Sirolimus enhances remission induction in patients with high risk acute myeloid leukemia and mTORC1 target inhibition

Abstract

Background Mammalian Target of Rapamycin Complex 1 (mTORC1) inhibitors enhance chemotherapy response in acute myelogenous leukemia (AML) cells in vitro. However whether inhibiting mTORC1 enhances clinical response to AML chemotherapy remains controversial. We previously optimized measurement of mTORC1's kinase activity in AML blasts during clinical trials using serial phospho-specific flow cytometry of formaldehyde-fixed whole blood or marrow specimens. To validate mTORC1 as a therapeutic target in AML, we performed two clinical trials combining an mTORC1 inhibitor (sirolimus) and MEC (mitoxantrone, etoposide, cytarabine) in patients with relapsed, refractory, or untreated high-risk AML. Methods Flow cytometric measurements of ribosomal protein S6 phosphorylation (pS6) were performed before and during sirolimus treatment to determine whether mTORC1 inhibition enriched for chemotherapy response. Results In 51 evaluable subjects, the overall response rate (ORR) to the combination regimen was 47% (95% confidence interval 33-61%, 33% CR, 2% CRi, 12% PR) and similar toxicity to historic experience with MEC alone. 37 subjects had baseline pS6 measured pre-sirolimus, of whom 27 (73%) exhibited mTORC1 activity. ORR was not significantly different between subjects with and without baseline mTORC1 activity (52% vs 40%, respectively, p = 0.20). The ORR among subjects with baseline target activation and mTORC1 inhibition during therapy was 71% (12/17) compared to 20% (2/10) in subjects without target inhibition. Conclusions Fixed, whole blood pS6 by flow cytometry may be a predictive biomarker for clinical response to mTORC1 inhibitor-based regimens. These data provide clinical confirmation that mTORC1 activation mediates chemotherapy resistance in patients with AML.

Keywords: Acute myeloid leukemia; Biomarker; Phospho-flow cytometry; mTOR; mTORC1.

Fig. 1

Treatment schema showing the sirolimus and MEC treatment regimen. Arrows indicate pharmacokinetic/pharmacodynamic sampling time points used on final analysis. To estimate peak concentration effects of sirolimus, drug concentration (and associated phospho-flow analysis) was also measured on a subset of subjects 2 hours following sirolimus dosing on study days 1 and 4, not shown

Fig. 2

(a) Paired analysis of pS6 positivity at baseline and trough level on day 4 in gated blasts. Median percent pS6 positivity was 9.7% (range 2.2 – 41.4%) at baseline and 3.0 (0.43 – 78.0%) at day 4 (Wilcoxon signed ranks test, p=0.004). (b) Percent change in pS6 positivity from baseline to day 4, for 27 patients with baseline pS6 activation. Patients are grouped by overall response. Responders include CR, CRi and PR. The solid line indicates no change from baseline. The dotted line indicates the cut point of >40% reduction used to distinguish sirolimus sensitive patients from sirolimus resistant patients.

Fig. 2

(a) Paired analysis of pS6 positivity at baseline and trough level on day 4 in gated blasts. Median percent pS6 positivity was 9.7% (range 2.2 – 41.4%) at baseline and 3.0 (0.43 – 78.0%) at day 4 (Wilcoxon signed ranks test, p=0.004). (b) Percent change in pS6 positivity from baseline to day 4, for 27 patients with baseline pS6 activation. Patients are grouped by overall response. Responders include CR, CRi and PR. The solid line indicates no change from baseline. The dotted line indicates the cut point of >40% reduction used to distinguish sirolimus sensitive patients from sirolimus resistant patients.

Fig. 3

Frequency of clinical responses to sirolimus plus MEC among subjects grouped for baseline mTORC1 activation and/or target inhibition as measured by percentage pS6 change in gated blasts by phospho-flow. Dotplots show gated blasts pS6 on vertical axis. Horizontal line to define pS6+ events was drawn for individual samples using dynamic controls. Biochemical sensitivity is defined by >40% reduction in pS6 on day 4, compared to baseline. CR= complete remission, CRi= complete remission with incomplete hematologic recovery, PR= partial remission, ORR= overall response rate

Fig. 4

Survival of patients treated with sirolimus plus MEC (a) Kaplan-Meier estimate of overall survival from first day of treatment. (b) Event free survival for 23 patients who responded to sirolimus plus MEC.

Fig. 4

Survival of patients treated with sirolimus plus MEC (a) Kaplan-Meier estimate of overall survival from first day of treatment. (b) Event free survival for 23 patients who responded to sirolimus plus MEC.