FLT3 inhibitors in acute myeloid leukemia

FLT3 mutations are one of the most common findings in acute myeloid leukemia (AML). FLT3 inhibitors have been in active clinical development. Midostaurin as the first-in-class FLT3 inhibitor has been approved for treatment of patients with FLT3-mutated AML. In this review, we summarized the preclinical and clinical studies on new FLT3 inhibitors, including sorafenib, lestaurtinib, sunitinib, tandutinib, quizartinib, midostaurin, gilteritinib, crenolanib, cabozantinib, Sel24-B489, G-749, AMG 925, TTT-3002, and FF-10101. New generation FLT3 inhibitors and combination therapies may overcome resistance to first-generation agents.     

Pre-B cell receptor signaling in acute lymphoblastic leukemia

B cell lineage ALL represents by far the most frequent malignancy in children and is also common in adults. Despite significant advances over the past four decades, cytotoxic treatment strategies have recently reached a plateau with cure rates at 80 percent for children and 55 percent for adults. Relapse after cytotoxic drug treatment, initial drug-resistance and dose-limiting toxicity are among the most frequent complications of current therapy approaches. For this reason, pathway-specific treatment strategies in addition to cytotoxic drug treatment seem promising to further improve therapy options for ALL patients. In a recent study on 111 cases of pre-B cell-derived human ALL, we found that ALL cells carrying a BCR-ABL1-gene rearrangement lack expression of a functional pre-B cell receptor in virtually all cases. In a proof-of-principle experiment, we studied pre-B cell receptor function during progressive leukemic transformation of pre-B cells in BCR-ABL1-transgenic mice: Interestingly, signaling from the pre-B cell receptor and the oncogenic BCR-ABL1 kinase are mutually exclusive and only "crippled" pre-B cells that fail to express a functional pre-B cell receptor are permissive to transformation by BCR-ABL1.     

Survey of activated FLT3 signaling in leukemia

Activating mutations of FMS-like tyrosine kinase-3 (FLT3) are found in approximately 30% of patients with acute myeloid leukemia (AML). FLT3 is therefore an attractive drug target. However, the molecular mechanisms by which FLT3 mutations lead to cell transformation in AML remain unclear. To develop a better understanding of FLT3 signaling as well as its downstream effectors, we performed detailed phosphoproteomic analysis of FLT3 signaling in human leukemia cells. We identified over 1000 tyrosine phosphorylation sites from about 750 proteins in both AML (wild type and mutant FLT3) and B cell acute lymphoblastic leukemia (normal and amplification of FLT3) cell lines. Furthermore, using stable isotope labeling by amino acids in cell culture (SILAC), we were able to quantified over 400 phosphorylation sites (pTyr, pSer, and pThr) that were responsive to FLT3 inhibition in FLT3 driven human leukemia cell lines. We also extended this phosphoproteomic analysis on bone marrow from primary AML patient samples, and identify over 200 tyrosine and 800 serine/threonine phosphorylation sites in vivo. This study showed that oncogenic FLT3 regulates proteins involving diverse cellular processes and affects multiple signaling pathways in human leukemia that we previously appreciated, such as Fc epsilon RI-mediated signaling, BCR, and CD40 signaling pathways. It provides a valuable resource for investigation of oncogenic FLT3 signaling in human leukemia.     

DNA profiling by arrayCGH in acute myeloid leukemia and myelodysplastic syndromes

Acute myeloid leukemia (AML) and myelodysplastic syndromes (MDS) represent two distinct but related myeloid haematological neoplasms. At diagnosis, a substantial proportion of cases show cytogenetic and molecular genetic markers whose range of specificity is highly variable. Most specific reciprocal translocations, as t(8;21)(q21;q21) or t(15;17)(q22;q21), have been extensively studied and are currently introduced in clinical diagnosis. Two other major groups remain to be better characterized at the genetic and genomic level: cases with normal karyotype and cases with complex aberrations. Comparative genomic hybridization (CGH) performed on chromosomes was the first approach taken and nearly 300 cases studied by this technique have already been reported. Array based CGH has also been applied to a smaller number of cases. Both types of genomic studies have confirmed that recurrent genomic losses and gains can almost exclusively be found in cases with complex karyotype. In most cases with normal karyotype (as well as in others with single chromosome aberrations as trisomy 8), arrayCGH has been able to unveil small DNA copy number changes whose recurrence is very low. Recently, single- nucleotide-polymorphism based arrays have been used in AML showing that loss of heterozygosity (LOH) is a common feature in normal karyotype leukemia.     

Over-expression of FoxM1 is associated with adverse prognosis and FLT3-ITD in acute myeloid leukemia

Forkhead box M1 (FoxM1) drives cell cycle progression and the prevention of growth arrest and is over-expressed in many human malignancies. However, the characteristics of FoxM1 in acute myeloid leukemia (AML) are not clearly understood. We investigated the expression level of FoxM1 and analyzed the correlation of FoxM1 expression with AML patient characteristics and prognoses. Changes in FoxM1 expression were detected after MV4–11 cells, which have an internal tandem duplication (ITD) of the fms-like tyrosine kinase 3 gene (FLT3-ITD), and control THP1 cells (encoding wild-type FLT3) were treated with the FLT3 receptor tyrosine kinase inhibitor AC220 (quizartinib) or FLT3 ligand (FL). Finally, we determined the apoptosis rates after the addition of the FoxM1 inhibitor thiostrepton (TST) to AML cells with or without FLT3-ITD. The expression of FoxM1 in AML patients was correlated with the presence of FLT3-ITD, genetic groups, and possibly overall survival. Inhibition of FLT3-ITD by AC220 down-regulated FoxM1 expression in MV4–11 cells, and stimulation of FLT3 by FL up-regulated FoxM1 expression in MV4–11 and THP1 cells. TST induced the apoptosis of MV4–11 and THP1 cells in a dose-dependent manner. Thus, FoxM1 is a potential prognostic marker and a promising therapeutic target in AML.     

Dendritic cell vaccination in acute myeloid leukemia

The prognosis of patients with acute myeloid leukemia (AML) remains dismal, with a 5-year overall survival rate of only 5.2% for the continuously growing subgroup of AML patients older than 65 years. These patients are generally not considered eligible for intensive chemotherapy and/or allogeneic hematopoietic stem cell transplantation because of high treatment-related morbidity and mortality, emphasizing the need for novel, less toxic, treatment alternatives. It is within this context that immunotherapy has gained attention in recent years. In this review, we focus on the use of dendritic cell (DC) vaccines for immunotherapy of AML. DC are central orchestrators of the immune system, bridging innate and adaptive immunity and critical to the induction of anti-leukemic immunity. We discuss the rationale and basic principles of DC-based therapy for AML and review the clinical experience that has been obtained so far with this form of immunotherapy for patients with AML.     

Functional Pathway Analysis Using SCNP of FLT3 Receptor Pathway Deregulation in AML Provides Prognostic Information Independent from Mutational Status

FMS-like tyrosine kinase 3 receptor (FLT3) internal tandem duplication (ITD) mutations result in constitutive activation of this receptor and have been shown to increase the risk of relapse in patients with acute myeloid leukemia (AML); however, substantial heterogeneity in clinical outcomes still exists within both the ITD mutated and unmutated AML subgroups, suggesting alternative mechanisms of disease relapse not accounted by FLT3 mutational status. Single cell network profiling (SCNP) is a multiparametric flow cytometry based assay that simultaneously measures, in a quantitative fashion and at the single cell level, both extracellular surface marker levels and changes in intracellular signaling proteins in response to extracellular modulators. We previously reported an initial characterization of FLT3 ITD-mediated signaling using SCNP. Herein SCNP was applied sequentially to two separate cohorts of samples collected from elderly AML patients at diagnosis. In the first (training) study, AML samples carrying unmutated, wild-type FLT3 (FLT3 WT) displayed a wide range of induced signaling, with a fraction having signaling profiles comparable to FLT3 ITD AML samples. Conversely, the FLT3 ITD AML samples displayed more homogeneous induced signaling, with the exception of patients with low (<40%) mutational load, which had profiles comparable to FLT3 WT AML samples. This observation was then confirmed in an independent (verification) cohort. Data from the second cohort were also used to assess the association between SCNP data and disease-free survival (DFS) in the context of FLT3 and nucleophosmin (NPM1) mutational status among patients who achieved complete remission (CR) to induction chemotherapy. The combination of SCNP read outs together with FLT3 and NPM1 molecular status improved the DFS prediction accuracy of the latter. Taken together, these results emphasize the value of comprehensive functional assessment of biologically relevant signaling pathways in AML as a basis for the development of highly predictive tests for guidance of post-remission therapy.     

Autocrine activation of the MET receptor tyrosine kinase in acute myeloid leukemia

Although the treatment of acute myeloid leukemia (AML) has improved substantially in the past three decades, more than half of all patients develop disease that is refractory to intensive chemotherapy. Functional genomics approaches offer a means to discover specific molecules mediating the aberrant growth and survival of cancer cells. Thus, using a loss-of-function RNA interference genomic screen, we identified the aberrant expression of hepatocyte growth factor (HGF) as a crucial element in AML pathogenesis. We found HGF expression leading to autocrine activation of its receptor tyrosine kinase, MET, in nearly half of the AML cell lines and clinical samples we studied. Genetic depletion of HGF or MET potently inhibited the growth and survival of HGF-expressing AML cells. However, leukemic cells treated with the specific MET kinase inhibitor crizotinib developed resistance resulting from compensatory upregulation of HGF expression, leading to the restoration of MET signaling. In cases of AML where MET is coactivated with other tyrosine kinases, such as fibroblast growth factor receptor 1 (FGFR1), concomitant inhibition of FGFR1 and MET blocked this compensatory HGF upregulation, resulting in sustained logarithmic cell killing both in vitro and in xenograft models in vivo. Our results show a widespread dependence of AML cells on autocrine activation of MET, as well as the key role of compensatory upregulation of HGF expression in maintaining leukemogenic signaling by this receptor. We anticipate that these findings will lead to the design of additional strategies to block adaptive cellular responses that drive compensatory ligand expression as an essential component of the targeted inhibition of oncogenic receptors in human cancers.     

(-)-Epigallocatechin gallate regulates CD3-mediated T cell receptor signaling in leukemia through the inhibition of ZAP-70 kinase

The zeta chain-associated 70-kDa protein (ZAP-70) of tyrosine kinase plays a critical role in T cell receptor-mediated signal transduction and the immune response. A high level of ZAP-70 expression is observed in leukemia, which suggests ZAP-70 as a logical target for immunomodulatory therapies. (-)-Epigallocatechin gallate (EGCG) is one of the major green tea catechins that is suggested to have a role as a preventive agent in cancer, obesity, diabetes, and cardiovascular disease. Here we identified ZAP-70 as an important and novel molecular target of EGCG in leukemia cells. ZAP-70 and EGCG displayed high binding affinity (Kd = 0.6207 micromol/liter), and additional results revealed that EGCG effectively suppressed ZAP-70, linker for the activation of T cells, phospholipase Cgamma1, extracellular signaling-regulated kinase, and MAPK kinase activities in CD3-activated T cell leukemia. Furthermore, the activation of activator protein-1 and interleukin-2 induced by CD3 was dose-dependently inhibited by EGCG treatment. Notably, EGCG dose-dependently induced caspase-mediated apoptosis in P116.cl39 ZAP-70-expressing leukemia cells, whereas P116 ZAP-70-deficient cells were resistant to EGCG treatment. Molecular docking studies, supported by site-directed mutagenesis experiments, showed that EGCG could form a series of intermolecular hydrogen bonds and hydrophobic interactions within the ATP binding domain, which may contribute to the stability of the ZAP-70-EGCG complex. Overall, these results strongly indicated that ZAP-70 activity was inhibited specifically by EGCG, which contributed to suppressing the CD3-mediated T cell-induced pathways in leukemia cells.     

The phosphoinositide 3-kinase/Akt pathway is activated by daunorubicin in human acute myeloid leukemia cell lines.

Daunorubicin induces apoptosis in myeloid leukemia cells by activation of neutral sphingomyelinase and ceramide generation occurring 4-10 min after daunorubicin addition. We show here that daunorubicin is able to increase the phosphoinositide 3-kinase activity and enhance intracellular phosphoinositide 3-kinase lipid products prior to ceramide generation. Daunorubicin activates Akt, a downstream phosphoinositide 3-kinase effector. Interestingly, the phosphoinositide 3-kinase inhibitors wortmannin and LY294002 accelerate daunorubicin-induced apoptosis in U937 cells. The phosphoinositide 3-kinase/Akt pathway has been involved in cell survival following serum deprivation, tumor necrosis factor alpha, anti-Fas and UV radiations. Our results suggest that anti-tumor agents such as daunorubicin may also activate anti-apoptotic signals that could contribute to drug resistance.     

Ring-shaped inclusions in an uncommon variety of acute myeloid leukemia (author's transl)

In cells of an acute, predominantly promyelocytic differentiated leukemia ring-shaped inclusions could be demonstrated by light- and electron-microscopic examinations. The patient showed good response to aggressive chemotherapy with complete remission. It is discussed, how far the presence of these morphological characteristics will have any bearing on the classification of this variety of leukemia.     

ATF3 coordinates serine and nucleotide metabolism to drive cell cycle progression in acute myeloid leukemia

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Prognostic significance of chromosome analysis in de novo acute myeloid leukemia (AML)

Between 1981 and 1986 cytogenetic studies of bone marrow and/or blood cells in 139 patients with de novo acute myeloid leukemia (AML) were performed. The overall incidence of chromosomal aberrations was 53%, and this was not significantly influenced by sex, age nor the FAB classification. The aberrations most often found were: complex anomalies (n = 13), t(8; 21) (n = 10), trisomy 8 (n = 9), monosomy 7 (n = 6), monosomy 5, 5q-, trisomy 11, 12p- (n = 4) and trisomy 6, 11q-, inv (n = 3). The prognostic significance of chromosomal findings was evaluated in 112 patients treated by combination chemotherapy. The chromosomal status NN, AN, AA did neither significantly influence complete remission rate (NN: 68%, AN: 71%, AA: 60%) nor mean survival (NN: 24, AN: 26.6, AA: 35.6 months). On the other hand, certain types of chromosomal anomalies were of prognostic value. CR was obtained in all 10 patients with t(8; 21) but only in 2 out of 9 patients with complex aberrations. Median duration of CR in patients with t(8; 21) was significantly longer than in patients with a normal karyotype (30 vs 7 months).