Loss of JAK1 Function Causes Radioresistance and G2/M Cell Cycle Defects Vulnerable to Kif18a Inhibition

Date of Award

Spring 1-1-2025

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Pharmacology

First Advisor

Contessa, Joseph

Abstract

Although Head and Neck Squamous Cell Carcinoma (HNSCC) is the seventh most common cancer worldwide, the high failure rates and the absence of new effective therapies has caused the survival rate for HNSCC to remain stagnant for the past decade. As radiation is a frontline treatment for HNSCC, we sought to uncover novel targets that affect cellular responses to radiation-induced DNA damage. To do this, we performed a CRISPR-Cas9 knockout screen in HNSCC cell lines using radiation as a selection pressure. Our results identified that loss of Janus kinase 1 (JAK1) caused resistance to radiation in Cal27 and Detroit562 HNSCC cell lines. We generated JAK1 knockout (KO) cell lines and confirmed that loss of JAK1 causes radioresistance in both in vitro and in vivo models by enhancing the DNA damage induced G2 cell cycle arrest and by slowing mitosis. We find that this enhanced G2 arrest allows the cells to avoid mitotic stress and mitotic catastrophe following radiation treatment, thereby promoting cell survival. We measure multiple cellular outcomes of this enhanced G2 arrest that further contribute to the radioresistance of the JAK1 KO cells including decreased micronuclei formation, a reduction in apoptotic signaling, and enhanced homologous recombination dependent DNA repair usage. We endeavored to overcome the radioresistance in the JAK1 KO cells by abrogating their enhanced G2 arrest using Wee1 inhibitors, which lead to constitutive activation of CDK1. To our surprise, treatment with the Wee1 inhibitor adavosertib was unable reverse the prolonged G2 arrest in the JAK1 KO cells despite efficacy in controls and sufficient dephosphorylation of Y15 in CDK1. This indicated that a signaling axis outside of the canonical CDK1-dependent checkpoint is responsible for the enhanced G2 arrest of the JAK1 KO cells. We find delayed activation of Aurora kinase A (AURKA) and Polo-like kinase 1 (PLK1) in the JAK1 KO cells, consistent with reports that AURKA and PLK1 reactivation are necessary to overcome a DNA damage induced G2 arrest. Given the inability to abrogate the prolonged JAK1 KO G2 arrest by activating CDK1, we sought to exploit this enhanced G2 phenotype for therapeutic benefit. Kif18a is a kinesin that is involved with the maintenance of complex genomes and mitotic spindle tension. As cells with >2N genomes have been reported to be sensitive to Kif18a inhibition, we tested the specific Kif18a inhibitor sovilnesib in both in vitro and in vivo JAK1 KO models. Indeed, we found that addition of sovilnesib to radiation treatment radiosensitized JAK1 KO cells and xenograft tumors, providing therapeutic recourse for this resistant population. Together, our results reveal a novel role for JAK1 in regulating cell cycle progression and the cellular response to radiation treatment, as well as identify a novel therapeutic approach to target these resistant cells.

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