Durham, William F.

Abstract Metastatic castration-resistant prostate cancer (mCRPC) is an aggressive malignancy with poor outcomes. To investigate novel therapeutic strategies, we characterized three new metastatic prostate cancer patient derived-tumor xenograft (PDTX) models and developed 3D spheroids from each to investigate molecular targeted therapy combinations including CDK4/6 inhibitors (CDK4/6i) with AKT inhibitors (ATKi). Metastatic prostate cancer tissue was collected and three PDTX models were established and characterized using whole-exome sequencing. PDTX 3D spheroids were developed from these three PDTXs to show resistance patterns and test novel molecular-targeted therapies. CDK4/6i's were combined with AKTi's to assess synergistic antitumor response to prove our hypothesis that blockade of AKT overcomes drug resistance to CDK4/6i. This combination was evaluated in PDTX three-dimensional (3D) spheroids and in vivo experiments with responses measured by tumor volumes, PSA, and Ga-68 PSMA-11 PET-CT imaging. We demonstrated CDK4/6i's with AKTi's possess synergistic antitumor activity in three mCRPC PDTX models. These models have multiple unique pathogenic and deleterious genomic alterations with resistance to single-agent CDK4/6i's. Despite this, combination therapy with AKTi's was able to overcome resistance mechanisms. The IHC and Western blot analysis confirmed on target effects, whereas tumor volume, serum PSA ELISA, and radionuclide imaging demonstrated response to therapy with statistically significant SUV differences seen with Ga-68 PSMA-11 PET-CT. These preclinical data demonstrating antitumor synergy by overcoming single-agent CDK 4/6i as well as AKTi drug resistance provide the rational for a clinical trial combining a CDK4/6i with an AKTi in patients with mCRPC whose tumor expresses wild-type retinoblastoma 1.

Abstract Metastatic melanoma (MM) is the most aggressive type of skin cancer contributing to approximately 80% of all skin cancer related deaths. In 2016, it was estimated that more 76,000 cases would be diagnosed in the United States. The high metastatic potential and aggressive clinical behavior of this disease makes it a major health problem. As a consequence there has been notable development in for novel targeted (i.e. BRAF inhibitors) and immune therapies (i.e. anti-PD1 and CTLA4 inhibitors). However, despite improvements in patient outcomes, most patients develop resistance within 6-11 months on dual BRAF/MEK inhibition, and 5 months on immune therapy, highlighting the need to identify new therapies that improve disease management and patient survival. Increased expression of CyclinD1 has been reported to occur as a mechanism of resistance to BRAF inhibition, resulting in reactivation of the canonical activating pathway in melanoma, MAPK. Thus, we hypothesize that combined inhibition of CDK4/6 and MAPK signaling will provide a novel anti-tumor synergy drug combination superior to current standard of care in BRAF mutant patient-derived preclinical models. Using MM human tissue from patients we developed 3 BRAF mutant preclinical models investigating a novel triple therapy drug combination BRAFi/MEKi/CDK4/6i. Protein analysis and drug proliferation assays were used to design the novel combination. In vivo, we found statistically significant differences in tumor inhibition between the novel combination and the current standard of care. The triple therapy also showed promising results, in vivo, when assessed as a salvage therapy following tumor escape from BRAFi/MEKi combination treatment. Citation Format: Antoneicka L. Harris, Michael Thompson, Svetomir Markovic, William Durham, Louis Dawson, Daniel Small, John A. Copland. Tumor regression in novel triple therapy BRAF mutant metastatic melanoma patient-derived preclinical models [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 LB-130. doi:10.1158/1538-7445.AM2017-LB-130

Abstract Acinar cell carcinoma of the pancreas (ACC) is an uncommon malignancy, accounting for less than 1% of all pancreatic neoplasms. Because of its rarity, only a few retrospective studies are available to help guide management. We previously reported the case of a patient with metastatic ACC who achieved prolonged survival with doxorubicin as a result of personalized treatment designed in part on the basis of molecular and in-vitro data collected on analysis of the tumor and primary cells in culture developed from the liver metastasis. We now report the characterization of a patient derived xenograft (PDX) mouse model originating from this patient's ACC liver metastasis tissue. Antitumor activity of multiple drugs (5-FU, irinotecan, oxaliplatin, gemcitabine, bevacizumab, erlotinib, doxorubicin and imatinib) used as single agent therapy is demonstrated. Of the targeted and cytotoxic therapies used, oxaliplatin produced a dramatic and prolonged response to therapy even after withdrawal of treatment. Bevacizumab produced a significant response, as well. Serum lipase and tissue amylase levels correlated with the antitumor response to therapy. Thus, we have developed and characterized an ACC PDX model that may be used in drug discovery for the treatment of this rare cancer for which no standard of care exists. Note: This abstract was not presented at the meeting. Citation Format: Laura A. Marlow, Adam C. Mathias, Louis K. Dawson, William F. Durham, Kenneth A. Meshaw, Robert J. Mullin, Aidan J. Synnott, Ricardo Paz-Fumagalli, Murli Krishna, Daniel Von Hoff, Daniel L. Small, Gerardo Colon-Otero, John A. Copland. Therapeutic responses in a novel patient-derived xenograft mouse model for rare acinar cell pancreatic carcinoma. [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 1475. doi:10.1158/1538-7445.AM2015-1475

Abstract Patient derived xenografts (PDX) mouse models for many types of cancer have demonstrated therapeutic responses similar to those seen in patients. Thus, resected patient tumor tissue directly implanted into immune compromised mice followed by therapy and tumor growth analysis is thought to be the closest preclinical model to predict patient therapeutic responses. To date, few models are available for the different histotypes of thyroid cancer derived from follicular thyrocytes; these include papillary, follicular, Hurthle cell, squamous and anaplastic thyroid carcinoma. We have developed eight PDX models in athymic nude mice representing all of these subtypes. The models have been extensively characterized for mutational status (i.e. BRAF, telomerase, RAS, PTEN, TP53, RB, etc) as well as validation by short tandem repeat (STR) analysis to match that of the originating patient tumor tissue. Squamous and anaplastic thyroid cancers are rare tumor types with no FDA approved therapies. Each model demonstrated their own unique responses to radiation, cytotoxic or molecular targeted therapies such as doxorubicin, sorafenib (tyrosine kinase inhibitor), cisplatin, paclitaxel, sunitinib (tyrosine kinase inhibitor), erlotinib (EGFR inhibitor), trametinib (MEK inhibitor) and vemurafenib (BRAF inhibitor). We expect that these models may provide useful in vivo models for thyroid cancer research as well as models for therapeutic guidance based upon histotype, mutational status and response to therapies. Citation Format: Laura A. Marlow, Adam C. Mathias, Louis K. Dawson, William F. Durham, Kenneth A. Meshaw, Robert J. Mullin, Daniel L. Small, Aidan J. Synnott, Dragana Milosevic, Brian C. Netzel, Stefan K. Grebe, Kevin Wu, Robert C. Smallridge, John A. Copland. Characterization of novel thyroid PDX models and their response to combination therapies. [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 1458. doi:10.1158/1538-7445.AM2015-1458

Abstract Patient derived xenografts (PDX) mouse models for many types of cancer have been developed recapitulating therapeutic responses in the patient for which the PDX model was derived. Essentially, live surgically resected or biopsy tumor tissues are directly implanted into immune incompetent mice to develop in vivo models to test therapies, follow tumor progression (gene mutations that occur with patient tumor progression do so in mice) or to better understand drug resistance. However, the use of PDX models also has its limitations and challenges. Our laboratory has attempted to develop PDX models in athymic nude mice for numerous cancer histotypes such as bladder, brain, breast, colon, kidney, lung, melanoma, ovarian, pancreas, sarcoma and thyroid. While melanoma and colon cancers have very high tumor take rates (73 - 100%), the other cancer histotypes do not (14 - 50%). We share our experience for each cancer histotype and the techniques used to enhance the success rates in athymic nude mice. We also address the other challenges that include expansion of tumors after cryopreservation and conversion of human tumors to mouse tumors and spontaneous lymphomas. We provide methods to test for human versus mouse content that include QPCR and immunohistochemistry Citation Format: Laura A. Marlow, James L. Miller, Brandy Edenfield, Adam C. Mathias, Louis K. Dawson, William F. Durham, Robert J. Mullin, Daniel L. Small, Aidan J. Synnott, Kevin Wu, John A. Copland. Methods for developing patient derived xenografts in athymic nude mice. [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 1453. doi:10.1158/1538-7445.AM2015-1453

Abstract The success of clinical trials for patients with malignant melanoma (MM) designed from the “promising” results using pre-clinical models is minimal. With its increase in incidence, there is a dire need to develop both pre-clinical models comparable to clinical practice that have the capability to translate from bench-to-bedside and novel therapeutic agents for this life-threatening form of skin cancer. To address this concern, we have developed a panel of pre-clinical MM models including patient matched cell lines, patient cell-line derived xenograft models and patient tumor tissue-derived xenograft models. Patient derived xenograft (PDX) models are purported to most closely replicate a patient's response to therapy. Since the development of patient-derived cell lines and tumors grown in mice were more efficacious, their genetic, phenotypic, and therapeutic responses were compared to those of their corresponding patient-matched PDX model. Each model was molecularly characterized to confirm a match to that of the originating patient tumor tissue via protein immunohistochemistry (IHC), mRNA quantitative polymerase chain reaction (qPCR), DNA short tandem repeat (STR) analysis, and Sanger sequencing. Each pre-clinical model provided a genetic match to their respective patient tumor sample. All pre-clinical models were positive for the melanoma-specific markers (S-100 beta, HMB-45, and Melan-A) confirming a melanoma phenotype. Soft agar studies and tumor growth of our PDX and patient cell-line derived xenograft models confirmed tumorigenicity within our models providing the foundation to perform therapeutic studies. Mutation status data (i.e. BRAF, RAS), patient therapeutic clinical history and oncogene overexpression analysis (pERK, pAKT, PI3K, pmTOR, etc.) provide the platform and rationale behind the use of specific therapeutic agents per model. BRAF, NRAS and KRAS mutations were identified along with elevated pERK, pAKT and pmTOR in the models. Targeted therapies (trametinib/MEK inhibitor; vemurafenib/BRAF inhibitor; MK-2206/pAKT inhibitor; and everolimus/mTOR inhibitor) demonstrated dose dependent inhibition of cell proliferation in each of the models based upon oncogenic expression or mutations. In vivo experiments are in progress, which will allow comparison of the three models. The next steps include combination therapy to identify antitumor synergy targeting oncogenic signaling pathways. Citation Format: Antoneicka L. Harris, Laura Marlow, Adam Mathias, Louis Dawson, William Durham, Kenneth Meshaw, Robert Mullin, Daniel Small, Aidan Synnott, Kevin Wu, Dragana Milosevic, Brian Netzel, Stefan Grebe, Svetomir Markovic, John Copland. Characterization of novel patient derived melanoma xenografts and cell lines in response to targeted therapies. [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 2417. doi:10.1158/1538-7445.AM2015-2417