An overview on the role of FLT3-tyrosine kinase receptor in acute myeloid leukemia: biology and treatment.

Hematopoiesis, the process by which the hematopoietic stem cells and progenitors differentiate into blood cells of various lineages, involves complex interactions of transcription factors that modulate the expression of downstream genes and mediate proliferation and differentiation signals. Despite the many controls that regulate hematopoiesis, mutations in the regulatory genes capable of promoting leukemogenesis may occur. The FLT3 gene encodes a tyrosine kinase receptor that plays a key role in controlling survival, proliferation and differentiation of hematopoietic cells. Mutations in this gene are critical in causing a deregulation of the delicate balance between cell proliferation and differentiation. In this review, we provide an update on the structure, synthesis and activation of the FLT3 receptor and the subsequent activation of multiple downstream signaling pathways. We also review activating FLT3 mutations that are frequently identified in acute myeloid leukemia, cause activation of more complex downstream signaling pathways and promote leukemogenesis. Finally, FLT3 has emerged as an important target for molecular therapy. We, therefore, report on some recent therapies directed against it.

Protein tyrosine phosphatase receptor type {gamma} is a functional tumor suppressor gene specifically downregulated in chronic myeloid leukemia.

Chronic myelogenous leukemia (CML) is the most common myeloproliferative disease. Protein tyrosine phosphatase receptor type γ (PTPRG) is a tumor suppressor gene and a myeloid cell marker expressed by CD34(+) cells. Downregulation of PTPRG increases colony formation in the PTPRG-positive megakaryocytic cell lines MEG-01 and LAMA-84 but has no effect in the PTPRG-negative cell lines K562 and KYO-1. Its overexpression has an oncosuppressive effect in all these cell lines and is associated with myeloid differentiation and inhibition of BCR/ABL-dependent signaling. The intracellular domain of PTPRG directly interacts with BCR/ABL and CRKL, but not with signal transducers and activators of transcription 5. PTPRG is downregulated at the mRNA and protein levels in leukocytes of CML patients in both peripheral blood and bone marrow, including CD34(+) cells, and is reexpressed following molecular remission of disease. Reexpression was associated with a loss of methylation of a CpG island of PTPRG promoter occurring in 55% of the patients analyzed. In K562 cell line, the DNA hypomethylating agent 5-aza-2'-deoxycytidine induced PTPRG expression and caused an inhibition of colony formation, partially reverted by downregulation of PTPRG expression. These findings establish, for the first time, PTPRG as a tumor suppressor gene involved in the pathogenesis of CML, suggesting its use as a potential diagnostic and therapeutic target.

Medium adsorbance fraction of reticulocyte and myeloperoxidase index may individuate a patient subset with a low risk of chemotherapy-related neutropenia.

In neoplastic patients chemotherapy frequently involves severe myeloid suppression. Sometimes myeloid suppression is the main cause of therapy recycling delay with severe and prolonged neutropenia, anaemia and thrombocytopenia. Our study aimed to verify whether there is a correlation between reticulocyte fractions, reticulocyte indices, myeloperoxidase index (MPXI) and post-chemotherapy myelopoietic function and severe post-chemotherapy neutropenia. A cohort of 112 patients was identified, 30 with lymphoma or myeloma and 82 with solid neoplasms with bone marrow micrometastases. The patients were treated with chemotherapy (CT). After CT, 60 patients had neutropenia (ANC <500/mcl) for a median of 7 days (range 3-21). Before CT, myelopoietic function was assessed by the above-mentioned parameters using a hematologic automated analyzer. We assigned patients with an MPXI-positive value and medium adsorbance fraction of reticulocyte (MFR) >10.7% a score of 1, and a score of 0 was assigned to the remaining patients. Patients with a score of 1 showed a lower number of neutropenic events (only 9 out of 36 patients) than those with a score of 0 (51 out of 76 patients), p<0.0001. MPXI and MFR may be used in the assessment of myelopoiesis before CT administration, independently of the type of tumor, CT regimen and number of CT cycle, with the aim of identifying a patient subset with a lower risk of developing neutropenia post-CT.

Molecular and chromosomal alterations: new therapies for relapsed acute myeloid leukemia.

Acute myeloid leukemia (AML) remains the most common form of leukemia and the most common cause of leukemia death. Although conventional chemotherapy can cure between 25 and 45% of AML patients, the majority of patients die after relapse or of complications associated with treatment. Thus, more specific and less toxic treatments for AML patients are needed, especially for elderly patients. An indispensable prerequisite to investigate tailored approaches for AML is the recent progress in the understanding the molecular features that distinguish leukemia progenitors from normal hematopoietic counterparts and the identification of a variety of dysregulated molecular pathways. This in turn would allow the identification of tumor-specific characteristics that provide a rational basis for the development of more tailored, and hence potentially more effective and less toxic, therapeutic approaches. In this review, we describe some of the signaling pathways that are aberrantly regulated in AML, with a specific focus on their pathogenetic and therapeutic significance, and we examine some recent therapies directed against these targets, used in clinical trial for relapsed patients or unfit for conventional chemotherapy.

Monitoring of FLT3 phosphorylation status and its response to drugs by flow cytometry in AML blast cells.

FLT3 mutation and overexpression in most acute myeloid leukaemia (AML) patients make this tyrosine kinase receptor an attractive therapeutic target. FLT3 kinase inhibitors are actually in clinical trials, thus it is critical to develop a reproducible and standardized method for screening of FLT3 activation and for monitoring its inhibition in response to drug in AML patients. We developed a flow cytometry method to analyse phosphorylated FLT3 (P-FLT3) in samples with <10(5) cells. The method was first validated in FLT3 wild-type (HL60/WT) and mutant (MV4-11/ITD(+)) as well as FLT3 negative (K562) cell lines. The method also proved to be reproducible in AML patient samples. Analysis was performed after exposure to drugs (CEP-701 and SU11657), in vitro and in vivo. In response to increasing drug concentrations, there was a linear reduction in P-FLT3. Intracellular flow cytometry analysis correlated with Western blot and XTT assays; flow cytometry data also correlated with FLT3 mutational status. The results highlight a rapid method to detect P-FLT3 protein at the single cell level by flow cytometry which enables an accurate assessment of FLT3 kinase activity in blast cells in response to novel tyrosine kinase inhibitors.

NPM1 mutations are more stable than FLT3 mutations during the course of disease in patients with acute myeloid leukemia.

NPM1 mutations have been reported to be the most frequent mutations in acute myeloid leukemia (AML). They are associated with a wide spectrum of morphologic subtypes of AML, normal karyotype and FLT3 mutations. The high frequency of NPM1 mutations might provide a suitable marker for monitoring residual disease of AML.

Persistent Cdk2 inactivation drives growth arrest of BCR-ABL-expressing cells in response to dual inhibitor of SRC and ABL kinases SKI606.

Complementary inhibition of tyrosine and SRC kinases implement dual SRC/ABL inhibitor effects in chronic myeloid leukemia (CML). Here, we show that one such inhibitor, SKI-606, induces persistent Cdk2 inactivation leading to growth arrest of BCR-ABL-expressing cells either IM-sensitive or driven to IM-resistance by other events than gene overexpression and point mutations. Inhibition of Akt serine/threonine kinase, a phosphatidylinositol 3 kinase (PI-3k) target that integrates p210 TK signaling with membrane-associated SRC kinases, is a central component of restored expression and subcellular redistribution of Cdk2 regulatory signals (p21 and p27 and Cdc25A phosphatase) in response to SKI-606. The putative roles of growth factor (namely IL-3) autocrine loop in BCR-ABL-expressing progenitor progression towards a drug-resistant phenotype are discussed.

Application of flow cytometry to molecular medicine: detection of tumor necrosis factor-related apoptosis-inducing ligand receptors in acute myeloid leukaemia blasts.

TRAIL (tumor necrosis factor-related apoptosis-inducing ligand), a cytokine belonging to the TNF (tumor necrosis factor) family, is currently regarded as a potential anti-cancer agent. Nevertheless, several types of cancer cells display a low sensitivity to TRAIL or are completely resistant to this pro-apoptotic cytokine. TRAIL signalling is dependent on four receptors. Two of them, death receptors 4 and 5 (DR4 and DR5), induce apoptosis, whereas decoy receptors 1 and 2 (DcR1 and DcR2) are unable to evoke cell death upon TRAIL binding. TRAIL resistance may be related to the expression of TRAIL decoy receptors. TRAIL has been proposed as a novel therapeutic agent for the treatment of haematological disorders, including acute myeloid leukaemia (AML). Surprisingly, however, very limited information is available concerning the expression of TRAIL receptors in AML blasts. Here, we have evaluated, using flow cytometry, TRAIL receptor surface expression and sensitivity to TRAIL-dependent apoptosis of AML blasts from 30 patients. We observed frequent expression of TRAIL DcR1 and DcR2, while expression of DR4 and DR5 was less frequent. Nevertheless, the expression of DR4 or DR5 in leukaemic cells was always matched by a similar expression of one of the decoy receptors. Leukaemic blasts were invariably resistant, even to a high concentration (1000 ng/ml) of TRAIL. We suggest that AML blasts are resistant to TRAIL apoptosis in vitro. Therefore, it is unlikely that TRAIL alone might be used in the future as an innovative pharmacological agent for the treatment of AML.

Phosphoinositide 3-kinase/Akt inhibition increases arsenic trioxide-induced apoptosis of acute promyelocytic and T-cell leukaemias.

Recent studies suggest that the prosurvival signal transduction pathway involving phosphoinositide 3-kinase (PI3K)/Akt can confer an aggressive, apoptosis-resistant phenotype to acute leukaemia cells. We have investigated the effect of modulating this signalling pathway on the sensitivity of leukaemic cell lines (NB-4, CEM, Jurkat, MOLT-4) and acute promyelocytic primary blasts to apoptosis induced by 1 micromol/l As2O3. Whereas parental NB-4 cells did not display any phosphorylated (active) Akt, CEM, Jurkat and MOLT-4 cells exhibited high levels of Akt activation. Consistently, treatment of NB-4 cells with pharmacological inhibitors of the PI3K/Akt pathway (LY294002, wortmannin) did not increase sensitivity of these cells to arsenic trioxide (As2O3), whereas siRNA knock-down of Akt enhanced As2O3-induced apoptosis of CEM, Jurkat and MOLT-4 cells. Overexpression of a constitutively active Akt cDNA rendered NB-4 cells less susceptible to As2O3. Upon prolonged exposure to As2O3, we isolated a NB-4 cell clone that was resistant to As2O3 and displayed high levels of active Akt. LY294002 treatment of acute promyelocytic primary blasts with elevated Akt phosphorylation levels resulted in an increased sensitivity to As2O3. These results may provide a rationale for the development of combined or sequential treatment with PI3K/Akt inhibitors to improve the efficacy of As2O3 on acute leukaemias and also to overcome As2O3 resistance.

Deguelin, A PI3K/AKT inhibitor, enhances chemosensitivity of leukaemia cells with an active PI3K/AKT pathway.

Activation of the phosphoinositide 3 kinase (PI3K)/Akt signalling pathway has been linked with resistance to chemotherapeutic drugs, and its downregulation, by means of PI3K inhibitors, lowers resistance to various types of therapy in tumour cell lines. Recently, it has been reported that deguelin, a naturally occurring rotenoid, is a powerful inhibitor of PI3K. We investigated whether or not deguelin could enhance the sensitivity to chemotherapeutic drugs of human U937 leukaemia cells and acute myeloid leukaemia (AML) blasts with an activated PI3K/Akt network. Deguelin (10 nmol/l) induced S phase arrest with interference of progression to G2/M, and at 100 nmol/l significantly increased apoptotic cell death of U937. At 10-100 nmol/l concentrations, deguelin downregulated Akt phosphorylation of leukaemia cells and markedly increased sensitivity of U937 cells to etoposide or cytarabine. A 10 nmol/l concentration of deguelin did not negatively affect the survival rate of human cord blood CD34+ cells, whereas it increased sensitivity of AML blasts to cytarabine. Deguelin was less toxic than wortmannin on erythropoietin- and stem cell factor-induced erythropoiesis from CD34+ progenitor cells. Overall, our results indicate that deguelin might be used in the future for increasing sensitivity to therapeutic treatments of leukaemia cells with an active PI3K/Akt signalling network.

Superiority of thalidomide and dexamethasone over vincristine-doxorubicindexamethasone (VAD) as primary therapy in preparation for autologous transplantation for multiple myeloma.

The aim of the present study was to compare thalidomide-dexamethasone (Thal-Dex) and vincristine-doxorubicin-dexamethasone (VAD) as primary therapy in preparation for autologous peripheral blood stem-cell (PBSC) transplantation for multiple myeloma (MM). For this purpose, we performed a retrospective matched case-control analysis of 200 patients who entered 2 consecutive studies from 1996 to 2004 and received Thal-Dex (n = 100) or VAD (n = 100) administered for 4 months before collection of PBSCs and autologous transplantation. Matching criteria included age, clinical stage, and serum beta2-microglobulin levels. In comparison with VAD, Thal-Dex resulted in a significantly higher response rate (52% versus 76%, respectively; P < .001) and effected more profound reduction in myeloma cell mass of both immunoglobulin G (IgG; P = .02) and IgA (P = .03) type. More frequent toxicities included nonfatal deep vein thrombosis with Thal-Dex (15%) and granulocytopenia with VAD (12%). In each of the 2 treatment groups, 91% of patients proceeded to PBSC mobilization. The median number of collected CD34+ cells was 7.85 x 10(6)/kg in the Thal-Dex group and 10.5 x 10(6)/kg in the control group. Thal-Dex may be considered an effective and relatively well-tolerated oral alternative to the more complex VAD regimen as front-line therapy for MM patients who are candidates for subsequent autologous transplantation.

No preferential sensitivity of t(8;21) acute myeloid leukemias to cytosine arabinoside in vitro: is intensity of therapy or high dose Ara-C crucial for response?

Acute myeloid leukemia (AML) patients with core binding factor abnormalities [inv(16) or t(8;21)] have a relatively good prognosis, especially patients with inv(16) when treated with high-dose cytosine arabinoside (AraC) containing regimens, whereas in the case of t(8;21) evidences in favor of such regimen are contrasting. We previously demonstrated that blast cells from inv(16)-positive AML patients are characterized by an increased sensitivity to AraC with higher incorporation of 3H AraC into DNA and the increase of induced apoptosis in vitro. In the present study we tested the sensitivity of leukemic cells from 15 t(8;21)-positive AML patients to AraC and compared it with the results obtained from cells of 74 patients with inv(16), "intermediate" or "unfavourable" karyotype at diagnosis (for a total of 89 patients). The incorporation of 3H AraC into DNA in cells with t(8;21) was significantly lower than in cells with inv(16) (P = 0.02) or normal karyotype (P = 0.04). Interestingly, the incorporation of the drug into DNA in t(8;21) cells was similar to those with "unfavourable" karyotype. Furthermore, AraC induced apoptosis in t(8;21)-positive AML cells was not increased. These data suggest that the mechanism of response to chemotherapy for t(8;21)-positive cells is probably different then in AML cells with inv(16), underlining the possible importance for patients carrying the t(8;21) of repeated high-dose regimens and not necessarily of high-dose AraC based ones.

Detection of serine 473 phosphorylated Akt in acute myeloid leukaemia blasts by flow cytometry.

The phosphoinositide 3-kinase/Akt signalling pathway is a recently recognized important parameter in the prognosis and the response to treatment of acute myeloid leukaemia (AML). Akt kinase is activated by phosphorylation on Thr 308 and Ser 473. Active Akt promotes cell growth and survival to apoptotic insults. Thus, it seems important to evaluate Akt phosphorylation in AML blasts. This work aimed to establish whether it was possible to detect Akt phosphorylation on Ser 473 of AML blasts by means of flow cytometry. High levels of Akt activity and phosphorylation were detected in 13 of 15 cases of AML. Flow cytometric analysis revealed similar patterns of Ser 473 expression as was observed with Akt kinase activity and Western blot analysis of Thr 308 and Ser 473 phosphorylation. Double immunostaining enabled the simultaneous flow cytometric detection of an AML-associated antigen (CD33) and Ser 473 phosphorylated Akt in leukaemic blast populations. Our results indicate that flow cytometry enabled the rapid and quantitative assessment of Ser 473 phosphorylated Akt of AML blasts that, when used in combination with cell surface staining, can provide more accurate phenotyping of AML blasts.

Rapid detection of Flt3 mutations in acute myeloid leukemia patients by denaturing HPLC.

BACKGROUND: fms-related tyrosine kinase 3 (Flt3) is the most commonly mutated gene in human acute myeloid leukemia (AML) and has been implicated in its pathogenesis. Because screening of Flt3 in AML patients by PCR followed by gel electrophoresis is time-consuming and fails to detect some very small internal tandem duplications (ITDs), we developed a method for screening of FLT3 receptor mutations with PCR plus denaturing HPLC (D-HPLC). METHODS: Total mRNAs extracted from 34 AML patients were first analyzed for the presence of juxtamembrane length mutations and tyrosine kinase domain point mutations by a conventional method involving PCR amplification, restriction enzyme digestion, and agarose gel electrophoresis (PCR-RED-AGE). Subsequently, the same patient panel was analyzed by D-HPLC, using specifically designed primers and optimized running temperatures for the length and point mutation analysis. RESULTS: Thirty-four patients were analyzed by PCR-RED-AGE; 9 were positive for known Flt3 mutations: 6 of 34 (18%) for ITDs in exon 14 and 3 of 34 (9%) for point mutations in exon 20. The same patient panel was analyzed by D-HPLC, and additional nucleotide changes were discovered; in total, 14 sequence variations were identified: 7 of 34 (21%) for ITDs in exon 14; 2 of 34 (6%) for point mutations in exon 20; 1 of 34 (3%) for a new point mutation in exon 16; and 4 of 34 (12%) for polymorphisms in exons 13 and 14. Direct sequencing analysis identified nucleotide alterations in each of the "D-HPLC positives" but in none of the "D-HPLC negatives", yielding a specificity and sensitivity of 100% for D-HPLC-based screening. CONCLUSIONS: This novel D-HPLC-based procedure, which is optimized for identification of new point mutations in the catalytic and regulatory domains of FLT3 receptor, could potentially be useful for studies involving precise genotype determination, which could be critical for selection of innovative AML therapies targeting the FLT3 protein.

Cyclin D1 overexpression is a favorable prognostic variable for newly diagnosed multiple myeloma patients treated with high-dose chemotherapy and single or double autologous transplantation.

We used a sensitive real-time reverse transcription-polymerase chain reaction assay to quantify cyclin D1 mRNA levels in bone marrow samples collected at diagnosis from 74 newly diagnosed multiple myeloma (MM) patients who were randomized to undergo either single or double autologous peripheral blood stem cell transplantation as part of first-line therapy for their malignancy. In 46 cases, fluorescence in situ hybridization (FISH) analysis and/or conventional cytogenetics were performed to detect chromosome 11 abnormalities. Patients with the t(11;14) or trisomy 11 significantly overexpressed cyclin D1 (P <.0001) in comparison with patients without 11q abnormalities, who had cyclin D1 mRNA levels similar to healthy donors. Overall, 32 (43%) of 74 patients showed cyclin D1 overexpression. No difference was found between cyclin D1-positive (group A) and cyclin D1-negative (group B) patients with respect to presenting clinical and laboratory characteristics, including chromosome 13 abnormalities, as well as to response to therapy and overall survival, both of which were calculated on an intent-to-treat basis. Patients who overexpressed cyclin D1 had significantly longer duration of remission in comparison with patients who did not (41 vs 26 months, respectively; P =.02). As a result, median event-free survival (EFS) was longer in group A than in group B (33 vs 24 months, respectively; P =.055). We concluded that cyclin D1 overexpression is closely associated with 11q abnormalities and identifies a subset of MM patients who are more likely to have prolonged duration of remission and EFS following autologous transplantation.