Significant benefits of AIP testing and clinical screening in familial isolated and young-onset pituitary tumors

Context Germline mutations in the aryl hydrocarbon receptor-interacting protein (AIP) gene are responsible for a subset of familial isolated pituitary adenoma (FIPA) cases and sporadic pituitary neuroendocrine tumors (PitNETs). Objective To compare prospectively diagnosed AIP mutation-positive (AIPmut) PitNET patients with clinically presenting patients and to compare the clinical characteristics of AIPmut and AIPneg PitNET patients. Design 12-year prospective, observational study. Participants & Setting We studied probands and family members of FIPA kindreds and sporadic patients with disease onset ≤18 years or macroadenomas with onset ≤30 years (n = 1477). This was a collaborative study conducted at referral centers for pituitary diseases. Interventions & Outcome AIP testing and clinical screening for pituitary disease. Comparison of characteristics of prospectively diagnosed (n = 22) vs clinically presenting AIPmut PitNET patients (n = 145), and AIPmut (n = 167) vs AIPneg PitNET patients (n = 1310). Results Prospectively diagnosed AIPmut PitNET patients had smaller lesions with less suprasellar extension or cavernous sinus invasion and required fewer treatments with fewer operations and no radiotherapy compared with clinically presenting cases; there were fewer cases with active disease and hypopituitarism at last follow-up. When comparing AIPmut and AIPneg cases, AIPmut patients were more often males, younger, more often had GH excess, pituitary apoplexy, suprasellar extension, and more patients required multimodal therapy, including radiotherapy. AIPmut patients (n = 136) with GH excess were taller than AIPneg counterparts (n = 650). Conclusions Prospectively diagnosed AIPmut patients show better outcomes than clinically presenting cases, demonstrating the benefits of genetic and clinical screening. AIP-related pituitary disease has a wide spectrum ranging from aggressively growing lesions to stable or indolent disease course

8-Chloroadenosine Sensitivity in Renal Cell Carcinoma Is Associated with AMPK Activation and mTOR Pathway Inhibition.

The adenosine analog 8-chloroadenosine has been shown to deplete ATP and inhibit tumor growth in hematological malignancies as well as in lung and breast cancer cell lines. We investigated effects of 8-chloroadenosine on clear cell (cc) renal cell carcinoma (RCC) cell lines. 8-chloroadenosine was effective against ccRCC cell viability in vitro, with IC50 ranging from 2 μM in the most sensitive CAKI-1 to 36 μM in the most resistant RXF-393. Proteomic analysis by reverse-phase protein array revealed that 8-chloroadenosine treatment leads to inhibition of the mTOR pathway. In time-course experiments, 8-chloroadenosine treatment rapidly activated AMPK, measured by AMPK and ACC phosphorylation, and subsequently caused dephosphorylation of p70S6K and ribosomal protein RPS6 in the sensitive cell lines. However, in the resistant cell lines, AMPK activity and the mTOR pathway were unaffected by the treatment. We also noted that the resistant cell lines had elevated basal levels of phospho RPS6 and AKT. Inhibition of PI3K pathway enhanced the efficacy of 8-chloroadenosine across all cell lines. Our observations indicate that 8-chloroadenosine activity is associated with inhibition of the mTOR pathway, and that phospho RPS6 and PI3K pathway activation status may determine resistance. Among solid tumors, RCC is one of the few susceptible to mTOR inhibition. We thus infer that 8-chloroadenosine may be effective in RCC by activating AMPK and inhibiting the mTOR pathway

Decrease in ATP and AMPK activation in sensitive and resistant cell lines.

<p><b>a.</b> ATP levels in sensitive and resistant ccRCC cell lines after 6 hours of 40 μM 8-chloroadenosine treatment. ATP levels decreased significantly in all cell lines (CAKI1 p<0.01, ACHN p = 0.02, RCC4 p<0.01, RXF393 p<0.01); however, ATP levels were not significantly different between the sensitive and resistant cell lines (p = n.s.). <b>b.</b> Western blots for phospho AMPK T172 and AMPK activity readout phospho ACC S79 after 0, 8, 16, and 24 hours of treatment with 40 μM 8-chloroadenosine. Vinculin is used as loading control.</p

Efficacy of 8-chloroadenosine in ccRCC cell lines.

<p><b>a.</b> ccRCC cell lines treated with 20 μM or 80 μM 8-chloroadenosine for 7 days. <b>b.</b> Cell cycle analysis of ccRCC cell lines treated with 40 μM 8-chloroadenosine for 48 hours. <b>c.</b> IC₅₀ values of ccRCC cell lines calculated in Calcusyn software.</p

PI3K pathway is active at baseline in 8-chloroadenosine resistant, but not in sensitive, cell lines.

<p><b>a</b>. RPPA heatmap showing proteins that are differentially expressed between the most sensitive and resistant cell lines at baseline prior to treatment. AKT phospho S473 and S308 were higher and phospho AMPK T172, ACC S79, and total PTEN were lower in the resistant cell lines. <b>b</b>. Western blots showing phospho and total AKT and S6 levels in sensitive and resistant cells before and after 40 μM 8-chloroadenosine treatment. Beta-actin served as loading control. <b>c</b>. Western blots showing phospho ERK T202/204 levels in sensitive and resistant cells before and after 40 μM 8-chloroadenosine treatment. Beta-actin served as loading control. <b>d</b>. Quantification of phospho AKT, S6, and ERK and total AKT and S6 levels.</p

mTOR pathway downregulation in sensitive cell lines upon treatment with 8-chloroadenosine.

<p><b>a</b>. RPPA heatmap showing expression changes that correlate with IC₅₀ values. ccRCC cells were treated with 40 μM 8-chloroadenosine for 24 hours. Graphs show changes in phospho S6 levels in sensitive and resistant cell lines. p-values are calculated using two-tailed paired t-test. coef. Pearson correlation coefficient between the IC₅₀ values and levels of proteins measured by RPPA. <b>b</b>. Western blot validating phospho S6 S235/236 downregulation in CAKI1 and ACHN cell lines upon 8-chloroadenosine treatment for 24 hours. <b>c</b>. Western blot showing phospho ACC S79 as readout of AMPK activity and phospho p70S6K T389 and phospho S6 S235/236 for mTOR pathway activity. ccRCC cells were treated with 40 μM 8-chloroadenosine for 1, 5, 10, 18, 35, 60, 120, or 300 minutes. Vinculin served as loading control.</p