Background: PI3K/mTOR signaling, a critical pathway in cell proliferation, metabolism, and apoptosis, is commonly dysregulated in ALL. A phase 1 trial of the mTOR inhibitor temsirolimus in combination with re-induction chemotherapy was performed in children with second or greater relapse of ALL. Methods: Temsirolimus was administered with 4-drug chemotherapy (UK R3 ALL re-induction; Parker, Lancet 2010). The starting dose level (DL1) of intravenous temsirolimus was 10mg/m² weekly x3 ; subsequent cohorts received temsirolimus 7.5mg/m² weekly x3 (DL0); or 7.5mg/m²weekly x2 (DL-1). PI3K pathway inhibition was measured by phosphoflow analysis (PFA) of peripheral blood. Results: Sixteen patients, age 1-21, [15 pre B-ALL (3 MLL infants, 2 Ph+); 1 T-ALL] were enrolled, 15 were evaluable. Dose-limiting toxicity (DLT) occurred in 2/5 patients at DL1; 3/6 at DL0 and 3/5 at DL-1. DLTs were hypertriglyceridemia, mucositis, gastric ulcer, hypertension with reversible posterior leukoencephalopathy, elevated GGT and alk phos, and severe infections including 1 death due to sepsis. Seven patients had a complete response, 3/7 had MRD < 0.01% at end therapy. Responses occurred at all dose levels of temsirolimus. Phospho(p)S6 and/or p4EBP1 were inhibited in a subset of patients with compensatory upregulation of pPI3K, pmTOR, and pAkt. High basal PI3K pathway signaling was observed in patients with poorer response to therapy. Conclusions: Temsirolimus in combination with UK R3 chemotherapy can induce responses in children with ALL; however, this intensive regimen is associated with unacceptable toxicity. A trial evaluating temsirolimus in combination with etoposide/cyclophosphamide in children with relapsed ALL is ongoing. Clinical trial information: NCT01403415