ß-Catenin-Driven Differentiation Is a Tissue-Specific Epigenetic Vulnerability in Adrenal Cancer.

Adrenocortical carcinoma (ACC) is a rare cancer in which tissue-specific differentiation is paradoxically associated with dismal outcomes. The differentiated ACC subtype CIMP-high is prevalent, incurable, and routinely fatal. CIMP-high ACC possess abnormal DNA methylation and frequent ß-catenin-activating mutations. Here, we demonstrated that ACC differentiation is maintained by a balance between nuclear, tissue-specific ß-catenin-containing complexes, and the epigenome. On chromatin, ß-catenin bound master adrenal transcription factor SF1 and hijacked the adrenocortical super-enhancer landscape to maintain differentiation in CIMP-high ACC; off chromatin, ß-catenin bound histone methyltransferase EZH2. SF1/ß-catenin and EZH2/ß-catenin complexes present in normal adrenals persisted through all phases of ACC evolution. Pharmacologic EZH2 inhibition in CIMP-high ACC expelled SF1/ß-catenin from chromatin and favored EZH2/ß-catenin assembly, erasing differentiation and restraining cancer growth in vitro and in vivo. These studies illustrate how tissue-specific programs shape oncogene selection, surreptitiously encoding targetable therapeutic vulnerabilities. SIGNIFICANCE: Oncogenic ß-catenin can use tissue-specific partners to regulate cellular differentiation programs that can be reversed by epigenetic therapies, identifying epigenetic control of differentiation as a viable target for ß-catenin-driven cancers.

MSI2 expression in adrenocortical carcinoma: Association with unfavorable prognosis and correlation with steroid and immune-related pathways.

Adrenocortical carcinoma (ACC) is a rare, but highly aggressive cancer of the adrenal cortex with a generally poor prognosis. Despite being rare, completely resected ACCs present a high risk of recurrence. Musashi-2 (MSI2) has recently been recognized as a potential prognostic biomarker and therapeutic target in many cancers. However, no studies have evaluated the clinical significance of MSI2 expression in ACC. Here, we addressed MSI2 expression and its association with ACC prognosis and clinicopathological parameters. MSI2 expression was analyzed in TCGA, GSE12368, GSE33371, and GSE49278 ACC datasets; and its correlation with other genes and immune cell infiltration were investigated by using the R2: Genomics Analysis and Visualization Platform and TIMER databases, respectively. Enrichment analysis was performed with the DAVID Functional Annotation Tool. Kaplan-Meier curves, log-rank tests, and Cox regression analyses were used to explore the prognostic role of MSI2 in ACC. Our findings demonstrated the potential value of MSI2 overexpression as an independent predictor of poor prognosis in patients with completely resected ACC (hazard ratio 6.715, 95% confidence interval 1.266 - 35.620, p =.025). In addition, MSI2 overexpression was associated with characteristics of unfavorable prognosis, such as cortisol excess (p = .002), recurrence (p =.003), and death (p =.015); positively correlated with genes related to steroid biosynthesis (p < .05); and negatively correlated with immune-related pathways (p < .05). Our findings demonstrate that MSI2 has value as a prognostic marker for completely resected ACC and reinforce the investigation of its role as a possible therapeutic target for patients with ACC.

Beta-Catenin Causes Adrenal Hyperplasia by Blocking Zonal Transdifferentiation.

Activating mutations in the canonical Wnt/ß-catenin pathway are key drivers of hyperplasia, the gateway for tumor development. In a wide range of tissues, this occurs primarily through enhanced effects on cellular proliferation. Whether additional mechanisms contribute to ß-catenin-driven hyperplasia remains unknown. The adrenal cortex is an ideal system in which to explore this question, as it undergoes hyperplasia following somatic ß-catenin gain-of-function (ßcat-GOF) mutations. Targeting ßcat-GOF to zona Glomerulosa (zG) cells leads to a progressive hyperplastic expansion in the absence of increased proliferation. Instead, we find that hyperplasia results from a functional block in the ability of zG cells to transdifferentiate into zona Fasciculata (zF) cells. Mechanistically, zG cells demonstrate an upregulation of Pde2a, an inhibitor of zF-specific cAMP/PKA signaling. Hyperplasia is further exacerbated by trophic factor stimulation leading to organomegaly. Together, these data indicate that ß-catenin drives adrenal hyperplasia through both proliferation-dependent and -independent mechanisms.

The aurora kinase inhibitor AMG 900 increases apoptosis and induces chemosensitivity to anticancer drugs in the NCI-H295 adrenocortical carcinoma cell line.

Adrenocortical tumor (ACT) is a malignancy with a low incidence rate and the current therapy for advanced disease has a limited impact on overall patient survival. A previous study from our group suggested that elevated expression of aurora-A and aurora-B is associated with poor outcome in childhood ACT. Similar results were also reported for adult ACTs. The present in-vitro study shows that AMG 900 inhibits aurora kinases in adrenocortical carcinoma cells. AMG 900 inhibited cell proliferation in NCI-H295 cells as well as in the ACT primary cultures and caused apoptosis in the cell line NCI-H295. Furthermore, it potentialized the mitotane, doxorubicin, and etoposide effects on apoptosis induction and acted synergistically with mitotane and doxorubicin in the inhibition of proliferation. In addition, we found that AMG 900 activated Notch signaling and rendered the cells sensitive to the combination of AMG 900 and Notch signaling inhibition. Altogether, these data show that aurora kinases inhibition using AMG 900 may be an adjuvant therapy to treat patients with invasive or recurrent adrenocortical carcinomas.

Mutations in the PCNA-binding site of CDKN1C inhibit cell proliferation by impairing the entry into S phase.

CDKN1C (also known as P57 (kip2) ) is a cyclin-dependent kinase inhibitor that functions as a negative regulator of cell proliferation through G1 phase cell cycle arrest. Recently, our group described gain-of-function mutations in the PCNA-binding site of CDKN1C that result in an undergrowth syndrome called IMAGe Syndrome (Intrauterine Growth Restriction, Metaphyseal dysplasia, Adrenal hypoplasia, and Genital anomalies), with life-threatening consequences. Loss-of-function mutations in CDKN1C have been identified in 5-10% of individuals with Beckwith-Wiedemann syndrome (BWS), an overgrowth disorder with features that are the opposite of IMAGe syndrome. Here, we investigate the effects of IMAGe-associated mutations on protein stability, cell cycle progression and cell proliferation. Mutations in the PCNA-binding site of CDKN1C significantly increase CDKN1C protein stability and prevent cell cycle progression into the S phase. Overexpression of either wild-type or BWS-mutant CDKN1C inhibited cell proliferation. However, the IMAGe-mutant CDKN1C protein decreased cell growth significantly more than both the wild-type or BWS protein. These findings bring new insights into the molecular events underlying IMAGe syndrome.

Update on the Use of l-Asparaginase in Infants and Adolescent Patients with Acute Lymphoblastic Leukemia.

Great improvements have been made in acute lymphoblastic leukemia (ALL) treatment in the past decades, especially due to the use of l-asparaginase (l-ASP). Despite the significant success rate, several side effects mainly caused by toxicity, asparaginase silent inactivation, and cellular resistance, encourage an open debate regarding the optimal dosage and formulation of l-ASP. Alternative sources of asparaginases have been constantly investigated in order to overcome hypersensitivity clinical toxicity. Additionally, genomic modulation as gene expression profiling, genetic polymorphisms, and epigenetic changes is also being investigated concerning their role in cellular resistance to l-ASP. Understanding the mechanisms that mediate the resistance to l-ASP treatment may bring new insights into ALL pathobiology and contribute to the development of more effective treatment strategies. In summary, this review presents an overview on l-ASP data and focuses on cellular mechanisms underlying resistance and alternative therapies for the use of asparaginase in childhood ALL treatment.

Zebularine induces chemosensitization to methotrexate and efficiently decreases AhR gene methylation in childhood acute lymphoblastic leukemia cells.

Acute lymphoblastic leukemia (ALL) is the most common hematologic malignancy in childhood. Despite the advances in treatment, about 20% of patients relapse and/or die, indicating the need for different therapies for this group. Zebularine (ZB) is a potent DNA methyltransferase (DNMT) inhibitor and has been associated with gene demethylation and enhancement of tumor chemosensitivity. This study aimed to evaluate the effects of ZB, alone or combined with chemotherapeutics (methotrexate and vincristine), on childhood ALL cell lines. Cell proliferation, apoptosis, and clonogenic capacity were studied in Jurkat and ReH cell lines. Bisulfite modification, followed by methylation-specific PCR was carried out to evaluate aryl hydrocarbon receptor (AhR) methylation status. Gene expression of DNMT1, DNMT3a, DNMT3b, and AhR was assessed using qRT-PCR. Both cell cultures were sensitive to ZB, showing a dose-dependent and time-dependent response (P<0.05). ZB induced apoptosis and decreased clonogenic capacity in both cell lines. Combination with methotrexate resulted in a strong synergistic effect, whereas combination with vincristine led to an antagonistic response in both cell lines. ZB treatment decreased gene expression of the three DNMTs and induced AhR gene promoter demethylation and its re-expression. These results indicate that ZB may be a promising drug for the adjuvant treatment of ALL, mainly when combined with methotrexate.

Hypoxia-related gene expression profile in childhood acute lymphoblastic leukemia: prognostic implications.

A cellular hypoxic condition is a key event in several human cancers, but knowledge about its role in childhood acute lymphoblastic leukemia (ALL) is very limited. In the present study, the gene expression profile of hypoxia-related genes (HIF1A, CA9, VEGF and SCL2A1) was evaluated in bone marrow samples of 113 pediatric patients. HIF1A mRNA up-regulation was significantly associated with higher 5-year event-free survival rates in patients with B-ALL as well as in the overall ALL population in both univariate and multivariate analysis (p = 0.023 and p = 0.041, respectively). In gene expression analysis, low oxygen levels promoted HIF1A activation in a time-dependent manner, in both ALL cell lines. In vitro cytotoxic assays suggested an initial trend toward hypoxia-related resistance in the first 24 h, but evaluation at later time points (48-72 h) clearly showed that there was no relevant difference in drug response when comparing hypoxic and normal oxygen level conditions. Modulation of mRNA expression of several hypoxia-related genes was also observed after hypoxic exposure in a cell specific manner, suggesting that HIF1A mRNA expression could play a different role in specific subtypes of leukemia. Despite the remaining questions regarding hypoxia-mediated mechanisms, these findings could be helpful to provide new insights into the role of hypoxia in childhood ALL.