Date of Graduation
12-2014
Document Type
Dissertation (PhD)
Program Affiliation
Cancer Biology
Degree Name
Doctor of Philosophy (PhD)
Advisor/Committee Chair
JEFFREY N. MYERS, MD, Ph.D.
Committee Member
WALTER N. HITTELMAN, Ph.D.
Committee Member
WILLIAM PLUNKETT, Ph.D.
Committee Member
JUNJIE CHEN, Ph.D.
Committee Member
ZAHID H. SIDDIK, Ph.D.
Abstract
Cisplatin, despite being the cornerstone chemotherapy for the treatment of head and neck squamous cell carcinoma (HNSCC), provides clinical benefits in just a subset of patients. This together with the lack of biomarkers predicting therapeutic responses, have led to unacceptably high rate of treatment failures in HNSCC. TP53 is the most frequently mutated gene in HNSCC, and the effect of p53 loss or mutation on cisplatin responses in HNSCC is poorly understood. In the current study, we hypothesized that HNSCC cells respond to cisplatin in a p53 dependent manner and unambiguously show that presence of wild-type TP53 (wtp53) confers sensitivity to cisplatin treatment in HNSCC cells, whereas mutation or loss of TP53 imparts resistance to cisplatin treatment. Moreover, we report that senescence, but not apoptosis is the prominent cellular response to cisplatin in wtp53 HNSCC cells and that the cisplatin resistance in p53-null or -mutant TP53 cells is due to their inability to evoke senescence response. In an effort to find strategies of overcoming cisplatin resistance in p53-deficient HNSCC cells, we found that a synthetic lethal strategy through targeted inhibition of checkpoint kinases Chk1/2 leads to cisplatin sensitization of p53-deficient cells through induction of mitotic death. Serendipitously, we also found out that a significant subset (roughly 20%) of HNSCC cell lines are acutely sensitive to single agent checkpoint inhibitors and that Chk1, but not Chk2, mediates this sensitivity. Characterization of phenotypic and molecular responses to Chk1 inhibition in the Chk sensitive and resistant cells revealed induction of early S phase arrest and DNA damage/replication stress markers in the Chk sensitive, but not resistant cells. In addition, we also found that inhibition of Chk1 kinase led to aberrant increase in origin of replication firings in the sensitive, but not resistant cells and that loss of cdk2 or treatment with Roscovitine rescues the lethal phenotype seen in the sensitive cells upon Chk1 inhibition. These results suggest that exquisite sensitivity to Chk1 inhibition in a subset of HNSCC cells could be due to these cells being in a pre-existing state of severe replication stress.
In summary, given the preponderance of p53 mutation in HNSCC and the widespread use of cisplatin in treating aggressive HNSCC, we provide preclinical evidence that cisplatin resistance of p53-deficient HNSCC cells can be overcome through inhibition of checkpoint kinases 1/2. These preclinical data suggest that Chk1/2 kinase is a promising therapeutic target in HNSCC and a precision approach using Chk inhibition in p53-mutant tumors may be feasible for the treatment of HNSCC. We also show that targeted inhibition of Chk1 alone imparts lethality in a significant subset of HNSCC cells by inducing an aberrant increase in origin firings. These results suggest that targeting Chk1 alone could be therapeutically beneficial in a significant subset of HNSCC. Identifying those patients with HNSCC that are particularly sensitive to Chk1 inhibition would enable more precise treatment selection thereby increasing the efficacy and decreasing the morbidity of treatment of patients with this disease.
Keywords
Chk1, p53, Chk2, senescence, origin firings