Maria Zajac-Kaye

Phase I trial of 17-dimethylaminoethylamino-17-demethoxygeldanamycin (17-DMAG), a heat shock protein inhibitor, administered twice weekly in patients with advanced malignancies

PURPOSE Phase I dose-escalation study to determine the toxicity and maximum tolerated dose (MTD) of 17-dimethylaminoethylamino-17-demethoxygeldanamycin (17-DMAG), a heat shock protein 90 (Hsp90) inhibitor, administered on a twice weekly schedule in patients with advanced cancer. EXPERIMENTAL DESIGN 17-DMAG was administered as a 1- to 2-h infusion twice weekly in 4-week cycles. An accelerated titration design was followed until toxicity was observed, at which point standard dose-escalation proceeded. MTD was defined as the dose at which no more than one of the six patients experienced a dose-limiting toxicity (DLT). Pharmacokinetics were assessed, and Hsp70 mRNA, whose gene product is a chaperone previously shown to be upregulated following the inhibition of Hsp90, was measured in peripheral blood mononuclear cells (PBMCs). RESULTS A total of 31 patients received 92 courses of treatment. The MTD was 21mg/m(2)/d; 20 patients were enrolled at this dose level. Nine patients had stable disease for a median of 4 (range 2-22) months. Both C(max) and AUC increased proportionally with dose. The most common toxicities were grade 1 or 2 fatigue, anorexia, nausea, blurred vision and musculoskeletal pain. DLTs were peripheral neuropathy and renal dysfunction. Expression of Hsp70 mRNA in PBMCs was highly variable. CONCLUSION Twice-weekly i.v. infusion of 17-DMAG is well tolerated, and combination phase I studies are warranted.

Current Understanding of the Molecular Biology of Pancreatic Neuroendocrine Tumors

Pancreatic neuroendocrine tumors (PanNETs) are complicated and often deadly neoplasms. A recent increased understanding of their molecular biology has contributed to expanded treatment options. DNA sequencing of samples derived from patients with PanNETs and rare genetic syndromes such as multiple endocrine neoplasia type 1 (MEN1) and Von Hippel-Lindau (VHL) syndrome reveals the involvement of MEN1, DAXX/ATRX, and the mammalian target of rapamycin (mTOR) pathways in PanNET tumorigenesis. Gene knock-out/knock-in studies indicate that inactivation of factors including MEN1 and abnormal PI3K/mTOR signaling uncouples endocrine cell cycle progression from the control of environmental cues such as glucose, leading to islet cell overgrowth. In addition, accumulating evidence suggests that further impairment of endothelial-endocrine cell interactions contributes to tumor invasion and metastasis. Recent phase III clinical trials have shown that therapeutic interventions, such as sunitinib and everolimus, targeting those signal transduction pathways improve disease-free survival rates. Yet, cure in the setting of advanced disease remains elusive. Further advances in our understanding of the molecular mechanisms of PanNETs and improved preclinical models will assist in developing personalized therapy utilizing novel drugs to provide prolonged control or even cure the disease.

Disruption of Myc-Tubulin Interaction by Hyperphosphorylation of c-Myc during Mitosis or by Constitutive Hyperphosphorylation of Mutant c-Myc in Burkitt's Lymphoma

ABSTRACT Somatic mutations at Thr-58 of c-Myc have been detected in Burkitts lymphoma (BL) tumors and have been shown to affect the transforming potential of the Myc oncoprotein. In addition, the N-terminal domain of c-Myc has been shown to interact with microtubules in vivo, and the binding of c-Myc to α-tubulin was localized to amino acids 48 to 135 within the c-Myc protein. We demonstrate that c-Myc proteins harboring a naturally occurring mutation at Thr-58 from BL cell lines have increased stability and are constitutively hyperphosphorylated, which disrupts the in vivo interaction of c-Myc with α-tubulin. In addition, we show that wild-type c-Myc–α-tubulin interactions are also disrupted during a transient mitosis-specific hyperphosphorylation of c-Myc, which resembles the constitutive hyperphosphorylation pattern of Thr-58 in BL cells.

DNA Methyltransferase Inhibitor, Zebularine, Delays Tumor Growth and Induces Apoptosis in a Genetically Engineered Mouse Model of Breast Cancer

Zebularine is a novel potent inhibitor of both cytidine deaminase and DNA methylation. We examined the effect of zebularine on mammary tumor growth in genetically engineered MMTV-PyMT transgenic mice that develop mammary tumors at 60 days of age with 100% penetrance. The MMTV-PyMT transgenic mice were randomized at 46 days of age into control (n = 25) and zebularine (n = 25) treatment groups and monitored for parameters of tumor growth. Zebularine was administered at 5 mg/mL in drinking water. We observed a significant delay in the growth of mammary tumors in zebularine-treated mice with a statistically significant reduction (P = 0.0135) in total tumor burden at 94 days of age when the mice were sacrificed. After 48 days of zebularine treatment, the tumors were predominantly necrotic compared with untreated animals. In addition, a high apoptotic index by terminal deoxynucleotidyl transferase–mediated dUTP nick end labeling assay was observed as early as 13 days following treatment. Immunoblot analysis showed depletion of DNMT1 and partial depletion of DNMT3b after zebularine treatment. Microarray analyses of global gene expression identified upregulation of twelve methylation-regulated genes as well as a set of candidate cancer genes that participate in cell growth and apoptosis. In summary, zebularine inhibits the growth of spontaneous mammary tumors and causes early onset of tumor cell necrosis and apoptosis in a genetically engineered mouse model of breast cancer. Defining the parameters of zebularine-mediated tumor inhibition may advance the future development of DNA methyltransferase inhibitors as an effective cancer treatment. Mol Cancer Ther; 11(2); 370–82. ©2011 AACR.

RET cooperates with RB/p53 inactivation in a somatic multi-step model for murine thyroid cancer

Mice bred to carry germline Rb and p53 null alleles are associated with a tumor spectrum that overlaps with the inherited multiple endocrine neoplasia-1 (MEN1) and MEN2 syndromes in humans, including medullary thyroid cancer (MTC). To study the genetic basis for these tumors, we microdissected MTC specimens or obtained fresh MTC tissue from nine independent Rb+/− p53+/− mice, amplified the region of the Ret gene known to be mutated in human MTC, and detected acquired missense Ret mutations in four different mice. These mutations were localized to a group of tandem cysteines which are analogous to activating germline mutations observed in human MEN2A and familial MTC (FMTC). To determine whether the remaining wild type Rb allele was inactivated in these murine MTC samples, we subjected tumor tissue to immunohistochemical staining with an Rb antibody, and demonstrated the absence of RB staining in murine MTC, while normal tissue retained RB nuclear staining. These findings demonstrate the ability of the gene knockout model to recapitulate somatic multi-step tumorigenesis and suggest that the development of a murine neuroendocrine tumor requires mutational dysregulation within both receptor tyrosine kinase and nuclear tumor suppressor gene pathways.

Enhanced activity of the CREB co-activator Crtc1 in LKB1 null lung cancer

Activation of Crtc1 (also known as Mect1/Torc1) by a t(11;19) chromosomal rearrangement underlies the etiology of malignant salivary gland tumors. As LKB1 is a target for mutational inactivation in lung cancer and was recently shown to regulate hepatic Crtc2/CREB transcriptional activity in mice, we now present evidence suggesting disruption of an LKB1/Crtc pathway in cancer. Although Crtc1 is preferentially expressed in adult brain tissues, we observed elevated levels of steady-state Crtc1 in thoracic tumors. In addition, we show that somatic loss of LKB1 is associated with underphosphorylation of endogenous Crtc1, enhanced Crtc1 nuclear localization and enhanced expression of the Crtc prototypic target gene, NR4A2/Nurr1. Inhibition of NR4A2 was associated with growth suppression of LKB1 null tumors, but showed little effect on LKB1-wildtype cells. These data strengthen the role of dysregulated Crtc as a bona fide cancer gene, present a new element to the complex LKB1 tumorigenic axis, and suggest that Crtc genes may be aberrantly activated in a wider range of common adult malignancies.

Poly(ADP-ribose) polymerase inhibition enhances p53-dependent and -independent DNA damage responses induced by DNA damaging agent.

Targeting DNA repair with poly(ADP-ribose) polymerase (PARP) inhibitors has shown a broad range of anti-tumor activity in patients with advanced malignancies with and without BRCA deficiency. It remains unclear what role p53 plays in response to PARP inhibition in BRCA-proficient cancer cells treated with DNA damaging agents. Using gene expression microarray analysis, we find that DNA damage response (DDR) pathways elicited by veliparib (ABT-888), a PARP inhibitor, plus topotecan comprise the G1/S checkpoint, ATM, and p53 signaling pathways in p53-wildtype cancer cell lines and BRCA1, BRCA2 and ATR pathway in p53-mutant lines. In contrast, topotecan alone induces the G1/S checkpoint pathway in p53-wildtype lines and not in p53-mutant cells. These responses are coupled with G2/G1 checkpoint effectors p21CDKN1A upregulation, and Chk1 and Chk2 activation. The drug combination enhances G2 cell cycle arrest, apoptosis and a marked increase in cell death relative to topotecan alone in p53-wildtype and p53-mutant or -null cells. We also show that the checkpoint kinase inhibitor UCN-01 abolishes the G2 arrest induced by the veliparib and topotecan combination and further increases cell death in both p53-wildtype and -mutant cells. Collectively, PARP inhibition by veliparib enhances DDR and cell death in BRCA-proficient cancer cells in a p53-dependent and -independent fashion. Abrogating the cell-cycle arrest induced by PARP inhibition plus chemotherapeutics may be a strategy in the treatment of BRCA-proficient cancer.

EGFRvIII undergoes activation-dependent downregulation mediated by the Cbl proteins

The overexpression or mutation of tyrosine kinases (TKs), such as the epidermal growth factor receptor (EGFR), can lead to the development of cancer. The most common mutation of the EGFR in glioblastomas is the deletion of exons 2–7 known as the EGFRvIII. This mutant receptor cannot bind EGF but, instead, is constitutively active. The Cbl family of ubiquitin ligases (Cbl, Cbl-b, and Cbl-c) targets the activated EGFR for degradation. As the EGFRvIII is transforming, we investigated whether it could be downregulated by the Cbl proteins. The overexpression of all three Cbl proteins resulted in the ubiquitination and degradation of the EGFRvIII. As with the wild-type EGFR, the TK-binding domain and the RING finger of Cbl-b are sufficient for the downregulation of the EGFRvIII. Also, we found that Cbl-b is recruited to the EGFRvIII and inhibits the transformation of NIH 3T3 cells by the EGFRvIII. Mutation of the Cbl-binding site (Y1045F) in the EGFRvIII inhibits its ubiquitination and downregulation by Cbl-b and enhances its ability to transform. Furthermore, the EGFR TK inhibitor, AG 1478, prevents the downregulation of the EGFRvIII by the Cbl proteins and antagonizes the ability of an immunotoxin directed against the EGFRvIII to kill cells expressing this receptor. In conclusion, the EGFRvIII does not transform by escaping regulation by Cbl proteins and this activation-induced downregulation of the EGFRvIII has an important role in mediating the toxicity of anti-EGFRvIII immunotoxins.

Disruption of the protein interaction between FAK and IGF-1R inhibits melanoma tumor growth.

FAK (focal adhesion kinase) and IGF-1R (insulin-like growth factor receptor-1) directly interact with each other and thereby activate crucial signaling pathways that benefit cancer cells. Inhibition of FAK and IGF-1R function has been shown to significantly decrease cancer cell proliferation and increase sensitivity to chemotherapy and radiation treatment. As a novel approach in human melanoma, we evaluated the effect of a small-molecule compound that disrupts the protein interaction of FAK and IGF-1R. Previously, using virtual screening and functional testing, we identified a lead compound (INT2–31) that targets the known FAK-IGF-1R protein interaction site. We studied the ability of this compound to disrupt FAK-IGF-1R protein interactions, inhibit downstream signaling, decrease human melanoma cell proliferation, alter cell cycle progression, induce apoptosis and decrease tumor growth in vivo. INT2–31 blocked the interaction of FAK and IGF-1R in vitro and in vivo in melanoma cells and tumor xenografts through precluding the activation of IRS-1, leading to reduced phosphorylation of AKT upon IGF-1 stimulation. As a result, INT2–31 significantly inhibited cell proliferation and viability (range 0.05–10 μM). More importantly, 15 mg/kg of INT2–31 given for 21 d via intraperitoneal injection disrupted the interaction of FAK and IGF-1R and effectively decreased phosphorylation of tumor AKT, resulting in significant melanoma tumor regression in vivo. Our data suggest that the FAK-IGF-1R protein interaction is an important target, and disruption of this interaction with a novel small molecule (INT2–31) has potential anti-neoplastic therapeutic effects in human melanoma.

Elevated levels of thymidylate synthase linked to neoplastic transformation of mammalian cells.

Thymidylate synthase (TS), an enzyme that is essential for DNA synthesis and repair has been identified as an important biomarker for colorectal and other human cancers. The elevated steady-state levels of TS found in many common human malignancies have been thought to represent a secondary event in tumor formation. However, it has recently been demonstrated that the deregulated levels of ectopic TS may also have a causal effect on tumorgenesis since overexpression of human TS transforms immortalized mammalian cells to a malignant phenotype. Since the levels of TS are regulated by E2F-1 and thus are linked to the cell cycle pathway, regulating TS activity may be an important factor for the control of cell cycle progression and for the development of therapeutic strategies and cancer prevention.