Kevetrin induces p53-dependent and independent cell cycle arrest and apoptosis in ovarian cancer cell lines representing heterogeneous histologies
April 4, 2017, 8:00 - 12:00 PM
Ashok Kumar1, David P. Brennan1, Karima Chafai-Fadela1, Sylvia A. Holden1, Siya Ram1, Geoffrey I. Shapiro2, Krishna Menon1. 1Cellceutix Corporation, Beverly, MA; 2Dana-Farber Cancer Institute, Boston, MA
A. Kumar: ; Cellceutix Corporation. D.P. Brennan: ; Cellceutix Corporation. K. Chafai-Fadela: ; Cellceutix Corporation. S.A. Holden: ; Cellceutix Corporation. S. Ram: ; Cellceutix Corporation. G.I. Shapiro: ; Dana-Farber Cancer Institute. K. Menon: ; Cellceutix Corporation.
Ovarian cancer (OC) is a molecularly and histologically heterogeneous disease; however, standard treatment is the same for all subtypes. High-grade serous OC initially responds to chemotherapy; however, low-grade serous and clear cell OC are relatively chemoresistant. Limited treatment options are available upon recurrence. p53 mutations are found in over 90% of high-grade serous OC. Low-grade serous OC harbor wild type p53, but contain other mutations. During later stages of OC, tumors are a heterogeneous population of mutant cells; thus, development of a novel drug that addresses these molecular differences is highly desirable. Previously, we showed that Kevetrin stabilized wild type p53 and induced transcriptional targets in human lung carcinoma. We sought to validate Kevetrin as a potential treatment for OC with varied p53 status. Endometrioid carcinoma (A2780, wt p53), atypical non-serous clear cell (SKOV-3, deleted p53), and high-grade serous (OVCAR-3, mutant p53) OC cell lines were treated with Kevetrin. Kevetrin induced apoptosis in all three OC cell lines, as assayed by cleavage of PARP and caspase-3. Studies showed significant increases in p53 and p21 protein levels in A2780 cells in 24 to 48 hours; however, in OVCAR-3, Kevetrin downregulated oncogenic mutant p53. RNA levels of p53 and p21 were quantified by qRT-PCR 8 to 72 hours after treatment. No significant changes were observed in p53 mRNA, whereas an increase in p21 mRNA was observed in all three cell lines. In A2780, Kevetrin also induced levels of PUMA in a dose-dependent manner. To establish that Kevetrin mediates increased p21 expression requiring p53 in A2780 cells, p53 was depleted by siRNA. p53 depletion reduced p21 expression, as assayed by FACS, indicating p53 directs p21 expression in a dose-dependent manner after Kevetrin treatment. In an in vivo xenograft study in immunocompromised nude mice bearing established A2780 tumors, Kevetrin treatment inhibited tumor growth at well-tolerated doses. The mode of action in vivo also showed enhanced expression of p21 in tumor tissue, indicating p53 pathway activation in A2780 tumors. In contrast, in SKOV-3 cells and xenografts, a p53 independent increase in p21 expression was observed. In OVCAR-3 cells,Kevetrin altered the expression of three microRNAs (miRNA-27a, miRNA-1274b, miRNA-25), that are known to be dysregulated in OC, in a time-dependent manner. To gain further insight into the mechanism of action in cells with diverse p53 status, RNA-Seq is being performed in the three cell lines and tumor tissue from mice before and after Kevetrin exposure. In summary, Kevetrin has promise as an effective therapeutic agent for endometrioid, non-serous clear cell, and high-grade serous OC, with molecularly diverse p53 status. A Phase 1 clinical study was completed and a Phase 2 clinical study in ovarian cancer is scheduled to begin January 2017.