONCLUSION: This study shows that a prediction of RNA expression classifiers can be made from H&E images, opening the door to simple, cheap and reliable biological stratification within routine workflows.

ATM Kinase Inhibition Preferentially Sensitises PTEN-Deficient Prostate Tumour Cells to Ionising Radiation.

Radical radiotherapy, often in combination with hormone ablation, is a safe and effective treatment option for localised or locally-advanced prostate cancer. However, up to 30% of patients with locally advanced PCa will go on to develop biochemical failure, within 5 years, following initial radiotherapy. Improving radiotherapy response is clinically important since patients exhibiting biochemical failure develop castrate-resistant metastatic disease for which there is no curative therapy and median survival is 8-18 months. The aim of this research was to determine if loss of PTEN (highly prevalent in advanced prostate cancer) is a novel therapeutic target in the treatment of advanced prostate cancer. Previous work has demonstrated PTEN-deficient cells are sensitised to inhibitors of ATM, a key regulator in the response to DSBs. Here, we have shown the role of PTEN in cellular response to IR was both complex and context-dependent. Secondly, we have confirmed ATM inhibition in PTEN-depleted cell models, enhances ionising radiation-induced cell killing with minimal toxicity to normal prostate RWPE-1 cells. Furthermore, combined treatment significantly inhibited PTEN-deficient tumour growth compared to PTEN-expressing counterparts, with minimal toxicity observed. We have further shown PTEN loss is accompanied by increased endogenous levels of ROS and DNA damage. Taken together, these findings provide pre-clinical data for future clinical evaluation of ATM inhibitors as a neoadjuvant/adjuvant in combination with radiation therapy in prostate cancer patients harbouring PTEN mutations.

Validation of the DNA Damage Immune Response Signature in Patients With Triple-Negative Breast Cancer From the SWOG 9313c Trial.

PURPOSE: To independently validate two biomarkers, a 44-gene DNA damage immune response (DDIR) signature and stromal tumor-infiltrating lymphocytes (sTILs), as prognostic markers in patients with triple-negative breast cancer (TNBC) treated with adjuvant doxorubicin (A) and cyclophosphamide (C) in SWOG 9313. METHODS: Four hundred twenty-five centrally determined patient cases with TNBC from S9313 were identified. DDIR signature was performed on RNA isolated from formalin-fixed paraffin-embedded tumor tissue, and samples were classified as DDIR negative or positive using predefined cutoffs. Evaluation of sTILs was performed as described previously. Markers were tested for prognostic value for disease-free survival (DFS) and overall survival (OS) using Cox regression models adjusted for treatment assignment, nodal status, and tumor size. RESULTS: Among 425 patients with TNBC, 33% were node positive. DDIR was tested successfully in 90% of patients (381 of 425), 62% of which were DDIR signature positive. DDIR signature positivity was associated with improved DFS (hazard ratio [HR], 0.67; 95% CI, 0.48 to 0.92; P = .015) and OS (HR, 0.61; 95% CI, 0.43 to 0.89; P = .010). sTILs density assessment was available in 99% of patients and was associated with improved DFS (HR, 0.70; 95% CI, 0.51 to 0.96; P = .026 for sTILs density ≥ 20% v < 20%) and OS (HR, 0.59; 95% CI, 0.41 to 0.85; P = .004 for sTILs density ≥ 20% v < 20%). DDIR signature score and sTILs density were moderately correlated (r = 0.60), which precluded statistical significance for DFS in a joint model. Three-year DFS and OS in a subgroup of patients with DDIR positivity and T1c/T2N0 disease were 88% and 94%, respectively. CONCLUSION: The prognostic role of sTILs and DDIR in early-stage TNBC was confirmed. DDIR signature conferred improved prognosis in two thirds of patients with TNBC treated with adjuvant AC. DDIR signature has the potential to stratify outcome and to identify patients with less projected benefit after AC chemotherapy.

Immune activation by DNA damage predicts response to chemotherapy and survival in oesophageal adenocarcinoma.

OBJECTIVE: Current strategies to guide selection of neoadjuvant therapy in oesophageal adenocarcinoma (OAC) are inadequate. We assessed the ability of a DNA damage immune response (DDIR) assay to predict response following neoadjuvant chemotherapy in OAC. DESIGN: Transcriptional profiling of 273 formalin-fixed paraffin-embedded prechemotherapy endoscopic OAC biopsies was performed. All patients were treated with platinum-based neoadjuvant chemotherapy and resection between 2003 and 2014 at four centres in the Oesophageal Cancer Clinical and Molecular Stratification consortium. CD8 and programmed death ligand 1 (PD-L1) immunohistochemical staining was assessed in matched resection specimens from 126 cases. Kaplan-Meier and Cox proportional hazards regression analysis were applied according to DDIR status for recurrence-free survival (RFS) and overall survival (OS). RESULTS: A total of 66 OAC samples (24%) were DDIR positive with the remaining 207 samples (76%) being DDIR negative. DDIR assay positivity was associated with improved RFS (HR: 0.61; 95% CI 0.38 to 0.98; p=0.042) and OS (HR: 0.52; 95% CI 0.31 to 0.88; p=0.015) following multivariate analysis. DDIR-positive patients had a higher pathological response rate (p=0.033), lower nodal burden (p=0.026) and reduced circumferential margin involvement (p=0.007). No difference in OS was observed according to DDIR status in an independent surgery-alone dataset.DDIR-positive OAC tumours were also associated with the presence of CD8+ lymphocytes (intratumoural: p<0.001; stromal: p=0.026) as well as PD-L1 expression (intratumoural: p=0.047; stromal: p=0.025). CONCLUSION: The DDIR assay is strongly predictive of benefit from DNA-damaging neoadjuvant chemotherapy followed by surgical resection and is associated with a proinflammatory microenvironment in OAC.

Molecular Subgroup of Primary Prostate Cancer Presenting with Metastatic Biology.

BACKGROUND: Approximately 4-25% of patients with early prostate cancer develop disease recurrence following radical prostatectomy. OBJECTIVE: To identify a molecular subgroup of prostate cancers with metastatic potential at presentation resulting in a high risk of recurrence following radical prostatectomy. DESIGN, SETTING, AND PARTICIPANTS: Unsupervised hierarchical clustering was performed using gene expression data from 70 primary resections, 31 metastatic lymph nodes, and 25 normal prostate samples. Independent assay validation was performed using 322 radical prostatectomy samples from four sites with a mean follow-up of 50.3 months. OUTCOME MEASUREMENTS AND STATISTICAL ANALYSIS: Molecular subgroups were identified using unsupervised hierarchical clustering. A partial least squares approach was used to generate a gene expression assay. Relationships with outcome (time to biochemical and metastatic recurrence) were analysed using multivariable Cox regression and log-rank analysis. RESULTS AND LIMITATIONS: A molecular subgroup of primary prostate cancer with biology similar to metastatic disease was identified. A 70-transcript signature (metastatic assay) was developed and independently validated in the radical prostatectomy samples. Metastatic assay positive patients had increased risk of biochemical recurrence (multivariable hazard ratio [HR] 1.62 [1.13-2.33]; p=0.0092) and metastatic recurrence (multivariable HR=3.20 [1.76-5.80]; p=0.0001). A combined model with Cancer of the Prostate Risk Assessment post surgical (CAPRA-S) identified patients at an increased risk of biochemical and metastatic recurrence superior to either model alone (HR=2.67 [1.90-3.75]; p<0.0001 and HR=7.53 [4.13-13.73]; p<0.0001, respectively). The retrospective nature of the study is acknowledged as a potential limitation. CONCLUSIONS: The metastatic assay may identify a molecular subgroup of primary prostate cancers with metastatic potential. PATIENT SUMMARY: The metastatic assay may improve the ability to detect patients at risk of metastatic recurrence following radical prostatectomy. The impact of adjuvant therapies should be assessed in this higher-risk population.

Activation of STING-Dependent Innate Immune Signaling By S-Phase-Specific DNA Damage in Breast Cancer.

Background: Previously we identified a DNA damage response-deficient (DDRD) molecular subtype within breast cancer. A 44-gene assay identifying this subtype was validated as predicting benefit from DNA-damaging chemotherapy. This subtype was defined by interferon signaling. In this study, we address the mechanism of this immune response and its possible clinical significance. Methods: We used immunohistochemistry (IHC) to characterize immune infiltration in 184 breast cancer samples, of which 65 were within the DDRD subtype. Isogenic cell lines, which represent DDRD-positive and -negative, were used to study the effects of chemokine release on peripheral blood mononuclear cell (PBMC) migration and the mechanism of immune signaling activation. Finally, we studied the association between the DDRD subtype and expression of the immune-checkpoint protein PD-L1 as detected by IHC. All statistical tests were two-sided. Results: We found that DDRD breast tumors were associated with CD4+ and CD8+ lymphocytic infiltration (Fisher's exact test P < .001) and that DDRD cells expressed the chemokines CXCL10 and CCL5 3.5- to 11.9-fold more than DNA damage response-proficient cells (P < .01). Conditioned medium from DDRD cells statistically significantly attracted PBMCs when compared with medium from DNA damage response-proficient cells (P < .05), and this was dependent on CXCL10 and CCL5. DDRD cells demonstrated increased cytosolic DNA and constitutive activation of the viral response cGAS/STING/TBK1/IRF3 pathway. Importantly, this pathway was activated in a cell cycle-specific manner. Finally, we demonstrated that S-phase DNA damage activated expression of PD-L1 in a STING-dependent manner. Conclusions: We propose a novel mechanism of immune infiltration in DDRD tumors, independent of neoantigen production. Activation of this pathway and associated PD-L1 expression may explain the paradoxical lack of T-cell-mediated cytotoxicity observed in DDRD tumors. We provide a rationale for exploration of DDRD in the stratification of patients for immune checkpoint-based therapies.

When the guardian becomes the enemy: Targeting ATM in PTEN-deficient cancers.

Ataxia telangiectasia mutated (ATM) is an important signaling molecule in the DNA damage response and inhibitors of ATM are under clinical development. We identified a synthetic lethal interaction between ATM inhibition and phosphatase and tensin homolog (PTEN) loss that was the result of increased oxidative stress. Inhibition of ATM therefore represents a novel strategy to target PTEN-associated cancers.

STAT3 regulated ARF expression suppresses prostate cancer metastasis.

Prostate cancer (PCa) is the most prevalent cancer in men. Hyperactive STAT3 is thought to be oncogenic in PCa. However, targeting of the IL-6/STAT3 axis in PCa patients has failed to provide therapeutic benefit. Here we show that genetic inactivation of Stat3 or IL-6 signalling in a Pten-deficient PCa mouse model accelerates cancer progression leading to metastasis. Mechanistically, we identify p19(ARF) as a direct Stat3 target. Loss of Stat3 signalling disrupts the ARF-Mdm2-p53 tumour suppressor axis bypassing senescence. Strikingly, we also identify STAT3 and CDKN2A mutations in primary human PCa. STAT3 and CDKN2A deletions co-occurred with high frequency in PCa metastases. In accordance, loss of STAT3 and p14(ARF) expression in patient tumours correlates with increased risk of disease recurrence and metastatic PCa. Thus, STAT3 and ARF may be prognostic markers to stratify high from low risk PCa patients. Our findings challenge the current discussion on therapeutic benefit or risk of IL-6/STAT3 inhibition.

Mechanistic Rationale to Target PTEN-Deficient Tumor Cells with Inhibitors of the DNA Damage Response Kinase ATM.

Ataxia telangiectasia mutated (ATM) is an important signaling molecule in the DNA damage response (DDR). ATM loss of function can produce a synthetic lethal phenotype in combination with tumor-associated mutations in FA/BRCA pathway components. In this study, we took an siRNA screening strategy to identify other tumor suppressors that, when inhibited, similarly sensitized cells to ATM inhibition. In this manner, we determined that PTEN and ATM were synthetically lethal when jointly inhibited. PTEN-deficient cells exhibited elevated levels of reactive oxygen species, increased endogenous DNA damage, and constitutive ATM activation. ATM inhibition caused catastrophic DNA damage, mitotic cell cycle arrest, and apoptosis specifically in PTEN-deficient cells in comparison with wild-type cells. Antioxidants abrogated the increase in DNA damage and ATM activation in PTEN-deficient cells, suggesting a requirement for oxidative DNA damage in the mechanism of cell death. Lastly, the ATM inhibitor KU-60019 was specifically toxic to PTEN mutant cancer cells in tumor xenografts and reversible by reintroduction of wild-type PTEN. Together, our results offer a mechanistic rationale for clinical evaluation of ATM inhibitors in PTEN-deficient tumors.

Identification and validation of an anthracycline/cyclophosphamide-based chemotherapy response assay in breast cancer.

BACKGROUND: There is no method routinely used to predict response to anthracycline and cyclophosphamide-based chemotherapy in the clinic; therefore patients often receive treatment for breast cancer with no benefit. Loss of the Fanconi anemia/BRCA (FA/BRCA) DNA damage response (DDR) pathway occurs in approximately 25% of breast cancer patients through several mechanisms and results in sensitization to DNA-damaging agents. The aim of this study was to develop an assay to detect DDR-deficient tumors associated with loss of the FA/BRCA pathway, for the purpose of treatment selection. METHODS: DNA microarray data from 21 FA patients and 11 control subjects were analyzed to identify genetic processes associated with a deficiency in DDR. Unsupervised hierarchical clustering was then performed using 60 BRCA1/2 mutant and 47 sporadic tumor samples, and a molecular subgroup was identified that was defined by the molecular processes represented within FA patients. A 44-gene microarray-based assay (the DDR deficiency assay) was developed to prospectively identify this subgroup from formalin-fixed, paraffin-embedded samples. All statistical tests were two-sided. RESULTS: In a publicly available independent cohort of 203 patients, the assay predicted complete pathologic response vs residual disease after neoadjuvant DNA-damaging chemotherapy (5-fluorouracil, anthracycline, and cyclophosphamide) with an odds ratio of 3.96 (95% confidence interval [Cl] =1.67 to 9.41; P = .002). In a new independent cohort of 191 breast cancer patients treated with adjuvant 5-fluorouracil, epirubicin, and cyclophosphamide, a positive assay result predicted 5-year relapse-free survival with a hazard ratio of 0.37 (95% Cl = 0.15 to 0.88; P = .03) compared with the assay negative population. CONCLUSIONS: A formalin-fixed, paraffin-embedded tissue-based assay has been developed and independently validated as a predictor of response and prognosis after anthracycline/cyclophosphamide-based chemotherapy in the neoadjuvant and adjuvant settings. These findings warrant further validation in a prospective clinical study.

Potentiation of inflammatory CXCL8 signalling sustains cell survival in PTEN-deficient prostate carcinoma.

BACKGROUND: Inflammation and genetic instability are enabling characteristics of prostate carcinoma (PCa). Inactivation of the tumour suppressor gene phosphatase and tensin homolog (PTEN) is prevalent in early PCa. The relationship of PTEN deficiency to inflammatory signalling remains to be characterised. OBJECTIVE: To determine how loss of PTEN functionality modulates expression and efficacy of clinically relevant, proinflammatory chemokines in PCa. DESIGN, SETTING, AND PARTICIPANTS: Experiments were performed in established cell-based PCa models, supported by pathologic analysis of chemokine expression in prostate tissue harvested from PTEN heterozygous (Pten(+/-)) mice harbouring inactivation of one PTEN allele. INTERVENTIONS: Small interfering RNA (siRNA)- or small hairpin RNA (shRNA)-directed strategies were used to repress PTEN expression and resultant interleukin-8 (CXCL8) signalling, determined under normal and hypoxic culture conditions. OUTCOME MEASUREMENTS AND STATISTICAL ANALYSIS: Changes in chemokine expression in PCa cells and tissue were analysed by real-time polymerase chain reaction (PCR), immunoblotting, enzyme-linked immunosorbent assay (ELISA), and immunohistochemistry; effects of chemokine signalling on cell function were assessed by cell cycle analysis, apoptosis, and survival assays. RESULTS AND LIMITATIONS: Transient (siRNA) or prolonged (shRNA) PTEN repression increased expression of CXCL8 and its receptors, chemokine (C-X-C motif) receptor (CXCR) 1 and CXCR2, in PCa cells. Hypoxia-induced increases in CXCL8, CXCR1, and CXCR2 expression were greater in magnitude and duration in PTEN-depleted cells. Autocrine CXCL8 signalling was more efficacious in PTEN-depleted cells, inducing hypoxia-inducible factor-1 (HIF-1) and nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) transcription and regulating genes involved in survival and angiogenesis. Increased expression of the orthologous chemokine KC was observed in regions displaying atypical cytologic features in Pten(+/-) murine prostate tissue relative to normal epithelium in wild-type PTEN (Pten(WT)) glands. Attenuation of CXCL8 signalling decreased viability of PCa cells harbouring partial or complete PTEN loss through promotion of G1 cell cycle arrest and apoptosis. The current absence of clinical validation is a limitation of the study. CONCLUSIONS: PTEN loss induces a selective upregulation of CXCL8 signalling that sustains the growth and survival of PTEN-deficient prostate epithelium.

Development and independent validation of a prognostic assay for stage II colon cancer using formalin-fixed paraffin-embedded tissue.

PURPOSE: Current prognostic factors are poor at identifying patients at risk of disease recurrence after surgery for stage II colon cancer. Here we describe a DNA microarray-based prognostic assay using clinically relevant formalin-fixed paraffin-embedded (FFPE) samples. PATIENTS AND METHODS: A gene signature was developed from a balanced set of 73 patients with recurrent disease (high risk) and 142 patients with no recurrence (low risk) within 5 years of surgery. RESULTS: The 634-probe set signature identified high-risk patients with a hazard ratio (HR) of 2.62 (P < .001) during cross validation of the training set. In an independent validation set of 144 samples, the signature identified high-risk patients with an HR of 2.53 (P < .001) for recurrence and an HR of 2.21 (P = .0084) for cancer-related death. Additionally, the signature was shown to perform independently from known prognostic factors (P < .001). CONCLUSION: This gene signature represents a novel prognostic biomarker for patients with stage II colon cancer that can be applied to FFPE tumor samples.

The tumour-suppressor function of PTEN requires an N-terminal lipid-binding motif.

The PTEN (phosphatase and tensin homologue deleted on chromosome 10) tumour-suppressor protein is a phosphoinositide 3-phosphatase which antagonizes phosphoinositide 3-kinase-dependent signalling by dephosphorylating PtdIns(3,4,5)P3. Most tumour-derived point mutations of PTEN induce a loss of function, which correlates with profoundly reduced catalytic activity. However, here we characterize a point mutation at the N-terminus of PTEN, K13E from a human glioblastoma, which displayed wild-type activity when assayed in vitro. This mutation occurs within a conserved polybasic motif, a putative PtdIns(4,5)P2-binding site that may participate in membrane targeting of PTEN. We found that catalytic activity against lipid substrates and vesicle binding of wild-type PTEN, but not of PTEN K13E, were greatly stimulated by anionic lipids, especially PtdIns(4,5)P2. The K13E mutation also greatly reduces the efficiency with which anionic lipids inhibit PTEN activity against soluble substrates, supporting the hypothesis that non-catalytic membrane binding orientates the active site to favour lipid substrates. Significantly, in contrast to the wild-type enzyme, PTEN K13E failed either to prevent protein kinase B/Akt phosphorylation, or inhibit cell proliferation when expressed in PTEN-null U87MG cells. The cellular functioning of K13E PTEN was recovered by targeting to the plasma membrane through inclusion of a myristoylation site. Our results establish a requirement for the conserved N-terminal motif of PTEN for correct membrane orientation, cellular activity and tumour-suppressor function.

Interfacial kinetic analysis of the tumour suppressor phosphatase, PTEN: evidence for activation by anionic phospholipids.

We investigated the kinetic behaviour and substrate specificity of PTEN (phosphatase and tensin homologue deleted on chromosome 10) using unilamellar vesicles containing substrate lipids in a background of phosphatidylcholine. PTEN displays the characteristics expected of an interfacial enzyme, since the rate of enzyme activity is dependent on the surface concentration of the substrate lipids used (mol fraction), as well as the bulk concentration. Surface-dilution analysis revealed the catalytic efficiency of PTEN for PtdIns(3,4,5) P (3) to be 200-fold greater than for either PtdIns(3,4) P (2) or PtdIns(3,5) P (2), and 1000-fold greater than for PtdIns3 P. The interfacial K (m) value of PTEN for PtdIns(3,4,5) P (3) was very low, reflecting the small proportions of this lipid that are present in cellular membranes. The catalytic-centre activity ( k (cat)) for PtdIns(3,4,5) P (3) was at least 200-fold greater than that for the water-soluble substrate Ins(1,3,4,5) P (4). The preference for lipid substrates may result from an interfacial activation of the enzyme, rather than processive catalysis of vesicular substrates. Moreover, both PtdIns(4,5) P (2) and univalent salts stimulated the activity of PTEN for PtdIns(3,4,5) P (3), but profoundly inhibited activity against Ins(1,3,4,5) P (4). The stimulatory effect of PtdIns(4,5) P (2) was greater in magnitude and more potent in comparison with other anionic phospholipid species. A mutation in the lipid-binding C2 domain (M-CBR3) that is biologically inactive did not alter overall catalytic efficiency in this model, but decreased the efficiency of the interfacial binding step, demonstrating its importance in the catalytic mechanism of PTEN.