Date of Award

Summer 2025

Document Type

Open Access Thesis

Department

Biological Sciences

First Advisor

Hexin Chen

Abstract

HER2-positive (HER2⁺) breast cancer is aggressive and often develops resistance to anti-HER2 therapies. CDK8 and CDK19, transcription-associated cyclin-dependent kinases (tCDKs) in the Mediator complex, are dysregulated in HER2⁺ breast cancer, correlate with poor survival, and support transcriptional escape driving therapeutic resistance. Previous studies Previous studies in immune-deficient models demonstrated that CDK8/19 inhibition (CDK8/19i) reduces pro-tumor macrophages (M2Φs), but its role in immune-competent tumors and innate immune–mediated tumor control remains unclear. Since HER2⁺ tumors are often immunogenic and anti-HER2 therapies rely on innate immune activation, reprogramming immunosuppressive M2Φ toward an M1Φ-like phenotype could enhance antitumor immunity. We hypothesized that CDK8/19i modulates the tumor immune microenvironment (TIME) promotes pro-inflammatory (pro-INF) M1Φ reprogramming through transcriptional and metabolic programs. In vivo, Senexin (SNX)631, a selective tCDK8/19i, suppressed tumor growth in HER2⁺ immune-replete mice but not in immunodeficient (NSG, Foxn1nu) models, confirming immune dependence. In our fourth model, Tamoxifen-inducible CDK8fl/fl; CDK19−/−; ROSA-CreERT2⁺ mice exhibited impaired tumor progression independent of drug treatment, demonstrating that immune-intrinsic CDK8/19 loss reshapes the TIME. Compared to controls, CDK8/19 KO tumors exhibited increased M1-like macrophages and reduced immature myeloid populations, shifting from suppressive, myeloid-dense niches to helper- and effector-enriched immune zones—consistent with immune re-education rather than direct cytotoxicity. In vitro, SNX631 consistently skewed polarization towards an M1Φ phenotype across BMDMs, RAW264.7 (RAW), and THP-1 models, exhibiting strong STAT1–CXCL10 axis activation and CCL5 induction, while decreasing M2 related genes. In RAW, we compared IFNγ-only, LPS-only, and IFNγ+LPS M1Φ-polarizing conditions to evaluate whether CDK8/19i uniformly skews polarization or exhibits stimulus-specific effects, to better mimic complex TIME signaling cues. This allowed most physiologically relevant model for downstream mechanistic studies. Temporal (30min or acute phase & 24hr sustained phase) and dose analyses revealed transcriptionally efficient programs shifting to metabolically reactive, stress-driven programs. At 24hrs, M1 and M2 biases demonstrated dose-dependent STAT3/6 reduction with increasing dosage. For single M1 biases, intermediate SNX631 doses (0.5–1 µM) preserved STAT1 Tyr701 signaling with increasing HIF1α and ROS, basal-to-moderate p62, generating a transcriptionally efficient (adaptive) M1 program. IFNγ+LPS preserved pro-longed STAT1 Tyr701 signaling with increasing HIF1α, p62 accumulation, lysosomal stress, and ROS remodeling suggesting a stress-driven, trained-like phenotype. M2 programs were dose-dependently dismantled, peaking at 1 µM with increased HIF1α, p62, and ROS. Mito-Tracker and mitophagy analyses confirmed enhanced mitochondrial biogenesis and fusion-lysosome coordination in M1Φ, contrasting with disrupted lysosomal flux and mitochondrial loss in M2Φs. Importantly, CDK8/19 act as immune-regulatory checkpoints, distinguished by M1 biases, in a controlled dosing and time-dependent manner. These findings support a model in which SNX631 shifts transcriptional efficiency to stress-driven, adaptive M1-like state, resembling trained innate features which may lock in and skew M1Φs in an antitumor state.

Rights

© 2025, Stephen O'Rourke

Download

Included in

Biology Commons

Share

COinS