(1102) Serendipitous Discovery of a Drug-responsive, Fast-growing Mouse Tumor for Preclinical Therapeutic Development

Objectives: Patient-derived xenografts (PDX) are widely acknowledged as invaluable tools for preclinical therapy development, as they closely mimic human disease both phenotypically and genetically. To successfully cultivate PDX tumors, it is required to utilize severely immunocompromised mice (NSG), as the immune systems of wild-type mice would attack human tumor, thwarting its growth. NSG mice are prone to spontaneous tumorigenicity. In our pursuit to investigate mTOR inhibitors in combination with SNX631-6, a CDK8/19 Mediator kinase inhibitor (CDK8/19i), we initially expanded what we believed to be a human breast cancer PDX. Upon thorough examination by RNASeq analysis and immunofluorescence staining for CD45 (LCA, Leukocyte Common Antigen) we identified the tumor tentatively as a lymphoma. Serendipitously, this newly established mouse tumor exhibited phenotypic characteristics beneficial to expediting in vivo drug sensitivity assays for novel therapeutics. Notably, this tumor model could swiftly progress from transplantation to reaching the endpoint tumor size in approximately 30 days, significantly shortening the time required for therapeutic testing compared to the several months typically needed for a PDX to grow. We chose to employ this new model to assess our novel CDK8/19i in combination with the newly FDA-approved mTOR inhibitor, Capivasertib, and earlier approved mTOR inhibitor, Everolimus. CDK8/19 Mediator kinase is a kinase that governs transcriptional changes, particularly those associated with metastasis and treatment resistance due to their dependance on transcriptional reprogramming. The mTOR pathway is dysregulated in many cancers, making it a prime target of interest in the realm of new therapeutics development. Our hypothesis posited that combining mTOR inhibitors with CDK8/19i would forestall resistance to mTOR-targeting drugs by preventing transcriptional reprogramming in mouse lymphoma tumors.

Methods: RNASeq: The raw data underwent processing using an algorithm developed to discern murine from human reads.

qPCR: RNA extraction from tumor samples was performed using a Qiagen extraction kit. Primers designed for human and mouse genes were employed to assess the composition of mouse versus human cells.

In vivo studies: We transplanted 2x2x2mm tumor fragments subcutaneously into the right flank of 60 NSG mice. The mice were randomized and subsequently administered either a single drug (Capivasertib, Everolimus, SNX631-6) or mTOR inhibitors in combination with CDK8/19 inhibitors. Daily observations of the mice were conducted to monitor for morbidity, and measurements of body weight and tumor volume were taken twice a week. Upon reaching humane endpoints, samples were collected for paraffin embedding and subjected to microscopic analysis.

IHC and Immunofluorescence: Tumor samples were fixed in 10% Formalin for 48 hours, followed by dehydration and embedding in paraffin for sectioning. Standard methods were employed for sectioning and staining of the tumors.

Results: As a single agent, Everolimus inhibited tumor growth for the first 40 days, after which all the tumors developed resistance. The addition of CDK8/19 inhibitors significantly prolonged the time to resistance (greater than 60 days). Capivasertib, at the dosages tested, did not exhibit any growth inhibition. SNX631-6 alone did not demonstrate significant reduction in tumor growth. However, combining SNX631-6 with Capivasertib led to tumor growth inhibition and a significant increase in event-free survival.

Conclusions: With our newly discovered model, we were able to demonstrate in vivo prevention of resistance to Everolimus using CDK8/19 inhibitors in a novel aggressive murine tumor. We also discovered synergistic growth inhibition of these tumors and extended animal survival by the combination of Capivasertib and CDK8/19 inhibitors. Our model offers a novel rapid approach for in vivo testing of new therapies.