Targeting PDZ-binding kinase is anti-tumorigenic in novel preclinical models of ACC

in Endocrine-Related Cancer
Correspondence should be addressed to K Kiseljak-Vassiliades: katja.kiseljak-vassiliades@ucdenver.edu
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Adrenocortical carcinoma (ACC) is an aggressive orphan malignancy with less than 35% 5-year survival and 75% recurrence. Surgery remains the primary therapy and mitotane, an adrenolytic, is the only FDA-approved drug with wide-range toxicities and poor tolerability. There are no targeted agents available to date. For the last three decades, H295R cell line and its xenograft were the only available preclinical models. We recently developed two new ACC patient-derived xenograft mouse models and corresponding cell lines (CU-ACC1 and CU-ACC2) to advance research in the field. Here, we have utilized these novel models along with H295R cells to establish the mitotic PDZ-binding kinase (PBK) as a promising therapeutic target. PBK is overexpressed in ACC samples and correlates with poor survival. We show that PBK is regulated by FOXM1 and targeting PBK via shRNA decreased cell proliferation, clonogenicity and anchorage-independent growth in ACC cell lines. PBK silencing inhibited pAkt, pp38MAPK and pHistone H3 altering the cell cycle. Therapeutically, targeting PBK with the small-molecule inhibitor HITOPK032 phenocopied PBK-specific modulation of pAkt and pHistone H3, but also induced apoptosis via activation of JNK. Consistent with in vitro findings, treatment of CU-ACC1 PDXs with HITOPK032 significantly reduced tumor growth by 5-fold (P < 0.01). Treated tumor tissues demonstrated increased rates of apoptosis and JNK activation, with decreased pAkt and Histone H3 phosphorylation, consistent with effects observed in ACC cell lines. Together these studies elucidate the mechanism of PBK in ACC tumorigenesis and establish the potential therapeutic potential of HITOPK032 in ACC patients.

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      Society for Endocrinology

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    PBK is overexpressed in ACC tumors and dictates survival outcomes. (A) PBK mRNA expression in ACC (n = 77) is 12-fold higher than normal adrenal tissues (n = 14) and 7-fold higher than adenomas (n = 22) in microarray gene expression studies. (B and C) Kaplan–Meier analysis of survival using GraphPad Prism with normalized RNA seq data from the TCGA cohort of ACC (n = 75) available in cbioportal. RNA expression was normalized using RSEM. 10% of the population in TCGA cohort had a Z score >2 and was designated to have PBK mRNA upregulation. High PBK expression associated with poor OS and poorer disease-free survival (DFS) (**P < 0.01). (D and E) PBK protein expression in ACC tumor tissues and ACC cell lines and PDXs. Quantification of E is shown on the right F. IHC with anti-PBK in normal adrenal show no nuclear staining but strong nuclear and diffused cytoplasmic staining in ACC.

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    PBK knockdown inhibits cell proliferation, clonogenicity and soft agar colony formation in ACC. (A and D) PBK silencing attenuates proliferation in CU-ACC1 and H295R cell lines. (B and E) PBK knockdown causes on average a ~2-fold decrease in colony formation in CU-ACC1 and on average a ~6-fold decrease in H295R. (C and F) Knocking down PBK significantly inhibits anchorage-independent growth by ~20-fold in CU-ACC1 and by (4 to 6) fold in H295R cell lines. (G) Immunoblot showing decrease in p-Akt, p-p38MAPK and no change in pERKf with PBK knockdown in CU-ACC1 and H295R cell lines. Data presented have been derived from at least three biological replicates. Data represented as mean ± s.e.m. (*P < 0.05, **P < 0.01). Quantification of immunoblots have been done using normalized densitometric values derived at least three biological repeats.

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    PBK silencing inhibits Histone H3 phosphorylation and alters cell cycle progression. (A) PBK knockdown decreased pHistone H3 in CU-ACC1 and H295R cell lines. Knockdown causes on average a 5-fold decrease (**P < 0.01) in CU-ACC1 and on average 2-fold decrease (*P < 0.05) in H295R as assessed by densitometry. (B) Flow cytometry analysis of asynchronous and double thymidine-blocked CU-ACC1 control shscr cells at 0 h post release showing a 1.6-fold increase in S phase cells in controls. (C) Cell cycle analysis post thymidine block through 0–18 h, original flow plots and a bar graph visualization of % cell progression through different time points. (D) Average percent of cells in G2/M and S phase through 0–18 h post release from double thymidine block collected from three experimental replicates. Significant changes in G2/M and S phase cell % in controls through 12–18 h have been denoted by **P < 0.01. Data have been derived from three experimental replicates and is represented as mean ± s.e.m.

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    HITOPK032 treatment inhibits tumorigenic properties and recapitulates effects of PBK silencing in ACC. (A, B and C) Cell lines show a dose-dependent response to HITOPK032 treatment at 600–1 µM range (**P < 0.01) by day 8. Graphs shown are representative experiments of three biological repeats; data presented as mean ± s.e.m. (D, E and F) Dose-dependent decrease in colony formation to doses of 50–300 nM of HITOPK032. (G) HITOPK032 decreases Akt and Histone H3 phosphorylation as shown via immunobloting. P values (**P < 0.01, *P < 0.05) have been calculated using normalized densitometric values derived from immunoblots of at least three biological repeats. All normalization has been done to the DMSO control. Data represented as mean ± s.e.m.

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    Apoptosis induced by HITOPK032 is in part mediated by JNK activation. (A) HITOPK032 at 1 µM or higher induces apoptosis in a dose dependent manner by 48 h in all cell lines. (B) HITOPK treatment induces JNK phosphorylation in CU-ACC1 and H295R cell lines. (C) Treating CU-ACC1 and H295R cell lines with 10 µM of the JNK inhibitor in presence of 4 µM HITOPK032 partially inhibits caspase 3/7 activity measured by the caspase 3/7 glo assay. P values (**P < 0.01, *P < 0.05) have been calculated from normalized RLU of at least three biological repeats. Quantification of immunoblots have been derived from normalized densitometric values from three independent replicates. All normalization has been done to the DMSO control. Data represented as mean ± s.e.m.

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    HITOPK032 inhibits tumorigenic growth in ACC. (A) HITOPK032 treatment in athymic nude mice bearing passage 14 CU-ACC1 PDXs shows significant decrease in tumor rate by day 14 and day 17 (**P < 0.01). (B) A representative image of tumors harvested at the end of the study. Treated group has smaller tumor size compared to control. (C) Graph showing growth rates of individual tumors following HITOPK032 treatment. (D) Average net weight of mice in treated and the control group shows no significant change in body weight. (E) IHC analysis of pHistone H3 in tissue sections of control and treated tumors. There is 1.9-fold decrease in histone 3 phosphorylation in treated group relative to control (**P < 0.01). (F) Immunoblot of representative tumor lysates from five control and six treated tumors collected from individual mice showing difference in cleaved PARP, cleaved caspase 3, pAkt and pJNK expression between control and treated group (*P < 0.05, **P < 0.01).

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