Lactate and choline metabolites detected in vitro by nuclear magnetic resonance spectroscopy are potential metabolic biomarkers for PI3K inhibition in pediatric glioblastoma.

The phosphoinositide 3-kinase (PI3K) pathway is believed to be of key importance in pediatric glioblastoma. Novel inhibitors of the PI3K pathway are being developed and are entering clinical trials. Our aim is to identify potential non-invasive biomarkers of PI3K signaling pathway inhibition in pediatric glioblastoma using in vitro nuclear magnetic resonance (NMR) spectroscopy, to aid identification of target inhibition and therapeutic response in early phase clinical trials of PI3K inhibitors in childhood cancer. Treatment of SF188 and KNS42 human pediatric glioblastoma cell lines with the dual pan-Class I PI3K/mTOR inhibitor PI-103, inhibited the PI3K signaling pathway and resulted in a decrease in phosphocholine (PC), total choline (tCho) and lactate levels (p<0.02) as detected by phosphorus (31P)- and proton (1H)-NMR. Similar changes were also detected using the pan-Class I PI3K inhibitor GDC-0941 which lacks significant mTOR activity and is entering Phase II clinical trials. In contrast, the DNA damaging agent temozolomide (TMZ), which is used as current frontline therapy in the treatment of glioblastoma postoperatively (in combination with radiotherapy), increased PC, glycerophosphocholine (GPC) and tCho levels (p<0.04). PI-103-induced NMR changes were associated with alterations in protein expression levels of regulatory enzymes involved in glucose and choline metabolism including GLUT1, HK2, LDHA and CHKA. Our results show that by using NMR we can detect distinct biomarkers following PI3K pathway inhibition compared to treatment with the DNA-damaging anti-cancer agent TMZ. This is the first study reporting that lactate and choline metabolites are potential non-invasive biomarkers for monitoring response to PI3K pathway inhibitors in pediatric glioblastoma

Model free approach to kinetic analysis of real-time hyperpolarized (13)c magnetic resonance spectroscopy data

Real-time detection of the rates of metabolic flux, or exchange rates of endogenous enzymatic reactions, is now feasible in biological systems using Dynamic Nuclear Polarization Magnetic Resonance. Derivation of reaction rate kinetics from this technique typically requires multi-compartmental modeling of dynamic data, and results are therefore model-dependent and prone to misinterpretation. We present a model-free formulism based on the ratio of total areas under the curve (AUC) of the injected and product metabolite, for example pyruvate and lactate. A theoretical framework to support this novel analysis approach is described, and demonstrates that the AUC ratio is proportional to the forward rate constant k. We show that the model-free approach strongly correlates with k for whole cell in vitro experiments across a range of cancer cell lines, and detects response in cells treated with the pan-class I PI3K inhibitor GDC-0941 with comparable or greater sensitivity. The same result is seen in vivo with tumor xenograft-bearing mice, in control tumors and following drug treatment with dichloroacetate. An important finding is that the area under the curve is independent of both the input function and of any other metabolic pathways arising from the injected metabolite. This model-free approach provides a robust and clinically relevant alternative to kinetic model-based rate measurements in the clinical translation of hyperpolarized (13)C metabolic imaging in humans, where measurement of the input function can be problematic

Metabolic changes following treatment of KNS42 pediatric glioblastoma cells with PI-103.

<p>Representative <i>in vitro</i> (A) ³¹P-NMR spectra and (B) expansion of ¹H-NMR spectra regions representing choline–containing metabolites (left) or lactate (Lac; right) of KNS42 aqueous cell extracts following 12 or 24 hours treatment with PI-103 (5×GI₅₀) compared to vehicle (DMSO) treated control.</p

Time-response analysis showing percentage changes in ³¹P-NMR-detected metabolite levels following treatment of KNS42 pediatric glioblastoma cells with PI-103 (5×GI₅₀).

<p>Data are expressed as percentage of treated to control (% T/C) and presented as the mean ± SD, n≥3.</p><p>PE in KNS42 cells was not consistently detectable due to the low levels of this metabolite and was not affected by treatment with PI-103.</p><p>Glycerophosphoethanolamine (GPE) level was not affected by treatment with PI-103.</p><p>Two-tailed unpaired <i>t</i> test was used to compare results in treated cells to controls.</p><p>*P<0.05.</p

Metabolic changes following treatment of SF188 pediatric glioblastoma cells with PI-103.

<p>Representative <i>in vitro</i> (A) ³¹P-NMR spectra and (B) expansion of ¹H-NMR spectra regions representing choline–containing metabolites (left) or lactate (Lac; right) of SF188 aqueous cell extracts following 16 or 24 hours treatment with PI-103 (5×GI₅₀) compared to vehicle (DMSO) treated control. UDPs = UDP sugars.</p

Time-response analysis of cell cycle effects following treatment of SF188 pediatric glioblastoma cells with PI-103 (5×GI₅₀).

<p>Data are expressed as % total cell count and presented as the mean ± SD, n≥3.</p><p>Two-tailed unpaired <i>t</i> test was used to compare results in treated cells to controls.</p><p>*P<0.05,</p><p>**P<0.005,</p><p>***P<0.0005.</p

Comparison of molecular and metabolic changes caused by 24 hours treatment of SF188 pediatric glioblastoma cells with PI-103 or TMZ at 2×GI₅₀.

<p>(A) Representative Western blots showing inhibition of the PI3K signaling pathway as indicated by decreased phosphorylation of AKT (Ser473) and RPS6 (Ser240/244) following treatment with PI-103 but not with TMZ. (B) Quantification of ¹H-NMR detected metabolite changes. Results are expressed as percentage of treated to control and presented as the mean ± SD (error bars) of at least three separate experiments. Statistically significantly different from the control *p≤0.05, **p≤0.01, ***p<0.005, ^¥p<0.0005; two-tailed unpaired <i>t</i> test was used for all comparisons.</p

Time-response analysis showing percentage changes in ¹H-NMR-detected metabolite levels following treatment of SF188 pediatric glioblastoma cells with PI-103 (5×GI₅₀).

<p>Data are expressed as percentage of treated to control (% T/C) and presented as the mean ± SD, n≥3.</p><p>Two-tailed unpaired <i>t</i> test was used to compare results in treated cells to controls.</p><p>*P<0.05,</p><p>**P<0.005,</p><p>***P<0.0005.</p

Molecular changes following treatment (48 h) with TMZ, PI-103 or PI-103+TMZ in KNS42 paediatric glioblastoma cells.

<p>(A) Representative Western blots showing decreases in molecular markers in the PI3K signalling pathway post-treatment relative to controls. (B) Representative Western blots showing induction of apoptosis as evidenced by the presence of cleaved PARP following treatment with TMZ or the combination of PI-103 with TMZ. GAPDH is used as a loading control. (C) A summary of the cell cycle distribution of control cells (DMSO), or following treatment.</p

Molecular and metabolic percentage changes caused by treatment of pediatric glioblastoma cells with GDC-0941 or PI-103 (5×GI₅₀, 24 hours).

<p>Representative Western blots showing inhibition of the PI3K signaling pathway as indicated by decreased phosphorylation of AKT (Ser473) and RPS6 (Ser240/244) post-treatment with GDC-0941 in: (A) SF188 or (B) KNS42 cells. Comparison of ¹H-NMR detected metabolite percentage changes caused by treatment with PI-103 or GDC-0941 in: (C) SF188 or (D) KNS42 cells. Results are expressed as percentage of treated to control and presented as the mean ± SD (error bars) of at least three separate experiments. Statistically significantly different from the control *p≤0.05, **p≤0.01, ***p<0.005, ^¥p<0.0005; two-tailed unpaired <i>t</i> test was used for all comparisons.</p