The development of new treatments for castrate resistant prostate cancer (CRPC) must address such challenges as intrinsic tumor heterogeneity and phenotypic plasticity. Combined PTEN/TP53 alterations represent a major genotype of CRPC (25–30%) and are associated with poor outcomes. Using tumor-derived, castration-resistant Pten/Tp53 null luminal prostate cells for comprehensive, high-throughput, mechanism-based screening, we identified several vulnerabilities among >1900 compounds, including inhibitors of: PI3K/AKT/mTOR, the proteasome, the cell cycle, heat shock proteins, DNA repair, NFκB, MAPK, and epigenetic modifiers. HSP90 inhibitors were one of the most active compound classes in the screen and have clinical potential for use in drug combinations to enhance efficacy and delay the development of resistance. To inform future design of rational drug combinations, we tested ganetespib, a potent second-generation HSP90 inhibitor, as a single agent in multiple CRPC genotypes and phenotypes. Ganetespib decreased growth of endogenous Pten/Tp53 null tumors, confirming therapeutic activity in situ. Fifteen human CRPC LuCaP PDX-derived organoid models were assayed for responses to 110 drugs, and HSP90 inhibitors (ganetespib and onalespib) were among the select group of drugs (<10%) that demonstrated broad activity (>75% of models) at high potency (IC50 <1 µM). Ganetespib inhibits multiple targets, including AR and PI3K pathways, which regulate mutually compensatory growth and survival signals in some forms of CRPC. Combined with castration, ganetespib displayed deeper PDX tumor regressions and delayed castration resistance relative to either monotherapy. In all, comprehensive data from near-patient models presents novel contexts for HSP90 inhibition in multiple CRPC genotypes and phenotypes, expands upon HSP90 inhibitors as simultaneous inhibitors of oncogenic signaling and resistance mechanisms, and suggests utility for combined HSP90/AR inhibition in CRPC.

Cancer genomic heterogeneity presents significant challenges for understanding oncogenic processes and for cancer’s clinical management. Variation in driver mutation frequency between patients with the same tumor type as well as within an individual patients’ cancer can shape the use of mutations as diagnostic, prognostic, and predictive biomarkers. We have characterized genomic heterogeneity between and within canine splenic hemangiosarcoma (HSA), a common naturally occurring cancer in pet dogs that is similar to human angiosarcoma (AS). HSA is a clinically, physiologically, and genomically complex canine cancer that may serve as a valuable model for understanding the origin and clinical impact of cancer heterogeneity. We conducted a prospective collection of 52 splenic masses from 43 dogs (27 HSA, 15 benign masses, and 1 stromal sarcoma) presenting for emergency care with hemoperitoneum secondary to a ruptured splenic mass. Multi-platform genomic analysis included matched tumor/normal targeted sequencing panel and exome sequencing. We found candidate somatic cancer driver mutations in 14/27 (52%) HSAs. Among recurrent candidate driver mutations, TP53 was most commonly mutated (30%) followed by PIK3CA (15%), AKT1 (11%), and CDKN2AIP (11%). We also identified significant intratumoral genomic heterogeneity, consistent with a branched evolution model, through multi-region exome sequencing of three distinct tumor regions from selected primary splenic tumors. These data provide new perspectives on the genomic landscape of this veterinary cancer and suggest a cross-species value for using HSA in pet dogs as a naturally occurring model of intratumoral heterogeneity.

Background Rabacfosadine (RAB), a novel antineoplastic agent conditionally licensed for the treatment of lymphoma in dogs, is efficacious in both naïve and previously treated dogs. Its use in combination with L‐asparaginase (L‐ASP) has not been studied. Hypothesis/Objectives To evaluate the safety and efficacy of L‐ASP given concurrently with RAB in dogs with relapsed multicentric lymphoma. Animals Fifty‐two dogs with relapse of lymphoma after treatment with at least 1 doxorubicin‐based chemotherapy protocol. Methods Open‐label, multicenter, prospective single‐arm clinical trial. Dogs were treated with RAB at 1.0 mg/kg IV every 21 days for up to a total of 5 doses. L‐asparaginase was administered at 400 IU/kg SQ concurrently with the first 2 treatments of RAB. Results The overall response rate (ORR) for all dogs was 67%, with 19 dogs (41%) achieving a complete response (CR). The median progression‐free survival time (MPFS) was 63 days (range 5‐428 days). Dogs experiencing a CR as their best response had an MPFS of 144 days (range 44‐428 days). Adverse events were similar to previous studies evaluating single agent RAB. Failure to achieve a CR and having previously received L‐ASP were negative prognostic factors on multivariate analysis. Conclusions and Clinical Importance Concurrent RAB/L‐ASP appears to be both efficacious and safe for treating relapsed multicentric lymphoma in dogs. Adverse events were most often mild and no unexpected toxicoses were observed.

Cancer genomic heterogeneity presents significant challenges for understanding oncogenic processes and for cancer’s clinical management. Variation in driver mutation frequency between patients with the same tumor type as well as within individual patients’ cancers can limit the power of mutations to serve as diagnostic, prognostic, and predictive biomarkers. We have characterized genomic heterogeneity between and within patients in canine splenic hemangiosarcoma (HSA), a common naturally occurring cancer in pet dogs that is similar to human angiosarcoma (AS). HSA is a clinically, physiologically, and genomically complex canine cancer that may serve as a valuable model for understanding the origin and clinical impact of cancer heterogeneity. We conducted a prospective collection of 52 splenic masses from 44 dogs (28 HSA, 15 benign masses, and 1 stromal sarcoma) presenting to emergency care with hemoperitoneum secondary to a ruptured splenic mass. Multi-platform genomic analysis included matched tumor/normal cancer gene panel and exome sequencing. We found candidate somatic cancer driver mutations in 14/28 (50%) HSAs. Among recurrent candidate driver mutations, TP53 was most commonly mutated (29%) followed by PIK3CA (14%), AKT1 (11%), and CDKN2AIP (11%). We also identified significant intratumoral genomic heterogeneity, consistent with a branched evolution model, through multi-region exome sequencing of three distinct tumor regions from selected primary splenic tumors. These data provide new perspective on the genomic landscape and comparative value of understanding HSA in pet dogs, particularly as a naturally occurring cancer bearing intratumoral heterogeneity.

Sorafenib is a multi-kinase small molecule inhibitor that targets serine/threonine and tyrosine kinases including the RAF kinase family, VEGFR-2, and PDGFR. The aim of this study was to evaluate the systemic pharmacokinetics of a previously defined tolerable oral dose of sorafenib in tumor-bearing dogs. Six client-owned dogs with a cytologic or histologic diagnosis of cancer were enrolled in this open-label, tolerability study. Dogs were administered sorafenib at an intended dose of 3 mg/kg and serum samples were obtained for analysis of sorafenib serum concentrations at 0, 1, 2, 6, 12, 24, 48, 72, 96, and 168 h post-drug administration. Median time to peak serum sorafenib concentration occurred at 4 h (range 2–12 h) resulting in an average serum concentration of 54.9 ± 33.5 ng/mL (118.2 ± 72.1 nM). Mean sorafenib levels declined by over 70% relative to peak serum concentrations by 24 h in all dogs, suggesting the value of at least twice daily administration. Doses of 3 mg/kg were well-tolerated and no patients in the study experienced adverse events that were attributable to sorafenib. Future trials in dogs with cancer are recommended at this dosing schedule to assess the effect of sorafenib administration on anti-tumor efficacy signals and relevant pharmacodynamic target modulation in vivo.

Comparative studies of naturally occurring canine cancers have provided new insight into many areas of cancer research. Development and validation of circulating tumor DNA (ctDNA) analysis in pet dogs can help address diagnostic needs in veterinary as well as human oncology. Dogs have high incidence of naturally occurring spontaneous cancers, demonstrate molecular heterogeneity and clonal evolution during therapy, allow serial sampling of blood from the same individuals during the course of disease progression, and have relatively compressed intervals for disease progression amenable to longitudinal studies. Here, we present a feasibility study of ctDNA analysis performed in 48 dogs including healthy dogs and dogs with either benign splenic lesions or malignant splenic tumors (hemangiosarcoma) using shallow whole genome sequencing (sWGS) of cell-free DNA. To enable detection and quantification of ctDNA using sWGS, we adapted two informatic approaches and compared their performance for the canine genome. At the time of initial clinical presentation, mean ctDNA fraction in dogs with malignant splenic tumors was 11.2%, significantly higher than dogs with benign lesions (3.2%; p = 0.001). ctDNA fraction was 14.3% and 9.0% in dogs with metastatic and localized disease, respectively (p = 0.227). In dogs treated with surgical resection of malignant tumors, mean ctDNA fraction decreased from 11.0% prior to resection to 7.9% post-resection (p = 0.047 for comparison of paired samples). Our results demonstrate that ctDNA analysis is feasible in dogs with hemangiosarcoma using a cost-effective approach such as sWGS. Additional studies are needed to validate these findings, and determine the role of ctDNA to assess burden of disease and treatment response in dogs with cancer.

Comparative studies of naturally-occurring canine cancers have provided new insight into many areas of cancer research. The inclusion of pet dogs in the development and validation of circulating tumor DNA (ctDNA) diagnostics may be uniquely informative for human translation for many reasons, including: high incidence of certain spontaneous cancers, repeated access to blood and tumor from the same individuals during the course of disease progression, and molecular heterogeneity of naturally-occurring cancers in dogs. Here, we present a feasibility study of ctDNA analysis performed in 9 healthy dogs and 39 dogs with either benign or malignant splenic tumors (hemangiosarcoma) using shallow whole genome sequencing (sWGS) of cell-free DNA. To enable detection and quantification of ctDNA using sWGS, we adapted two informatic approaches and compared their performance for the canine genome. At presentation, mean ctDNA tumor fraction in dogs with malignant splenic tumors was 11.2%, significantly higher than dogs with benign lesions (3.2%; p 0.001), achieving an AUC of 0.84. ctDNA tumor fraction was 14.3% and 9.0% in dogs with metastatic and localized disease, respectively although this difference was not statistically significant (p 0.227). In paired analysis, ctDNA fraction decreased from 11.0% to 7.9% after resection of malignant tumors (p 0.047). Our results demonstrate that ctDNA analysis is feasible in dogs with hemangiosarcoma using a cost-effective approach such as sWGS. Future studies are underway to validate these findings, and further evaluate the role of ctDNA to assess burden of disease and treatment response during drug development.

Androgen-deprivation therapy (ADT) remains the gold-standard therapy for prostate cancer (PrCa), and although ADT is initially effective, most men progress to castrate-resistant prostate cancer (CRPC) within 2-3 years. Advanced CRPC is challenging to treat because intrinsic tumor heterogeneity and phenotypic plasticity engender short-lived responses and underlie resistance to conventional therapies. Combined PTEN/TP53 alterations represent a major genotype of advanced CRPC (25-30%) and are associated with poor clinical outcomes. Established PrCa cell lines do not accurately represent the heterogeneity of advanced CRPC, and therefore, nonbiased pharmacogenomics screens have not been done. The development of clinically representative, tractable models suitable for high-throughput target identification and validation is crucial for advancing novel CRPC therapies to the clinic. A comprehensive nonbiased high-throughput screen performed on seven cell lines derived from a genetically engineered mouse model (GEMM) of Pten/Tp53 null PrCa identified strongly active compounds, including inhibitors of PI3K/AKT/mTOR signaling, the proteasome, cell cycle regulatory proteins, heat shock proteins, DNA repair signaling, NFKB signaling, MAPK signaling, and several types of epigenetic modifiers. HSP90 inhibitors were one of the most efficacious classes of compounds in the screen, and ganetespib, a clinically used second-generation HSP90 inhibitor with a favorable safety profile, was the most potent. Although HSP90 inhibitors have yet to be successful as single agents, they have not been thoroughly investigated in clinically representative models of advanced PrCa and have shown potential as “network drugs,” prompting our investigations into their utility in polytherapy. We first validated ganetespib as a single agent, where it displayed strong activity against several GEMM-derived and LuCaP PDX-derived organoid models encompassing genotypic, phenotypic, and lineage heterogeneity. These 10 novel LuCaP PDX-derived organoids are representative of the numerous categories of CRPC, including adenocarcinomas with wild-type AR, adenocarcinomas with altered AR, adenocarcinoma with neuroendocrine features, and neuroendocrine disease. Single-agent ganetespib was also strongly inhibitory in vivo, decreasing growth of Pten/Tp53 null endogenous GEMM tumors as well as a human PDX tumor. Mechanistic interrogation of cell lines, organoids, and tumors exposed to ganetespib revealed inhibition of targets from several inter-related networks including AR and pAKT, two central and mutually compensatory growth and survival pathways for PrCa. The efficacy of ganetespib against a diverse group of CRPC organoids and the simultaneous inhibition of PrCa survival signaling suggested it may work well in combination. We performed a proof-of-principle high-throughput matrix screen on organoids derived from a Pten/Tp53 null GEMM and identified docetaxel and etoposide to be synergistic when combined with ganetespib. Preclinical in vivo studies to validate these findings are ongoing. In all, comprehensive data from multiple near-patient models suggest novel contexts for second-generation HSP90-directed intervention against a variety of CRPC genotypes and phenotypes and expand upon the potential of HSP90 inhibitors to simultaneously inhibit oncogenic signaling and compensatory resistance mechanisms. Citation Format: Keith H. Jansson, John B. Tucker, Lauren E. Stahl, John K. Simmons, Caitlyn Fuller, Michael L. Beshiri, Supreet Agarwal, Yasmine Abbey, Lei Fang, Paul G. Hynes, Alilin Aian Neil, Jacob Cawley, Ross Lake, Crystal Tran, Caitlin M. Tice, JuanJuan Yin, Xiahu Zhang, Rajarshi Guha, Shelley Hoover, R. Mark Simpson, Holly Nguyen, Eva Corey, Craig J. Thomas, David Proia, Kathleen Kelly. A high-throughput screen identifies HSP90 inhibitors as potent therapeutics across multiple clinically representative organoid models of advanced prostate cancer [abstract]. In: Proceedings of the AACR Special Conference: Prostate Cancer: Advances in Basic, Translational, and Clinical Research; 2017 Dec 2-5; Orlando, Florida. Philadelphia (PA): AACR; Cancer Res 2018;78(16 Suppl):Abstract nr B018.