Kelly Waldeck

The Heat Shock Protein 90 Inhibitor, 17-Allylamino-17-demethoxygeldanamycin, Enhances Osteoclast Formation and Potentiates Bone Metastasis of a Human Breast Cancer Cell Line

Breast cancer metastasis to the bone occurs frequently, causing numerous complications including severe pain, fracture, hypercalcemia, and paralysis. Despite its prevalence and severity, few effective therapies exist. To address this, we examined whether the heat shock protein 90 (Hsp90) inhibitor, 17-allylamino-17-demethoxygeldanamycin (17-AAG), would be efficacious in inhibiting breast cancer metastasis to bone. Utilizing the human breast cancer subline, MDA-MB-231SA, previously in vivo selected for its enhanced ability to generate osteolytic bone lesions, we determined that 17-AAG potently inhibited its in vitro proliferation and migration. Moreover, 17-AAG significantly reduced MDA-MB-231SA tumor growth in the mammary-fat pad of nude mice. Despite these findings, 17-AAG enhanced the incidence of bone metastasis and osteolytic lesions following intracardiac inoculation in the nude mouse. Consistent with these findings, 17-AAG enhanced osteoclast formation 2- to 4-fold in mouse bone marrow/osteoblast cocultures, receptor activator of nuclear factor kappaB ligand (RANKL)-stimulated bone marrow, and RAW264.7 cell models of in vitro osteoclastogenesis. Moreover, the drug enhanced osteoclastogenesis in human cord blood progenitor cells, demonstrating that its effects were not limited to mouse models. In addition to 17-AAG, other Hsp90 inhibitors, such as radicicol and herbimycin A, also enhanced osteoclastogenesis. A pro-osteolytic action of 17-AAG independent of tumor presence was also determined in vivo, in which 17-AAG-treated tumor-naive mice had reduced trabecular bone volume with an associated increase in osteoclast number. Thus, HSP90 inhibitors can stimulate osteoclast formation, which may underlie the increased incidence of osteolysis and skeletal tumor incidence caused by 17-AAG in vivo. These data suggest an important contraindication to the Hsp90 targeted cancer therapy currently undergoing clinical trial.

Loss of CDKN2A expression is a frequent event in primary invasive melanoma and correlates with sensitivity to the CDK4/6 inhibitor PD0332991 in melanoma cell lines

We have investigated the potential for the p16‐cyclin D‐CDK4/6‐retinoblastoma protein pathway to be exploited as a therapeutic target in melanoma. In a cohort of 143 patients with primary invasive melanoma, we used fluorescence in situ hybridization to detect gene copy number variations (CNVs) in CDK4, CCND1, and CDKN2A and immunohistochemistry to determine protein expression. CNVs were common in melanoma, with gain of CDK4 or CCND1 in 37 and 18% of cases, respectively, and hemizygous or homozygous loss of CDKN2A in 56%. Three‐quarters of all patients demonstrated a CNV in at least one of the three genes. The combination of CCND1 gain with either a gain of CDK4 and/or loss of CDKN2A was associated with poorer melanoma‐specific survival. In 47 melanoma cell lines homozygous loss, methylation or mutation of CDKN2A gene or loss of protein (p16INK4A) predicted sensitivity to the CDK4/6 inhibitor PD0332991, while RB1 loss predicted resistance.

Relationship between Epidermal Growth Factor Receptor Status, p16 INK4A , and Outcome in Head and Neck Squamous Cell Carcinoma

Background: Human papilloma virus (HPV) infection is a powerful prognostic biomarker in head and neck squamous cell carcinoma (HNSCC). Increased epidermal growth factor receptor (EGFR) gene copy number and protein expression have been reported to be negative predictors of outcome. This study examined the relationship between HPV status, EGFR gene copy number, EGFR protein expression, and clinical outcome in HNSCC patients treated with chemoradiation. Methods: HPV status was determined using p16INK4A immunohistochemistry (IHC), EGFR gene copy number was evaluated with FISH, and EGFR protein expression by IHC in 212 subjects. Results:EGFR FISH was positive in 41 of 204 (20%) patients and was negatively correlated with failure-free survival (FFS; HR = 1.84, P = 0.027) and overall survival (OS; HR = 1.78, P = 0.082). For p16INK4A, 85 of 200 (42.5%) patients were found to be p16 positive, including 75 of 131 (57%) with oropharyngeal cancer. Patients with p16-positive oropharyngeal cancer had significantly improved FFS (HR = 0.28, P < 0.001) and OS (HR = 0.31, P = 0.002). Only 2 of 126 (1.6%) oropharyngeal cancer patients were found to be p16+/EGFR FISH+. EGFR IHC was positive in 81 of 93 (87%) of patients and was associated with poorer FFS (HR = 1.98, P = 0.35) and OS (HR = 2.52, P = 0.22). Conclusions: Increased EGFR gene copy number is largely restricted to p16INK4A-negative oropharyngeal cancer. Although p16INK4A and EGFR FISH are both predictive of outcome in univariate analyses, only p16INK4A remains independently predictive. Impact: Knowledge of HPV and EGFR status can have implications for treatment options and prognosis in HNSCC. Cancer Epidemiol Biomarkers Prev; 20(6); 1230–7. ©2011 AACR.

UV-Associated Mutations Underlie the Etiology of MCV-Negative Merkel Cell Carcinomas

Merkel cell carcinoma (MCC) is an uncommon, but highly malignant, cutaneous tumor. Merkel cell polyoma virus (MCV) has been implicated in a majority of MCC tumors; however, viral-negative tumors have been reported to be more prevalent in some geographic regions subject to high sun exposure. While the impact of MCV and viral T-antigens on MCC development has been extensively investigated, little is known about the etiology of viral-negative tumors. We performed targeted capture and massively parallel DNA sequencing of 619 cancer genes to compare the gene mutations and copy number alterations in MCV-positive (n = 13) and -negative (n = 21) MCC tumors and cell lines. We found that MCV-positive tumors displayed very low mutation rates, but MCV-negative tumors exhibited a high mutation burden associated with a UV-induced DNA damage signature. All viral-negative tumors harbored mutations in RB1, TP53, and a high frequency of mutations in NOTCH1 and FAT1. Additional mutated or amplified cancer genes of potential clinical importance included PI3K (PIK3CA, AKT1, PIK3CG) and MAPK (HRAS, NF1) pathway members and the receptor tyrosine kinase FGFR2. Furthermore, looking ahead to potential therapeutic strategies encompassing immune checkpoint inhibitors such as anti-PD-L1, we also assessed the status of T-cell-infiltrating lymphocytes (TIL) and PD-L1 in MCC tumors. A subset of viral-negative tumors exhibited high TILs and PD-L1 expression, corresponding with the higher mutation load within these cancers. Taken together, this study provides new insights into the underlying biology of viral-negative MCC and paves the road for further investigation into new treatment opportunities.

The mTORC1 Inhibitor Everolimus Prevents and Treats Eμ-Myc Lymphoma by Restoring Oncogene-Induced Senescence

UNLABELLED MYC deregulation is common in human cancer. IG-MYC translocations that are modeled in Eμ-Myc mice occur in almost all cases of Burkitt lymphoma as well as in other B-cell lymphoproliferative disorders. Deregulated expression of MYC results in increased mTOR complex 1 (mTORC1) signaling. As tumors with mTORC1 activation are sensitive to mTORC1 inhibition, we used everolimus, a potent and specific mTORC1 inhibitor, to test the requirement for mTORC1 in the initiation and maintenance of Eμ-Myc lymphoma. Everolimus selectively cleared premalignant B cells from the bone marrow and spleen, restored a normal pattern of B-cell differentiation, and strongly protected against lymphoma development. Established Eμ-Myc lymphoma also regressed after everolimus therapy. Therapeutic response correlated with a cellular senescence phenotype and induction of p53 activity. Therefore, mTORC1-dependent evasion of senescence is critical for cellular transformation and tumor maintenance by MYC in B lymphocytes. SIGNIFICANCE This work provides novel insights into the requirements for MYC-induced oncogenesis by showing that mTORC1 activity is necessary to bypass senescence during transformation of B lymphocytes. Furthermore, tumor eradication through senescence elicited by targeted inhibition of mTORC1 identifies a previously uncharacterized mechanism responsible for significant anticancer activity of rapamycin analogues and serves as proof-of-concept that senescence can be harnessed for therapeutic benefit

18F-FLT PET as a Surrogate Marker of Drug Efficacy During mTOR Inhibition by Everolimus in a Preclinical Cisplatin-Resistant Ovarian Tumor Model

Targeting the mammalian target of rapamycin (mTOR) pathway is a potential means of overcoming cisplatin resistance in ovarian cancer patients. Because mTOR inhibition affects cell proliferation, we aimed to study whether 3′-deoxy-3′-18F-fluorothymidine (18F-FLT) PET could be useful for monitoring early response to treatment with mTOR inhibitors in an animal model of cisplatin-resistant ovarian tumor. Methods: BALB/c nude mice bearing subcutaneous human SKOV3 ovarian cancer xenografts were treated with either the mTOR inhibitor everolimus (5 mg/kg) or vehicle, and 18F-FLT PET was performed at baseline, day 2, and day 7 of treatment. 18F-FLT uptake was evaluated by calculation of mean standardized uptake value (SUVmean) corrected for partial-volume effect. Ex vivo immunohistochemistry studies were performed on separate cohorts of mice treated as above and sacrificed at the same time points as for the PET studies. The ex vivo analysis included bromodeoxyuridine incorporation as a marker of cell proliferation, and phosphorylation of ribosomal protein S6 as a downstream marker of mTOR activation. Results: During the treatment period, no significant change in tumor 18F-FLT uptake was observed in the vehicle group, whereas in everolimus-treated mice, 18F-FLT SUVmean decreased by 33% (P = 0.003) at day 2 and 66% (P < 0.001) at day 7, compared with baseline. Notably, the reduction of 18F-FLT uptake observed at day 2 in the everolimus group preceded changes in tumor volume, and a significant difference in 18F-FLT uptake was observed between vehicle and drug-treated tumors at both day 2 (P = 0.0008) and day 7 (P = 0.01). In ex vivo studies, everolimus treatment resulted in a 98% reduction in phosphorylated ribosomal protein S6 immunostaining at day 2 (P = 0.02) and 91% reduction at day 7 (P = 0.003), compared with the vehicle group. Bromodeoxyuridine incorporation was reduced by 65% at day 2 (not significant) and by 41% at day 7 (P = 0.02) in drug versus vehicle groups. Conclusion: Reduction in 18F-FLT uptake correlates well with the level of mTOR inhibition by everolimus in the SKOV3 ovarian tumor model. These data suggest that early treatment monitoring by 18F-FLT PET may be of use in future preclinical or clinical trials evaluating treatment of cisplatin-resistant ovarian tumors by mTOR inhibitors.

The genomic landscape of phaeochromocytoma

Phaeochromocytomas (PCCs) and paragangliomas (PGLs) are rare neural crest‐derived tumours originating from adrenal chromaffin cells or extra‐adrenal sympathetic and parasympathetic tissues. More than a third of PCC/PGL cases are associated with heritable syndromes involving 13 or more known genes. These genes have been broadly partitioned into two groups based on pseudo‐hypoxic and receptor tyrosine kinase (RTK) signalling pathways. Many of these genes can also become somatically mutated, although up to one third of sporadic cases have no known genetic driver. Furthermore, little is known of the genes that co‐operate with known driver genes to initiate and drive tumourigenesis. To explore the genomic landscape of PCC/PGL, we applied exome sequencing, high‐density SNP‐array analysis, and RNA sequencing to 36 PCCs and four functional PGL tumours. All tumours displayed low mutation frequency, in contrast to frequent large segmental copy‐number alterations, aneuploidy, and evidence for chromothripsis in one case. Multi‐region sampling of one benign familial PCC tumour provided evidence for the timing of mutations during tumourigenesis and ongoing clonal evolution. Thirty‐one of 40 (77.5%) cases could be explained by germline or somatic mutations or structural alterations affecting known PCC/PGL genes. Deleterious somatic mutations were also identified in known tumour‐suppressor genes associated with genome maintenance and epigenetic modulation. A multitude of other genes were also found mutated that are likely important for normal neuroendocrine cell function. We revisited the gene‐expression subtyping of PCC/PGL by integrating published microarray data with our RNA‐seq data, enabling the identification of six robust gene‐expression subtypes. The majority of cases in our cohort with no identifiable driver mutation were classified into a gene‐expression subtype bearing similarity to MAX mutant PCC/PGL. Our data suggest there are yet unknown PCC/PGL cancer genes that can phenocopy MAX mutant PCC/PGL tumours. This study provides new insight into the molecular diversity and genetic origins of PCC/PGL tumours. Copyright

Long term, continuous exposure to panobinostat induces terminal differentiation and long term survival in the TH-MYCN neuroblastoma mouse model

Neuroblastoma is the most common extra‐cranial malignancy in childhood and accounts for ∼15% of childhood cancer deaths. Amplification of MYCN in neuroblastoma is associated with aggressive disease and predicts for poor prognosis. Novel therapeutic approaches are therefore essential to improving patient outcomes in this setting. The histone deacetylases are known to interact with N‐Myc and regulate numerous cellular processes via epigenetic modulation, including differentiation. In this study, we used the TH‐MYCN mouse model of neuroblastoma to investigate the antitumor activity of the pan‐HDAC inhibitor, panobinostat. In particular we sought to explore the impact of long term, continuous panobinostat exposure on the epigenetically driven differentiation process. Continuous treatment of tumor bearing TH‐MYCN transgenic mice with panobinostat for nine weeks led to a significant improvement in survival as compared with mice treated with panobinostat for a three‐week period. Panobinostat induced rapid tumor regression with no regrowth observed following a nine‐week treatment period. Initial tumor response was associated with apoptosis mediated via upregulation of BMF and BIM. The process of terminal differentiation of neuroblastoma into benign ganglioneuroma, with a characteristic increase in S100 expression and reduction of N‐Myc expression, occurred following prolonged exposure to the drug. RNA‐sequencing analysis of tumors from treated animals confirmed significant upregulation of gene pathways associated with apoptosis and differentiation. Together our data demonstrate the potential of panobinostat as a novel therapeutic strategy for high‐risk neuroblastoma patients.

Preclinical characterization of 18F-D-FPHCys, a new amino acid-based PET tracer

PurposeThe imaging potential of a new 18F-labelled methionine derivative, S-(3-[18F]fluoropropyl)-d-homocysteine (18F-D-FPHCys), and its selectivity for amino acid transporter subtypes were investigated in vitro and by imaging of human tumour xenografts.MethodsExpression of members of the system L (LAT isoforms 1–4 and 4F2hc) and ASCT (ASCT isoforms 1 and 2) amino acid transporter subclasses were assessed by quantitative real-time PCR in four human tumour models, including A431 squamous cell carcinoma, PC3 prostate cancer, and Colo 205 and HT-29 colorectal cancer lines. The first investigations for the characterization of 18F-D-FPHCys were in vitro uptake studies by comparing it with [1-14C]-l-methionine (14C-MET) and in vivo by PET imaging. In addition, the specific involvement of LAT1 transporters in 18F-D-FPHCys accumulation was tested by silencing LAT1 mRNA transcription with siRNAs. To determine the proliferative activity in tumour xenografts ex vivo, Ki-67 staining was used as a biomarker.ResultsA431 cells showed the highest 18F-D-FPHCys uptake in vitro and in vivo followed by Colo 205, PC3 and HT-29. A similar pattern of retention was observed with 14C-MET. 18F-D-FPHCys retention was strongly correlated with LAT1 expression both in vitro (R2 = 0.85) and in vivo (R2 = 0.99). Downregulation of LAT1 by siRNA inhibited 18F-D-FPHCys uptake, demonstrating a clear dependence on this transporter for tumour uptake. Furthermore, 18F-D-FPHCys accumulation mirrored cellular proliferation.ConclusionThe favourable properties of 18F-D-FPHCys make this tracer a promising imaging probe for detection of tumours as well as for the noninvasive evaluation and monitoring of tumour growth.

Abstract 1800: The PARP inhibitor, rucaparib enhances the antitumor activity of177Lu-DOTA-octreotate radionuclide therapy in preclinical models of neuroendocrine tumor

Purpose: Neuroendocrine tumors (NET) are a relatively rare, but heterogeneous group of tumors that arise predominantly in the gastrointestinal tract and bronchopulmonary system. While peptide receptor radionuclide therapy (PRRT) targeting somatostatin receptor 2 (SSTR2) has proved valuable in the treatment of NET, more effective clinical regimens are needed. In this study we investigated the efficacy of the combination of 177Lu-DOTA-octreotate (LuTate) PRRT and a poly (ADP-ribose) polymerase (PARP) inhibitor in well characterised preclinical NET models. Methods: A panel of seven cell lines with neuroendocrine features was characterised for SSTR2 expression, in vivo tumor growth properties, 68Ga-DOTA-octreotate (GaTate) uptake by positron emission tomography (PET) and sensitivity to LuTate. Representative tumor models were then selected to investigate the radiosensitizing potential of the PARP inhibitor, rucaparib when given in combination with LuTate. Results: Wide variation in SSTR2 mRNA and protein expression was observed between the various cell lines. Furthermore, immunohistochemical analysis revealed diffuse cytoplasmic expression of SSTR2 in vitro in contrast to strong cell membrane expression in vivo. Consistent with the in vivo cellular localisation, three tumor models demonstrated high tumor uptake of GaTate, an SSTR2 binding PET tracer used for the diagnosis and staging of NET. All three models responded to treatment with 20 MBq LuTate although the extent of the response was not predicted by SSTR2 expression or GaTate uptake. Treatment of A427-SSTR2 tumour bearing mice with two fortnightly cycles of 25 MBq Lutate in combination with rucaparib (15 mg/kg QD, IP) on days 1-5 of each cycle was well tolerated as assessed by the absence of any body weight loss. LuTate therapy alone induced tumour regression (56%) to day 32 after which the tumours began to regrow. Tumour regression in the combination treated group reached a maximum of 86% on day 35 (P Citation Format: Carleen Cullinane, Kelly Waldeck, Peter Eu, Rodney J. Hicks. The PARP inhibitor, rucaparib enhances the antitumor activity of 177Lu-DOTA-octreotate radionuclide therapy in preclinical models of neuroendocrine tumor. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1800. doi:10.1158/1538-7445.AM2015-1800