Effect of purified fractions from cell culture supernate of high-density pre-B acute lymphoblastic leukemia cells (ALL3) on the growth of ALL3 cells at low density.

The mechanisms underlying the aberrant growth and interactions between cells are not understood very well. The pre-B acute lymphoblastic leukemia cells directly obtained from an adult patient grow very poorly or do not grow at all at low density (LD), but grow better at high starting cell density (HD). We found that the LD ALL3 cells can be stimulated to grow in the presence of diffusible, soluble factors secreted by ALL3 cells themselves growing at high starting cell density. We then developed a biochemical purification procedure that allowed us to purify the factor(s) with stimulatory activity and analyzed them by nanoliquid chromatography-tandem mass spectrometry (nanoLC-MS/MS). Using nanoLC-MS/MS we have identified several proteins which were further processed using various bioinformatics tools. This resulted in eight protein candidates which might be responsible for the growth activity on non-growing LD ALL3 cells and their involvement in the stimulatory activity are discussed.

The possible roles of B-cell novel protein-1 (BCNP1) in cellular signalling pathways and in cancer.

B-cell novel protein-1 (BCNP1) or Family member of 129C (FAM129C) was identified as a B-cell-specific plasma-membrane protein. Bioinformatics analysis predicted that BCNP1 might be heavily phosphorylated. The BCNP1 protein contains a pleckstrin homology (PH) domain, two proline-rich (PR) regions and a Leucine Zipper (LZ) domain suggesting that it may be involved in protein-protein interactions. Using The Cancer Genome Atlas (TCGA) data sets, we investigated the correlation of alteration of the BCNP1 copy-number changes and mutations in several cancer types. We also investigated the function of BCNP1 in cellular signalling pathways. We found that BCNP1 is highly altered in some types of cancers and that BCNP1 copy-number changes and mutations co-occur with other molecular alteration events for TP53 (tumour protein P53), PIK3CA (Phosphatidylinositol-4,5-Bisphosphate 3-Kinase, Catalytic Subunit Alpha), MAPK1 (mitogen-activated protein kinase-1; ERK: extracellular signal regulated kinase), KRAS (Kirsten rat sarcoma viral oncogene homolog) and AKT2 (V-Akt Murine Thymoma Viral Oncogene Homolog 2). We also found that PI3K (Phoshoinositide 3-kinase) inhibition and p38 MAPK (p38 mitogen-activated protein kinase) activation leads to reduction in phosphorylation of BCNP1 at serine residues, suggesting that BCNP1 phosphorylation is PI3K and p38MAPK dependent and that it might be involved in cancer. Its degradation depends on a proteasome-mediated pathway.

AFM study shows prominent physical changes in elasticity and pericellular layer in human acute leukemic cells due to inadequate cell-cell communication.

Biomechanical properties of single cells in vitro or ex vivo and their pericellular interfaces have recently attracted a lot of attention as a potential biophysical (and possibly prognostic) marker of various diseases and cell abnormalities. At the same time, the influence of the cell environment on the biomechanical properties of cells is not well studied. Here we use atomic force microscopy to demonstrate that cell-cell communication can have a profound effect on both cell elasticity and its pericellular coat. A human pre-B p190BCR/ABL acute lymphoblastic leukemia cell line (ALL3) was used in this study. Assuming that cell-cell communication is inversely proportional to the distance between cells, we study ALL3 cells in vitro growing at different cell densities. ALL3 cells demonstrate a clear density dependent behavior. These cells grow very well if started at a relatively high cell density (HD, >2 × 105 cells ml-1) and are poised to grow at low cell density (LD, <1 × 104 cells ml-1). Here we observe ∼6× increase in the elastic (Young's) modulus of the cell body and ∼3.6× decrease in the pericellular brush length of LD cells compared to HD ALL3 cells. The difference observed in the elastic modulus is much larger than typically reported for pathologically transformed cells. Thus, cell-cell communication must be taken into account when studying biomechanics of cells, in particular, correlating cell phenotype and its biophysical properties.

Exosome mediated growth effect on the non-growing pre-B acute lymphoblastic leukemia cells at low starting cell density.

Tumors contain heterogeneous cell populations and achieve dominance by functioning as collective systems. The mechanisms underlying the aberrant growth and interactions between cells are not very well understood. The pre-B acute lymphoblastic leukemia cells we studied were obtained directly from a patient with Ph+ ALL. A new Ph+ ALL cell line (ALL3) was established from the leukemic cells growing as ascitic cells in his pleural fluid. The patient died of his disease shortly after the cells were obtained. ALL3 cells grow well at high cell densities (HD), but not at low cell densities. ALL3 cells are very sensitive to potent tyrosine kinase inhibitors (TKIs) such as Dasatinib and PD166325, but less sensitive to AMN 107, Imatinib, and BMS 214662 (a farnesyl transferase inhibitor). Here, we show that the growth of the LD ALL3 cells can be stimulated to grow in the presence of diffusible, soluble factors secreted by ALL3 cells themselves growing at high density. We also show that exosomes, part of the secretome components, are also able to stimulate the growth of the non-growing LD ALL3 cells and modulate their proliferative behavior. Characterization of the exosome particles also showed that the HD ALL3 cells are able to secret them in large quantities and that they are capable of inducing the growth of the LD ALL3 cells without which they will not survive. Direct stimulation of non-growing LD ALL3 cells using purified exosomes shows that the ALL3 cells can also communicate with each other by means of exchange of exosomes independently of direct cell-cell contacts or diffusible soluble stimulatory factors secreted by HD ALL3 cells.

Defective quorum sensing of acute lymphoblastic leukemic cells: evidence of collective behavior of leukemic populations as semi-autonomous aberrant ecosystems.

Quorum sensing (QS) is a generic term used to describe cell-cell communication and collective decision making by bacterial and social insects to regulate the expression of specific genes in controlling cell density and other properties of the populations in response to nutrient supply or changes in the environment. QS mechanisms also have a role in higher organisms in maintaining homeostasis, regulation of the immune system and collective behavior of cancer cell populations. In the present study, we used a p190(BCR-ABL) driven pre-B acute lymphoblastic leukemia (ALL3) cell line derived from the pleural fluid of a terminally ill patient with ALL to test the QS hypothesis in leukemia. ALL3 cells don't grow at low density (LD) in liquid media but grow progressively faster at increasingly high cell densities (HD) in contrast to other established leukemic cell lines that grow well at very low starting cell densities. The ALL3 cells at LD are poised to grow but shortly die without additional stimulation. Supernates of ALL3 cells (HDSN) and some other primary cells grown at HD stimulate the growth of the LD ALL3 cells without which they won't survive. To get further insight into the activation processes we performed microarray analysis of the LD ALL3 cells after stimulation with ALL3 HDSN at days 1, 3, and 6. This screen identified several candidate genes, and we linked them to signaling networks and their functions. We observed that genes involved in lipid, cholesterol, fatty acid metabolism, and B cell activation are most up- or down-regulated upon stimulation of the LD ALL3 cells using HDSN. We also discuss other pathways that are differentially expressed upon stimulation of the LD ALL3 cells. Our findings suggest that the Ph+ ALL population achieves dominance by functioning as a collective aberrant ecosystem subject to defective quorum-sensing regulatory mechanisms.

Biophysical differences between chronic myelogenous leukemic quiescent and proliferating stem/progenitor cells.

The treatment of chronic myeloid leukemia (CML), a clonal myeloproliferative disorder has improved recently, but most patients have not yet been cured. Some patients develop resistance to the available tyrosine kinase treatments. Persistence of residual quiescent CML stem cells (LSCs) that later resume proliferation is another common cause of recurrence or relapse of CML. Eradication of quiescent LSCs is a promising approach to prevent recurrence of CML. Here we report on new biophysical differences between quiescent and proliferating CD34+ LSCs, and speculate how this information could be of use to eradicate quiescent LSCs. Using AFM measurements on cells collected from four untreated CML patients, substantial differences are observed between quiescent and proliferating cells in the elastic modulus, pericellular brush length and its grafting density at the single cell level. The higher pericellular brush densities of quiescent LSCs are common for all samples. The significance of these observations is discussed.

Cancer secretomes and their place in supplementing other hallmarks of cancer.

The secretome includes all macromolecules secreted by cells, in particular conditions at defined times, allowing cell-cell communication. Cancer cell secretomes that are altered compared to normal cells have shown significant potential for elucidating cancer biology. Proteins of secretomes are secreted by various secretory pathways and can be studied using different methods. Cancer secretomes seem to play an important role in known hallmarks of cancers such as excessive proliferation, reduced apoptosis, immune invasion, angioneogenesis, alteration in energy metabolism, and development of resistance against anti-cancer therapy [1, 2]. If a significant role of an altered secretome can be identified in cancer cells, using advanced mass spectrometry-based techniques, this may allow researchers to screen and characterize the secretome proteins involved in cancer progression and open up new opportunities to develop new therapies. We aim to elaborate upon recent advances in cancer cell secretome analysis using different proteomics techniques. In this review, we highlight the role of the altered secretome in contributing to already recognized and emerging hallmarks of cancer and we discuss new challenges in the field of secretome analysis.

Automated mass spectrometry-based functional assay for the routine analysis of the secretome.

The secretome represents the set of proteins secreted into the extracellular space of cells. These proteins have been shown to play a major role in cell-cell communication. For example, recent observations revealed the presence of diffusible factors with proliferative properties in the secretome of cancer cells. Thus, a qualitative and quantitative analysis of the secretome could lead to the identification of these factors and consequently to the development of new therapeutic strategies. Here, we provide an automated simple and effective strategy to identify novel targets in the secretome of specifically treated cells using liquid chromatography-tandem mass spectrometry (LC-MS/MS). Furthermore, we explore the supportive role of mass spectrometry (MS) in the development of functional assays of identified secreted target molecules. Simplicity is achieved by growing cells in medium free of serum, which eliminates the need to remove the most abundant serum proteins and at the same time reduces disturbing matrix effects. Upon identification of these factors, their validation and characterization will follow. Moreover, this approach can also lead to the identification of proteins abnormally secreted, shed, or oversecreted by cells as response to a stimulus. Furthermore, we also discuss the problems that one may encounter. Finally, we discuss the broad application of automated MS-based proteomics, particularly in cancer research, highlighting new horizons for the use of MS.

Imatinib upregulates compensatory integrin signaling in a mouse model of gastrointestinal stromal tumor and is more effective when combined with dasatinib.

Activating mutations in the Kit receptor tyrosine kinase are associated with gastrointestinal stromal tumor (GIST). Imatinib inhibits Kit and is front-line therapy for GIST. However, imatinib most often elicits a partial response or stable disease, and most GIST patients who initially respond to imatinib eventually acquire resistance. Thus, improved treatment strategies for GIST are needed. We investigated the role of Src family kinases (SFK) in tumorigenesis in a mouse model of human GIST. The SFKs Src and Lyn were active in GIST, and surprisingly, imatinib treatment stimulated their phosphorylation/activation. We show that integrin signaling activates focal adhesion kinase and, consequently, SFKs in GIST and that imatinib enhances integrin signaling, implying a role for the extracellular matrix and integrin signaling in tumor maintenance and imatinib resistance. Dasatinib, an inhibitor of SFKs and Kit, inhibited SFK and focal adhesion kinase activation in GIST but also inhibited Kit and Kit-dependent downstream signaling pathways including phosphoinositide 3-kinase and mitogen-activated protein kinase, but not signal transducer and activator of transcription (STAT) signaling. Whereas dasatinib and imatinib alone both produced a minimal histopathologic response, combination therapy improved their efficacy, leading to increased necrosis in GIST. These results highlight the importance of SFK and STAT signaling in GIST and suggest that the clinical efficacy of imatinib may be limited by the stimulation of integrin signaling.

Structure-activity relationships of 6-(2,6-dichlorophenyl)-8-methyl-2-(phenylamino)pyrido[2,3-d]pyrimidin-7-ones: toward selective Abl inhibitors.

We report the design, synthesis, and structure-activity relationship (SAR) of a series of novel pyrido[2,3-d]pyrimidin-7-one compounds as potent Abl kinase inhibitors. We evaluate their specificity profile against a panel of human recombinant kinases, as well as their biological profile toward a panel of well-characterized cancer cell lines. Our study reveals that substitutions in the 3- and 4-positions of the phenylamino moiety lead to improved potency and improved selectivity both in target-based and cell-based assays. Altogether, our results provide an insight into the SAR of pyrido[2,3-d]pyrimidin-7-ones for the development of drug candidates with improved potency and selectivity for the targeted treatment of CML.

Mechanisms of sunitinib resistance in gastrointestinal stromal tumors harboring KITAY502-3ins mutation: an in vitro mutagenesis screen for drug resistance.

PURPOSE: Although tyrosine kinase inhibitors have improved survival in advanced gastrointestinal stromal tumor (GIST), complete response is rare and most patients eventually fail the first-line treatment with imatinib. Sunitinib malate is the only approved second-line therapy for patients with imatinib-resistant or imatinib-intolerant GIST. The clinical benefit of sunitinib is genotype-dependent in regards to both primary and secondary mutations, with GIST patients harboring the KIT(AY502-3ins) exon 9 mutation being the most sensitive. EXPERIMENTAL DESIGN: As sunitinib resistance is now emerging, our goal was to investigate mechanisms of progression and to test the efficacy of novel tyrosine kinase inhibitor on these resistant mutants in vitro. N-ethyl-N-nitrosourea mutagenesis of Ba/F3 cells expressing the KIT(AY502-3ins) mutant was used to investigate novel patterns of resistant mutations evolving in the presence of sunitinib. RESULTS: Tumors from patients who developed sunitinib resistance after at least 1 year of radiographic response were analyzed, showing similar findings of a primary KIT(AY502-3ins) mutation and a secondary mutation in the KIT activation loop. Ba/F3 cells expressing these sunitinib-resistant double mutants showed sensitivity to both dasatinib and nilotinib. CONCLUSIONS: Sunitinib resistance in GIST shares similar pathogenetic mechanisms identified in imatinib failure, with acquisition of secondary mutations in the activation domain after an extended initial response to the drug. Moreover, in vitro mutagenesis with or without N-ethyl-N-nitrosourea of Ba/F3 cells expressing KIT(AY502-3ins) showed acquisition of secondary mutations restricted to the second kinase domain of KIT. In contrast, in vitro resistance to imatinib produces a broader spectrum of secondary mutations including mutations in both KIT kinase domains.

Eradication of chronic myeloid leukemia stem cells: a novel mathematical model predicts no therapeutic benefit of adding G-CSF to imatinib.

Imatinib mesylate induces complete cytogenetic responses in patients with chronic myeloid leukemia (CML), yet many patients have detectable BCR-ABL transcripts in peripheral blood even after prolonged therapy. Bone marrow studies have shown that this residual disease resides within the stem cell compartment. Quiescence of leukemic stem cells has been suggested as a mechanism conferring insensitivity to imatinib, and exposure to the Granulocyte-Colony Stimulating Factor (G-CSF), together with imatinib, has led to a significant reduction in leukemic stem cells in vitro. In this paper, we design a novel mathematical model of stem cell quiescence to investigate the treatment response to imatinib and G-CSF. We find that the addition of G-CSF to an imatinib treatment protocol leads to observable effects only if the majority of leukemic stem cells are quiescent; otherwise it does not modulate the leukemic cell burden. The latter scenario is in agreement with clinical findings in a pilot study administering imatinib continuously or intermittently, with or without G-CSF (GIMI trial). Furthermore, our model predicts that the addition of G-CSF leads to a higher risk of resistance since it increases the production of cycling leukemic stem cells. Although the pilot study did not include enough patients to draw any conclusion with statistical significance, there were more cases of progression in the experimental arms as compared to continuous imatinib. Our results suggest that the additional use of G-CSF may be detrimental to patients in the clinic.

A solid-phase Bcr-Abl kinase assay in 96-well hydrogel plates.

Regulated phosphorylation by protein tyrosine kinases (PTKs), such as c-Abl, is critical to cellular homeostasis. In turn, once deregulated as in the chronic myeloid leukemia (CML) fusion protein Bcr-Abl, PTKs can promote cancer onset and progression. The dramatic success of the Bcr-Abl inhibitor imatinib as therapy for CML has inspired interest in other PTKs as targets for cancer drug discovery. Here we report a novel PTK activity and inhibition screening method using hydrogel-immobilized peptide substrates. Using acrylate crosslinkers, we tether peptides via terminal cysteines to thiol-presenting hydrogels in 96-well plates. These surfaces display low background and high reproducibility, allowing semiquantitative detection of peptide phosphorylation by recombinant c-Abl or by Bcr-Abl activity in cell extracts using traditional anti-phosphotyrosine immunodetection and chemifluorescence. The capabilities of this assay are demonstrated by performing model screens for inhibition with several commercially available PTK inhibitors and a collection of pyridopyrimidine Src/Abl dual inhibitors. This assay provides a practical method to measure the activity of a single kinase present in a whole cell lysate with high sensitivity and specificity as a valuable means for efficient small molecule screening.

Mutations in the EGFR kinase domain mediate STAT3 activation via IL-6 production in human lung adenocarcinomas.

Persistently activated or tyrosine-phosphorylated STAT3 (pSTAT3) is found in 50% of lung adenocarcinomas. pSTAT3 is found in primary adenocarcinomas and cell lines harboring somatic-activating mutations in the tyrosine kinase domain of EGFR. Treatment of cell lines with either an EGFR inhibitor or an src kinase inhibitor had no effect on pSTAT3 levels, whereas a pan-JAK inhibitor (P6) blocked activation of STAT3 and inhibited tumorigenesis. Cell lines expressing these persistently activated mutant EGFRs also produced high IL-6 levels, and blockade of the IL-6/gp130/JAK pathway led to a decrease in pSTAT3 levels. In addition, reduction of IL-6 levels by RNA interference led to a decrease in tumorigenesis. Introduction of persistently activated EGFR into immortalized breast epithelial cells led to tumorigenesis, IL-6 expression, and STAT3 activation, all of which could be inhibited with P6 or gp130 blockade. Furthermore, inhibition of EGFR activity in multiple cell lines partially blocked transcription of IL-6 and concurrently decreased production and release of IL-6. Finally, immunohistochemical analysis revealed a positive correlation between pSTAT3 and IL-6 positivity in primary lung adenocarcinomas. Therefore, mutant EGFR could activate the gp130/JAK/STAT3 pathway by means of IL-6 upregulation in primary human lung adenocarcinomas, making this pathway a potential target for cancer treatment.

Synthesis and biological evaluation of a fluorine-18 derivative of dasatinib.

Tyrosine kinases often play pivotal roles in the pathogenesis of cancer and are good candidates for therapeutic intervention and targeted molecular imaging. The precursor synthesis, radiosynthesis, and biological characterization of a fluorine-18 analog of dasatinib, a multitargeted kinase inhibitor, are reported. Compound 5 potently inhibits Abl, Src, and Kit kinases and inhibits K562 and M07e/p210bcr-abl human leukemic cell growth. Using positron emission tomography, we visualized K562 tumor xenografts in mice with [18F]-5.

Sorafenib inhibits the imatinib-resistant KITT670I gatekeeper mutation in gastrointestinal stromal tumor.

PURPOSE: Resistance is commonly acquired in patients with metastatic gastrointestinal stromal tumor who are treated with imatinib mesylate, often due to the development of secondary mutations in the KIT kinase domain. We sought to investigate the efficacy of second-line tyrosine kinase inhibitors, such as sorafenib, dasatinib, and nilotinib, against the commonly observed imatinib-resistant KIT mutations (KIT(V654A), KIT(T670I), KIT(D820Y), and KIT(N822K)) expressed in the Ba/F3 cellular system. EXPERIMENTAL DESIGN: In vitro drug screening of stable Ba/F3 KIT mutants recapitulating the genotype of imatinib-resistant patients harboring primary and secondary KIT mutations was investigated. Comparison was made to imatinib-sensitive Ba/F3 KIT mutant cells as well as Ba/F3 cells expressing only secondary KIT mutations. The efficacy of drug treatment was evaluated by proliferation and apoptosis assays, in addition to biochemical inhibition of KIT activation. RESULTS: Sorafenib was potent against all imatinib-resistant Ba/F3 KIT double mutants tested, including the gatekeeper secondary mutation KIT(WK557-8del/T670I), which was resistant to other kinase inhibitors. Although all three drugs tested decreased cell proliferation and inhibited KIT activation against exon 13 (KIT(V560del/V654A)) and exon 17 (KIT(V559D/D820Y)) double mutants, nilotinib did so at lower concentrations. CONCLUSIONS: Our results emphasize the need for tailored salvage therapy in imatinib-refractory gastrointestinal stromal tumors according to individual molecular mechanisms of resistance. The Ba/F3 KIT(WK557-8del/T670I) cells were sensitive only to sorafenib inhibition, whereas nilotinib was more potent on imatinib-resistant KIT(V560del/V654A) and KIT(V559D/D820Y) mutant cells than dasatinib and sorafenib.

L576P KIT mutation in anal melanomas correlates with KIT protein expression and is sensitive to specific kinase inhibition.

Activating mutations in either BRAF or NRAS are seen in a significant number of malignant melanomas, but their incidence appears to be dependent to ultraviolet light exposure. Thus, BRAF mutations have the highest incidence in non-chronic sun damaged (CSD), and are uncommon in acral, mucosal and CSD melanomas. More recently, activating KIT mutations have been described in rare cases of metastatic melanoma, without further reference to their clinical phenotypes. This finding is intriguing since KIT expression is downregulated in most melanomas progressing to more aggressive lesions. In this study, we investigated a group of anal melanomas for the presence of BRAF, NRAS, KIT and PDGFRA mutations. A heterozygous KIT exon 11 L576P substitution was identified in 3 of 20 cases tested. The 3 KIT mutation-carrying tumors were strongly immunopositive for KIT protein. No KIT mutations were identified in tumors with less than 4+ KIT immunostaining. NRAS mutation was identified in one tumor. No BRAF or PDGFRA mutations were identified in either KIT positive or negative anal melanomas. In vitro drug testing of stable transformant Ba/F3 KIT(L576P) mutant cells showed sensitivity for dasatinib (previously known as BMS-354825), a dual SRC/ABL kinase inhibitor, and imatinib. However, compared to an imatinib-sensitive KIT mutant, dasatinib was potent at lower doses than imatinib in the KIT(L576P) mutant. These results suggest that a subset of anal melanomas show activating KIT mutations, which are susceptible for therapy with specific kinase inhibitors.

Quantification of change in phosphorylation of BCR-ABL kinase and its substrates in response to Imatinib treatment in human chronic myelogenous leukemia cells.

Phosphorylation by the constitutively activated BCR-ABL tyrosine kinase is associated with the pathogenesis of the human chronic myelogenous leukemia (CML). It is difficult to characterize kinase response to stimuli or drug treatment because regulatory phosphorylation events are largely transient changes affecting low abundance proteins. Stable isotope labeling with amino acids in cell culture (SILAC) has emerged as a pivotal technology for quantitative proteomics. By metabolically labeling proteins with light or heavy tyrosine, we are able to quantify the change in phosphorylation of BCR-ABL kinase and its substrates in response to drug treatment in human CML cells. In this study, we observed that BCR-ABL kinase is phosphorylated at tyrosines 393 and 644, and that SH2-domain containing inositol phosphatase (SHIP)-2 and downstream of kinase (Dok)-2 are phosphorylated at tyrosine 1135 and 299, respectively. Based on the relative intensity of isotopic peptide pairs, we demonstrate that the level of phosphorylation of BCR-ABL kinase as well as SHIP-2 and Dok-2 is reduced approximately 90% upon treatment with Imatinib, a specific inhibitor of BCR-ABL kinase. Furthermore, proteins, such as SHIP-1, SH2-containing protein (SHC) and Casitas B-lineage lymphoma proto-oncogene (CBL), are also regulated by Imatinib. These results demonstrate the simplicity and utility of SILAC as a method to quantify dynamic changes in phosphorylation at specific sites in response to stimuli or drug treatment in cell culture.