Clinical responses achieved with FLT3 kinase inhibitors in acute myeloid leukemia (AML) are typically transient and partial. Thus, there is a need for identification of molecular mechanisms of ...clinical resistance to these drugs. In response, we characterized MOLM13 AML cell lines made resistant to two structurally-independent FLT3 inhibitors.
MOLM13 cells were made drug resistant via prolonged exposure to midostaurin and HG-7-85-01, respectively. Cell proliferation was determined by Trypan blue exclusion. Protein expression was assessed by immunoblotting, immunoprecipitation, and flow cytometry. Cycloheximide was used to determine protein half-life. RT-PCR was performed to determine FLT3 mRNA levels, and FISH analysis was performed to determine FLT3 gene expression.
We found that MOLM13 cells readily developed cross-resistance when exposed to either midostaurin or HG-7-85-01. Resistance in both lines was associated with dramatically elevated levels of cell surface FLT3 and elevated levels of phosphor-MAPK, but not phospho-STAT5. The increase in FLT3-ITD expression was at least in part due to reduced turnover of the receptor, with prolonged half-life. Importantly, the drug-resistant phenotype could be rapidly reversed upon withdrawal of either inhibitor. Consistent with this phenotype, no significant evidence of FLT3 gene amplification, kinase domain mutations, or elevated levels of mRNA was observed, suggesting that protein turnover may be part of an auto-regulatory pathway initiated by FLT3 kinase activity. Interestingly, FLT3 inhibitor resistance also correlated with resistance to cytosine arabinoside. Over-expression of FLT3 protein in response to kinase inhibitors may be part of a novel mechanism that could contribute to clinical resistance.
Novel drug discoveries have shifted the treatment paradigms of most hematological malignancies, including multiple myeloma (MM). However, this plasma cell malignancy remains incurable, and novel ...therapies are therefore urgently needed. Whole-genome transcriptome analyses in a large cohort of MM patients demonstrated that alterations in pre-mRNA splicing (AS) are frequent in MM. This manuscript describes approaches to identify disease-specific alterations in MM and proposes RNA-based therapeutic strategies to eradicate such alterations. As a "proof of concept", we examined the causes of aberrant HMMR (Hyaluronan-mediated motility receptor) splicing in MM. We identified clusters of single nucleotide variations (SNVs) in the HMMR transcript where the altered splicing took place. Using bioinformatics tools, we predicted SNVs and splicing factors that potentially contribute to aberrant HMMR splicing. Based on bioinformatic analyses and validation studies, we provided the rationale for RNA-based therapeutic strategies to selectively inhibit altered HMMR splicing in MM. Since splicing is a hallmark of many cancers, strategies described herein for target identification and the design of RNA-based therapeutics that inhibit gene splicing can be applied not only to other genes in MM but also more broadly to other hematological malignancies and solid tumors as well.
Current evidence suggests a significant role of aberrant splicing in the development and maintenance of malignancy. This multistep, tightly regulated epigenetic process leads to the production of ...abnormal proteins with abnormal functions contributing to underlying mechanisms of malignant transformation. Splicing patterns in malignant cells can be altered not only by the mutations detected on the aberrantly spliced gene, but also by the mutations detected on the genes encoding splicing factors. For example, aberrant pre-mRNA splicing, leading to intracellular or extracellular HA synthesis by HASs, contributes to the initiation and progression of various types of cancer. The influence of intracellular HA appears to be particularly significant and is promoted by aberrant splicing. In this review we report a model describing oncogenic potential of aberrant splicing, with a focus on HAS1 and intracellular HA. We also suggest that the influence of splicing mutations on malignant disease is likely multifactorial. For the triple axis of HA, HAS1 and RHAMM, mutations in HAS1 provide an indicator that these aberrations contribute to the events that lead to malignancy through increased risk and predisposition. Here, we also summarize the impact of splicing abnormalities on cancer and the possible oncogenic impact of aberrantly spliced HAS1. In conclusion, we emphasize that specific gene splice variants and the splicing process itself offer potential targets for novel drug treatment strategies.
It is becoming increasingly apparent that splicing defects play a key role in cancer, and that alterations in genomic splicing elements promote aberrant splicing. Alternative splicing increases the ...diversity of the human transcriptome and increases the numbers of functional gene products. However, dysregulation that leads to aberrant pre-mRNA splicing can contribute to cancer. Hyaluronan (HA), known to be an important component of cancer progression, is synthesized by hyaluronan synthases (HASs). In cancer cells, hyaluronan synthase 1 (HAS1) pre-mRNA is abnormally spliced to generate a family of aberrant splice variants (HAS1Vs) that synthesize extracellular and intracellular HA. HAS1Vs are clinically relevant, being found almost exclusively in malignant cells. Expression of aberrant HAS1Vs predicts poor survival in multiple myeloma. In this review, we summarize the unusual properties of HAS1Vs and their relationship to cancer. HAS1Vs form heterogeneous multimers with normally spliced HAS1 as well as with each other and with HAS3. Aberrant variants of HAS1 synthesize HA. Extracellular HA synthesized by HAS1Vs is likely to promote malignant spread. We speculate that synthesis of intracellular HA plays a fundamental and early role in oncogenesis by promoting genetic instability and the emergence of viable cancer variants that lead to aggressive disease.
Clinical studies of patients with chronic myeloid leukemia revealed that a common pattern of response is a dramatic fall in the circulating population of blast cells, with a minimal or delayed ...decrease in marrow blasts, suggesting a protective environment. These observations suggest that a greater understanding of the interaction of stromal cells with leukemic cells is essential. Here, we present an in vivo system for monitoring relative tumor accumulation in leukemic mice and residual disease in leukemic mice treated with a tyrosine kinase inhibitor and an in vitro system for identifying integral factors involved in stromal-mediated cytoprotection. Using the in vivo model, we observed high tumor burden/residual disease in tissues characterized as significant sources of hematopoiesis-promoting stroma, with bone marrow stroma most frequently showing the highest accumulation of leukemia in untreated and nilotinib-treated mice as well as partial protection of leukemic cells from the inhibitory effects of nilotinib. These studies, which showed a pattern of leukemia distribution consistent with what is observed in imatinib- and nilotinib-treated chronic myeloid leukemia patients, were followed by a more in-depth analysis of stroma-leukemia cell interactions that lead to protection of leukemia cells from nilotinib-induced cytotoxicity. For the latter, we used the human BCR-ABL-positive cell line, KU812F, and the human bone marrow stroma cell line, HS-5, to more closely approximate the bone marrow-associated cytoprotection observed in drug-treated leukemia patients. This in vitro system helped to elucidate stromal-secreted viability factors that may play a role in stromal-mediated cytoprotection of tyrosine kinase inhibitor-treated leukemia cells.
The study of the dielectric properties of micrometer- or nanometer-scale particles is of particular interest in present-day applications of biomedical engineering. Electrokinetics utilises ...electrically energised microelectrode structures within microfluidic chambers to noninvasively probe the physiological structure of live cancer cells.
A system is described that combines the three complementary techniques of dielectrophoresis (DEP), travelling wave dielectrophoresis (TWD) and electrorotation (ROT) for the first time on a single, integrated chip (3×6 mm).
The chip employs planar microelectrode arrays fabricated on a silicon substrate to facilitate the synthesis of the various nonuniform electric fields required for the controlled manipulation, measurement and characterization of mammalian cells.
A study of the dielectric properties of human malignant cells (Daudi and NCI-H929) was performed to demonstrate the potential and the versatility of the system in providing a fully programmable microsystem.
Genetic variations in the hyaluronan synthase 1 gene (HAS1) influence HAS1 aberrant splicing. HAS1 is aberrantly spliced in malignant cells from multiple myeloma (MM) and Waldenstrom ...macroglobulinemia (WM), but not in their counterparts from healthy donors. The presence of aberrant HAS1 splice variants predicts for poor survival in multiple myeloma (MM). We evaluated the influence of inherited HAS1 single nucleotide polymorphisms (SNP) on the risk of having a systemic B cell malignancy in 1414 individuals compromising 832 patients and 582 healthy controls, including familial analysis of an Icelandic kindred. We sequenced HAS1 gene segments from 181 patients with MM, 98 with monoclonal gammopathy of undetermined significance (MGUS), 72 with Waldenstrom macroglobulinemia (WM), 169 with chronic lymphocytic leukemia (CLL), as well as 34 members of a monoclonal gammopathy-prone Icelandic family, 212 age-matched healthy donors and a case-control cohort of 295 breast cancer patients with 353 healthy controls. Three linked single nucleotide polymorphisms (SNP) in HAS1 intron3 are significantly associated with B-cell malignancies (range p = 0.007 to p = 10(-5)), but not MGUS or breast cancer, and predict risk in a 34 member Icelandic family (p = 0.005, Odds Ratio = 5.8 (OR)), a relatively homogeneous cohort. In contrast, exon3 SNPs were not significantly different among the study groups. Pooled analyses showed a strong association between the linked HAS1 intron3 SNPs and B-cell malignancies (OR = 1.78), but not for sporadic MGUS or for breast cancer (OR<1.0). The minor allele genotypes of HAS1 SNPs are significantly more frequent in MM, WM, CLL and in affected members of a monoclonal gammopathy-prone family than they are in breast cancer, sporadic MGUS or healthy donors. These inherited changes may increase the risk for systemic B-cell malignancies but not for solid tumors.
Although the imatinib based therapy of chronic myeloid leukemia (CML) represents a triumph of medicine, not all patients with CML benefit from this drug due to the development of resistance and ...intolerance. The interruption of imatinib treatment is often followed by clinical relapse, suggesting a failure in the killing of residual leukaemic stem cells. There is need to identify alternative selective molecular targets for this disease and develop more effective therapeutic approaches. Alternative pre-mRNA splicing (AS) is an epigenetic process that greatly diversifies the repertoire of the transcriptome. AS orchestrates interactions between various types of proteins and between proteins and nucleic acids. Changes caused by individual splicing events in the cells are small, however, "splicing programs" typically react to these individual changes with considerable effects in cell proliferation, cell survival, and apoptosis. Current evidence suggests a pivotal role of AS in leukemias, particularly in myelodisplastic syndrome (MDS) and chronic lymphocyte leukemia (CLL). From these studies and studies in other malignances, it is clear that splicing abnormalities play a significant role in malignant transformation. Evaluation of AS events in CML can be used to identify novel disease markers and drugsensitive targets to overcome the limits of the small molecule inhibitors currently used for treating patients with CML. The use of aberrant splice variants as disease markers has been reported, however, little is known about the use of splicing abnormalities as drug targets in CML. Herein we discuss potential therapeutic approaches that can be used to target splicing abnormalities in CML.
Multiple myeloma (MM) plasma cells (PCs) express receptor for hyaluronan-mediated motility (RHAMM), a hyaluronan-binding, cytoskeleton and centrosome protein. The most abundant RHAMM isoforms in MM ...are full-length RHAMM (RHAMMFL) and the splice variant RHAMM-exon4. We separately examined the significance of RHAMM expression, and isoform balance, in 2 groups of MM patients. In oligonucleotide microarray experiments (n = 210, Arkansas), increasing RHAMM mRNA expression in MM PCs is strongly associated with osteolytic bone lesions (P < .001), and event-free (P = .05) and overall (P = .04) survival. Semiquantitative determination of RHAMM isoform expression (Alberta, Canada) used capillary electrophoretic detection and measurement of RHAMM-exon4/RHAMMFL reverse-transcriptase-polymerase chain reaction (RT-PCR) products. RHAMM isoforms are rarely expressed concurrently in single MM PCs; the pattern of isoform expression, at the single-cell level, is approximated in larger numbers of cells by the RHAMM-exon4/RHAMMFL ratio. Absolute RHAMM expression and the RHAMM-exon4/RHAMMFL ratio are only partially correlated in MM PCs; in cell lines, absolute RHAMM expression is elevated in mitosis, while RHAMM ratios remain stable. Temporal examination of MM patients' peripheral blood reveals that the RHAMM-exon4/RHAMMFL ratio increases with disease burden. The RHAMM-exon4/RHAMMFL ratio in diagnostic bone marrow samples (n = 101, Alberta) is an independent prognostic factor. Thus, expression and splicing of RHAMM are important molecular determinants of disease severity in MM. (Blood. 2004;104:1151-1158)
Fluorescently labeled peanut lectin agglutinin (PNA-FITC) was used to identify a subtype of mitochondria-rich (MR) cells in the gills of freshwater rainbow trout. In situ binding of PNA-FITC was ...visualized by inverted fluorescence microscopy and found to bind to cells on the trailing edge of the filament epithelium as demonstrated by differential interference contrast optics. The amount of PNA-FITC binding on the filament epithelium increased with cortisol pretreatment concomitant with an increased chloride cell fractional area as demonstrated by scanning electron microscopy. Dispersed gill cells were isolated by trypsinization and separated using a discontinuous Percoll density gradient. Cells migrating to the 1.06-1.09 g/ml interface were found to be MR as demonstrated by staining with the vital mitochondrial dye 4-(4-(dimethylamino)styryl)-N-methylpyridinium iodide and transmission electron microscopy (TEM). However, only approximately 40% of the MR cells were found to bind PNA-FITC. Cortisol pretreatment increased the relative numbers of MR cells isolated from the dispersed gill cell population, but the relative proportions of PNA binding cells remained unchanged. Ultrastructural analysis of isolated cells in the TEM demonstrated that the MR cell fraction was comprised of a mixed population of chloride cells and pavement cells.
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