Much attention has recently been focused on PIM kinases as potential targets for the treatment of hematopoietic malignancies and some solid cancers. Using protein stability shift assays, we identified a family of imidazo [1,2-b]pyridazines to specifically interact with and inhibit PIM kinases with low nanomolar potency. The high-resolution crystal structure of a PIM1 inhibitor complex revealed that imidazo[1,2-b]pyrridazines surprisingly interact with the NH 2 -terminal lobe helix AC rather than with the kinase hinge region. Thus, the identified inhibitors are ATP competitive but not ATP mimetic compounds, explaining their enhanced selectivity with respect to conventional type I kinase inhibitors. One of the identified imidazo[1,2-b]pyridazines (K00135) was further tested in several hematopoietic cellular systems. First, K00135 dosedependently impaired survival of murine Ba/F3 cells that have been rendered cytokine independent by overexpression of human PIMs. Second, K00135 impaired survival and clonogenic growth of a panel of human acute leukemia cells. Third, exposure of K00135 significantly suppressed in vitro growth of leukemic blasts from five acute myelogenous leukemia patients but not of normal umbilical cord blood mononuclear cells. In vitro kinase assays and immunoblotting using lysates from human MV4;11 leukemic cells showed inhibition of phosphorylation of known PIM downstream targets, such as BAD and eukaryotic translation initiation factor 4E-binding protein 1, by K00135. Taken together, we report a family of small molecules that selectively interact and block PIM kinases and could serve as a lead to develop new targeted antileukemic therapeutics. [Cancer Res 2007;67(14):6916-24]

We assessed incidence and risk factors of cardiovascular events in 265 patients undergoing allogeneic hematopoietic stem-cell transplantation (HSCT) between 1980 and 2000 and who survived at least 2 years. Results were compared with a cohort of 145 patients treated during the same period with autologous HSCT. The median age of patients with allogeneic HSCT at last follow-up was 39 years, and median follow-up was 9 years. Eighteen (6.8%) patients after allogeneic and 3 (2.1%) patients after autologous HSCT experienced an arterial event. The cumulative incidence of first arterial event after allogeneic HSCT was 22.1% (95% CI, 12.0-40.9) at 25 years. The cumulative incidence 15 years after allogeneic HSCT was 7.5% as compared with 2.3% after autologous HSCT. Adjusting for age, risk of an arterial event was significantly higher after allogeneic HSCT (RR 6.92; P ‫؍‬ .009). In multivariate analysis, allogeneic HSCT (RR: 14.5; P ‫؍‬ .003), and at least 2 of 4 cardiovascular risk factors (hypertension, dyslipidemia, diabetes, obesity) (RR:

Acute promyelocytic leukemia (APL) is highly curable with the combination of alltrans retinoic acid (ATRA) and anthracyclinebased chemotherapy (CT), but very longterm results of this treatment, when CT should be added to ATRA and the role of maintenance treatment, remain uncertain. In our APL93 trial that included 576 newly diagnosed APL patients, with a median follow-up of 10 years, 10-year survival was 77%. Maintenance treatment significantly reduced 10-year cumulative incidence of relapses, from 43.2% to 33%, 23.4%, and 13.4% with no maintenance, maintenance using intermittent ATRA, continuous 6 mercaptopurine plus methotrexate, and both treatments, respectively (P < .001). Maintenance particularly benefited patients with white blood cell (WBC) count higher than 5 ؋ 10 9 /L (5000/L). Early addition of CT to ATRA significantly improved 10-year event-free survival (EFS), but without significant effect on overall survival (OS). The 10-year cumulative incidence of deaths in complete response (CR), resulting mainly from myelosuppression, was 5.7%, 15.4%, and 21.7% in patients younger than 55, 55 to 65, and older than 65 years, respectively, supporting the need for less myelosuppressive treatments, particularly for consolidation therapy. This study is registered at http:// clinicaltrials.gov as NCT00599937. (Blood.

This pilot study tested feasibility of natural killer cell purification and infusion (NK-DLI) in patients after haploidentical hematopoietic stem cell transplantation (HSCT). The aim was to obtain X1.0 Â 10 7 /kg CD56 þ /CD3À NK cells and o1.0 Â 10 5 /kg CD3 þ T cells. Mononuclear cells were collected by 10 l leukapheresis. A two-step ex vivo procedure was used to purify NK cells, using an immunomagnetic T-cell depletion, followed by NK-cell enrichment. Five patients with high-risk myeloid malignancies were included, presenting 3-12 months after a haploidentical HSCT with mixed chimerism (3), impending graft failure (1) or early relapse (1). The purified product contained a median of 1.61 Â 10 7 /kg (range 0.21-2.2) NK cells and 0.29 Â 10 5 /kg (0.11-1.1) T cells. A purity of NK cells of 97% (78-99), a recovery of 35.5% (13-75), and a T-cell depletion of 3.55 log (2.9-4.5) was achieved. Infusions were well tolerated and none of the patients developed graft-versus-host disease. We observed an increase in donor chimerism in 2/5, stable mixed chimerism, decreasing chimerism and relapse of AML in one patient each. Selection of NK-DLI is technically feasible. NK cells are well tolerated when used as adoptive immunotherapy in recipients of haploidentical HSCT.

All-trans retinoic acid (ATRA) plus anthracycline chemotherapy is the reference treatment of newly diagnosed acute promyelocytic leukemia (APL), whereas the role of cytosine arabinoside (AraC) remains disputed. We performed a joint analysis of patients younger than 65 years included in Programa para el Estudio de la Terapéutica en Hemopatía Maligna (PETHEMA) LPA 99 trial, where patients received no AraC in addition to ATRA, high cumulative dose idarubicin, and mitoxantrone, and APL 2000 trial, where patients received AraC in addition to ATRA and lower cumulative dose daunorubicin. In patients with white blood cell (WBC) count less than 10 ؋ 10 9 /L, complete remission (CR) rates were similar, but 3-year cumulative incidence of relapse (CIR) was significantly lower in LPA 99 trial: 4.2% versus 14.3% (P ‫؍‬ .03), although 3-year survival was similar in both trials. This suggested that AraC is not required in APL with WBC count less than 10 ؋ 10 9 /L, at least in trials with high-dose anthracycline and maintenance treatment. In patients with WBC of 10 ؋ 10 9 /L or more, however, the CR rate (95.1% vs 83.6% P ‫؍‬ .018) and 3-year survival (91.5% vs 80.8%, P ‫؍‬ .026) were significantly higher in APL 2000 trial, and there was a trend for lower 3-year CIR (9.9% vs 18.5%, P ‫؍‬ .12), suggesting a beneficial role for AraC in those patients. IntroductionAcute promyelocytic leukemia (APL) is a distinct subtype of acute myeloid leukemia characterized by its morphology, t(15; 17) translocation leading to PML-RARa fusion gene, and by a life-threatening coagulopathy. 1-5 All-trans retinoic acid (ATRA) combined with anthracycline-based chemotherapy yield complete remission (CR) rate in approximately 90% of newly diagnosed APLs and 25% to 30% subsequently relapse. 6 Maintenance treatment (especially combining continuous 6-mercaptopurine [6MP] and methotrexate [MTX] to ATRA) appears to further reduce the relapse risk to 10% to 15%. 6-9 Pretreatment white blood cell (WBC) count is the main factor associated with relapse in APL and a predictive model for relapse risk (Sanz score) based on pretreatment WBC and platelet counts allowing for the distinction among low-risk patients (with WBC count Ͻ 10 ϫ 10 9 /L and platelet count Ͼ 40 ϫ 10 9 /L), intermediaterisk patients (with WBC count Ͻ 10 ϫ 10 9 /L and platelet count Ͻ 40 ϫ 10 9 /L), and high-risk patients (with WBC count Ն 10 ϫ 10 9 /L). 10 Another sizable subset of patients die in CR from complications of consolidation treatment, mainly from infection due to chemotherapy-induced myelosuppression. 7,[11][12][13] To decrease mortality in CR, the Programa para el Estudio de la Terapéutica en Hemopatía Maligna (PETHEMA) group reduced the intensity of consolidation chemotherapy by avoiding cytosine arabinoside (AraC) in the chemotherapy regimen. 8,9 They observed CR rates comparable with regimens using a combination of AraC with anthracycline, a lower rate of death in CR (2%) and a low cumulative incidence of relapse (10%). On the other hand, the French-Belgian-Swiss APL group, i...

Adoptive immunotherapy with allogeneic purified natural killer (NK) cell products might exert graft-versus-tumor alloreactivity with little risk of GVHD. In a prospective phase II study in two centers, we administered purified NK cell products to high-risk patients treated with haploidentical T-cell-depleted SCT. Sixteen patients received a total of 29 NK cell infusions on days þ 3, þ 40 and þ 100 after transplantation. Median doses (and ranges) of infused NK-and T-cells per product were 1.21 (0.3-3.8) Â 10 7 /kg and 0.03 (0.004-0.72) Â 10 5 /kg, respectively. With a median follow-up of 5.8 years 4/16 patients are alive. Cause of death was relapse in five, GVHD in three, graft failure in three, and transplant related neurotoxicity in one patient. Four patients developed acute GVHDXgrade II, all receiving a total of X0.5 Â 10 5 T cells/kg. Compared with historical controls, NK cell infusions had no apparent effect on the rates of graft failure or relapse. Adoptive transfer of allogeneic NK cells is safe and feasible, but further studies are needed to determine the optimal dose and timing of NK cell therapy. Moreover, NK cell activation/expansion may be required to attain clinical benefit, while careful consideration must be given to the number of T cells infused.

AU-rich elements within the 3' untranslated region of transcripts of lymphokines and some protooncogenes serve as signal for rapid mRNA degradation. By using an AUUUA matrix, we have affinity-purified a 32-kDa protein, microsequenced it, and cloned the corresponding cDNA. In vitro, the recombinant protein bound specifically to AU-rich transcripts, including those for interleukin 3, granulocyte/macrophage colony-stimulating factor, c-fos, and c-myc. Sequence analysis revealed an unexpected homology to enoylCoA hydratase (EC 4.2.1.17), and the recombinant protein showed a low degree of the enzymatic activity. Thus, this gene, designated AUH, encodes an RNA binding protein with intrinsic enzymatic activity. Protein immobilized on an AUUUA matrix was enzymatically active, suggesting that hydratase and AU-binding functions are located on distinct domains within a single polypeptide.

Allogeneic natural killer (NK) cells are used for adoptive immunotherapy after stem cell transplantation. In order to overcome technical limitations in NK cell purification and activation, the following study investigates the impact of different variables on NK cell recovery, cytotoxicity, and T-cell depletion during good manufacturing practice (GMP)-grade NK cell selection. Forty NK cell products were derived from 54 unstimulated donor leukaphereses using immunomagnetic CD3 T-cell depletion, followed by a CD56 cell enrichment step. For T-cell depletion, either the depletion 2.1 program in single or double procedure (D2.11depl, n = 18; D2.12depl, n = 13) or the faster depletion 3.1 (D3.1, n = 9) was used on the CliniMACS instrument. Seventeen purified NK cell products were activated in vitro by IL-2 for 12 days. The whole process resulted in a median number of 7.59 × 108 CD56+CD3− cells with both purity and viability of 94%, respectively. The T-cell depletion was significantly better using D2.11depl/2depl compared to D3.1 (log 4.6/log 4.9 vs. log 3.7; p < 0.01) and double procedure in two stages led always to residual T cells below 0.1%. In contrast D3.1 was superior to D2.11depl/2depl with regard to recovery of CD56+CD3− NK cells (68% vs. 41%/38%). Concomitant monocytes and especially IL-2 activation led to increased NK cell activity against malignant target cells compared to unstimulated NK cells, which correlated with both up-regulation of natural cytotoxicity receptors and intracellular signaling. Overall, wide variations in the NK cell expansion rate and the distribution of NK cell subpopulations were found. In conclusion, our results indicate that GMP-grade purification of NK cells might be improved by a sequential processing of T-cell depletion program D2.1 and D3.1. In addition NK cell expansion protocols need to be further optimized.