Impaired long-term expansion and self-renewal potential of pediatric acute myeloid leukemia-initiating cells by PTK787/ZK 222584.

Although most children with acute myeloid leukemia (AML) achieve complete remission, the relapse rate is 30% to 40%. Because it is thought that leukemia-initiating cells (LIC) are responsible for AML relapses, targeting these cells might improve outcome. Treatment of pediatric AML blasts with the receptor tyrosine kinase (RTK) inhibitor PTK787/ZK 222584 (PTK/ZK) induces cell death in vitro. However, the role of PTK/ZK inhibition on outgrowth of (pediatric) LICs is unknown. In this study, we cultured CD34+ cells from pediatric patients with AML on MS5 stromal cells in long-term cocultures. In analogy to adult AML, long-term expansion of leukemic cells up to 10 weeks could be generated in 9 of 13 pediatric AMLs. Addition of PTK/ZK to long-term cocultures significantly inhibited leukemic expansion in all samples, ranging from 4% to 80% growth inhibition at week 5 compared with untreated samples. In 75% of the samples, the inhibitory effect was more pronounced at week 10. Proteome profiler array analysis of downstream kinases revealed that PTK/ZK reduced activation of PI3K/Akt kinase signaling. Although main targets of PTK/ZK are VEGF receptors (VEGFR), no effect was seen on outgrowth of LICs when cultured with bevacizumab (monoclonal VEGFA-antibody), specific antibodies against VEGFR2 or VEGFR3, or exposed to stroma-derived VEGFA. These data suggest that the effect of PTK/ZK on LICs is not only dependent on inhibition of VEGFA/VEGFR signaling. Taken together, our data elucidated antileukemic properties of PTK/ZK in long-term expansion cultures, and suggest that targeting multiple RTKs by PTK/ZK might be a potential effective approach in eradicating (pediatric) LICs.

High acute myeloid leukemia derived VEGFA levels are associated with a specific vascular morphology in the leukemic bone marrow.

BACKGROUND: Acute Myeloid Leukemia (AML) bone marrow biopsies at diagnosis display enhanced angiogenesis and increased VEGFA expression. In a xenograft mouse model it was described that availability of free VEGFA versus bound VEGFA is related to different vascular morphology. In this study we investigate the relationship between vascular morphology within AML bone marrow biopsies and AML derived VEGFA levels. METHODS: Vessel count and surface area (Chalkley count) were calculated in AML bone marrow biopsies at diagnosis (n = 32), at remission (n = 8) and Normal Bone Marrow (n = 32) using immunohistochemical staining for FVIII, CD31, CTIV, SMA and VEGFA. VEGFA protein levels were measured. RESULTS: High vessel count was associated with an immature vessel status. Combining vessel count and Chalkley count different vessel morphology patterns were quantified within AML bone marrow biopsies. Three different subgroups could be distinguished. The subgroup (37.5% of the samples) exhibiting a high vessel count and vessels with predominantly large lumen (normal Chalkley count) was associated with high secreted VEGFA protein levels. CONCLUSION: Different vasculature patterns are seen in AML bone marrow biopsies, defined by combining number and size of vessel. These quantified morphology patterns, combined with VEGFA levels, might be of value in the success of VEGF/VEGFR-signaling interference approaches.

A new perspective on transcriptional system regulation (TSR): towards TSR profiling.

It has been hypothesized that the net expression of a gene is determined by the combined effects of various transcriptional system regulators (TSRs). However, characterizing the complexity of regulation of the transcriptome is a major challenge. Principal component analysis on 17,550 heterogeneous human microarray experiments revealed that 50 orthogonal factors (hereafter called TSRs) are able to capture 64% of the variability in expression in a wide range of experimental conditions and tissues. We identified gene clusters controlled in the same direction and show that gene expression can be conceptualized as a process influenced by a fairly limited set of TSRs. Furthermore, TSRs can be linked to biological functions, as we demonstrate a strong relation between TSR-related gene clusters and biological functionality as well as cellular localization, i.e. gene products of similarly regulated genes by a specific TSR are located in identical parts of a cell. Using 3,934 diverse mouse microarray experiments we found striking similarities in transcriptional system regulation between human and mouse. Our results give biological insights into regulation of the cellular transcriptome and provide a tool to characterize expression profiles with highly reliable TSRs instead of thousands of individual genes, leading to a >500-fold reduction of complexity with just 50 TSRs. This might open new avenues for those performing gene expression profiling studies.

Endogenous vascular endothelial growth factor-C expression is associated with decreased drug responsiveness in childhood acute myeloid leukemia.

PURPOSE: We hypothesized that downstream effects of endogenous vascular endothelial growth factor (VEGF)/VEGF receptor signaling on acute myelogenous leukemia (AML) cell survival resulted in increased in vitro cellular drug resistance and a longer time to kill most leukemic cells in vivo upon drug exposure. EXPERIMENTAL DESIGN: In primary AML cells from pediatric patients, VEGFA and VEGFC mRNA expression and in vitro cellular resistance to nine cytotoxic drugs were studied. As in vivo equivalents for in vitro drug resistance, in vivo AML blast reduction upon drug exposure, measured as blast cell reduction on day 15 in the bone marrow and as time in days from diagnosis to complete remission (CR) were used. RESULTS: Increased endogenous VEGFC levels significantly correlated with increased in vitro resistance for six typical AML drugs in primary AML cells from pediatric patients. Patients with >5% blasts on day 15 showed a 12.9-fold increase in the median VEGFC level compared with patients with < or =5% blasts (P = 0.002). Time to reach CR was studied using linear regression analysis with VEGFC, age at diagnosis, sex, treatment protocol, FAB type, cytogenetic risk profile, and WBC counts as variables. There was a significant positive independent association between VEGFC levels and time to CR (b = 6.02, SE = 1.58, P < or = 0.0001, n = 72). CONCLUSIONS: These results suggest for the first time that higher endogenous VEGFC levels of AML cells are related to decreased in vitro and in vivo drug responsiveness.

Addition of PTK787/ZK 222584 can lower the dosage of amsacrine to achieve equal amounts of acute myeloid leukemia cell death.

Acute myeloid leukemia (AML) is a disease with a poor prognosis. It has been demonstrated that AML cells express the vascular endothelial growth factors, VEGFA and VEGFC, as well as kinase insert domain-containing receptor (VEGFR2), the main receptor for downstream effects, resulting in an autocrine pathway for cell survival. This study investigates the role of the VEGFR inhibitor PTK787/ZK 222584 in leukemic cell death, and the possibility of an additional effect on cell death by a chemotherapeutic drug, amsacrine. In three AML cell lines and 33 pediatric AML patient samples, we performed total cell-kill assays to determine the percentages of cell death achieved by PTK787/ZK 222584 and/or amsacrine. Both drugs induced AML cell death. Using a response surface analysis, we could show that, in cell lines as well as in primary AML blasts, an equal magnitude of leukemic cell death could be obtained when lower doses of the more toxic amsacrine were combined with low dosages of the less toxic VEGFR inhibitor. This study shows that PTK787/ZK 222584 might have more clinical potential in AML when combined with a chemotherapeutic drug such as amsacrine. In future, it will be interesting to study whether the complications and the long-term effects of chemotherapy can be reduced by lowering the dosages of amsacrine, and by replacing it with other drugs with lower toxicity profiles, such as PTK787/ZK 222584.