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Purpose: Dasatinib is a dual Src/Abl inhibitor recently approved for Bcr-Abl+ leukemias with resistance or intolerance to prior therapy. Because Src kinases contribute to multiple blood cell functions by triggering a variety of signaling pathways, we hypothesized that their molecular targeting might lead to growth inhibition in acute myeloid leukemia (AML).Experimental Design: We studied growth factor–dependent and growth factor–independent leukemic cell lines, including three cell lines expressing mutants of receptor tyrosine kinases (Flt3 or c-Kit) as well as primary AML blasts for responsiveness to dasatinib.Results: Dasatinib resulted in the inhibition of Src family kinases in all cell lines and blast cells at ∼1 × 10−9 mol/L. It also inhibited mutant Flt3 or Kit tyrosine phosphorylation at ∼1 × 10−6 mol/L. Mo7e cells expressing the activating mutation (codon 816) of c-Kit were most sensitive to growth inhibition with a GI50 of 5 × 10−9 mol/L. Primary AML blast cells exhibited a growth inhibition of −6 mol/L. Cell lines that showed growth inhibition at ∼1 × 10−6 mol/L showed a G1 cell cycle arrest and correlated with accumulation of p21 and p27 protein. The addition of rapamycin or cytotoxic agents enhanced growth inhibition. Dasatinib also caused the apoptosis of Mo7e cells expressing oncogenic Kit.Conclusions: Although all of the precise targets for dasatinib are not known, this multikinase inhibitor causes either growth arrest or apoptosis in molecularly heterogeneous AML. The addition of cytotoxic or targeted agents can enhance its effects. Clin Cancer Res; 16(4); 1149–58

Supplementary Data from Dasatinib Inhibits the Growth of Molecularly Heterogeneous Myeloid Leukemias

Purpose To determine the efficacy and safety of clofarabine in pediatric patients with refractory or relapsed acute myeloid leukemia (AML). Patients and Methods A phase II, open-label, multicenter study was conducted with single-agent clofarabine in pediatric patients with refractory or relapsed AML. Clofarabine was administered intravenously over 2 hours at the pediatric maximum-tolerated dose (MTD) of 52 mg/m2 daily for 5 consecutive days. Cycles were repeated every 2 to 6 weeks. Responses determined by an independent response review panel. Results The 42 patients treated on the study had a median age of 13 years (range, 2 to 22 years) and had received a median number of two (range, one to five) prior regimens. The response rate was 26% and included one complete response without platelet recovery and 10 partial responses. The median duration of response was 20 weeks (range, 2 to ≥ 156 weeks). Six of 28 patients who were refractory to the immediately preceding therapy achieved response. Thirteen patients (31%), including seven responders, proceeded to hematopoietic stem-cell transplantation (HSCT) after treatment with clofarabine and survived between 24 to ≥ 160 weeks. Five patients (12%) remain alive post-transplantation at ≥ 63, ≥ 71, ≥ 86, ≥ 114, and ≥ 130 weeks. The most common grade 3 or greater adverse events without regard to causality were febrile neutropenia, catheter-related infection, epistaxis, hypotension, nausea, and fever. Transient elevation of liver enzymes and hypokalemia occurred frequently. Five patients died within 30 days of clofarabine administration secondary to progressive disease, and another five died as a result of an adverse event. Conclusion Clofarabine is active in pediatric patients with multiply relapsed or refractory AML. Responses allowed several refractory patients to proceed to HSCT. The toxicity profile was expected in this patient population.

Conformational changes accompanying P-glycoprotein (Pgp) mediated drug transport are reflected by changes in the avidity of certain monoclonal antibodies (mAbs). More of the UIC2 mAb binds to Pgp-expressing cells in the presence of substrates or modulators [Mechetner, E.B., Schott, B., Morse, S.B., Stein, W., Druley, T., Dvis, K.A., Tsuruo, T. & Roninson, I.B. (1997) Proc. Natl Acad. Sci. USA 94, 12908-12913], while the binding of other mAbs (e.g. MM12.10, MRK16, 4E3) is not conformation sensitive. Pre-staining of Pgp+ cells with UIC2 decreased the subsequent binding of MM12.10 mAb by about 30-40%, suggesting that there are Pgp molecules available for both UIC2 and MM12.10, and others accessible only for MM12.10. In the presence of certain substrates/modulators such as vinblastin, cyclosporin A or valinomycin, the MM12.10 reactivity was completely abolished by preincubation with UIC2. However, verapamil, Tween-80 and nifedipine did not influence the ratio of bound mAbs significantly. This is the first assay to our knowledge, sharply distinguishing two classes of modulators. The conformational changes accompanying the mAb competition phenomenon appear to be closely related, though not identical to those accompanying the UIC2-shift, as suggested by the simultaneous assessment of the two phenomena.

We administered high doses of calcitriol (up to 32 micrograms per day) to an infant with malignant osteopetrosis, in an attempt to stimulate bone resorption. The patient was placed on a low-calcium diet to prevent hypercalcemia. Measures of bone turnover increased during calcitriol therapy; hydroxyproline excretion rose from 140 to 1358 micrograms per milligram of creatinine per 24 hours, with parallel increases in the ratio of calcium to creatinine in the urine, urinary gamma-carboxyglutamic acid, serum osteocalcin, and serum alkaline phosphatase. A pretreatment bone-biopsy specimen contained no osteoclasts with ruffled borders, a feature of active osteoclasts. After 11 days of calcitriol, ruffled borders were noted. After three months, numerous osteoclasts with ruffled borders and associated bony disruption were evident. Before therapy, the patient's monocytes were incapable of in vitro bone resorption, but after calcitriol, their resorptive capacity was increased to 3.3 times control levels. These data demonstrate that calcitriol increased bone mineral and matrix turnover in our patient. However, during the three months of calcitriol therapy there was only slight clinical improvement in her severe disease. Early and sustained treatment with calcitriol may be useful in osteopetrosis.