Vanitha Ramakrishnan

Loss of E-Cadherin Promotes Ovarian Cancer Metastasis via α5-Integrin, which Is a Therapeutic Target

E-cadherin loss is frequently associated with ovarian cancer metastasis. Given that adhesion to the abdominal peritoneum is the first step in ovarian cancer dissemination, we reasoned that down-regulation of E-cadherin would affect expression of cell matrix adhesion receptors. We show here that inhibition of E-cadherin in ovarian cancer cells causes up-regulation of alpha(5)-integrin protein expression and transcription. When E-cadherin was blocked, RMUG-S ovarian cancer cells were able to attach and invade more efficiently. This greater efficiency could, in turn, be inhibited both in vitro and in vivo with an alpha(5)beta(1)-integrin-blocking antibody. When E-cadherin is silenced, alpha(5)-integrin is up-regulated through activation of an epidermal growth factor receptor/FAK/Erk1-mitogen-activated protein kinase-dependent signaling pathway and not through the canonical E-cadherin/beta-catenin signaling pathway. In SKOV-3ip1 ovarian cancer xenografts, which express high levels of alpha(5)-integrin, i.p. treatment with an alpha(5)beta(1)-integrin antibody significantly reduced tumor burden, ascites, and number of metastasis and increased survival by an average of 12 days when compared with IgG treatment (P < 0.0005). alpha(5)-Integrin expression was detected by immunohistochemistry in 107 advanced stage ovarian cancers using a tissue microarray annotated with disease-specific patient follow-up. Ten of 107 tissues (9%) had alpha(5)-integrin overexpression, and 39% had some level of alpha(5)-integrin expression. The median survival for patients with high alpha(5)-integrin levels was 26 months versus 35 months for those with low integrin expression (P < 0.05). Taken together, we have identified alpha(5)-integrin up-regulation as a molecular mechanism by which E-cadherin loss promotes tumor progression, providing an explanation for how E-cadherin loss increases metastasis. Targeting this integrin could be a promising therapy for a subset of ovarian cancer patients.

Biomarker Analyses from a Placebo-Controlled Phase II Study Evaluating Erlotinib ± Onartuzumab in Advanced Non-Small-Cell Lung Cancer: MET Expression Levels Are Predictive of Patient Benefit

Purpose: In a recent phase II study of onartuzumab (MetMAb), patients whose non–small cell lung cancer (NSCLC) tissue scored as positive for MET protein by immunohistochemistry (IHC) experienced a significant benefit with onartuzumab plus erlotinib (O+E) versus erlotinib. We describe development and validation of a standardized MET IHC assay and, retrospectively, evaluate multiple biomarkers as predictors of patient benefit. Experimental Design: Biomarkers related to MET and/or EGF receptor (EGFR) signaling were measured by IHC, FISH, quantitative reverse transcription PCR, mutation detection techniques, and ELISA. Results: A positive correlation between IHC, Western blotting, and MET mRNA expression was observed in NSCLC cell lines/tissues. An IHC scoring system of MET expression taking proportional and intensity-based thresholds into consideration was applied in an analysis of the phase II study and resulted in the best differentiation of outcomes. Further analyses revealed a nonsignificant overall survival (OS) improvement with O+E in patients with high MET copy number (mean ≥5 copies/cell by FISH); however, benefit was maintained in “MET IHC-positive”/MET FISH-negative patients (HR, 0.37; P = 0.01). MET, EGFR, amphiregulin, epiregulin, or HGF mRNA expression did not predict a significant benefit with onartuzumab; a nonsignificant OS improvement was observed in patients with high tumor MET mRNA levels (HR, 0.59; P = 0.23). Patients with low baseline plasma hepatocyte growth factor (HGF) exhibited an HR for OS of 0.519 (P = 0.09) in favor of onartuzumab treatment. Conclusions: MET IHC remains the most robust predictor of OS and progression-free survival benefit from O+E relative to all examined exploratory markers. Clin Cancer Res; 20(17); 4488–98. ©2014 AACR.

Expression of the zinc transporter ZnT4 is decreased in the progression from early prostate disease to invasive prostate cancer.

We have utilized oligonucleotide microarrays to identify novel genes of potential clinical and biological importance in prostate cancer. RNA from 74 prostate cancers and 164 normal body samples representing 40 different tissues were analysed using a customized Affymetrix GeneChip® oligonucleotide microarray representative of over 90% of the expressed human genome. The gene for the zinc transporter ZnT4 was one of several genes that displayed significantly higher expression in prostate cancer compared to normal tissues from other organs. A polyclonal antipeptide antibody was used to demonstrate ZnT4 expression in the epithelium of all 165 elements of benign and 326 elements of localized prostate cancers examined and in nine of 10 advanced prostate cancer specimens by immunohistochemistry. Interestingly, decreased intensity of ZnT4 immunoreactivity occurred in the progression from benign to invasive localized prostate cancer and to metastatic disease. Immunofluorescence analysis and surface biotinylation studies of cells expressing ZnT4 localised the protein to intracellular vesicles and to the plasma membrane. These findings are consistent with a role for ZnT4 in vesicular transport of zinc to the cell membrane and potentially in efflux of zinc in the prostate.

A function blocking anti-mouse integrin α5β1 antibody inhibits angiogenesis and impedes tumor growth in vivo

BackgroundIntegrins are important adhesion molecules that regulate tumor and endothelial cell survival, proliferation and migration. The integrin α5β1 has been shown to play a critical role during angiogenesis. An inhibitor of this integrin, volociximab (M200), inhibits endothelial cell growth and movement in vitro, independent of the growth factor milieu, and inhibits tumor growth in vivo in the rabbit VX2 carcinoma model. Although volociximab has already been tested in open label, pilot phase II clinical trials in melanoma, pancreatic and renal cell cancer, evaluation of the mechanism of action of volociximab has been limited because this antibody does not cross-react with murine α5β1, precluding its use in standard mouse xenograft models.MethodsWe generated a panel of rat-anti-mouse α5β1 antibodies, with the intent of identifying an antibody that recapitulated the properties of volociximab. Hybridoma clones were screened for analogous function to volociximab, including specificity for α5β1 heterodimer and blocking of integrin binding to fibronectin. A subset of antibodies that met these criteria were further characterized for their capacities to bind to mouse endothelial cells, inhibit cell migration and block angiogenesis in vitro. One antibody that encompassed all of these attributes, 339.1, was selected from this panel and tested in xenograft models.ResultsA panel of antibodies was characterized for specificity and potency. The affinity of antibody 339.1 for mouse integrin α5β1 was determined to be 0.59 nM, as measured by BIAcore. This antibody does not significantly cross-react with human integrin, however 339.1 inhibits murine endothelial cell migration and tube formation and elicits cell death in these cells (EC50 = 5.3 nM). In multiple xenograft models, 339.1 inhibited the growth of established tumors by 40–60% (p < 0.05) and this inhibition correlates with a concomitant decrease in vessel density.ConclusionThe results herein demonstrate that 339.1, like volociximab, exhibits potent anti-α5β1 activity and confirms that inhibition of integrin α5β1 impedes angiogenesis and slows tumor growth in vivo.

Antibodies to TWEAK Receptor Inhibit Human Tumor Growth through Dual Mechanisms

Purpose: Targeted therapeutics have significantly changed the outcome for patients diagnosed with cancer. Still, effective therapeutic intervention does not exist for many cancers and much remains to be done. The objective of this study was to identify novel genes that potentially regulate tumor growth, to target these gene products with monoclonal antibodies, and to examine the therapeutic potential of these antibodies. Experimental Design: Using cDNA microarray analysis, we identified genes overexpressed in several solid malignancies. We generated a mouse monoclonal antibody, 19.2.1, and its humanized counterpart, PDL192, to one such target, TweakR (TWEAK receptor, Fn14, TNFRSF12A, CD266), and characterized the antitumor activities in vitro and in mouse xenograft models. Results: Both 19.2.1 (mouse IgG2a) and PDL192 (human IgG1), like TWEAK, the natural ligand of TweakR, inhibited the growth of several TweakR-expressing cancer cell lines in anchorage-dependent and anchorage-independent assays in vitro. Both antibodies showed significant antitumor activity in multiple mouse xenograft models. PDL192 and 19.2.1 also induced antibody-dependent cellular cytotoxicity (ADCC) of cancer cell lines in vitro. A chimeric version of 19.2.1 containing the mouse IgG1 Fc region (19.2.1×G1) exhibited significantly less ADCC than 19.2.1. However, 19.2.1×G1 showed differential activity in vivo, with activity equivalent to 19.2.1 in one model, but significantly less efficacy than 19.2.1 in a second model. These results indicate that PDL192 and 19.2.1 mediate their antitumor effects by signaling through TweakR, resulting in reduced tumor cell proliferation, and by ADCC. Clin Cancer Res; 16(2); 497–508

DCDT2980S, an Anti-CD22-Monomethyl Auristatin E Antibody–Drug Conjugate, Is a Potential Treatment for Non-Hodgkin Lymphoma

Antibody–drug conjugates (ADC), potent cytotoxic drugs linked to antibodies via chemical linkers, allow specific targeting of drugs to neoplastic cells. We have used this technology to develop the ADC DCDT2980S that targets CD22, an antigen with expression limited to B cells and the vast majority of non-Hodgkin lymphomas (NHL). DCDT2980S consists of a humanized anti-CD22 monoclonal IgG1 antibody with a potent microtubule-disrupting agent, monomethyl auristatin E (MMAE), linked to the reduced cysteines of the antibody via a protease cleavable linker, maleimidocaproyl-valine-citrulline-p-aminobenzoyloxycarbonyl (MC-vc-PAB). We describe the efficacy, safety, and pharmacokinetics of DCDT2980S in animal models to assess its potential as a therapeutic for the treatment of B-cell malignancies. We did not find a strong correlation between in vitro or in vivo efficacy and CD22 surface expression, nor a correlation of sensitivity to free drug and in vitro potency. We show that DCDT2980S was capable of inducing complete tumor regression in xenograft mouse models of NHL and can be more effective than rituximab plus combination chemotherapy at drug exposures that were well tolerated in cynomolgus monkeys. These results suggest that DCDT2980S has an efficacy, safety, and pharmacokinetics profile that support potential treatment of NHL. Mol Cancer Ther; 12(7); 1255–65. ©2013 AACR.

Investigational antibody-drug conjugates for hematological malignancies

Importance of the field: Antibody-drug conjugates (ADCs) consist of potent cytotoxic drugs linked to antibodies via chemical linkers. ADCs facilitate the specific targeting of drugs to neoplastic cells. This technology is showing efficacy with manageable toxicity for the treatment of hematological malignancies. Areas covered in this review: ADCs for the treatment of hematological malignancies are in pre-clinical and early clinical trials. This review describes these ADCs in detail and explores the challenges of optimizing the use of this technology. What the reader will gain: The reader should understand that, although ADCs are conceptually simple, the application of this idea to practice has not been straightforward, and the challenges of developing ADCs include identifying targets with appropriate expression profiles and biology, developing successful linker chemistries, and the selection of a potent cytotoxic drug. Take home message: Hematological malignancies are particularly suited to the development of ADC therapeutics as their surface proteins are well characterized, and the consequences of expression of the target in the normal tissue like the bone marrow results in manageable toxicities since, in many cases, the normal tissue can regenerate. While this technology is complex, the ADCs for hematological malignancies currently in clinical use show great promise.

Potential Mechanisms for Thrombocytopenia Development with Trastuzumab Emtansine (T-DM1)

Purpose: Trastuzumab-emtansine (T-DM1) is an antibody–drug conjugate (ADC) comprising the cytotoxic agent DM1 conjugated to trastuzumab with a stable linker. Thrombocytopenia was the dose-limiting toxicity in the phase I study, and grade ≥3 thrombocytopenia occurred in up to 13% of patients receiving T-DM1 in phase III studies. We investigated the mechanism of T-DM1–induced thrombocytopenia. Experimental Design: The effect of T-DM1 on platelet function was measured by aggregometry, and by flow cytometry to detect the markers of activation. The effect of T-DM1 on differentiation and maturation of megakaryocytes (MK) from human hematopoietic stem cells was assessed by flow cytometry and microscopy. Binding, uptake, and catabolism of T-DM1 in MKs, were assessed by various techniques including fluorescence microscopy, scintigraphy to detect T-[H3]-DM1 and 125I-T-DM1, and mass spectrometry. The role of FcγRIIa was assessed using blocking antibodies and mutant constructs of trastuzumab that do not bind FcγR. Results: T-DM1 had no direct effect on platelet activation and aggregation, but it did markedly inhibit MK differentiation via a cytotoxic effect. Inhibition occurred with DM1-containing ADCs but not with trastuzumab demonstrating a role for DM1. MKs internalized these ADCs in a HER2-independent, FcγRIIa-dependent manner, resulting in intracellular release of DM1. Binding and internalization of T-DM1 diminished as MKs matured; however, prolonged exposure of mature MKs to T-DM1 resulted in a disrupted cytoskeletal structure. Conclusions: These data support the hypothesis that T-DM1–induced thrombocytopenia is mediated in large part by DM1-induced impairment of MK differentiation, with a less pronounced effect on mature MKs. Clin Cancer Res; 21(1); 123–33. ©2014 AACR.

Evaluation and Clinical Analyses of Downstream Targets of the Akt Inhibitor GDC-0068

Purpose: The oncogenic PI3K/Akt/mTOR pathway is an attractive therapeutic target in cancer. However, it is unknown whether the pathway blockade required for tumor growth inhibition is clinically achievable. Therefore, we conducted pharmacodynamic studies with GDC-0068, an ATP competitive, selective Akt1/2/3 inhibitor, in preclinical models and in patients treated with this compound. Experimental Design: We used a reverse phase protein array (RPPA) platform to identify a biomarker set indicative of Akt inhibition in cell lines and human-tumor xenografts, and correlated the degree of pathway inhibition with antitumor activity. Akt pathway activity was measured using this biomarker set in pre- and post-dose tumor biopsies from patients treated with GDC-0068 in the dose escalation clinical trial. Results: The set of biomarkers of Akt inhibition is composed of 10 phosphoproteins, including Akt and PRAS40, and is modulated in a dose-dependent fashion, both in vitro and in vivo. In human-tumor xenografts, this dose dependency significantly correlated with tumor growth inhibition. Tumor biopsies from patients treated with GDC-0068 at clinically achievable doses attained a degree of biomarker inhibition that correlated with tumor growth inhibition in preclinical models. In these clinical samples, compensatory feedback activation of ERK and HER3 was observed, consistent with preclinical observations. Conclusion: This study identified a set of biomarkers of Akt inhibition that can be used in the clinical setting to assess target engagement. Here, it was used to show that robust Akt inhibition in tumors from patients treated with GDC-0068 is achievable, supporting the clinical development of this compound in defined patient populations. Clin Cancer Res; 19(24); 6976–86. ©2013 AACR.

Abstract B154: A first-in-human trial of GDC-0068: A novel, oral, ATP-competitive Akt inhibitor, demonstrates robust suppression of the Akt pathway in surrogate and tumor tissues.

GDC-0068 is a highly selective ATP-competitive small molecule that inhibits all three isoforms of Akt with IC50 values of 5 to 30 nM. GDC-0068 selectively inhibits cancer cells with activated Akt signaling (e.g. via PTEN loss or PIK3CA mutations). Patients with advanced solid tumors were treated with GDC-0068 using a 3+3 escalation design. GDC-0068 was dosed PO QD on a 21-day on, 7-day off schedule; endpoints included safety, pharmacokinetics (PK) and determination of pathway knockdown. Pharmacodynamics (PD) endpoints in surrogate tissue [platelet rich plasma (PRP)] were evaluated in all patients. In addition, at least two patients per cohort had pre- and on-treatment (day 15) tumor biopsies at doses ≥100mg QD. The tumor tissue samples were evaluated using reverse phase protein array (RPPA). Thirty patients were enrolled across 7 cohorts (25, 50, 100, 200, 400, 600 and 800 mg QD). GDC-0068 was generally well-tolerated at doses ≤ 600 mg. Preliminary PK analyses show a dose proportional increase in exposure over the dose range tested. In addition, clinical exposures at doses ≥ 200 mg QD met or exceeded GDC-0068 exposures associated with tumor stasis in multiple PTEN null preclinical xenograft models. PK/PD evaluation showed dose-dependent Akt pathway inhibition in the PRP assay, with ≥70% inhibition of pGSK3 in all patients at doses ≥ 200 mg QD. Pre- and on-treatment tumor biopsies showed ≥50% decrease in pPRAS40 and cyclin D1 in multiple patients at doses of 200 mg QD and higher. In addition, evidence of feedback activation of the MAP kinase pathway after treatment with GDC-0068 was observed in biopsy samples. One patient with PTEN low/ PIK3CA H1047R/ KRAS wt CRC had prolonged stable disease and showed Akt pathway suppression by multiple downstream markers. GDC-0068 is well tolerated at doses ≤ 600 mg with a favorable safety profile and dose proportional pharmacokinetics. Treatment with GDC-0068 results in substantial pathway knockdown in both surrogate and tumor tissues at doses ≥ 200 mg QD. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr B154.