Faulty Metabolism: A Potential Instigator of an Aggressive Phenotype in Cdk5-dependent Medullary Thyroid Carcinoma.

Mechanistic modeling of cancers such as Medullary Thyroid Carcinoma (MTC) to emulate patient-specific phenotypes is challenging. The discovery of potential diagnostic markers and druggable targets in MTC urgently requires clinically relevant animal models. Here we established orthotopic mouse models of MTC driven by aberrantly active Cdk5 using cell-specific promoters. Each of the two models elicits distinct growth differences that recapitulate the less or more aggressive forms of human tumors. The comparative mutational and transcriptomic landscape of tumors revealed significant alterations in mitotic cell cycle processes coupled with the slow-growing tumor phenotype. Conversely, perturbation in metabolic pathways emerged as critical for aggressive tumor growth. Moreover, an overlapping mutational profile was identified between mouse and human tumors. Gene prioritization revealed putative downstream effectors of Cdk5 which may contribute to the slow and aggressive growth in the mouse MTC models. In addition, Cdk5/p25 phosphorylation sites identified as biomarkers for Cdk5-driven neuroendocrine tumors (NETs) were detected in both slow and rapid onset models and were also histologically present in human MTC. Thus, this study directly relates mouse and human MTC models and uncovers vulnerable pathways potentially responsible for differential tumor growth rates. Functional validation of our findings may lead to better prediction of patient-specific personalized combinational therapies.

A multiplexed assay for quantifying immunomodulatory proteins supports correlative studies in immunotherapy clinical trials.

Introduction: Immunotherapy is an effective treatment for a subset of cancer patients, and expanding the benefits of immunotherapy to all cancer patients will require predictive biomarkers of response and immune-related adverse events (irAEs). To support correlative studies in immunotherapy clinical trials, we are developing highly validated assays for quantifying immunomodulatory proteins in human biospecimens. Methods: Here, we developed a panel of novel monoclonal antibodies and incorporated them into a novel, multiplexed, immuno-multiple reaction monitoring mass spectrometry (MRM-MS)-based proteomic assay targeting 49 proteotypic peptides representing 43 immunomodulatory proteins. Results and discussion: The multiplex assay was validated in human tissue and plasma matrices, where the linearity of quantification was >3 orders of magnitude with median interday CVs of 8.7% (tissue) and 10.1% (plasma). Proof-of-principle demonstration of the assay was conducted in plasma samples collected in clinical trials from lymphoma patients receiving an immune checkpoint inhibitor. We provide the assays and novel monoclonal antibodies as a publicly available resource for the biomedical community.

Targeted Mass Spectrometry Enables Multiplexed Quantification of Immunomodulatory Proteins in Clinical Biospecimens.

Immunotherapies are revolutionizing cancer care, producing durable responses and potentially cures in a subset of patients. However, response rates are low for most tumors, grade 3/4 toxicities are not uncommon, and our current understanding of tumor immunobiology is incomplete. While hundreds of immunomodulatory proteins in the tumor microenvironment shape the anti-tumor response, few of them can be reliably quantified. To address this need, we developed a multiplex panel of targeted proteomic assays targeting 52 peptides representing 46 proteins using peptide immunoaffinity enrichment coupled to multiple reaction monitoring-mass spectrometry. We validated the assays in tissue and plasma matrices, where performance figures of merit showed over 3 orders of dynamic range and median inter-day CVs of 5.2% (tissue) and 21% (plasma). A feasibility study in clinical biospecimens showed detection of 48/52 peptides in frozen tissue and 38/52 peptides in plasma. The assays are publicly available as a resource for the research community.

Targeted Mass Spectrometry Enables Quantification of Novel Pharmacodynamic Biomarkers of ATM Kinase Inhibition.

The ATM serine/threonine kinase (HGNC: ATM) is involved in initiation of repair of DNA double-stranded breaks, and ATM inhibitors are currently being tested as anti-cancer agents in clinical trials, where pharmacodynamic (PD) assays are crucial to help guide dose and scheduling and support mechanism of action studies. To identify and quantify PD biomarkers of ATM inhibition, we developed and analytically validated a 51-plex assay (DDR-2) quantifying protein expression and DNA damage-responsive phosphorylation. The median lower limit of quantification was 1.28 fmol, the linear range was over 3 orders of magnitude, the median inter-assay variability was 11% CV, and 86% of peptides were stable for storage prior to analysis. Use of the assay was demonstrated to quantify signaling following ionizing radiation-induced DNA damage in both immortalized lymphoblast cell lines and primary human peripheral blood mononuclear cells, identifying PD biomarkers for ATM inhibition to support preclinical and clinical studies.