Abstract. Binding of GTP induces ot subunits of heterotrimeric G proteins to take on an active conformation, capable of regulating effector molecules. We expressed epitope-tagged versions of the ct subunit (txs) of Gs in genetically us-deficient $49 cyc-cells. Addition of a hemagglutinin (HA) epitope did not alter the ability of wild type t~s to mediate hormonal stimulation of adenylyl cyclase or to attach to cell membranes. The HA epitope did, however, allow a mAb to immunoprecipitate the recombinant protein (HA-o~) quantitatively from cell extracts. We activated the epitope-tagged t~ in intact cells by: (a) exposure of cells to cholera toxin, which activates ot~ by covalent modification; (b) mutational replacement of arginine-201 in HA-ct~ by a cysteine residue, to create HA-ct~-R201C; like the cholera toxin-catalyzed modification, this mutation activates c~ by slowing its intrinsic GTPase activity; and (c) treatment of cells with the ~-adrenoceptor agonist, isoproterenol, which promotes binding of GTP to ets, thereby activating adenylyl cyclase. Both cholera toxin and the R201C mutation accelerated the rate of degradation of as (0.03 h -1) by three-to fourfold and induced a partial shift of the protein from a membrane bound to a soluble compartment. At steady state, 80% of HA-o~c R201C was found in the soluble fraction, as compared to 10% of wild type HA-ors. Isoproterenol rapidly (in <2 min) caused 20% of HA-o~s to shift from the membrane-bound to the soluble compartment. Cholera toxin induced a 3.5-fold increase in the rate of degradation of a second mutant, HA-c~,-G226A, but did not cause it to move into the soluble fraction; this observation shows that loss of membrane attachment is not responsible for the accelerated degradation of o~ in response to activation. Taken together, these findings show that activation of or, induces a conformational change that loosens its attachment to membranes and increases its degradation rate. HETEROTRIM~RIC G proteins transduce signals from cell-surface receptors to membrane-bound etfector molecules, including adenyly cyclase, phospholipase C, and ion channels (2,8,32). Agonist-bound receptors activate G proteins by promoting exchange of GTP for GDP bound to the ct subunit of the heterotrimer, ot-GTP rapidly dissociates from the ~3' complex and can interact with the effector until its intrinsic GTPase converts it to ot-GDP, allowing re-association with ~3'. Experirnents in broken cells, confirmed and refined in studies using biochemically pure components, show that the nucleotide-bound state of the ot subunit determines its interactions with other proteins.How do changes in the active state of G protein o~ subunits affect their behavior in the more complex environment of an intact cell? Attempts to answer this question require experimental models that allow manipulation of the nucleotidebound state of intracellular c~ subunits and assessments of their number, subcellular distribution, turnover, and associations with other proteins. Here we report an attempt to devise and...

The clinical benefit of adding FMS-like tyrosine kinase-3 (FLT3)-directed small molecule therapy to standard first-line treatment of acute myeloid leukemia (AML) has not yet been established. As part of the UK AML15 and AML17 trials, patients with previously untreated AML and confirmed FLT3-activating mutations, mostly younger than 60 years, were randomly assigned either to receive oral lestaurtinib (CEP701) or not after each of 4 cycles of induction and consolidation chemotherapy. Lestaurtinib was commenced 2 days after completing chemotherapy and administered in cycles of up to 28 days. The trials ran consecutively. Primary endpoints were overall survival in AML15 and relapse-free survival in AML17; outcome data were meta-analyzed. Five hundred patients were randomly assigned between lestaurtinib and control: 74% had -internal tandem duplication mutations, 23%-tyrosine kinase domain point mutations, and 2% both types. No significant differences were seen in either 5-year overall survival (lestaurtinib 46% vs control 45%; hazard ratio, 0.90; 95% CI 0.70-1.15; = .3) or 5-year relapse-free survival (40% vs 36%; hazard ratio, 0.88; 95% CI 0.69-1.12; = .3). Exploratory subgroup analysis suggested survival benefit with lestaurtinib in patients receiving concomitant azole antifungal prophylaxis and gemtuzumab ozogamicin with the first course of chemotherapy. Correlative studies included analysis of in vivo FLT3 inhibition by plasma inhibitory activity assay and indicated improved overall survival and significantly reduced rates of relapse in lestaurtinib-treated patients who achieved sustained greater than 85% FLT3 inhibition. In conclusion, combining lestaurtinib with intensive chemotherapy proved feasible in younger patients with newly diagnosed -mutated AML, but yielded no overall clinical benefit. The improved clinical outcomes seen in patients achieving sustained FLT3 inhibition encourage continued evaluation of FLT3-directed therapy alongside front-line AML treatment. The UK AML15 and AML17 trials are registered at www.isrctn.com/ISRCTN17161961 and www.isrctn.com/ISRCTN55675535 respectively.

This randomized, open-label, phase 2b study (NCT01565668) evaluated the efficacy and safety of 2 dosing regimens of quizartinib monotherapy in patients with relapsed/refractory (R/R) -internal tandem duplication (ITD)-mutated acute myeloid leukemia (AML) who previously underwent transplant or 1 second-line salvage therapy. Patients (N = 76) were randomly assigned to 30- or 60-mg/day doses (escalations to 60 or 90 mg/day, respectively, permitted for lack/loss of response) of single-agent oral quizartinib dihydrochloride. Allelic frequency of at least 10% was defined as-ITD-mutated disease. Coprimary endpoints were composite complete remission (CRc) rates and incidence of QT interval corrected by Fridericia's formula (QTcF) of more than 480 ms (grade 2 or greater). Secondary endpoints included overall survival (OS), duration of CRc, bridge to transplant, and safety. CRc rates were 47% in both groups, similar to earlier reports with higher quizartinib doses. Incidence of QTcF above 480 ms was 11% and 17%, and QTcF above 500 ms was 5% and 3% in the 30- and 60-mg groups, respectively, which is less than earlier reports with higher doses of quizartinib. Median OS (20.9 and 27.3 weeks), duration of CRc (4.2 and 9.1 weeks), and bridge to transplant rates (32% and 42%) were higher in the 60-mg groups than in the 30-mg group. Dose escalation occurred in 61% and 14% of patients in the 30- and 60-mg groups, respectively. This high clinical activity of quizartinib at the evaluated doses is consistent with previous reports with an improved safety profile. Need to dose-escalate more than half of patients who received quizartinib 30 mg also supports further investigation of treatment with quizartinib 60 mg/day.

Purpose: Flavopiridol is a cyclin-dependent kinase inhibitor that is cytotoxic to leukemic blasts.In a phase I study of flavopiridol followed by 1-h-D-arabinofuranosylcytosine (ara-C) and mitoxantrone, overall response rate for adults with relapsed and refractory acute myelogenous leukemias (AML) was 31%. We have now completed a phase II study of sequential flavopiridol, ara-C, and mitoxantrone in 62 adults with poor-risk AML. Experimental Design: Flavopiridol (50 mg/m 2 ) was given by 1-h infusion daily  3 beginning day 1followed by 2 gm/m 2 /72 h ara-C beginning day 6 and 40 mg/m 2 mitoxantrone on day 9. Results: Flavopiridol caused a z50% decrease in peripheral blood blasts in 44% by median day 2 and z80% decrease in 26% by day 3. Self-limited tumor lysis occurred in 53%. Three (5%) died during therapy (2 multiorgan failure and 1 fungal pneumonia). Complete remissions (CR) were achieved in 12 of 15 (75%) newly diagnosed secondary AML, 18 of 24 (75%) first relapse after short CR (median CR, 9 months, including prior allotransplant), and 2 of 13 (15%) primary refractory but 0 of 10 multiply refractory AML. Disease-free survival for all CR patients is 40% at 2 years, with newly diagnosed patients having a 2-year disease-free survival of 50%. Conclusions: Flavopiridol has anti-AML activity directly and in combination with ara-C and mitoxantrone. This timed sequential regimen induces durable CRs in a significant proportion of adults with newly diagnosed secondary AML (including complex cytogenetics) and adults with AML in first relapse after short first CR.