Preclinical Characterization of an Antibody-Drug Conjugate Targeting CS-1 and the Identification of Uncharacterized Populations of CS-1-Positive Cells.

Multiple myeloma is a hematologic cancer that disrupts normal bone marrow function and has multiple lines of therapeutic options, but is incurable as patients ultimately relapse. We developed a novel antibody-drug conjugate (ADC) targeting CS-1, a protein that is highly expressed on multiple myeloma tumor cells. The anti-CS-1 mAb specifically bound to cells expressing CS-1 and, when conjugated to a cytotoxic pyrrolobenzodiazepine payload, reduced the viability of multiple myeloma cell lines in vitro In mouse models of multiple myeloma, a single administration of the CS-1 ADC caused durable regressions in disseminated models and complete regression in a subcutaneous model. In an exploratory study in cynomolgus monkeys, the CS-1 ADC demonstrated a half-life of 3 to 6 days; however, no highest nonseverely toxic dose was achieved, as bone marrow toxicity was dose limiting. Bone marrow from dosed monkeys showed reductions in progenitor cells as compared with normal marrow. In vitro cell killing assays demonstrated that the CS-1 ADC substantially reduced the number of progenitor cells in healthy bone marrow, leading us to identify previously unreported CS-1 expression on a small population of progenitor cells in the myeloid-erythroid lineage. This finding suggests that bone marrow toxicity is the result of both on-target and off-target killing by the ADC.

BMI1 silencing enhances docetaxel activity and impairs antioxidant response in prostate cancer.

The BMI1 oncogene promotes prostate cancer (PC) progression. High B-cell-specific Moloney murine leukemia virus integration site 1 (BMI1) expression predicts poor prognosis in PC patients. Recent evidence suggests that BMI1 may also play a role in docetaxel chemoresistance. However, mechanisms and clinical significance of BMI1-related chemoresistance have not been investigated. For this purpose, BMI1 was silenced in 2 PC cell lines (LNCaP and DU 145). Cell proliferation and apoptosis after docetaxel treatment were measured. Guanine oxidation was assessed by in-cell western. Global gene expression analysis was performed on BMI1 silenced cells. Oncomine database was used to compare in vitro data with gene expression in PC samples. BMI1 silencing had no effect on cell proliferation but significantly enhanced docetaxel-induced antitumor activity. Gene expression analysis demonstrated that BMI1 silencing downregulates a set of antioxidant genes. Docetaxel treatment increased guanine oxidation, whereas the antioxidant N-acetyl cysteine rescued docetaxel-induced cell death. Examination of clinical datasets revealed a positive correlation of BMI1 and antioxidant gene expression. BMI1-controlled antioxidant genes were predictive of poor prognosis in PC patients. In conclusion, BMI1 enhances antioxidant response, thereby allowing PC survival after docetaxel-based chemotherapy. BMI1-controlled antioxidant genes are overexpressed in aggressive PC and should be tested as predictors of chemotherapy failure.

Identification of vitronectin as an extrinsic inducer of cancer stem cell differentiation and tumor formation.

There is mounting evidence that tumors are initiated by a rare subset of cells called cancer stem cells (CSCs). CSCs are generally quiescent, self-renew, form tumors at low numbers, and give rise to the heterogeneous cell types found within a tumor. CSCs isolated from multiple tumor types differentiate both in vivo and in vitro when cultured in serum, yet the factors responsible for their differentiation have not yet been identified. Here we show that vitronectin is the component of human serum driving stem cell differentiation through an integrin alpha V beta 3-dependent mechanism. CSCs cultured on vitronectin result in downregulation of stem cell genes, modulation of differentiation markers, and loss of beta-catenin nuclear localization. Blocking integrin alpha V beta 3 inhibits differentiation and subsequently tumor formation. Thus, CSCs must be engaged by one or more extracellular signals to differentiate and initiate tumor formation, defining a new axis for future novel therapies aimed at both the extrinsic and intracellular pathways.

Invasive prostate cancer cells are tumor initiating cells that have a stem cell-like genomic signature.

Development of metastasis is a leading cause of cancer-induced death. Acquisition of an invasive tumor cell phenotype suggests loss of cell adhesion and basement membrane breakdown during a process termed epithelial-to-mesenchymal transition (EMT). Recently, cancer stem cells (CSC) were discovered to mediate solid tumor initiation and progression. Prostate CSCs are a subpopulation of CD44(+) cells within the tumor that give rise to differentiated tumor cells and also self-renew. Using both primary and established prostate cancer cell lines, we tested the assumption that CSCs are more invasive. The ability of unsorted cells and CD44-positive and -negative subpopulations to undergo Matrigel invasion and EMT was evaluated, and the gene expression profiles of these cells were analyzed by microarray and a subset confirmed using QRT-PCR. Our data reveal that a subpopulation of CD44(+) CSC-like cells invade Matrigel through an EMT, while in contrast, CD44(-) cells are non-invasive. Furthermore, the genomic profile of the invasive cells closely resembles that of CD44(+)CD24(-) prostate CSCs and shows evidence for increased Hedgehog signaling. Finally, invasive cells from DU145 and primary prostate cancer cells are more tumorigenic in NOD/SCID mice compared with non-invasive cells. Our data strongly suggest that basement membrane invasion, an early and necessary step in metastasis development, is mediated by these potential cancer stem cells.

Effects of manganese superoxide dismutase silencing on androgen receptor function and gene regulation: implications for castration-resistant prostate cancer.

PURPOSE: Advanced prostate cancer is first treated with androgen deprivation therapy. However, tumors become resistant to and grow despite castrate levels of testosterone. Growth and proliferation of CRPC is mediated by gain-of-function changes in the AR and AR reactivation. Expression of manganese superoxide dismutase (SOD2), which regulates cellular ROS, is markedly down-regulated in CRPC when compared with hormone-responsive tumors. EXPERIMENTAL DESIGN: Here, we knocked down SOD2 expression in AR-expressing LNCaP prostate cancer cells and determined gene expression changes, transcription factor binding, and AR transcription activity in SOD2 knockdown cells. RESULTS: SOD2 knockdown results in an increase in ROS. Gene expression changes induced by SOD2 knockdown results in the up-regulation of genes that are also androgen responsive and 46% of genes up-regulated 2-fold by the androgen ligand R1881 are also up-regulated to the same extent with SOD2 knockdown. The induction of many of these genes with SOD2 knockdown, such as VEGFA and FKBP5, is reversible with the antioxidant N-acetylcysteine, suggesting that this mechanism is directly linked to ROS. Furthermore, an array for transcription factor DNA-binding activity shows that SOD2 knockdown induces DNA binding by several transcription factors, including AR. SOD2 knockdown-induced AR activation was confirmed by electrophoretic mobility shift assay and luciferase activity, and both were readily reversible with N-acetylcysteine. CONCLUSIONS: These findings show that down-regulation of SOD2 induces AR activity in a ROS-dependent manner, and suggest that there may be a role for antioxidant therapy in CRPC.

Integrated molecular profiling of SOD2 expression in multiple myeloma.

Reactive oxygen species are known to be involved in several cellular processes, including cell signaling. SOD2 is a key enzyme in the conversion of reactive oxygen species and has been implicated in a host of disease states, including cancer. Using an integrated, whole-cell approach encompassing epigenetics, genomics, and proteomics, we have defined the role of SOD2 in multiple myeloma. We show that the SOD2 promoter is methylated in several cell lines and there is a correlative decrease in expression. Furthermore, myeloma patient samples have decreased SOD2 expression compared with healthy donors. Overexpression of SOD2 results in decreased proliferation and altered sensitivity to 2-methoxyestradiol-induced DNA damage and apoptosis. Genomic profiling revealed regulation of 65 genes, including genes involved in tumorigenesis, and proteomic analysis identified activation of the JAK/STAT pathway. Analysis of nearly 400 activated transcription factors identified 31 transcription factors with altered DNA binding activity, including XBP1, NFAT, forkhead, and GAS binding sites. Integration of data from our gestalt molecular analysis has defined a role for SOD2 in cellular proliferation, JAK/STAT signaling, and regulation of several transcription factors.

Reversal of p53 epigenetic silencing in multiple myeloma permits apoptosis by a p53 activator.

Inactivation of the p53 pathway is a common feature of neoplasia. Dysregulation of the p53 pathway has been shown to involve mutations of p53, increased expression of the p53 inhibitor HDM-2, or epigenetic silencing of the p53 promoter. In multiple myeloma, a neoplasia of terminally differentiated B cells, p53 mutations and deletions are relatively rare and occur in late stage disease. Here, we show that the p53 promoter is hypermethylated in several multiple myeloma cell lines in comparison to normal plasma cells. Two cell lines containing mutant p53, Lp-1 and OPM-2, show a methylation pattern that suggests that they contain one methylated and one unmethylated mutant allele. Two other cell lines, KMS-11 and OPM-2, show hypermethylation of p53 with a lack of expression. In all cell lines tested, treatment with a demethylating agents results in higher expression of p53. Furthermore, following increased expression of p53, treatment of the myeloma cell lines with a p53 activating peptide induces apoptosis. Therefore, combinatorial treatment with demethylating agents followed by delivery of a p53 activating peptide may be an effective therapeutic strategy against multiple myeloma.

DNA hypermethylation and partial gene silencing of human thymine- DNA glycosylase in multiple myeloma cell lines.

Multiple myeloma (MM) has prominent features of karyotypic instability at the earliest stage, leading to extreme genetic abnormalities as the disease progresses. These successive genetic alterations can be attributed, in part, to defects in DNA repair pathways. A possible mechanism of dysregulation of the DNA repair pathway is epigenetic gene silencing. Therefore, we sought to determine the methylation status of enzymes involved in the base excision repair pathway in multiple myeloma cell lines. Here, we report the aberrant DNA methylation of TDG, one of the enzymes involved in base excision repair of damaged DNA, in several multiple myeloma cell lines but not in normal human plasma cells. DNA hypermethylation of TDG in the MM cell lines leads to lower gene expression levels that results in less efficient DNA repair activity in response to hydrogen peroxideinduced DNA damage. Expression of exogenous TDG can functionally compensate for lower repair activities of damaged DNA in the KAS-6/1 myeloma cell line, which has extensive DNA hypermethylation of the TDG promoter. Hypermethylation of DNA damage repair genes in MM cell lines may provide an explanation for the frequent genomic instability, as well as point mutations, that are encountered in MM.