D. Marc Rosen

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.

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.

Targeting Carcinoma-Associated Fibroblasts Within the Tumor Stroma With a Fibroblast Activation Protein-Activated Prodrug

BACKGROUND Fibroblasts undergo a morphological transformation to a reactive phenotype in the tumor microenvironment characterized by the expression of proteins such as fibroblast activation protein (FAP), a post-prolyl endopeptidase with expression largely restricted to carcinoma-associated fibroblasts. Thapsigargin (TG) is a highly toxic natural plant product that triggers a rise in intracellular calcium levels and apoptosis. FAP is therefore a provocative target for the activation of prodrugs consisting of a FAP-specific peptide coupled to a potent cytotoxic analog of TG. METHODS The efficacy of FAP-activated peptidyl-TG prodrugs was tested in vitro in cell proliferation assays and effects on intracellular calcium in human cancer cell lines. The effects of FAP-activated prodrugs on tumor growth and host toxicity were tested in Balb-C nude MCF-7 and LNCaP xenograft mice (n = 9-11 per group). P values were calculated using permutation tests based on 50 000 permutations. Mixed effects models were used to account for correlations among replicate measures. All statistical tests were two-sided. RESULTS FAP-activated prodrugs killed human cancer cells at low nanomolar concentrations (MCF-7 cells: IC(50) = 3.5 nM). Amino acid-12ADT analogs from FAP-cleaved prodrugs, but not uncleaved prodrugs, produced a rapid rise in intracellular calcium within minutes of exposure. Immunohistochemical analysis of xenografts exposed to FAP-prodrugs documented stromal-selective cell death of fibroblasts, pericytes, and endothelial cells of sufficient magnitude to inhibit growth of MCF-7 and LNCaP xenografts with minimal systemic toxicity, whereas non-FAP cleavable prodrugs were inactive. MCF-7 and LNCaP xenografts treated with a FAP-activated prodrug had maximal treated-to-control tumor volume ratios of 0.36 (treated: mean = 0.206 mm(3), 95% CI = 0.068 to 0.344 mm(3); control: mean = 0.580 mm(3), 95% CI = 0.267 to 0.893 mm(3)) and 0.24 (treated: mean = 0.131 mm(3), 95% CI = 0.09 to 0.180 mm(3); control: mean = 0.543 mm(3), 95% CI = 0.173 to 0.913 mm(3)), respectively, on day 21 after therapy. CONCLUSIONS This study validates the proteolytic activity of FAP as a target for the activation of a systemically delivered cytotoxic prodrug and demonstrates that targeted killing of cells within the stromal compartment of the tumor microenvironment can produce a therapeutic response.

Use of methotrexate-based peptide substrates to characterize the substrate specificity of prostate-specific membrane antigen (PSMA).

Prostate-Specific Membrane Antigen (PSMA) is a glutamate carboxypeptidase II that is highly expressed by both normal and malignant prostate epithelial cells and by the neovasculature of many tumor types but is not expressed by endothelial cells in normal tissue. PSMA possesses the hydrolytic properties of an N-acetylated ?-linked acidic dipeptidase (NAALADase) and also functions as a pteroyl poly-?-glutamyl carboxypeptidase (i.e. folate hydrolase). Therefore, PSMA can be targeted for activation of peptide-based prodrugs within the extracellular fluid of prostate cancers. In this study, methotrexate-based peptide analogs were evaluated to identify PSMA selective substrates that are also stable to non-specific hydrolysis in human and mouse plasma. These methotrexate analogs were also characterized for in vitro toxicity against PSMA and non-PSMA producing human cancer cell lines. Analogs containing ?-linked glutamate residues were most efficiently hydrolyzed by PSMA, but were unstable in plasma. Analogs containing both ?- and ?-linked acidic amino acids were less efficiently hydrolyzed by PSMA but were most stable in plasma. Analogs were 5-10 fold more selectively toxic in vitro in the presence of active PSMA. These studies have identified PSMA selective, plasma stable peptide substrates that can be incorporated into prodrugs targeted for activation by PSMA within prostate cancer sites.

Dual inhibition of mitogen-activated protein kinase kinase and mammalian target of rapamycin in differentiated and anaplastic thyroid cancer.

CONTEXT Differentiated thyroid cancer and anaplastic thyroid cancer tumors frequently have activation of the ras/raf /MAPK kinase (MEK)/ERK and phosphatidylinositol 3-kinase (PI-3K)/AKT/mammalian target of rapamycin (mTOR) signaling pathways. OBJECTIVE The objective of the study was to investigate the efficacy of MEK and mTOR inhibitors in preclinical thyroid cancer treatment models with defined mutation status. EXPERIMENTAL DESIGN The MEK inhibitor AZD6244 (ARRY-142886) and mTOR inhibitor rapamycin were tested separately and in combination in 10 differentiated thyroid cancer and anaplastic thyroid cancer cell lines and in a xenograft model for evidence of pathway inhibition, growth inhibition, apoptosis, and long-range adaptation and resistance. RESULTS Seven of 10 tested lines had evidence of significant basal activity of the PI-3K/AKT/mTOR pathway, with elevated phosphorylated AKT and phosphorylated p70 S6 kinase. Activation of ras/RAF/MEK/ERK was equally common in this panel. All 10 lines exhibited better than 60% growth inhibition with combined MEK and mTOR inhibition, including lines with BRAF, Ret-PTC, ras, and PTEN mutations. Rapamycin or AZD6244 alone achieved this threshold in six and two lines, respectively. Dual-pathway inhibition in the Ret-PTC mutant cell line TPC1 caused an intense G(1) arrest in cell culture and reversible cytostatic inhibition in a xenograft model. We did not observe significant feedback up-regulation of AKT activation in either acute or prolonged exposures. CONCLUSION These preclinical results support the inclusion of thyroid cancer patients in early-phase clinical trials combining RAS/RAF/MEK/ERK and PI-3K/AKT/mTOR pathway inhibition.

PIK3CA and AKT1 Mutations Have Distinct Effects on Sensitivity to Targeted Pathway Inhibitors in an Isogenic Luminal Breast Cancer Model System

Purpose: Activating mutations in the phosphoinositide-3-kinase (PI3K)/AKT/mTOR pathway are present in the majority of breast cancers and therefore are a major focus of drug development and clinical trials. Pathway mutations have been proposed as predictive biomarkers for efficacy of PI3K-targeted therapies. However, the precise contribution of distinct PI3K pathway mutations to drug sensitivity is unknown. Experimental Design: We describe the creation of a physiologic human luminal breast cancer cell line model to study the phenotype of these mutations using the MCF-7 cell line. We used somatic cell gene targeting to “correct” PIK3CA E545K-mutant alleles in MCF-7 cells to wild-type sequence. The AKT1 E17K hotspot mutation was knocked in on this wild-type background. Results: Loss of mutant PIK3CA dramatically reduced phosphorylation of AKT proteins and several known AKT targets, but other AKT target proteins and downstream effectors of mTOR were not affected. PIK3CA wild-type cells exhibited reduced proliferation in vitro and in vivo. Knockin of the AKT1 E17K hotspot mutation on this PIK3CA wild-type background restored pathway signaling, proliferation, and tumor growth in vivo. PIK3CA, but not AKT1 mutation, increased sensitivity to the PI3K inhibitor GDC-0941 and the allosteric AKT inhibitor MK-2206. Conclusions: AKT1 E17K is a bona fide oncogene in a human luminal breast cancer context. Distinct PI3K pathway mutations confer differential sensitivity to drugs targeting the pathway at different points and by distinct mechanisms. These findings have implications for the use of tumor genome sequencing to assign patients to targeted therapies. Clin Cancer Res; 19(19); 5413–22. ©2013 AACR.

A prostate-specific antigen-activated N-(2-hydroxypropyl) methacrylamide copolymer prodrug as dual-targeted therapy for prostate cancer

Prostate cancer targeted peptide prodrugs that are activated by the serine protease activity of prostate-specific antigen (PSA) are under development in our laboratory. To enhance delivery and solubility of these prodrugs, macromolecular carriers consisting of N-(2-hydroxypropyl) methacrylamide (HPMA)–based copolymers were covalently coupled to a PSA-activated peptide prodrug. HPMA copolymers are water-soluble, nonimmunogenic synthetic carriers that exhibit promise for drug delivery applications. These macromolecular copolymers enter the interstitium of solid tumors by the enhanced permeability and retention effect. The PSA-activated peptide substrate imparts selectivity because it is specifically hydrolyzed to release a cytotoxin at the site of prostate tumor. Enzymatically active PSA is present in high amounts in the extracellular fluid of a tumor, but PSA is inactivated in blood by binding to serum protease inhibitors. As an initial proof of concept, the HPMA copolymer was synthesized with a peptide substrate (HSSKLQ) bound to a fluorophore, 7-amino-4-methylcoumarin (AMC). PSA cleavage of the HPMA-HSSKLQ-AMC copolymer was observed, which led to the synthesis of an HPMA-based copolymer with the prodrug SSKYQ-L12ADT [HPMA–morpholinocarbonyl-Ser-Ser-Lys-Tyr-Gln-Leu-12-aminododecanoyl thapsigargin (JHPD)]. L12ADT is a potent analogue of the highly cytotoxic natural product thapsigargin. HPMA-JHPD was hydrolyzed by PSA in vitro and was toxic to prostate cancer cells in the presence of active PSA. The HPMA-JHPD produced no systemic toxicity when given at a 500 μmol/L L12ADT equivalent dose. Analysis of tumor tissue from mice treated with a single or multiple dose of the HPMA-JHPD copolymer showed release and accumulation of the L12ADT toxin within the tumor tissue. [Mol Cancer Ther 2007;6(11):2928–37]

Single copies of mutant KRAS and mutant PIK3CA cooperate in immortalized human epithelial cells to induce tumor formation

The selective pressures leading to cancers with mutations in both KRAS and PIK3CA are unclear. Here, we show that somatic cell knockin of both KRAS G12V and oncogenic PIK3CA mutations in human breast epithelial cells results in cooperative activation of the phosphoinositide 3-kinase (PI3K) and mitogen-activated protein kinase (MAPK) pathways in vitro, and leads to tumor formation in immunocompromised mice. Xenografts from double-knockin cells retain single copies of mutant KRAS and PIK3CA, suggesting that tumor formation does not require increased copy number of either oncogene, and these results were also observed in human colorectal cancer specimens. Mechanistically, the cooperativity between mutant KRAS and PIK3CA is mediated in part by Ras/p110α binding, as inactivating point mutations within the Ras-binding domain of PIK3CA significantly abates pathway signaling. In addition, Pdk1 activation of the downstream effector p90RSK is also increased by the combined presence of mutant KRAS and PIK3CA. These results provide new insights into mutant KRAS function and its role in carcinogenesis.

MACROD2 overexpression mediates estrogen independent growth and tamoxifen resistance in breast cancers

Significance Despite the widespread use and success of tamoxifen for treating ER-positive breast cancers, overcoming resistance to this drug remains an unmet need in clinical breast oncology. The results presented in this study demonstrate that overexpression of a novel gene, MACROD2, can mediate tamoxifen resistance and estrogen independent growth in human breast cancers, and that amplification of MACROD2 in primary breast tumors is associated with worse overall survival. Tamoxifen is effective for treating estrogen receptor-alpha (ER) positive breast cancers. However, few molecular mediators of tamoxifen resistance have been elucidated. Here we describe a previously unidentified gene, MACROD2 that confers tamoxifen resistance and estrogen independent growth. We found MACROD2 is amplified and overexpressed in metastatic tamoxifen-resistant tumors. Transgene overexpression of MACROD2 in breast cancer cell lines results in tamoxifen resistance, whereas RNAi-mediated gene knock down reverses this phenotype. MACROD2 overexpression also leads to estrogen independent growth in xenograft assays. Mechanistically, MACROD2 increases p300 binding to estrogen response elements in a subset of ER regulated genes. Primary breast cancers and matched metastases demonstrate MACROD2 expression can change with disease evolution, and increased expression and amplification of MACROD2 in primary tumors is associated with worse overall survival. These studies establish MACROD2 as a key mediator of estrogen independent growth and tamoxifen resistance, as well as a potential novel target for diagnostics and therapy.

HER2 missense mutations have distinct effects on oncogenic signaling and migration

Significance The discovery of human epidermal growth factor receptor 2 (HER2) missense mutations in breast and other cancers potentially make such tumors susceptible to current and future HER2-targeted therapies. However, the majority of HER2 mutations occur in HER2 nonamplified cancers, and whether these mutations will predict for sensitivity to HER2-directed therapies remains unknown. Using genome editing, the data presented here suggest that HER2 missense mutations are functionally distinct and require additional oncogenic input to impart cancerous phenotypes. These results suggest that HER2 missense mutations by themselves may not be reliable predictors of response to HER2-targeted therapies, a hypothesis currently being tested in genomically driven clinical trials. Recurrent human epidermal growth factor receptor 2 (HER2) missense mutations have been reported in human cancers. These mutations occur primarily in the absence of HER2 gene amplification such that most HER2-mutant tumors are classified as “negative” by FISH or immunohistochemistry assays. It remains unclear whether nonamplified HER2 missense mutations are oncogenic and whether they are targets for HER2-directed therapies that are currently approved for the treatment of HER2 gene-amplified breast cancers. Here we functionally characterize HER2 kinase and extracellular domain mutations through gene editing of the endogenous loci in HER2 nonamplified human breast epithelial cells. In in vitro and in vivo assays, the majority of HER2 missense mutations do not impart detectable oncogenic changes. However, the HER2 V777L mutation increased biochemical pathway activation and, in the context of a PIK3CA mutation, enhanced migratory features in vitro. However, the V777L mutation did not alter in vivo tumorigenicity or sensitivity to HER2-directed therapies in proliferation assays. Our results suggest the oncogenicity and potential targeting of HER2 missense mutations should be considered in the context of cooperating genetic alterations and provide previously unidentified insights into functional analysis of HER2 mutations and strategies to target them.