The insulin-like growth factor-I receptor kinase inhibitor NVP-AEW541 induces apoptosis in acute myeloid leukemia cells exhibiting autocrine insulin-like growth factor-I secretion.

Insulin-like growth factor-I (IGF-I) and its receptor (IGF-IR) have been implicated in the pathophysiology of many human cancers, including those of hematopoietic lineage. We investigated the therapeutic potential of the novel IGF-IR tyrosine kinase activity inhibitor, NVP-AEW541, on human acute myeloid leukemia (AML) cells. NVP-AEW541 was tested on a HL60 cell subclone, which is dependent on autocrine secretion of IGF-I for survival and drug resistance, as well as primary drug resistant leukemia cells. NVP-AEW541 treatment (24 h) induced dephosphorylation of IGF-IR. NVP-AEW541 also caused Akt dephosphorylation and changes in the expression of key regulatory proteins of the cell cycle. At longer incubation times (48 h), NVP-AEW541-induced apoptotic cell death, as demonstrated by caspase-3 cleavage. Apoptosis was accompanied by decreased expression of anti-apoptotic proteins. NVP-AEW541 enhanced sensitivity of HL60 cells to either cytarabine or etoposide. Moreover, NVP-AEW541 reduced the clonogenic capacity of AML CD34(+) cells cultured in the presence of IGF-I. Chemoresistant AML blasts displayed enhanced IGF-I secretion, and were sensitized to etoposide-induced apoptosis by NVP-AEW541. Our findings indicate that NVP-AEW541 might be a promising therapeutic agent for the treatment of those AML cases characterized by IGF-I autocrine secretion.

Multidrug resistance-associated protein 1 expression is under the control of the phosphoinositide 3 kinase/Akt signal transduction network in human acute myelogenous leukemia blasts.

A high incidence of relapses following induction chemotherapy is a major hindrance to patient survival in acute myelogenous leukemia (AML). There is strong evidence that activation of the phosphoinositide 3 kinase (PI3K)/Akt signaling network plays a significant role in rendering AML blasts drug resistant. An important mechanism underlying drug resistance is represented by overexpression of membrane drug transporters such as multidrug resistance-associated protein 1 (MRP1) or 170-kDa P-glycoprotein (P-gp). Here, we present evidence that MRP1, but not P-gp, expression is under the control of the PI3K/Akt axis in AML blasts. We observed a highly significant correlation between levels of phosphorylated Akt and MRP1 expression in AML cells. Furthermore, incubation of AML blasts with wortmannin, a PI3K pharmacological inhibitor, resulted in lower levels of phosphorylated Akt, downregulated MRP1 expression, and decreased Rhodamine 123 extrusion in an in vitro functional dye efflux assay. We also demonstrate that wortmannin-dependent PI3K/Akt inhibition upregulated p53 protein levels in most AML cases, and this correlated with diminished MRP1 expression and enhanced phosphorylation of murine double minute 2 (MDM2). Taken together, these data suggest that PI3K/Akt activation may lead to the development of chemoresistance in AML blasts through a mechanism involving a p53-dependent suppression of MRP1 expression.

Frequent elevation of Akt kinase phosphorylation in blood marrow and peripheral blood mononuclear cells from high-risk myelodysplastic syndrome patients.

The serine/threonine kinase Akt, a downstream effector of phosphatidylinositol 3-kinase (PI3K), is known to play an important role in antiapoptotic signaling and has been implicated in the aggressiveness of a number of different human cancers including acute myeloid leukemia (AML). The progression of myelodysplastic syndromes (MDSs) to AML is thought to be associated with abrogation of apoptotic control mechanisms. However, little is known about signal transduction pathways which may be involved in enhanced survival of MDS cells. In this report, we have performed immunocytochemical and flow cytometric analysis to evaluate the levels of activated Akt in bone marrow or peripheral blood mononuclear cells from patients diagnosed with MDS. We observed high levels of Ser473 phosphorylated Akt (p-Akt) staining in 90% of the cases (n=22) diagnosed as high-risk MDS, whereas mononuclear cells from normal bone marrow or low-risk MDS patients showed low or absent Ser473 p-Akt staining. Furthermore, all high-risk MDS patients also demonstrated high expression of the Class I PI3K p110delta catalytic subunit and a decreased expression of PTEN. Taken together, our results suggest that Akt activation might be one of the factors contributing to the decreased apoptosis rate observed in patients with high-risk MDS.

Clinical efficacy and antiangiogenic activity of thalidomide in myelofibrosis with myeloid metaplasia. A pilot study.

Increased neoangiogenesis has been reported in myelofibrosis with myeloid metaplasia (MMM). Thus we studied the effects of thalidomide, an antiangiogenic drug, in 12 MMM patients. Before treatment, all the cases showed a significantly increased micro-vessel density (MVD); in all eight tested cases bFGF and VEGF plasma levels were higher than controls. All patients presented disease progression in the last 3 months with standard therapy, regarding splenomegaly, anemia and/or thrombocytopenia and/or hyperleukocytosis. Thalidomide was administered at daily doses increasing from 100 to 600 mg. Eleven out of 12 patients were evaluable. No progression of disease was seen during the treatment in any case. In particular, spleen size decreased in 7/11 patients, anemia improved in 3/4 (two are now transfusion independent), thrombocytopenia in 2/2 and hyperleukocytosis in 2/5 patients. Side-effects were frequent, although not severe. After treatment, VEGF and bFGF plasma levels varied widely and in selected cases decreased. In particular, VEGF and/or bFGF decreased in 4/5 responders and in 1/3 non-responders. Moreover, MVD significantly decreased in all the responders evaluated after treatment. We conclude that thalidomide is a feasible therapy in MMM patients and looks promising at least to control the growth progression of disease.

Pulsed ATRA as single therapy restores long-term remission in PML-RARalpha-positive acute promyelocytic leukemia patients: real time quantification of minimal residual disease. A pilot study.

All-trans retinoic acid (ATRA), alone or combined with chemotherapy (CHT) is widely used to induce complete remission (CR) in newly diagnosed acute promyelocytic leukemia (APL). If used alone, ATRA results in a substantial proportion of CRs. To maintain remission further, ATRA is commonly used with cycles of CHT, frequently followed by autologous (auto) or allogeneic (allo) stem cell transplantation (SCT), as early reports have shown that the continuous administration of ATRA as single therapy almost invariably leads to relapse in a short period of time (months). Pharmacokinetic studies have shown that induced resistance to ATRA is frequently suppressed by the intermittent use of the drug. In this study we applied an intermittent therapeutic protocol with ATRA in five APL patients who were either molecularly refractory after combined ATRA/CHT treatment, or relapsed, or at diagnosis, but not eligible for the combination treatment because of previous toxicity. They were treated with ATRA (45 mg/m2/day) for 21 days. The treatment was then prolonged continuously for 1 week every 2 weeks. Molecular analysis was performed by qualitative and quantitative reverse transcription-polymerase chain reaction (RT-PCR). All patients obtained molecular remission, as assessed by qualitative RT-PCR, in a median of 3 months (range 1-15). Quantitative RT-PCR confirmed these data, showing a progressive reduction (1 or 2 logs) to a 'negligible quantity' of PML-RARalpha fusion transcript (ratio PML-RARalpha/ABL x 10(4) ABL < 10(-1)) in all but one patient treated with pulsed ATRA therapy. These data were confirmed with qualitative and quantitative RT-PCR. After a median follow-up of 17 months from the start of ATRA therapy, 4/5 patients (80%) are in continuous complete molecular remission. To our knowledge, this is the first clinical observation that intermittent ATRA therapy (without chemotherapy) is effective not only in inducing but also in maintaining long-term molecular remission in APL patients. This approach could therefore be effective, if confirmed in larger series, in relapsed/refractory patients unsuitable for high-dose therapy and SCT; it may be proposed as induction therapy for selected older APL patients if considered not to be eligible for combined ATRA/CHT due to inadequate performance status or concurrent disease.

Salvage therapy with thalidomide in multiple myeloma patients relapsing after autologous peripheral blood stem cell transplantation.

BACKGROUND AND OBJECTIVES: The introduction of high-dose therapy with stem cell support has significantly improved the outcome of patients with multiple myeloma (MM) in terms of increased complete remission (CR) rate and extended survival, both disease-free and overall. Few options, however, are presently available for patients who relapse after single or double autologous stem cell transplantation (SCT). Thalidomide, a glutamic acid derivative with anti-angiogenetic properties, has been recently proposed as salvage treatment for such patients. The present study was aimed at evaluating thalidomide as single agent therapy for patients who had previously received autologous peripheral blood stem cell transplantation. DESIGN AND METHODS: From October 1999 to August 2000, 11 patients (7 males/4 females) who had relapsed after single (n = 4) or double (n = 7) autologous peripheral blood SCT were enrolled in the trial. Thalidomide, always employed as a single agent, was initially administered at a dose of 100 mg/day; if well tolerated, the dose was increased serially by 200 mg every other week to a maximum of 800 mg/day. RESULTS: The median administered dose was 600 mg/day. WHO grade > II toxic effects were constipation, lethargy, and leukopenia. Four patients (36%) showed > 50% reduction in serum M protein concentration and 4 showed > 25% reduction, for a total response rate averaging 72%. After a median follow-up of 5 months, 3 out of 8 responding patients are alive and progression-free and 5 patients have relapsed. INTERPRETATION AND CONCLUSIONS; These data confirm that thalidomide is active in poor-prognosis MM patients such as those relapsing after autologous SCT, and could thus deserve further testing in combination therapy.

Chemotherapy of secondary leukemias.

Chemotherapy of secondary leukemias is currently still considered to be associated with poor results. However, recent data suggest that the response to remission induction may substantially differ according to the previous medical history of the patients. Therapy related leukemia, arising following exposure to previous alkylating agents or radiotherapy, is often associated with chromosomal abnormalities involving chromosomes 5 and 7 and has a particularly bad response, whereas AML after exposure to epipodophyllotoxins or topoisomerase-II active agents could have a somewhat better response. Acute promyelocytic leukemia secondary to treatment of a primary malignant neoplasm seems to be associated with a better response if compared to other cytotypes of AML or to AML arising after transformation of myelodysplasia. However, here the literature data are not in full agreement, as different kinds of approaches have been applied. In fact, even if the problems encountered in treating patients with secondary leukemia are similar to those seen in patients with AML arising in a background of myelodysplasia (resistant disease and prolonged cytopenia after treatment), there are data suggesting that the use of high dose ara-C, with or without fludarabine, can circumvent resistance in a small but significant number of cases. One of the unsolved problems which still remains is how to consolidate the CR induced with high dose ara-C or with cycles based on anthracycline derivatives. In addition, another question relates to the categories of patients in whom chemotherapy may change the expected survival. Intensive post-remission chemotherapy, with or without autologous HSCT, may constitute an appropriate alternative for patients lacking a suitable sibling donor or for older patients who are in remission after chemotherapy and also able to tolerate other cycles of intensive chemotherapy. In this respect, the specific cytogenetic abnormality involved should be considered the most important prognostic factor for response and disease free survival; patients with abnormalities of chromosome 5 and 7 have a particularly low possibility of response and duration of CR. Furthermore, it is still debatable whether patients, especially the elderly, with these characteristics should go through a series of conventional treatments or just receive supportive treatment. On the other hand, patients with better prognostic factors should be entitled to further intensive treatments, taking into account possible delayed recovery and/or possible less successful collection of peripheral or marrow stem cells.

All-trans retinoic acid significantly reduces the incidence of early hemorrhagic death during induction therapy of acute promyelocytic leukemia.

Early hemorrhagic death (within the first 10 d of treatment [EHD]) is reported as the main cause of death during induction therapy for acute promyelocytic leukemia (APL). In order to evaluate possible differences in the incidence of EHD during induction regimens based on all-trans retinoic acid (ATRA), we retrospectively analyzed a consecutive series of 86 APL patients, diagnosed and treated at our Institution from 1982. Forty-three patients received combination chemotherapy with anthracyclines and cytosine arabinoside (January 1982 to December 1991), while induction of the remaining 43 was based on ATRA alone or on a combination of ATRA and anthracyclines (January 1992 to October 1996). There were significantly less induction deaths in the ATRA group [9 (chemotherapy group-CT) vs. 2 (ATRA group-RA) overall and 8(CT) vs. 1(RA) of EHD; p = 0.01]. Hemostatic evaluations showed an earlier reduction of D-dimer in the ATRA group. No cases of morphological resistance were observed in the ATRA group after induction. In addition, the number of relapses occurring in the first 24 months from the achievement of complete remission (CR) was significantly lower in the ATRA group (15 vs. 7; p = 0.01), with a disease free survival at 2 yr of 67% vs. 31%. In conclusion, ATRA appears to be able to significantly reduce the incidence of EHD, increasing the number of possible long-term remissions.