Single-Cell Pharmacodynamic Monitoring of S6 Ribosomal Protein in AML Blasts During Trials Combining Sirolimus and Intensive Chemotherapy: Target Inhibition Enhances Response

Mammalian target of rapamycin (mTOR) inhibitors enhance cytotoxic chemotherapy effects in primary acute leukemia samples in preclinical assays, prompting multi-center evaluation of regimens combining mTOR inhibitors plus induction chemotherapy in AML. As mTOR is frequently but not uniformly activated in primary AML samples, it is unclear which patients benefit from this targeted approach. Thus, we sought to monitor mTOR kinase activity during therapy to determine whether target activation and/or inhibition predicted clinical response. We previously reported the feasibility of real-time, pharmacodynamic monitoring of ribosomal S6 phosphorylation (pS6) in leukemic blasts using flow cytometry of fixed whole blood (Perl et al, ASH 2009, #413). mTOR directly regulates the p70S6 kinase and its phosphorylation of S6 at serines 235/6 is inhibited by rapamycin. Thus pS6 provides a surrogate marker of mTOR kinase activity. Fixing whole blood and/or marrow preserves phosphorylation states in the presence of administered signal transduction inhibitors, thus avoiding cell-processing effects. Here we update our experience monitoring pS6 in AML blasts by flow during clinical trials combining sirolimus and AML induction chemotherapy.

Subjects had relapsed/refractory AML or untreated AML with unfavorable risk factors (e.g. therapy-related, prior MDS or MPN, or age >60 with non-favorable karyotype) and received oral sirolimus (12 mg on day 1, then 4 mg daily on days 2–9) plus MEC (mitoxantrone 8 mg/m²/day, etoposide 100 mg/m²/day, cytarabine 1 gm/m²/d on days 4–8) on one of two successive clinical trials. Clinical response was assessed at hematologic recovery or day 42 using IWG criteria (CR, CRp, PR vs. non-response). Pharmacodynamic samples were collected from blood or marrow at baseline, 2 hours post-sirolimus dose on days 1 and 4, and at trough on day 4 (prior to chemotherapy administration). Concurrent blood rapamycin concentration was measured by immunoassay or HPLC. Whole blood/marrow fixation was performed using published methods (Chow & Hedley, Cytometry A, 2005). Positive gates for pS6 were created by comparing blasts in ex vivo stimulated (phorbol ester/PMA) and inhibited (rapamycin) conditions and/or autofluorescence (FMO) controls.

27 subjects provided paired day 1 and day 4 flow samples and were evaluable for clinical response at the time of submission. Mean peak and trough rapamycin levels were 22.8 and 9.2 ng/ml, respectively, and did not differ among clinically responding and non-responding subjects. 17/27 (63%) subjects' blasts had constitutive S6 phosphorylation at baseline. Consistent with prior reports, pS6 was heterogeneous and typically present in a subset of blasts. In these 17 subjects, we observed a median of 13% pS6+ blasts (mean 14, range 2–33). 14/17 showed a therapy-induced reduction in pS6+ blasts to a mean of 4.5% (median 2.5, range 0.4–22) on day 4. The remainder had either no change or increased pS6+ blasts. Comparing the percentage of pS6+ cells on day 4 to baseline, the median reduction in pS6 differed among clinically responding and non-responding subjects (72% and 43%, respectively). The clinical response rate was 9/17 (53%, 6 CR 3 PR) among subjects with baseline S6 phosphorylation and 4/10 (40%, 3 CR, 1 PR) in patients with no baseline S6 phosphorylation. Subjects with >50% reduction in pS6 blasts on day 4 were considered to be biochemically sensitive to rapamycin, while subjects with <50% reduction or increased pS6 were considered rapamycin-resistant. The response rate in rapamycin-sensitive patients was 8/12 (67%), while in resistant subjects was 1/5 (20%). 5/15 (33%) subjects who either lacked baseline pS6 or were rapamycin resistant responded to the regimen.

Data from these ongoing trials suggest that sirolimus plus MEC preferentially benefits the subset of patients with demonstrable baseline mTOR activation. The greatest response is seen when mTOR is activated in leukemic cells at baseline and its function is potently inhibited during therapy. Future trials may benefit from enrichment for subjects with mTOR activation and/or rapamycin sensitivity assessment. These data provide in vivo evidence that mTOR inhibitors augment cytotoxic chemotherapy effect in AML and demonstrate the utility of fixed whole blood flow cytometry for real-time pharmacodynamic evaluation of novel signal transduction inhibitors.

Off Label Use: Rapamycin. FDA approved for solid organ transplant. Investigational use for treatment of leukemia. Carroll:Agios Pharmaceuticals: Research Funding; TetraLogic Pharmaceuticals: Research Funding; Sanofi Aventis Corporation: Research Funding; Glaxo Smith Kline, Inc.: Research Funding.