Author ORCID Identifier

https://orcid.org/0000-0002-3892-1564

Semester

Fall

Date of Graduation

2024

Document Type

Dissertation

Degree Type

PhD

College

School of Medicine

Department

Microbiology, Immunology, and Cell Biology

Committee Chair

Lori Hazlehurst

Committee Co-Chair

Scott Weed

Committee Member

Scott Weed

Committee Member

Michael Ruppert

Committee Member

Ivan Martinez

Committee Member

Werner Geldenhuys

Abstract

Lung cancer remains one of the deadliest cancers. Novel, paradigm shifting treatments including immunotherapy and targeted therapies have recently been developed to cull the deadly effects of lung cancer, but many challenges remain. There remains a significant unmet need to accurately predict and optimally select for patients who will respond to immune checkpoint inhibitors (ICI) treatment. In Chapter 2 of this dissertation, we investigated a novel live single cell cytokine profiling lab-on-chip platform, IsoLight, using peripheral CD4+ and CD8+ T-cells for ICI biomarker development. A total of 55,175 single T-lymphocytes were analyzed in this proof-of-concept study. We found that an increase in overall polyfunctionality (PolyFx) and polyfunctional strength index (PSI) in single CD8+ T-cells, but not CD4+ T-cells, after 2 cycles of ICI correlated significantly with treatment response, with better performance than PD-L1 TPS (p=0.082) alone. When combined with PD-L1 TPS, the combined index correlated with ICI treatment response with kappa coefficient of 1.0 (p=0.003). Moreover, induction of CD4+ T-cells PSI (ΔPSI>5) correlated with a strong trend of long-term treatment survival outcomes benefits (PFS; 3.9-fold; OS; 3-fold). Chapter 3 focuses on oncogene-addictive ALK-fusion NSCLC as our model for precision therapy drug resistance. We investigated the molecular genesis and evolution of rapid-onset precision therapy drug-tolerant persistence, using both genomics-matching cell lines, and patient-derived (Ma-ALK001.S) cell line and xenograft models. Together, we identified the tumoral autocrine TGFb2-PRC-2-EZH2/UTX-HOXB3 mediated signaling cascade as the key non-genetic epigenetic mechanism of the rapid-onset of ALK-TKI drug-tolerant persister cells, as early as between 8 hr to 14 days. Importantly, we found that EZH2/UTX could be leveraged as therapeutic vulnerabilities of the drug-tolerant persisters. Bulk RNA-sequencing and scRNA-seq bioinformatics analysis on the Ma-ALK001.S PDC under ALK-TKI alectinib identified 3 stages of transcriptomic reprogramming: Genesis (0, 3, 8 hr), Early (3, 6, 9 days), and Late stage (30, 60, 120 days). Pathway analysis nominated a crucial role of cAMP-PKA upregulated signaling in the persister cell states genesis, especially during the 8 hr - 3 days post-alectinib of plasticity reprogramming, associating with dominance of G2/M and G0/G1 cell cycle checkpoint arrest states. Importantly, pharmacologic inhibition with adenylyl cyclase activator forskolin, and inhibitor KH7 provides a promising preemptive treatment approach for drug-tolerant persistence for clinical outcomes improvement in oncogene-addictive NSCLC precision therapy. In summary, we developed strategies to optimize treatment response and clinical outcomes in both ICI- and precision therapy-treated NSCLCs.

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