David A. Alcorta

T (brachyury) gene duplication confers major susceptibility to familial chordoma

Using high-resolution array-CGH, we identified unique duplications of a region on 6q27 in four multiplex families with at least three cases of chordoma, a cancer of presumed notochordal origin. The duplicated region contains only the T (brachyury) gene, which is important in notochord development and is expressed in most sporadic chordomas. Our findings highlight the value of screening for complex genomic rearrangements in searches for cancer-susceptibility genes.

Molecular characterization of putative chordoma cell lines.

Immortal tumor cell lines are an important model system for cancer research, however, misidentification and cross-contamination of cell lines are a common problem. Seven chordoma cell lines are reported in the literature, but none has been characterized in detail. We analyzed gene expression patterns and genomic copy number variations in five putative chordoma cell lines (U-CH1, CCL3, CCL4, GB60, and CM319). We also created a new chordoma cell line, U-CH2, and provided genotypes for cell lines for identity confirmation. Our analyses revealed that CCL3, CCL4, and GB60 are not chordoma cell lines, and that CM319 is a cancer cell line possibly derived from chordoma, but lacking expression of key chordoma biomarkers. U-CH1 and U-CH2 both have gene expression profiles, copy number aberrations, and morphology consistent with chordoma tumors. These cell lines also harbor genetic changes, such as loss of p16, MTAP, or PTEN, that make them potentially useful models for studying mechanisms of chordoma pathogenesis and for evaluating targeted therapies.

Identification of repurposed small molecule drugs for chordoma therapy

Chordoma is a rare, slow growing malignant tumor arising from remnants of the fetal notochord. Surgery is the first choice for chordoma treatment, followed by radiotherapy, although postoperative complications remain significant. Recurrence of the disease occurs frequently due to the anatomy of the tumor location and violation of the tumor margins at the initial surgery. Currently, there are no effective drugs available for patients with chordoma. Due to the rarity of the disease, there is limited opportunity to test agents in clinical trials and no concerted effort to develop agents for chordoma in the pharmaceutical industry. To rapidly and efficiently identify small molecules that inhibit chordoma cell growth, we screened the NCGC Pharmaceutical Collection (NPC) containing approximately 2800 clinically approved and investigational drugs at 15 different concentrations in chordoma cell lines, U-CH1 and U-CH2. We identified a group of drugs including bortezomib, 17-AAG, digitoxin, staurosporine, digoxin, rubitecan, and trimetrexate that inhibited chordoma cell growth, with potencies from 10 to 370 nM in U-CH1 cells, but less potently in U-CH2 cells. Most of these drugs also induced caspase 3/7 activity with a similar rank order as the cytotoxic effect on U-CH1 cells. Cantharidin, digoxin, digitoxin, staurosporine, and bortezomib showed similar inhibitory effect on cell lines and 3 primary chordoma cell cultures. The combination treatment of bortezomib with topoisomerase I and II inhibitors increased the therapeutic potency in U-CH2 and patient-derived primary cultures. Our results provide information useful for repurposing currently approved drugs for chordoma and potential approach of combination therapy.

X-Ray Psoralen Activated Cancer Therapy (X-PACT).

This work investigates X-PACT (X-ray Psoralen Activated Cancer Therapy): a new approach for the treatment of solid cancer. X-PACT utilizes psoralen, a potent anti-cancer therapeutic with current application to proliferative disease and extracorporeal photopheresis (ECP) of cutaneous T Cell Lymphoma. An immunogenic role for light-activated psoralen has been reported, contributing to long-term clinical responses. Psoralen therapies have to-date been limited to superficial or extracorporeal scenarios due to the requirement for psoralen activation by UVA light, which has limited penetration in tissue. X-PACT solves this challenge by activating psoralen with UV light emitted from novel non-tethered phosphors (co-incubated with psoralen) that absorb x-rays and re-radiate (phosphoresce) at UV wavelengths. The efficacy of X-PACT was evaluated in both in-vitro and in-vivo settings. In-vitro studies utilized breast (4T1), glioma (CT2A) and sarcoma (KP-B) cell lines. Cells were exposed to X-PACT treatments where the concentrations of drug (psoralen and phosphor) and radiation parameters (energy, dose, and dose rate) were varied. Efficacy was evaluated primarily using flow cell cytometry in combination with complimentary assays, and the in-vivo mouse study. In an in-vitro study, we show that X-PACT induces significant tumor cell apoptosis and cytotoxicity, unlike psoralen or phosphor alone (p<0.0001). We also show that apoptosis increases as doses of phosphor, psoralen, or radiation increase. Finally, in an in-vivo pilot study of BALBc mice with syngeneic 4T1 tumors, we show that the rate of tumor growth is slower with X-PACT than with saline or AMT + X-ray (p<0.0001). Overall these studies demonstrate a potential therapeutic effect for X-PACT, and provide a foundation and rationale for future studies. In summary, X-PACT represents a novel treatment approach in which well-tolerated low doses of x-ray radiation are delivered to a specific tumor site to generate UVA light which in-turn unleashes both short- and potentially long-term antitumor activity of photo-active therapeutics like psoralen.

Abstract 2219: Cancer prevention using a novel fatty acid synthase inhibitor

Tumor growth and survival is dependent upon de novo fatty acid synthesis regulated via preferential upregulation of fatty acid synthase (FASN) in tumor cells. Here a novel FASN inhibitor that has shown antitumor activity in established tumor models (Alwarawrah Y et al., 2016), now shows promise blocking oncogene-induced cell transformation and significantly delaying time to tumor development in a prevention study using a GEMM of triple negative breast cancer. Non-malignant, immortalized MCF10A human breast epithelial cells were infected with lentivirus encoding for the highly oncogenic 110 kDa truncated form of HER2, herein referred to as p110, under a doxycycline inducible promoter. Lentivirus containing a sham insert served as a control. Forty-eight hours after lentiviral infection, p110 was induced by dox and cells treated with FASN (2 and 4 μM) or vehicle alone for 21 days, and then assessed for cell growth and evidence of senescence. The C3TAg GEMM model of triple negative breast cancer was used to study FASN in the prevention setting. The mice (FVB/N background) express C(3)SV40 T-antigen resulting in the inactivation of p53 and Rb. Eighteen mice were randomized to receive FASN treatment (15mg/kg/ 5 days per week) or control (non-treatment [NT]). The mice were randomized and started treatment at 6 weeks of age, and continued until tumors reached an average size of 87.5mm3. All mice were monitored (5 times/week) for weight loss and time to tumor development. Expression of p110 led to the transformation of MCF10A cells, as evidenced by the formation of plaques in cell culture. Treatment with FASN blocked transformation in p110 expressing MCF10A cells and instead induced growth arrest, expression of senescence associated β-gal, and morphologic appearance consistent with cell senescence. Analysis of viable p110 expressing MCF10A cells that were maintained in the continuous presence of FASN for 60 days led to the identification of a potential mechanism of therapeutic resistance. In the C3TAg in vivo prevention study, FASN resulted in a significant prolongation in time to tumor development (Log-rank (Mantel-Cox); p A novel small molecule FASN inhibitor blocked oncogenic cell transformation and induced a state of senescence. Moreover, FASN significantly delayed the onset of mammary tumors in a GEMM model of aggressive triple negative breast cancer. Identification of a mechanism(s) enabling tumor cells to survive long term FASN inhibition will provide rationale combination strategies to induce permanent senescence or apoptosis, and improve the efficacy of FASN inhibitors as preventative agents. Citation Format: Neil L. Spector, Tim Haystead, Sumin Zhao, Yazan Alwarawrah, David Alcorta, William Kim, Jose R. Roques, Michael Trinkler, David B. Darr. Cancer prevention using a novel fatty acid synthase inhibitor [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2219. doi:10.1158/1538-7445.AM2017-2219

WE-FG-BRA-01: Cancer Treatment Utilizing Photo-Activation of Psoralen with KV X-Rays

PURPOSE This work investigates X-PACT (X-ray Psoralen Activated Cancer Therapy): a new approach for the treatment of cancer. X-PACT utilizes psoralen, a potent anti-cancer therapeutic with immunogenic anti-cancer potential. Psoralen therapies have been limited due to the requirement for psoralen activation by UVA light. X-PACT solves this challenge by activating psoralen with UV light emitted from novel non-tethered phosphors (co-incubated with psoralen) that absorb x-rays and reradiate (phosphoresce) at UV wavelengths. METHODS The efficacy of X-PACT was evaluated in both in-vitro and in-vivo settings. In-vitro studies utilized breast (4T1), glioma (CT2A) and sarcoma (KP-B) cell lines. Cells were exposed to X-PACT treatments where the concentrations of drug (psoralen and phosphor) and radiation parameters (energy, dose, and dose rate) were varied. Efficacy was evaluated primarily using flow cell cytometry to investigate treatment induced apoptosis. Methylene blue staining, and WST assays were also used. X-PACT was then evaluated in an in-vivo pilot study on BALBc mice with syngeneic 4T1 tumors, including control arms for X-PACT components. Analysis focused on tumor growth delay. RESULTS A multivariable regression analysis of 36 independent in-vitro irradiation experiments demonstrated that X-PACT induces significant tumor cell apoptosis and cytotoxicity on all three tumor cell lines in-vitro (p<0.0001). Neither psoralen nor phosphor alone had a strongly significant effect. The in-vivo studies show a pronounced tumor growth delay when compared to controls (42% reduction at 25 days, p=0.0002). CONCLUSIONS These studies demonstrate for the first time a therapeutic effect for X-PACT, and provide a foundation and rationale for future studies. X-PACT represents a novel treatment approach in which well-tolerated low doses of x-ray radiation generate UVA light in-situ (including deep seated lesions) which in-turn photo-activates powerful anticancer therapeutics which may lead to short and long term therapeutic effect. This work was supported by Immunolight Llc.

SU-E-T-93: Activation of Psoralen at Depth Using Kilovoltage X-Rays: Physics Considerations in Implementing a New Teletherapy Paradigm

Purpose: Psoralen is a UV-light activated anti-cancer biotherapeutic used for treating skin lesions (PUVA) and advanced cutaneous T-cell lymphoma (ECP). To date psoralen has not been used to treat deep seated tumors due to difficulty in generating UV-light at depth. We recently demonstrated psoralen activation at depth by introducing energy converting particles that absorb kV x-ray radiation and re-emit UV-light. Our in-vitro work found that 0.2–1Gy using 40–100kVp x-rays combined with psoralen and particles can induce a substantial apoptotic response beyond that expected from the sum of individual components. In preparation for a phase I clinical trial of canine companion animals, we address the physics and dosimetry considerations for applying this new teletherapy paradigm to an in-vivo setting. Methods: The kV on-board imaging (OBI) system mounted on a medical linear accelerator (Varian) was commissioned to deliver the prescribed dose (0.6Gy) using 80 and 100kVp. Dosimetric measurements included kVp, HVL, depth dose, backscatter factors, collimator and phantom scatter factors, field size factors, and blade leakage. Absolute dosimetry was performed following AAPM TG61 recommendations and verified with an independent kV dose meter. We also investigated collimated rotational delivery to minimize skin dose using simple dose calculations on homogeneous cylindrical phantoms. Results: Single beam delivery is feasible for shallow targets (<5cm) without exceeding skin tolerance, while a rotational delivery may be utilized for deeper targets; skin dose is ∼75% of target dose for 80kVp collimated rotational delivery to a 3cm target within a 20cm phantom. Heat loading was tolerable; 0.6Gy to 5cm can be delivered before the anode reaches 75% capacity. Conclusion: KV teletherapy for Psoralen activation in deep seated tissue was successfully commissioned for a Varian OBI machine for use in a phase I clinical trial in canines. Future work will use Monte Carlo dosimetry to investigate dose in presence of bone. Research funded by Immunolight LLC. H. Walder, Z. Fathi, & W. Beyer are employees of Immunolight LLC which holds a patent on the technology. Drs. Adamson and Oldham are consultants to Immunolight LLC.

Abstract 352: Murine model of chordoma: Sonic Hedgehog promoter-driven Cre activation of Brachyury (T) expression induces spinal disk abnormalities and perinatal lethal developmental defects.

Introduction: Chordoma is a rare malignancy arising in the axial skeleton from notochord remnants. The T-box transcription factor, Brachyury (T), is associated with familial chordoma, is expressed in over 90% of chordomas, and is a driver of cell growth in cultured chordoma cells. Suppression of T expression or function in vitro results in down-regulation of cell growth pathways and reduction in matrix formation. To further understand the mechanisms of T9s oncogenic properties, we sought to generate a murine model of constitutive, notochord-directed T expression. Methods: To produce elevated expression of T in the notochord-derived (ND) cells, we generated a murine transgenic animal in which a cassette (T/GFP) containing the β-actin promoter, a transcriptional stop sequence flanked by LoxP sites (FS), the murine T cDNA, an intragenic ribosomal entry sequence and a GFP cDNA was homologously recombined into the ROSA26 locus. We termed these animals FS-T/GFP mice. FS-T/GFP mice were mated to mice with Cre expression regulated by the developmentally-timed and tissue-specific control of the Sonic Hedgehog promoter (Shh-Cre mice). Results: Excision of the FS sequence by Cre was confirmed to result in constitutive expression of T and GFP in 293 cells prior to transgenic creation, and subsequently in cultured fibroblasts from ear punches of the transgenic animals. Mice containing homozygous FS-T/GFP at ROSA26 were healthy with no apparent physiologic or developmental defects and with the expected Mendelian ratios of sex and genotype. A cross of FS-T/GFP with Shh-Cre mice resulted in T/Cre pups that died perinatally with grossly evident heart, lung and kidney defects. In addition, these animals showed incomplete fusion of the vertebral bodies, and dysmorphic spinal disks. Elevated T protein levels and GFP co-expression in disk cells was demonstrated by immunofluorescent detection in tissue sections. Summary: Constitutive expression of T in ND lineage cells results in alteration of the morphology of intervertebral disks possibly indicative of a proliferative effect of Brachyury on ND cells. Expression of T in “off-target” tissues that express Sonic Hedgehog likely results in perinatal death. Citation Format: David A. Alcorta, Jennifer A. Maier, Brian D. Harfe, Michael J. Kelley. Murine model of chordoma: Sonic Hedgehog promoter-driven Cre activation of Brachyury (T) expression induces spinal disk abnormalities and perinatal lethal developmental defects. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 352. doi:10.1158/1538-7445.AM2013-352 Note: This abstract was not presented at the AACR Annual Meeting 2013 because the presenter was unable to attend.