Daniel B. Lipka

FLT3 as a therapeutic target in AML: still challenging after all these years

Mutations within the FMS-like tyrosine kinase 3 (FLT3) gene on chromosome 13q12 have been detected in up to 35% of acute myeloid leukemia (AML) patients and represent one of the most frequently identified genetic alterations in AML. Over the last years, FLT3 has emerged as a promising molecular target in therapy of AML. Here, we review results of clinical trials and of correlative laboratory studies using small molecule FLT3 tyrosine kinase inhibitors (TKIs) in AML patients. We also review mechanisms of primary and secondary drug resistance to FLT3-TKI, and from the data currently available we summarize lessons learned from FLT3-TKI monotherapy. Finally, for using FLT3 as a molecular target, we discuss novel strategies to overcome treatment failure and to improve FLT3 inhibitor therapy.

Identification of Regulatory Networks in HSCs and Their Immediate Progeny via Integrated Proteome, Transcriptome, and DNA Methylome Analysis

In this study, we present integrated quantitative proteome, transcriptome, and methylome analyses of hematopoietic stem cells (HSCs) and four multipotent progenitor (MPP) populations. From the characterization of more than 6,000 proteins, 27,000 transcripts, and 15,000 differentially methylated regions (DMRs), we identified coordinated changes associated with early differentiation steps. DMRs show continuous gain or loss of methylation during differentiation, and the overall change in DNA methylation correlates inversely with gene expression at key loci. Our data reveal the differential expression landscape of 493 transcription factors and 682 lncRNAs and highlight specific expression clusters operating in HSCs. We also found an unexpectedly dynamic pattern of transcript isoform regulation, suggesting a critical regulatory role during HSC differentiation, and a cell cycle/DNA repair signature associated with multipotency in MPP2 cells. This study provides a comprehensive genome-wide resource for the functional exploration of molecular, cellular, and epigenetic regulation at the top of the hematopoietic hierarchy.

Disclosure of Candidate Genes in Acute Myeloid Leukemia With Complex Karyotypes Using Microarray-Based Molecular Characterization

PURPOSE To identify novel genomic regions of interest in acute myeloid leukemia (AML) with complex karyotypes, we applied comparative genomic hybridization to microarrays (array-CGH), allowing high-resolution genome-wide screening of genomic imbalances. PATIENTS AND METHODS Sixty AML cases with complex karyotypes were analyzed using array-CGH; parallel analysis of gene expression was performed in a subset of cases. RESULTS Genomic losses were found more frequently than gains. The most frequent losses affected 5q (77%), 17p (55%), and 7q (45%), and the most frequent genomic gains 11q (40%) and 8q (38%). Critical segments could be delineated to genomic fragments of only 0.8 to a few megabase-pairs of DNA. In lost/gained regions, gene expression profiling detected a gene dosage effect with significant lower/higher average gene expression levels across the genes located in the respective regions. Furthermore, high-level DNA amplifications were identified in several regions: 11q23.3-q24.1 (n = 7), 21q22 (n = 6), 11q23.3 (n = 5), 13q12 (n = 3), 8q24 (n = 3), 9p24 (n = 2), 12p13 (n = 2), and 20q11 (n = 2). Parallel analysis of gene expression in critical amplicons displayed overexpressed candidate genes (eg, C8FW and MYC in 8q24). CONCLUSION In conclusion, a large spectrum of genomic imbalances, including novel recurring changes in AML with complex karyotypes, was identified using array-CGH. In addition, the combined analysis of array-CGH data with gene expression profiles allowed the detection of candidate genes involved in the pathogenesis of AML.

DNA methylation dynamics during B cell maturation underlie a continuum of disease phenotypes in chronic lymphocytic leukemia

Charting differences between tumors and normal tissue is a mainstay of cancer research. However, clonal tumor expansion from complex normal tissue architectures potentially obscures cancer-specific events, including divergent epigenetic patterns. Using whole-genome bisulfite sequencing of normal B cell subsets, we observed broad epigenetic programming of selective transcription factor binding sites coincident with the degree of B cell maturation. By comparing normal B cells to malignant B cells from 268 patients with chronic lymphocytic leukemia (CLL), we showed that tumors derive largely from a continuum of maturation states reflected in normal developmental stages. Epigenetic maturation in CLL was associated with an indolent gene expression pattern and increasingly favorable clinical outcomes. We further uncovered that most previously reported tumor-specific methylation events are normally present in non-malignant B cells. Instead, we identified a potential pathogenic role for transcription factor dysregulation in CLL, where excess programming by EGR and NFAT with reduced EBF and AP-1 programming imbalances the normal B cell epigenetic program.

LS104, a non-ATP-competitive small-molecule inhibitor of JAK2, is potently inducing apoptosis in JAK2V617F-positive cells

The activating JAK2V617F mutation has been described in the majority of patients with BCR-ABL-negative myeloproliferative disorders (MPD). In this report, we characterize the small-molecule LS104 as a novel non-ATP-competitive JAK2 inhibitor: Treatment of JAK2V617F-positive cells with LS104 resulted in dose-dependent induction of apoptosis and inhibition of JAK2 autophosphorylation and of downstream targets. Activation of these targets by JAK2 was confirmed in experiments using small interfering RNA. LS104 inhibited JAK2 kinase activity in vitro. This effect was not reversible using elevated ATP concentrations, whereas variation of the kinase substrate peptide led to modulation of the IC50 value for LS104. In line with these data, combination treatment using LS104 plus an ATP-competitive JAK2 inhibitor (JAK inhibitor I) led to synergistically increased apoptosis in JAK2V617F-positive cells. Furthermore, LS104 strongly inhibited cytokine-independent growth of endogenous erythroid colonies isolated from patients with JAK2V617F-positive MPD in vitro, whereas there was no significant effect on growth of myeloid colonies obtained from normal controls. Based on these data, we have recently started a phase I clinical trial of LS104 for patients with JAK2V617F-positive MPDs. To the best of our knowledge, this is the first report on a non-ATP-competitive kinase inhibitor being tested in a clinical trial. [Mol Cancer Ther 2008;7(5):1176–84]

DNMT and HDAC inhibitors induce cryptic transcription start sites encoded in long terminal repeats

Several mechanisms of action have been proposed for DNA methyltransferase and histone deacetylase inhibitors (DNMTi and HDACi), primarily based on candidate-gene approaches. However, less is known about their genome-wide transcriptional and epigenomic consequences. By mapping global transcription start site (TSS) and chromatin dynamics, we observed the cryptic transcription of thousands of treatment-induced non-annotated TSSs (TINATs) following DNMTi and HDACi treatment. The resulting transcripts frequently splice into protein-coding exons and encode truncated or chimeric ORFs translated into products with predicted abnormal or immunogenic functions. TINAT transcription after DNMTi treatment coincided with DNA hypomethylation and gain of classical promoter histone marks, while HDACi specifically induced a subset of TINATs in association with H2AK9ac, H3K14ac, and H3K23ac. Despite this mechanistic difference, both inhibitors convergently induced transcription from identical sites, as we found TINATs to be encoded in solitary long terminal repeats of the ERV9/LTR12 family, which are epigenetically repressed in virtually all normal cells.

The kinase inhibitor LS104 induces apoptosis, enhances cytotoxic effects of chemotherapeutic drugs and is targeting the receptor tyrosine kinase FLT3 in acute myeloid leukemia

Activating mutations of FLT3 are found in approximately one-third of acute myeloid leukemia (AML)-cases and are considered to represent an attractive therapeutic target. In this study, we report that the hydroxystyryl-acrylonitrile compound LS104 inhibits proliferation and induces potent cytotoxic effects in FLT3 expressing leukemic cells in vitro. Immunoblot and phosphoprotein-FACS analysis demonstrated inhibiton of phosphorylation of FLT3-ITD and of its downstream targets. In pharmacokinetic studies, a rapid and dose dependent cellular uptake of LS104 lasting up to 11h could be demonstrated. Combination of LS104 with chemotherapeutic agents markedly enhanced cytotoxic effects. Recently, a phase I clinical trial investigating LS104 in refractory/relapsed hematologic malignancies has been initiated.

Epigenetic stress responses induce muscle stem-cell ageing by Hoxa9 developmental signals

The functionality of stem cells declines during ageing, and this decline contributes to ageing-associated impairments in tissue regeneration and function. Alterations in developmental pathways have been associated with declines in stem-cell function during ageing, but the nature of this process remains poorly understood. Hox genes are key regulators of stem cells and tissue patterning during embryogenesis with an unknown role in ageing. Here we show that the epigenetic stress response in muscle stem cells (also known as satellite cells) differs between aged and young mice. The alteration includes aberrant global and site-specific induction of active chromatin marks in activated satellite cells from aged mice, resulting in the specific induction of Hoxa9 but not other Hox genes. Hoxa9 in turn activates several developmental pathways and represents a decisive factor that separates satellite cell gene expression in aged mice from that in young mice. The activated pathways include most of the currently known inhibitors of satellite cell function in ageing muscle, including Wnt, TGFβ, JAK/STAT and senescence signalling. Inhibition of aberrant chromatin activation or deletion of Hoxa9 improves satellite cell function and muscle regeneration in aged mice, whereas overexpression of Hoxa9 mimics ageing-associated defects in satellite cells from young mice, which can be rescued by the inhibition of Hoxa9-targeted developmental pathways. Together, these data delineate an altered epigenetic stress response in activated satellite cells from aged mice, which limits satellite cell function and muscle regeneration by Hoxa9-dependent activation of developmental pathways.

Identification of DNA methylation changes at cis-regulatory elements during early steps of HSC differentiation using tagmentation-based whole genome bisulfite sequencing

Epigenetic alterations during cellular differentiation are a key molecular mechanism which both instructs and reinforces the process of lineage commitment. Within the haematopoietic system, progressive changes in the DNA methylome of haematopoietic stem cells (HSCs) are essential for the effective production of mature blood cells. Inhibition or loss of function of the cellular DNA methylation machinery has been shown to lead to a severe perturbation in blood production and is also an important driver of malignant transformation. HSCs constitute a very rare cell population in the bone marrow, capable of life-long self-renewal and multi-lineage differentiation. The low abundance of HSCs has been a major technological barrier to the global analysis of the CpG methylation status within both HSCs and their immediate progeny, the multipotent progenitors (MPPs). Within this Extra View article, we review the current understanding of how the DNA methylome regulates normal and malignant hematopoiesis. We also discuss the current methodologies that are available for interrogating the DNA methylation status of HSCs and MPPs and describe a new data set that was generated using tagmentation-based whole genome bisulfite sequencing (TWGBS) in order to comprehensively map methylated cytosines using the limited amount of genomic DNA that can be harvested from rare cell populations. Extended analysis of this data set clearly demonstrates the added value of genome-wide sequencing of methylated cytosines and identifies novel important cis-acting regulatory regions that are dynamically remodeled during the first steps of haematopoietic differentiation.

Intracellular retention of ABL kinase inhibitors determines commitment to apoptosis in CML cells.

Clinical development of imatinib in CML established continuous target inhibition as a paradigm for successful tyrosine kinase inhibitor (TKI) therapy. However, recent reports suggested that transient potent target inhibition of BCR-ABL by high-dose TKI (HD-TKI) pulse-exposure is sufficient to irreversibly commit cells to apoptosis. Here, we report a novel mechanism of prolonged intracellular TKI activity upon HD-TKI pulse-exposure (imatinib, dasatinib) in BCR-ABL-positive cells. Comprehensive mechanistic exploration revealed dramatic intracellular accumulation of TKIs which closely correlated with induction of apoptosis. Cells were rescued from apoptosis upon HD-TKI pulse either by repetitive drug wash-out or by overexpression of ABC-family drug transporters. Inhibition of ABCB1 restored sensitivity to HD-TKI pulse-exposure. Thus, our data provide evidence that intracellular drug retention crucially determines biological activity of imatinib and dasatinib. These studies may refine our current thinking on critical requirements of TKI dose and duration of target inhibition for biological activity of TKIs.