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Clinical Trial
, 18 (6), 1716-25

Single-cell Pharmacodynamic Monitoring of S6 Ribosomal Protein Phosphorylation in AML Blasts During a Clinical Trial Combining the mTOR Inhibitor Sirolimus and Intensive Chemotherapy

Affiliations
Clinical Trial

Single-cell Pharmacodynamic Monitoring of S6 Ribosomal Protein Phosphorylation in AML Blasts During a Clinical Trial Combining the mTOR Inhibitor Sirolimus and Intensive Chemotherapy

Alexander E Perl et al. Clin Cancer Res.

Abstract

Purpose: Integration of signal transduction inhibitors into chemotherapy regimens generally has generally not led to anticipated increases in response and survival. However, it remains unclear whether this is because of inadequate or inconsistent inhibition of target or other complex biology. The mTOR signaling pathway is frequently activated in acute myelogenous leukemia (AML) and we previously showed the safety of combining the mTOR inhibitor, sirolimus, with mitoxantrone, etoposide, and cytarabine (MEC) chemotherapy. However, we did not reliably determine the extent of mTOR inhibition on that study. Here, we sought to develop an assay that allowed us to serially quantify the activation state of mTOR kinase during therapy.

Experimental design: To provide evidence of mTOR kinase activation and inhibition, we applied a validated whole blood fixation/permeabilization technique for flow cytometry to serially monitor S6 ribosomal protein (S6) phosphorylation in immunophenotypically identified AML blasts.

Results: With this approach, we show activation of mTOR signaling in 8 of 10 subjects' samples (80%) and conclusively show inhibition of mTOR in the majority of subjects' tumor cell during therapy. Of note, S6 phosphorylation in AML blasts is heterogeneous and, in some cases, intrinsically resistant to rapamycin at clinically achieved concentrations.

Conclusions: The methodology described is rapid and reproducible. We show the feasibility of real-time, direct pharmacodynamic monitoring by flow cytometry during clinical trials combining intensive chemotherapy and signal transduction inhibitors. This approach greatly clarifies pharmacokinetic/pharmacodynamic relationships and has broad application to preclinical and clinical testing of drugs whose direct or downstream effects disrupt PI3K/AKT/mTOR signaling.

Figures

Figure 1
Figure 1. treatment schema
A 12 mg loading dose of oral sirolimus was administered on day 1, followed by 8 daily doses of 4 mg sirolimus. Mitoxantrone 100 mg/m2/day, etoposide 100 mg/m2/day, and cytarabine 1000 mg/m2/day were each administered over 1 hour for five doses each starting after sirolimus administration on study day 4. Samples were obtained for pharmacodynamic measurement and rapamycin concentration measurement on day 4 prior to sirolimus dosing. A second rapamycin level was obtained prior to sirolimus dosing on day 7.
Figure 2
Figure 2. Identification of blast gate and establishment of positive gates for phospho-S6
Top: Color-enhanced contour plot showing CD45 by side scatter or CD33 pattern of this 100 μL sample of peripheral blood obtained on trial. Total white cell count is 5 × 103/μL with 23% blasts by light microscopy. Although not seen in all samples, this subject’s granulocytes show relatively low side scatter, consistent with a history of antecedent myelodysplasia and peripheral blood smear findings of hypogranular neutrophils. Blasts show dim CD33 expression, confirming myeloid lineage. CD34 was not expressed on this sample’s blasts but on other samples helped confirm immaturity (not shown). Bottom: In the same sample’s blasts, heterogeneity of S6 phosphorylation is noted, unlike granulocyte or lymphocytes. Too few monocytes were present for meaningful data evaluation in this population. PMA stimulated cells (right column) define a positive region for S6 phosphorylated cell events. The majority of blasts at baseline show no constitutive phosphorylation, but a small population has variable, but constitutive activation. Ex vivo rapamycin treatment of the baseline blood sample (middle column) inhibits mTOR and markedly reduces the percentage of blasts with constitutive S6 phosphorylation.
Figure 3
Figure 3. Ex vivo inhibition of mTOR predicts in vivo effects of oral sirolimus therapy
Top: Exposing aliquots of a representative baseline sample (see figure 2) to a increasing concentrations of rapamycin ex vivo suggest that target inhibition occurs at a whole blood concentration between 10 and 20 nM (9.1–18.2 ng/mL). Of note, higher concentrations do not further impair S6 phosphorylation, predicting that mTOR inhibition in this sample will occur at clinically achievable concentrations. Bottom: Serially obtained samples demonstrate abrogation of S6 phosphorylation in vivo during oral sirolimus therapy. Measured rapamycin concentration on day 4 was 4.7 ng/mL (5.1 nM). Exposure of the day 4 sample to ex vivo rapamycin in excess shows no significant additional effect, suggesting that near maximal target inhibition occurred in vivo. Note: although all samples shown in this figure were obtained from the same patient’s sample, the staining and cytometer data acquisition for the two experiments were not performed simultaneously. This accounts for minor variation in phospho-S6 staining across the ex vivo and in vivo experiments. Within experiments, cell samples were exposed to a single staining antibody cocktail, such that dynamic changes in phospho-signal can directly compared.
Figure 4
Figure 4
Patterns of AML blasts’ pharmacodynamic responses to oral sirolimus therapy Subjects’ samples showed either constitutive S6 phosphorylation at baseline (n=8) or no obvious phosphorylation at baseline (n=2). The effects of 72 hours of oral sirolimus therapy are shown as well as the measured rapamycin concentrations at the time of pharmacodynamic monitoring and clinical responses. Reduction in S6 phosphorylation (>50% reduction in percentage of cells in S6 + gate) defined rapamycin sensitivity. Abbreviations: CR= complete remission, PR= partial remission, NR= no response

Comment in

  • Phospho-specific Flow: Fixating on the Target
    M Levis. Clin Cancer Res 18 (6), 1493-5. PMID 22302900.
    Targeted therapies are all the rage in oncology research these days. The problem remains as to how to confirm that the target is actually being hit in vivo. This report d …

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